TITLES AND AUTHORS OF LECTURES AND SYMPOSIA PRESENTATIONS
Plenary Lectures
PL1 - Exploring ensemble representations of olfactory stimuli: making scents of
intertwined spatial and temporal codes - J.G. Hildebrand, H. Lei, J.A. Riffell,
C.E. Reisenman
PL2 - Solitary Chemosensory Cells: An Ancient Vertebrate Chemosense Guarding The
Airways In Extant Mammals - Tom Finger
Firmenich Award
F-Aw - Dynamic ensemble odor coding in the mammalian olfactory bulb - Carleton A
Symposium 1
S1 Bimodal function of Drosophila odorant receptors - Dieter Wicher, Ronny
Schäfer, Marcus C. Stensmyr, Bill S. Hansson
S2 Insect chemosensation via ligand-gated cation channels - Koji Sato and
Kazushige Touhara
S3 Activation of Grueneberg ganglion cells in mice by alarm pheromones Brechbühl J, Hurni N and Broillet MC
S4 Molecular mechanisms of adaptation in mouse olfactory sensory neurons - Zhao
H, Song Y, Cygnar KD, Stephan AB, Hirsh S, Shum E, and Reisert J
S5 –
The styryl dye FM1-43 inhibits olfactory transduction in larval Xenopus laevis Breunig E, Kludt E, Manzini I, Schild D and Czesnik D
Symposium 2
S6 Visualizing stimulus convergence in amygdala neurons during taste aversion
learning - Ilene L. Bernstein and Sabiha Barot
S7 Translational Control in the Gustatory Cortex Determines the Stability of a Taste
Memory - Rosenblum K
S8 Integration in central neurons of gustatory information from four appendages in
the moth Heliothis virescens - Pål Kvello, Kari Jørgensen and Hanna Mustaparta
S9 Taste post-prandial categorization during endotoxin challenge: neural and
behavioral correlates - Pacheco-López, G.
S10 - Reversible inactivation of the dorsal hippocampus in aged rats and taste learning
- Gámiz F and Gallo M
S11 - Taste Memory impairments in Alzheimer-mice model is related with dopamine
release failure - Bermúdez-Rattoni F and Guzman-Ramos KR
Symposium 3
S12 - Sweet proteins, structure, production and function - Göran Hellekant
S13 - Efficient production and characterization of the sweet-tasting brazzein secreted
by the yeast pichia pastoris - Loïc Briand
S14 - Lactic acid bacteria as hosts for the expression of brazzein - Berlec A, Jevnikar
Z, Tompa G, Rogelj I, Štrukelj B
S15 -
Analyses of new brazzein derivatives - Tiffany Cragin, , D. Eric Walters, R. Jin,
Jon N. Rumbley, and Göran Hellekant
Symposium 4
S16 - The complexity of sensory perception in insects: electrophysiologial evidences De Cristofaro A, Anfora G, Germinara GS, Ioriatti C, Rotundo G, Tasin M,
Vitagliano S
S17 - The complexity of sensory perception in insects: the study case of codling moth
- Anfora G, Tasin M, De Cristofaro A
S18 - The complexity of sensory perception in insects: study of an aphid-plant-fungal
system - Ganassi S, Claudio A, De Cristofaro A, Andolfi A, Evidente A,
Sabatini MA
Symposium 5
S19 - ORL420, an example of a broadly-tuned human olfactory receptor - Veithen A,
Wilkin F, Philipeau M and Chatelain P
S20 - Birth of an olfactory receptor clade in the teleost lineage - Ashiq Hussain, Luis
Saraiva, Yen Yen Kwan, Sigrun Korsching
S21 - Localization of olfactory receptors heterologously expressed in S. cerevisiae
cells - Sanz G, Persuy MA, Vidic J, Wade F, Longin C, Corcelli A, Lobasso S,
Monnerie R, and Pajot E
S22 - Axon wiring and neural map formation in the mouse olfactory system - Sakano
H
S23 - The OR37 subfamily: establishment of the clustered expression pattern Strotmann J, Bader A, and Breer H
S24 - Olfaction Targeted - Mombaerts P
Symposium 6
S25 - The ESP peptide family and its role in chemical communication - Kazushige
Touhara
S26 - Dissecting an olfactory subsystem: GC-D neurons and the necklace glomeruli Munger SD
S27 - A new receptor family in the vomeronasal organ - Rivière S, Challet L, Fluegge
D, Spehr M, Rodriguez I
Symposium 7
S28 - Studying the functional integration of adult-born neurons in the mouse olfactory
bulb - Carleton A
S29 - Learning-dependent neurogenesis in the olfactory bulb determines remote
olfactory memory - Sultan S, Mandairon N, Kermen F, Garcia S, Sacquet J,
Didier A
S30 - Integration and survival of newly-formed neurons in the AOB of adult mice Paolo Peretto
Symposium 8
S31 - Electronic Nose Based Applications in Medicine - A.D’Amico
S32 - Detecting wound infection - Krishna C. Persaud, Anna Maria Pisanelli, Arthur
Bailey
S33 - Odor Communication for Olfactory Display - Jeong-Do Kim, Hyung-Gi Byun,
Sang-Goog Lee, Chang-Hoon Park, Jin-Ho Ahn and Jang-Hoon Lee
S34 Integration olfactory display into a video game for exercise - Changhoon Park,
Jin-Ho Ahn, Hyung-Gi Byun, Jeong-Do Kim, Sang-Goog Lee
S35 - Methods for building computer–controlled, fMRI–compatible, inexpensive
olfactometers suitable for event-related imaging designs - Lundström JN,
Gordon AR, Alden EC, Albrech J, Boesveldt S
Symposium 9
S36 - Scientific and nonscientific wine descriptors in wine language - Adrienne Lehrer
S37 - Communicating Taste - Bieler LM, Diederich C
S38 - Lexicon in the Sense of Smell: Etymology vs. Motivation. A Comparison
Between English and French - Rémi Digonnet
Symposium 10
S39 - Taste and food acceptance: sweet, bitter, salty and umami tastes, a complex
relationship - Piero A. Temussi
S40 - Sweet taste: from receptor to perception - Wolfgang Meyerhof, Marcel Winnig,
Bernd Bufe
S41 - Downstream signaling effectors in sweet taste - Kinnamon S, and Chaudhari N
Symposium 11
S42 - Adventurous TRPs: Chemoreception at the interface of nutritional and
biomedical sciences - Appendino G
S43 - Some like it cool : novel technologies for testing the menthol receptor - Sabrina
Corazza, Michela Stucchi
S44 - Function of TRP channels and cannabinoid CB1 receptors, and their interactions,
in chemoreception - Vincenzo Di Marzo
S45 - The TRPA1 channel: a sensor for oxidative stress and more - Pierangelo
Geppetti
Symposium 12
S46 - Context-dependent modulation of activity in the vertebrate olfactory epithelium Eisthen HL
S47 - Specialist Drosophila Avoids Costs of Appetitive Olfactory Learning - Teun
Dekker, Barbara Randlkofer, Peter Anderson, Holger Daniels, Stephan Knapek,
Bill S. Hansson
S48 – Olfactory plasticity: a key to survival for honey bee queens? - Vanina Vergoz,
Kyle T. Beggs, Lisa H. Geddes, Alastair Aiken, Jamie McQuillan and Alison R.
Mercer
S49 - Mating-dependent plasticity of the olfactory system in an insect - Anton S,
Barrozo RB, Jarriault D, and Gadenne C
S50 - Pheromone-plant odour interactions and mating effects in the antennal lobe of
Agrotis ipsilon males - Deisig N, Anton S, Gadenne C
S51 -
Increased dopamine after mating impairs olfaction and prevents odor
interference with pregnancy - Che Serguera, Viviana Triaca, Jakki Kelly-Barrett,
Mumna Al Banchaabouchi & Liliana Minichiello
LIST OF POSTERS
Section “Signal transduction, St”
1A - Highly specific responses to amine odorants of individual olfactory receptor neurons in situ
- Gliem S, Schild D and Manzini I
2A - Expression of the adiponectin receptor 1 in the olfactory mucosa of mice - Burry M-I, Hass
N, Schwarzenbacher K, Breer H
3A - Taste signaling elements in the gastrointestinal tract - Hass N, Schwarzenbacher K, Breer H
4A - Grueneberg ganglion - a dual sensory organ? - Fleischer J, Mamasuew K, Breer H
5A - Outgrowing olfactory axons contain the Reelin receptor VLDLR and navigate through the
reelin-rich cribriform mesenchyme - Schnaufer C, Breer H, Fleischer J
6A - Properties of the Ca2+-dependent chloride current in olfactory receptor neurons (ORNs) of
the moth S littoralis - Grauso M, Pézier A, Rospars J-P, Lucas P
7A - Characteriz ation of clonal cell lines derived from mouse taste buds and their responsivity to
gustatory stimuli. - Miyamoto T, Nishiyama M, Sako H, Tomooka
8A - TMEM16B is a novel Ca2+-activated Cl- channel candidate in vertebrate olfactory
transduction - Pifferi S, Dibattista M, Menini A
9A - The calcium-activated chloride current in the cilia of intact mouse olfactory sensory neurons
- Sagheddu C, Boccaccio A, Menini A
Section “Taste 1: periphery , T-1“
10A - Signalling Mechanisms in Mouse Bitter Responsive Taste Cells - Narukawa M,
Minamisawa E, Hayashi Y
11A - PKD1L3 and PKD2L1 in human sour taste perception - Stähler F, Nitschke S, Demgensky
S, Raguse JD, Meyerhof W
12A - Expression of taste reception-related molecules in circumvallate papilla and
gastrointestinal tract of developing rat - Wakisaka S, Okada H, Kadono K, Honma S,
Maeda T
13A - Relationship between salivary zinc and taste sensitivity - Tomassini Barbarossa I, Padiglia
A, Zonza A, Atzori E, Angioy AM
14A - Identification of vesicular nucleotide transporter (VNUT) in taste cells - Iwatsuki K,
Ichikawa R , Hiasa M, Moriyama Y, Torii K, Uneyama H
15A - The extra-oral taste buds in crucian carp - Kvernenes SA, Isachsen, C-J, Døving KB
16A - Bitter taste transduction involves potassium and calcium conductance in the blowfly
Protophormia terraenovae - Masala C, Solari P, Sollai G, Crnjar R, Liscia A
17A - Variations in Human Tas1R1 and Tas1R3 taste receptors affect taste responses to MSG in
functional assays - Raliou M, Grauso M, Schlegel-Lepoupon C, Trotier D, Nespoulous C,
Débat H, Pernollet JC, Faurion A, MontmayeurJP &, Briand L
18A - Vesicular Glutamate transporters localization in the rat lingual papillae- Zerari-Mailly F,
Braud A, Azerad J, Boucher Y
Section “Trigeminal Chemosensation, TC”
19A - TrpM5 channel in solitary chemosensory cells is necessary for the trigeminal respiratory
depression elicited by the bitter compound denatonium benzoate - Tizzano M,
Vandenbeuch A, Silver WL, Kinnamon SC, Finger TE
20A - Synthesis and biological activity on TRPA1 receptor of perillaketone analogues - Bassoli
A, Borgonovo G, Morini G, Calamello C, Di Marzo V, De Petrocellis L
Section “Taste 2 : CNS, T-2“
21A - Anatomical and functional relationships between trigeminal dental primary afferents and
lingual gustatory afferents within the NTS Braud A, Zerari-Mailly F, Felizardo R, Boucher
Y
22A - Effects of flavour prenatal exposure on taste related behavior during adolescence in rats Márquez R, Gámiz F, De la Casa G, Gallo M
23A - Behavioral assessment of the effect of a long-chain polyuns fatty acid supplemented diet in
aged rats - Díaz A, Ramírez M, Manrique T, Barranco A, Rueda R, Gallo M
24A - Hunger and Satiety Modulate Abnormal Brain Response to Taste in Restrained Eaters Murphy C and Kemmotsu N
Section “Aquatic organisms, Ao”
25A – Chemical alarm signals are not inducing morphology changes in crucian carp - Stabell OB,
Faeravaag AC, Tuvikene a
26A – Olfactory coding neurons of the black bullhead catfish Ameiurus melas - Valentini T. and
Dolenšek J.
Section “Olfaction 3: molecular aspects, O-3”
1B - The purinergic system of the olfactory epithelium: effects on sustentacular cells and
olfactory epithelium progenitors - Manzini I, Hassenklöver T, Kurtanska S, Junek S,
Bartoszek I, Schild D
2B - Inhibition of olfactory cytochrome P450-dependent activities modifies the electroolfactogram responses to quinoline and coumarin in rats - Thiebaud N, Veloso Da Silva
S, Jakob I, Sicard G, Artur Y, Heydel JM, Le Bon AM
3B - Retinoic acid-regulated gene expression and survival - Hörnberg M, Öztokatli H, Gussing
F, Berghard A, Bohm S
4B - Odorant receptors in olfactory cilia and axons - Strotmann J, Eberle J, Levai O, Breer H
5B - Uptake of odorant binding proteins in the olfactory epithelium - Brose H, Breer H,
Strotmann
J
6B - The Calmodulin-binding protein Neurogranin identifies a subpopulation of mature
granule interneurons in the adult mouse olfactory bulb - Gribaudo S, Bovetti S, Garzotto
D, Fasolo A, De Marchis S
7B - Rat olfactory epithelium RNA profiles are age related - Rimbault M, Robin S, Galibert F
8B - Determination of a number of olfactory receptor/ligand pairs of importance for explosives
and human odor detection - Robin S, Rimbault M, Benbernou N, Galibert F
9B - Characterization of olfactory receptors expression in BON cells - Sanz G, Legenre E,
Dewaele A, Pajot E
10B - Modulation of rat olfactory mucosa gene expression profile by nutritional status: a
preliminary microarray approach- Badonnel K, Durieux D, Salesse R, Caillol M and Baly
C
11B - The RFamide peptides influence feeding, locomotion, and odor response - Ruthann
Nichols
Section “Invertebrate chemosensation, Ic”
12B - Molecular and cellular basis of thermoreception of the malaria mosquito A gambiae - Qiu
YT, Wang G, Lu T, Kwon H-W, Pitts J, van Loon JJA, Takken W, Zwiebel LJ
13B - Olfactoy function in a viviparous - Poddighe S, Dekker T, Scala A, Angioy AM
14B - Novel natural ligands for OSNs in the coeloconic sensilla of Drosophila - Ibba I, Dekker
T, Cadeddu F, Hansson B, Angioy AM
15B - Functional study of gustatory receptors in Drosophila - Faucher C, Chyb S
16B - Implication of a putative UGT in Drosophila male pheromonal discrimination - Fraichard
S, Legendre A, Artur Y, Ferveur JF, Heydel JM
17B - Experience-dependent plasticity in the olfactory system of a moth - Kauer I, Minoli S,
Colson V, Marion-Poll F, Anton S
18B - A unique arrangement of male specific glomeruli in Helicoverpa assulta as compared to
related species and hybrid offsprings - Zhao XC, Dong J, Wang CZ, Berg BG
19B - Visualization of inhibitory interneurons in the olfactory pathways of the mot - Hoel Fjaerli
H, Halvorsen JM, Zhao XC, Berg BG
20B - Pheromones and biodiversity - monitoring change in a changing world - Larsson MC,
Svensson GP, Ryrholm N
21B - Olfactory responses of H. obsoletus to host plants volatile compounds - Riolo P, Minuz RL,
Anfora G, Rossi Stacconi MV , Isidoro1 N, Romani R
22B - Olfactory coding in trichoid sensilla of Anopheles gambiae s.s. - Suer RA, Qiu YT, van
Loon JJA, W. Takken
23B - Odorant Capture: Heterogeneity of Cuticular Lipid Coatings on Moth Trichoid Sensilla Baker TC
24B - Representation and discrimination of pheromones and plant odours in the antennal lobe of
codling moth males and females - Trona F, Ignell R, Witzgall P
Section “Olfaction 1: psychophysics, O-1”
1C - Memory and discrimination in three senses: olfaction, vision and kinesthesis - Møller P,
Piper D, Hartvig D, Mojet J, Köster EP
2C - A human pheromone affects central nervous face processing - Frey MCM, Weyers P, Pauli
P, Mühlberger A
3C - Odour-evoked Autobiographical Memory: Age and gender differences along the life spanZucco GM, Aiello L, Turuani L
4C - Do children remember the sensory properties of food as adults do?- Laureati M, Pagliarini
E, Mojet J, Köster EP
5C - Beyond the inheritance of olfactory preferences: How cognitive factors can implicitly
modulate preferences for odours - Coppin G, Delplanque S, Cayeux I, Porcherot C 2,
Margot C 2, Velazco MI 2, Sander D
6C - Short and long-term behavioural and physiological effects of a postnatal olfactory
deprivation - Gilbert C, Giroud S, Padzys GS, Martrette J-M, Boivin S, Trabalon
7C - Relationships between odour properties as a function of hedonic category - Djordjevic J,
Goodman A, Olsson MJ
8C - Crossmodal associations between vision and olfaction: Evidence from eye movements Seigneuric A, Durand K, Jiang T, Baudouin J-Y, Benoist Schaal B
9C - The Effect of Odor Image on Identification of City Gas Odor - Tomoko Matsubasa T and
Gomi Y
10C - Influence of information-manipulation of the same odor stimulus on cardiovascular
response - Kobayashi T, Akiyama Y, Nagano Y, Saito S, Kobayakawa T
11C - Does fetal and neonatal exposure to odour variety affect later response to novelty in mice?
- Hébert B, Lara E, Schaal B, Patris B
12C - Impact of pre-exposure on the perception of odorant mixtures in humans - Sinding C,
Coureaud G, Bervialle B, Schaal B, Thomas-Danguin T
13C - Characterization of industrial and agricultural odors in field studies using semantic
differential scaling - Sucker K, Both R, Müller F, Hangartner M, Winneke G
14C - ‘Health sniffers’: Ability, use and perceived importance of smell in a group of health
professionals - Johnston ANB, Fleischmann J, Mackay-Sim A
15C - Valence of odor mixtures - Olsson MJ, Brodin M, Moeller P
16C - Speed of processing olfactory information is reduced in old adults - Poncelet J, Rouby C,
Abriat A, Moustafa Bensafi
Section “Olfaction 2: CNS, O-2”
17C - Inheritance of pheromone neuroanatomy and physiology in European corn borer Kárpáti Z, Hansson BS, Dekker T
18C - Amygdalo-cortical interaction during odor fear acquisition session: Neurochem and
electrophys investigation - Hegoburu C, Sevelinges Y, Gervais R, Parrot S, Mouly AM
19C - How nasal airflow shapes odor representation at the olfactory bulb level - Courtiol E,
Thevenet M, Messaoudi B, Garcia S, Buonviso N
20C - Neuromodulators in the antennal lobe of the heliothine moth - Berg BG, Zhao XC
21C - Relationsh betw intrac activities, oscilllocal field pot and resp in olf bulb of freely breath
anesthet rat -Briffaud V, Fourcaud TN.,Cenier T,Garcia S,Buonviso N, Amat C
22C - Reading Vital Information from a Pattern-Independent Olfactory Neural Code - Haddad
R and Sobel N
23C - Beyond olfactory value to olfactory decision-making - Rolls ET, Grabenhorst F, Deco G
24C - Manganese-enhanced fMRI in olfaction: optimisation of Mn dose with minimal
deleterious effects upon odour induced behaviour in rats - Lehallier B, Ben Moussa A,
Ben Hassen W, Coureaud G, Rampin O, Schaal B, Maurin Y, Bonny JM
25C - Event related potentials in response to odor mixtures and single components - Brodin M,
Olsson MJ, Hummel T
26C - Newborn rabbits perceive odour mixtures elementally or configurally according to their
experience - Coureaud G, Sinding C, Crepeaux G, Schaal B, Thomas-Danguin T
27C - HCN channels in the olfactory bulb - Aho AM, Fried HU, Müller F, Kaupp UB
Pheromones: vertebrates (P)
1D - Social and hormonal status influence the emission of urinary molecules in the mouse Bondi’ M, Da Dalt L, Redaelli M, Gabai G, Zagotto G, Cavaggioni A, Mucignat C
2D - The importance of volatile and involatile urine components in stimulating female attraction
to individual male mice - Cheetham SA, Beynon RJ, Hurst JL
3D – Combinatorial and coordinated expression of the mouse major proteins (MUPs) may reflect
a modular principle of the olfactory - Novikov SN, Ermakova II, Fedorova EM, Mylnikov
SV
4D - Temporal changes in deposited urine scent marks - Platt JE, Armstrong SD, Cheetham SA,
Robertson DHL, Hurst JL, Beynon RJ
5D – Neurogenesis in the AOB of adult mice: Role of pheromonal perception on survival of
newborn neurons - Oboti L, Schellino R, Fasolo A, Peretto P
6D - Dissecting the complexity of major urinary protein signals in rodents - Hurst JL,
Cheetham SA, Davidson AJ, Armstrong SD, Garratt MG, Beynon RJ
Olfaction/Taste: flavour, fragance (O/T-f)
7D - Temporal contrast of salt delivery in mouth increases salt perception - Busch JLHC,
Tournier C, Knoop JE, Kooyman G, Smit G
8D - Correlation of In Vitro and In Vivo Activities of New Umami Tasting Neomenthyl
Derivatives - Ley JP, Stähler F, Batram C, Backes M, Paetz S, Looft J, Meyerhof W,
Krammer G
9D - Gas chromatography/sniffing port analysis for screening of impact odorants in maple
syrups - Fortin J, Sabik H, Martin N
10D- Taste and relativity in handbooks of physiology- Sylvain Farge
11D - Smell and taste of Perilla frutescens described by sensory and instrumental evaluation Laureati M and Pagliarini E
12D - Decrease in conditioned flavour preference after post-training CS-exposure seems to be
due to conditioned inhibition rather than to extinction: US devaluation effect failure and
evidence of retardation - Gonzalez F and Garcia-Burgos D
13D - Odour-Taste interactions: Sensory modulation or perceptual integration - Godinot N,
Pelletier C, Labbe D, Martin N
14D - Between Ions Discrimination Capacities Of The Gustatory System: The Tastes Of
Drinking Water - Puget S, Cure Y, Béno N, Piriou P, Guichard E, Thomas-Danguin T
15D - Food habit and taste perception: a North and South Vietnam comparative study - Patris B,
Do Vihn B, Nguyen D, Valentin D
16D - Infant preferences for food odours over the first 2 years of life - Monnery-Patris S, Rigal
N, Marlier L, Schaal B, Issanchou
17D - The influence of culture on the structure of odour-elicited emotions - Ferdenzi C,
Grandjean D, Roberts SC, Schirmer A, Delplanque S, Chrea C, Cayeux, Margot C,
Velazco MI, Sander D, Scherer KR
Olfaction/Taste: dysfunction (O/T-d)
18D - The Identification and Discrimination of umami taste in German and Norwegian
population - Singh B, Schuster B, Seo H
19D - Olfactory dysfunction in allergic fungal sinusitis - Philpott CM, Lai L, Zheng G,
Homayoon B, Clark A, Javer AR
A B S T R A C T S
PL1 - Exploring ensemble representations of olfactory stimuli: making scents of intertwined spatial and temporal codes
J.G. Hildebrand, H. Lei, J.A. Riffell, C.E. Reisenman
University of Arizona, Tucson, AZ, USA
We study the olfactory system and its role in controlling specific behaviors of the giant sphinx moth Manduca sexta. An advantage of
insects as models for olfactory research is that they offer opportunities to study odor stimuli of known chemistry and importance for
behavior. Thus, a principal goal of our work is to understand neurobiological mechanisms through which information about specific,
behaviorally significant olfactory stimuli is encoded, processed, and integrated with inputs of other modalities in the brain and how
odor information ultimately initiates and controls the moth’s characteristic, natural behavioral responses
We have focused on two odor-dependent behaviors -- mate-seeking in males and interactions with hostplants in females -- and on the
antennal lobe (AL), the primary olfactory center in the moth’s brain. Primary olfactory centers in most vertebrates and invertebrates
are characterized by glomeruli, condensed-neuropil structures in which primary-afferent and central neural elements interact
synaptically, but the functional significance of these structures has been uncertain until recently. In Manduca, sexually dimorphic
glomeruli have been identified, and their functions have been probed extensively. Research on these glomeruli has taught us much
about olfactory information processing. Findings about the morphology, development, and physiology of glomeruli and the neurons
innervating them will be emphasized in this presentation.
The spatial and temporal patterns of neural activity elicited by different odor stimuli in the primary olfactory center in the brain have
been investigated in diverse animals for more than half a century. In an ongoing effort to decipher the neural-coding strategies
underlying the central processing of olfactory information, an increasing number of studies in the vertebrate olfactory bulb (OB) and
insect antennal lobe (AL) rely on imaging methods that reveal odor-dependent patterns of activity across olfactory glomeruli.
Olfactory stimuli possessing different chemical features evoke unique but spatially overlapping patterns or activity, whereas the
patterns for stimuli with similar chemical structures are less distinguishable. Despite the power of this approach, activity labeling is
limited in that it cannot resolve the rapid changes in neuronal firing that are embedded in these spatial patterns but undoubtedly also
contribute to odor discrimination. Similarly, electrophysiological studies often are performed without regard to the spatially welldefined maps of olfactory glomeruli. A method that combines the spatial resolution of activity labeling with the temporal resolution of
microelectrode recording is needed to fill the gap. We use multichannel recording with silicon multiprobes to explore the dynamic,
spatially organized neural activity patterns evoked by different odor stimuli across anatomically and functionally defined glomeruli in
the olfactory lobes in the brain of an experimentally favorable model system, the giant hawkmoth Manduca sexta. We find that
correlated firing of action potentials within and among glomeruli contributes to coding of different odorants and that coherent firing is
important for encoding the behavioral significance of odor mixtures.
Supported by NIH grant R01-DC00275.1
PL2 - Solitary Chemosensory Cells: An Ancient Vertebrate Chemosense Guarding The Airways In Extant Mammals
Tom Finger
Rocky Mountain Taste & Smell Center - Univ. Colorado Denver
Our chemosensory capabilities are not solely due to the senses of taste and smell but include chemical responses by the trigeminal
nerve which innervates both the oral and nasal cavities. Although trigeminal chemosensitivity is usually attributed to chemosensitive
ion channels (e.g. Trp channels, ASCICs) on the nerve fibers themselves, the broad chemoreceptive range of this system is increased
by the presence of solitary chemosensory (or chemoreceptor) cells (SCCs) scattered within the nasal epithelium. Similar SCCs are
present in the epidermis as well as the respiratory passages of all non-mammalian aquatic vertebrates and represent a primitive
vertebrate chemosensory modality. In all vertebrates, SCCs are elongate specialized epithelial cells extending beyond the surface of
the epithelium and a basal process innervated by fine caliber somatosensory (or general visceral) afferent fibers. SCCs, including those
in the respiratory epithelia, express most components of the GPCR taste transduction cascade including T2Rs, gustducin, PLCbeta2
and TrpM5, a downstream transduction channel. Unlike T2R-expressing taste cells, the nasal SCCs form clear synaptic contacts with
the trigeminal sensory fibers and utilize conventional synaptic vesicular release systems to communicate with the afferent nerves. The
expression of T2Rs by nasal SCCs suggests that these cells mediate detection of potential airway irritants that activate the T2R
transduction cascade. In the taste system, activation of T2Rs gives rise to the sensation of bitterness, but in the nasal cavity, activation
of the same molecular receptor cascade would be expected to activate trigeminal pain fibers and therefore give rise to the sensation of
irritation. Consistent with this is the finding that bitter-tasting compounds, e.g. denatonium, evoke trigeminally-mediated reflex apnea
similar to the response of nasal application of capsaicin or acids. Since SCCs synapse onto trigeminal chemosensitive fibers, these are
two routes to activation of the trigeminal pain fibers: 1) via activation of SCCs which in turn would activate the nerve fibers, or 2)
direct action of the irritant substance on the free nerve endings. Experiments utilizing TrpM5-null mice dissociate these two avenues
of trigeminal activation. Genetic deletion of TrpM5 disrupts transmission from SCC to trigeminal afferent, but does not interfere with
direct activation of the trigeminal fibers via TrpV1 (capsaicin receptor) or ASCICs. Consistent with this hypothesis, denatonium fails
to elicit trigeminally-mediated changes in respiration in TrpM5-KO mice although acids and capsaicin still elicit responses. In
summary, SCCs in the upper respiratory tract of mammals are remnants of a primitive vertebrate chemosensory modality. Despite
relying on a taste-like transduction cascade, these SCCs are not part of the taste system, but instead mediate respiratory reflexes
appropriate for the avoidance of respiratory irritants.
F-Aw - Dynamic ensemble odor coding in the mammalian olfactory bulb
Carleton A
Department of Neurosciences, Faculty of medicine, University of Geneva, 1 rue Michel-Servet, 1211 Genève 4, Switzerland
How neural networks encode sensory information is a key question in neuroscience. In the olfactory system, sensory inputs
convergence onto defined glomeruli maps is the first basis of odor coding. How sensory information is then read and transferred by the
first relay network (the olfactory bulb) remains largely debated. Several coding theories were proposed based on neuronal firing rates
(“rate” codes) or on spike timing (“temporal” codes). In order to understand the actual coding principles in the mammalian olfactory
system, we performed in this study in vivo imaging guided tetrode recordings of ensembles of olfactory bulb projection neurons.
We show that neurons respond to different odors and concentrations with complex firing patterns that evolve rapidly in time. While no
obvious single neuron coding principles emerged from the data, we found that neural ensemble activity could encode efficiently odor
identity and intensity. We could quantitatively test the performance of different coding hypotheses (that is the quantity of information
conveyed by different codes). Using a population activity-based odor prediction algorithm and Fourier analysis, we evaluate the
amount of information contained at different resolutions of the activity sequences. We observe that the amount of odor information is
maximal in time scales close to breathing cycle period, but it is also present at finer temporal resolutions. We showed that, although
ensemble activity is highly dynamic, spike timing based information is almost completely redundant to firing rate-based information in
ensemble codes.
We further show that an ensemble rate code permits to predict the identity and intensity of the odorant with high accuracy within a
time window of 200 ms corresponding to the speed of discrimination measured in rodent. In addition, rate coding (contrary to
temporal coding) can fully adapt to variations in the animal sniffing frequency, which is known to be strongly fluctuating in rodents.
Finally, we observed fast and slow dynamics which are characterized by a reshaping of ensemble activity over time below and above
breathing frequency. Interestingly, slow dynamics have been proposed to improve processing of the odor information in the fish
olfactory system. In the mouse olfactory bulb, we found that ensemble activity does not evolve in time towards less correlated and
thereby more easily separable representations of different odors.
Interestingly, these results show some parallel between the function of mammalian and insect olfactory systems, as some dynamical
properties of mammalian odor representations could be demonstrated similar to those recently observed in the insect equivalent of
olfactory bulb. But they also show some divergence with some coding principles reported in fish.
S1 - Bimodal function of Drosophila odorant receptors
Dieter Wicher, Ronny Schäfer, Marcus C. Stensmyr, Bill S. Hansson
Max Planck Institute for Chemical Ecology, Hans-Knöll-St. 8, D-07745 Jena, Germany
Odor information is detected by binding of molecules to odorant receptors (ORs) belonging to the G-protein-coupled receptor (GPCR)
family. These couple to G-proteins, most of which stimulate cAMP production. This interaction opens cyclic nucleotide-gated ion
channels and depolarise the olfactory sensory neuron. Insect OR proteins lack any sequence similarity to other GPCRs and show an
inverted orientation in the plasma membrane. The ORs are dimers composed of an odor-specific OR protein and a ubiquitously
expressed chaperone protein (as Or83b in Drosophila). As G-proteins are expressed in insect OR neurons, and olfactory perception is
modified by mutations affecting the cAMP transduction pathway, this study was aimed at elucidating the function of insect ORs.
Therefore we expressed a receptor (Or22a) and Or83b in HEK293 cells and performed calcium imaging and patch clamp experiments.
Odor stimulation produced non-selective cation currents activated via both an ionotropic and a metabotropic pathway, and a
subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone lead to functional ion channels insensitive to
odorants, but directly activated by intracellular cAMP or cGMP. Furthermore, application of cAMP to excised inside-out patches
caused a current production with a concentration of only 1 nM for half-maximal response. Insect ORs thus form ligand-gated channels
as well as complexes of odorant sensing units and cyclic nucleotide-activated nonselective cation channels.
This study was supported by the Max Planck Society.
10
S2 - Insect chemosensation via ligand-gated cation channels
Koji Sato and Kazushige Touhara
Department of Integrated Biosciences, The University of Tokyo
The G-protein-signaling cascade is a conserved molecular basis for the chemical senses among various organisms from nematode to
primates. In vertebrates, the binding of chemosignals to olfactory or gustatory receptors leads to activation of G-protein-mediated
second messenger pathways, resulting in the opening of second messenger-gated cation channels that convey sodium and calcium
currents, and thereby transform chemical signals into electrical neural activity. In contrast, although insect olfactory receptors (ORs)
are classified into the seven-transmembrane receptor family, they lack homology to the G-protein-coupled receptor family and possess
a distinct membrane topology with the intracellular N-terminus. Moreover, the functional insect OR consists of a heteromeric complex
together with the Or83b family co-receptor, implicating that insects utilize a unique signal transduction mechanism different from that
in vertebrates (Nakagawa et al., 2005). Indeed, we found that stimulation of Xenopus oocytes expressing insect OR complex with the
cognate ligand elicited unknown odor-induced inward cation currents at the resting potential, suggesting that insect OR complex
conferred a novel cation channel activity. To characterize the channel properties, we next expressed various combinations of insect OR
complex in mammalian cell lines. HeLa and HEK293T cells expressing fruit fly, silkmoth, and mosquito OR complex exhibited
extracellular calcium influx and nonselective cation conductance upon odorant stimulation (Sato et al., 2008). Inhibitors for known Gprotein-coupled second messenger pathways had no effect on the odorant response. Interestingly, particular combinations of OR
complex were sensitive to 8-bromo-cyclic nucleotides. The odorant response kinetics was completely different from that in vertebrate
olfactory receptor neurons. Insect ORs expressed in HeLa cells showed no desensitization with faster response latency than that of the
odor response of vertebrate olfactory sensory neurons. Ion permeability and the degree of response inhibition by a calcium channel
blocker were different, depending on the OR subunit composition. These results suggested that OR complexes directly elicited Gprotein independent responses. The evidence for the current production by OR complex itself was obtained from the outside-out
single-channel recording of HEK293T cell membrane containing the OR complex. The pA order of spontaneous cation conductance
was recorded, and its open probability increased upon stimulation with the cognate ligand. Conductance of fruits fly ORs was larger
than that of mosquito ORs at –60 mV. Our study demonstrates a novel molecular aspect of seven-transmembrane receptors in that
insect OR complex are spontaneously-active odor-gated ion channels that likely regulate the receptor potential of olfactory receptor
neurons.
The study was supported by grants from PROBRAIN and MEXT
S3 - Activation of Grueneberg ganglion cells in mice by alarm pheromones
Brechbühl J, Hurni N and Broillet MC
Department of Pharmacology and Toxicology, University of Lausanne, Bugnon 27, CH-1005 Lausanne, Switzerland
Alarm pheromones are widely used throughout the plant and animal kingdoms. Species such as fish, insects, and mammals signal
danger to conspecifics by releasing volatile alarm molecules. These substances signal injury, distress or the presence of predators. In
animals, the conspecific response to these signals is usually freezing, attack or dispersal. Thus far, neither the chemicals nor their
bodily source have been isolated or identified in mammals. Alarm pheromones may be low molecular weight and highly volatile
substances to allow fast diffusion from the source but a quick decline once the danger has past. We found that alarm pheromones are
recognized in the mouse by the Grueneberg ganglion, the most recently discovered olfactory subsystem.
This organ was first described by Hans Grueneberg in 1973, as a 'ganglion' of unknown function. With the limited histological
methods available at the time, Grueneberg concluded that this ganglion-like cell mass might belong to the complex of the Nervus
terminalis. These cells were “rediscovered” in 2005-2006 thanks to the inspection of whole-mount specimens from one particular
gene-targeted mouse strain called OMP-GFP. These mice express GFP as a histological reporter under the control of the OMP
promoter. OMP (olfactory marker protein) is a mature olfactory-sensory neuron-specific marker. In memory of the original work of
Grueneberg, this structure kept its original name. The Grueneberg ganglion is an arrow-shaped neuronal structure at the anterior end of
the nasal cavity that lines both sides of the nasal septum. The paired organ is located in a shallow depression of the cartilage. In an
adult mouse, from 300 to 500 cells can be found in each Grueneberg ganglion. We showed by electron microscopy,
immunohistochemistry and electrophysiology that a Grueneberg ganglion comprises two populations of cells : neurons and glial cells.
Grueneberg ganglion neurons bear multiple primary cilia, putative site of chemosensory transduction. We have now localized by
immunohistochemistry the expression of membrane proteins that might be implicated in the specific chemosensory function taking
place in this ganglion from birth on: danger detection via alarm pheromone recognition. Alarm pheromones evoked calcium responses
in Grueneberg ganglion neurons in vitro and induced freezing behavior in vivo, which completely disappeared when the Grueneberg
ganglion degenerated after axotomy. Chemosensing by the Grueneberg ganglion is a typical example of how the nervous system has
adapted to sense and react to a very important social cue. Alarm pheromone sensing is an instinctive or innate behaviour involved in
natural selection.
11
S4 - Molecular mechanisms of adaptation in mouse olfactory sensory neurons
Zhao H (1), Song Y (1), Cygnar KD (1), Stephan AB (1), Hirsh S (1), Shum E (1), and Reisert J (2)
1. Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA; 2. Monell Chemical Senses Center,
Philadelphia, PA 19104, USA
Adaptation is a common feature of sensory transduction, referring to the phenomenon and ability of sensory receptor cells in altering
the sensitivity and response kinetics due to experience of stimulation. Olfactory sensory neurons rapidly reduce sensitivity to repeated
odor exposure. Such adaptation is thought to result from a cohort of regulatory mechanisms that alter activities of various signal
transduction components from odorant receptor to the olfactory cyclic nucleotide-gated (CNG) channels. However, the relative
contribution of individual regulatory mechanisms to adaptation remains poorly understood. Our recent investigations using molecular
genetic approach in mice allowed new insight into mechanisms by which olfactory sensory neurons regulate their sensitivity and
response kinetics. We provided data that challenge the classical model praising the dominant role of calcium/calmodulin-mediated
desensitization of the olfactory CNG channel in adaptation of olfactory sensory neurons. We introduced a mutation in the olfactory
CNG channel subunit CNGB1b that rendered the channel resistant to rapid desensitization by calcium/calmodulin. Mutant olfactory
sensory neurons showed normal sensitivity, slower response termination, but normal adaptation to repeated stimulation. The results
suggested that calcium/calmodulin-mediated CNG channel desensitization is less important in regulating the sensitivity to recurring
stimulation than previously thought and instead functions primarily to allow rapid response termination. We also provided data that
suggest the phosphodiesterase 1C (PDE1C), a calcium/calmodulin-stimulated phosphodiesterase and the only phosphodiesterase found
in the cilia of olfactory sensory neurons, may contribute to adaptation of olfactory sensory neurons. We disrupted Pde1c gene and
observed that mutant olfactory sensory neurons showed reduced sensitivity and attenuated adaptation to repeated stimulation.
Furthermore, we conducted a mass spectrometry-based proteomic assay of the cilial membrane proteins of olfactory sensory neurons.
This assay led to identification of Anoctamin 2 as the calcium-activated chloride channel that may account for signal amplification in
olfactory transduction. This assay also revealed a potassium-dependent sodium/calcium exchanger NCKX4 that might contribute to
removal of calcium from olfactory cilia. Molecular identification these components provide new targets of investigation for their
involvement in adaptation of olfactory sensory neurons.
Supported by the Whitehall Foundation, NIH DC006178 and DC007395, and the Monell Chemical Senses Cen
S5 - The styryl dye FM1-43 inhibits olfactory transduction in larval Xenopus laevis
Breunig E (1), Kludt E (1), Manzini I (1,2), Schild D (1,2) and Czesnik D (1)
1 Department of Neurophysiology and Cellular Biophysics, 2 DFG Research Center for Molecular Physiology of the Brain (CMPB),
University of Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
FM1-43 among other styryl dyes is mainly used in neurosciences to monitor membrane trafficking because of its lipophilic binding
properties. In addition to the slow uptake of the dye mediated by endocytosis, FM1-43 rapidly and specifically labels sensory cells like
hair cells in the inner ear and primary sensory neurons in the dorsal root ganglion. The dye enters the cells by passing open channels
like the mechanotransducer channel in hair cells, or the vanilloid receptor TRPV1 or the purinergic receptor P2X2 in cell culture. In
this study we observed FM1-43 labeling of a subpopulation of receptor neurons in the olfactory epithelium when larval Xenopus
laevis were exposed to FM1-43-containing water in vivo. The fast and cytosolic staining of the cells cannot be explained by
endocytosis. Indeed, dye uptake is prevented by divalent cations. This already indicates, that FM1-43 enters olfactory receptor neurons
at their apical parts and that a cation channel is involved in dye uptake. Further, FM1-43 blocks olfactory transduction and thus,
reduces odor-induced responses dramatically.
Supported by DFG Research Center Molecular Physiology of the Brain (CMPB)
12
S6 - Visualizing stimulus convergence in amygdala neurons during taste aversion learning
Ilene L. Bernstein and Sabiha Barot
University of Washington
A central feature of models of associative memory formation is the reliance on information convergence from pathways responsive to
the conditioned stimulus (CS) and unconditioned stimulus (US). In particular, cells receiving coincident input are held to be critical for
subsequent plasticity. Yet identification of neurons in the mammalian brain that respond to such coincident inputs during a learning
event remains elusive. We used Arc catFISH (Cellular compartmental Analysis of Temporal gene transcription by Fluorescence in situ
hybridization) to locate populations of neurons in the mammalian brain that respond to both CS and US during training in a one-trial
learning task, conditioned taste aversion (CTA). Utilizing catFISH as a functional imager allowed us to visualize patterns of neuronal
activation in response to the CS and US in animals that were awake and acquiring a one-trial learned association. Further, they provide
evidence that, during CTA acquisition, CS and US information converge on a subset of cells in the BLA when presentation of the
stimuli is effective in promoting learning (novel CS-US pairing) but not when the same stimuli are presented in an ineffective manner
(familiar CS-US pairing; backward US-CS pairing). The development of a behavioral and analytical protocol that pinpoints neurons
receiving convergent stimulus information during learning provides a critical tool for subsequent characterization of the
neurochemical and neuroanatomical characteristics of these neurons and ultimately for the identification of downstream signalling
events that depend on coincident activation. In addition, the finding that backward pairing does not yield coincident activation despite
exposure to identical stimuli raises fundamental questions about underlying mechanisms. We propose a model in which potentiation of
US responses by novel CS presentation is key to coincident activation and its sensitivity to temporal order. Specifically, the model
proposes that the elevation observed in US responding is due to a subset of neurons in BLA that receive strong CS input and weak US
input. When the US is presented alone, the stimulus fails to excite these neurons. However when a novel CS precedes the US, these
neurons become more sensitive to subsequent US input and convergent activation is seen. This model is supported by the consistent
observation that the number of neurons responding to the US is enhanced when it is preceded by a novel CS but not when it was
preceded by a familiar CS (or followed by a novel CS). Indeed, this enhancement in US responding is correlated with the appearance
of convergently activated neurons, suggesting that the same process may be responsible for both.
S7 - Translational Control in the Gustatory Cortex Determines the Stability of a Taste Memory
Rosenblum K
Department of Neurobiology, Faculty of Science, University of Haifa, Haifa, Israel.
The off-line processing of acquired sensory information in the mammalian cortex is an example for the unique way biology creates to
compute and store information which guides behavior. The relatively short temporal phase in the process (i.e. hours following
acquisition) is defined biochemically by its sensitivity to protein synthesis inhibitors. Until recently this negative definition of
molecular consolidation did not reveal the details of the endogenous processes taking place, minutes to hours, in the neurons and
circuit underlying a given memory. We use taste learning paradigms in order to study this process of molecular consolidation in the
gustatory cortex.
Recent results, from our laboratory, obtained from genetic, pharmacological, biochemical, electrophysiological and behavioral studies
demonstrate that translational control, at the initiation and elongation phases of translation, plays a key role in the process of molecular
consolidation. Moreover, this spatially and temporally regulated translation control modifies both general and synaptic protein
expression that is crucial for memory stabilization. We propose a model to explain the interplay between regulation of initiation and
elongation phases of translation and demonstrate that in certain situations cognitive enhancement can be achieved.
References
Antion MD, Merhav M, Hoeffer CA, Reis G, Kozma SC, Thomas G, Schuman EM, Rosenblum K, Klann E. Removal of S6K1 and
S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity. Learning and Memory, 2008, 15(1) 29-38.
Cohen-Matsliah SI, Seroussi Y, Rosenblum K and Barkai E. Persistent ERK activation maintains learning-induced long-lasting
modulation of synaptic connectivity. Learning and Memory, 2008, 15(10) 756-761.
Elkobi A, Ehrlich I, Belelovsky K, Barki-Harrington L and Rosenblum K. ERK-dependent PSD-95 induction in the gustatory cortex is
necessary for taste learning, but not retrieval. Nature Neuroscience, 2008, 11(10) 1149-1151.
Lavagni P, Indrigo M, Colombo G, Martegani E, Rosenblum K, Gnesutta N and Zippel R. Identification of novel RasGRF1 interacting
partners by large-scale proteomic analysis. Journal of Molecular Neuroscience, 2009, 37(3) 212-224 .
Liraz O, Rosenblum K and Barkai E. CAMKII activation is not required for maintenance of learning-induced enhancement of
neuronal excitability. PLoS ONE., 2009, 4(1) e4289.
Merhav M and Rosenblum K. Facilitation of taste memory acquisition by experiencing previous novel taste is protein-synthesis
dependent. Learning and Memory, 2008, 15(7) 501-507.
13
S8 - Integration in central neurons of gustatory information from four appendages in the moth Heliothis virescens
Pål Kvello, Kari Jørgensen and Hanna Mustaparta
Norwegian University of Science and Technology, Department of Biology-Neuroscience Unit, Trondheim, Norway.
Discrimination between edible and noxious food is crucial for survival in all organisms. Gustatory receptors detecting phagostimulants
and deterrents in insects correspond to the sweet and bitter receptors in mammals. Whereas integration of gustatory information in the
central nervous system is well studied in mammals, this kind of knowledge in insects is only fragmentary in a few species. The
particular organization of the gustatory receptor neurons on several appendages in insects, like the antennae, the proboscis and the tarsi
of the moth Heliothis virescens, has raised the question whether the information about the different tastants from the four appendages
are integrated in central neurons of the primary gustatory areas in the suboesophageal ganglion (SOG) and tritocerebrum. We here
present data from intracellular recordings of central gustatory neurons (CGNs) tested for the phagostimulants, sucrose and water, and
the deterrent, quinine, as well as mechanosensory stimuli applied to each of the four appendages; the left and right antenna, the
proboscis and the right tarsus. Each of the stimuli elicits a specific appetitive or aversive behaviour depending on which appendage is
stimulated; phagostimulants and deterrents applied to the antennae eliciting orientation towards versus away from the tastant, as well
as extension versus recoiling of proboscis (Jørgensen et al 2007). Proboscis stimulation maintain proboscis extension and intiate
consumption versus inhibit consumption and initiate recoiling. In previous studies of this moth we have shown that the four stimuli
activate separate receptor neurons of the contact chemosensilla, each sensillum containing 2-3 gustatory and one mechanosensory
neuron(s) (Jørgensen et al 2007; Kvello et al 2006). In the present study we have shown that the CGNs responded with varying tuning
breadth to stimulation with more than one stimulus as well as to stimulation of more than one appendage. In an ensemble of 13 CGNs
tested for all stimuli applied to all appendages, each of the stimuli generated unique, but partially overlapping patterns of activity
within and across appendages. This suggests that tastant quality and location is coded by ensembles of CGNs. Staining revealed that
the majority of the CGNs were confined locally within the SOG/tritocerebrum, whereas others projected in the deuto- and
protocerebrum, as well as in the connectives leading to the frontal ganglion and the thoracic ganglia. Registration of some CGNs into
the standard brain atlas showed overlap with the previously described projections of the gustatory receptor neurons (Jørgensen et al
2006; Kvello et al 2006). Whereas some CGNs could be ascribed to involvement in specific appetitive or aversive behaviours, most of
them seemed to be multifunctional, i.e. each belonging to different networks executing specific functions.
S9 - Taste post-prandial categorization during endotoxin challenge: neural and behavioral correlates.
Pacheco-López, G.
ETH (Swiss Federal Institute of Technology)-Zurich, Physiology and Behavior Group. CH-8603, Switzerland
Appetitive and consummatory feeding behavior are significantly affected by immune-derived molecules elicited by peripheral immune
challenges (e.g., viruses, bacteria or tumor cells). In addition to illness-anorexia, when relevant gustatory/olfactory stimuli precede
immune activation, strong associative learning occurs, even after a single pairing trial. Importantly, postprandial categorization of new
food, as safe or dangerous, may significantly affect individual food selection long after the association phase, i.e. conditioned taste
avoidance/aversion (CTA). Single administration of bacterial lipopolysaccharide (LPS) induces a rapid peripheral increase in proinflammatory cytokines (e.g. tumor necrosis factor: TNF-alpha, interleukin-1beta, and interleukin 6). However, repeated
lipopolysaccharide administrations induce a state of endotoxin-tolerance characterized by a drastic reduction in cytokines response. In
this regard, the single contingent pairing of a taste (conditioned stimulus) with a first encounter to lipopolysaccharide (unconditioned
stimulus) induces a strong and long lasting associative learning, evidenced by a reduction of the appetitive and ingestive behavior
(conditioned response) after subsequent re-exposures to the conditioned stimulus. In contrast, our recent data clearly indicate that the
immune history affects associative learning when the unconditioned stimulus employed is transduced (i.e. filtered) by the immune
system (Pacheco-López et al., Physiology & Behavior 93 (2008), 261-266). Saccharin-lipopolysaccharide associative learning in
endotoxin-tolerant animal is weakened (i.e. low avoidance behavior and fast extinction) than in lipopolysaccharide-naïve animals,
indicating that in endotoxin-tolerant animals lipopolysaccharide i.p. injection was differently perceived by the central nervous system
and thus associated in a different way. Peripheral immune data and electrophysiological recordings supporting differential neural
encoding of immune challenges will be presented and discussed, indicating that similar immune challenges might results in different
neuro (behavioral)-endocrine consequences depending on the immune history of each individual. In conclusion, the capacity to
associate a specific stimulus such as environmental clues or flavors (conditioned stimulus) with a certain immune status or immune
response against e.g. allergens, toxins or antigens (unconditioned stimulus) seems to be of high adaptive value. Thus, it can be
hypothesized that this capacity was acquired during evolution as an adaptive strategy in order to protect the organism and prepare it
for danger.
14
S10 - Reversible inactivation of the dorsal hippocampus in aged rats and taste learning.
Gámiz, F and Gallo, M
Department of Experimental Psychology and Physiology of Behavior. Institute of Neurosciences. University of Granada. Spain.
Aging offers a privileged opportunity to study the brain memory systems plasticity induced by a long-life learning experience together
with changes in several body functions and new adaptive requirements. Previous research in our lab has drawn a rather complex
picture of taste learning in aged rats (Manrique et al., Chem. Senses, 2007, 32(1) 111-117 ). First, some complex hippocampal taste
learning phenomena are absent in intact naïve aged rats, while permanent dorsal hippocampal lesions may facilitate in aged rats nonhippocampal phenomena such as the temporal context dependency of CTA (Manrique, et al., Hippocampus, 2009, 19, 57-65). In
addition, previous CTA modify to a greater extent later taste-related responses in aged rats than in adult rats (Morón and Gallo, 2007,
Physiol. Behav., 90, 308-317). Therefore, we have investigated if temporary reversible hippocampal inactivation by tetrodotoxin
(TTX) could induce a similar functional reorganization of the brain memory circuits during aging and its potential interaction with the
effect of previous learning experiences. Following a five days habituation to drink twice a day, according to Manrique et al.
(Neurobiol. Learn. and Mem., 2004, 82, 77-80), aged Wistar rats (24-month-old) were subjected to a latent inhibition procedure in
which two taste preexposures and five one-bottle extinction tests were applied either at the same or a different time of day of CTA.
The results confirmed that naïve aged rats had similar taste aversions if they were conditioned at either the familiar or the unfamiliar
time-of-day, independently of the taste solution used (sodium saccharin 0.1%; cider vinegar 3%). No effect of bilateral dorsal
hippocampus reversible inactivation by TTX (10ng per hemisphere) was seen. However, in aged rats previously trained in taste
learning the temporal context recovered the ability to modulate CTA. Rats conditioned at a different time of preexposure had a greater
aversion than those conditioned at the same time of day, thus exhibiting a disruption of the latent inhibition effect as previously shown
in adult rats. In these rats, bilateral TTX inactivation of the dorsal hippocampus during CTA acquisition, but not during testing,
reversed the effect of the time-of-day shift unveiling a non-hippocampal temporal context dependency of taste aversion. Thus, those
rats conditioned and tested at the same time of day exhibited stronger aversions than those conditioned and tested at a different time of
day. In all, these data support a peculiar organization of the memory systems during aging that cannot be explained by a general
cognitive decline or exclusively by the decay of the hippocampal function, and it should also take into account the individual previous
learning experiences.
Grants HUM 02763 (Junta de Andalucía. Spain) and PSIC2008-03933 (MICINN. Spain), with FEDER funding
S11 - Taste Memory impairments in Alzheimer-mice model is related with dopamine release failure.
Bermúdez-Rattoni, F and Guzman-Ramos, KR.
Unidad Neurociencias. Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. México.
The Alzheimer disease (AD) is clinically diagnosed by a progressive loss of certain mental abilities, which coincide with the selective
dysfunction and damage of neurons in the limbic structures and association cortices critical for cognition and memory. The first signs
of cognitive decline observed in AD include difficulties with the acquisition of new information. This cognitive impairment has been
correlated to significant protein depositions, like Beta-amyloid and tau. Recently, a triple transgenic mice model of AD has been
engineered in which the Beta-amyloid and tau appear at AD-relevant brain regions. In these transgenic mice the cognitive impairments
appear in the first months of age, which have been related to Beta-amyloid accumulation. As the disease progresses, structural changes
start to appear due to accumulation of tau protein, which in turn exacerbate the cognitive decline. In our laboratory, we have worked
out a behavioral model based on the recognition of stimulus, and we have developed a technique, which couples capillary
electrophoresis with in vivo microdialysis. With this technique we were able to determine almost in real-time the simultaneous release
of several neurotransmitters, like dopamine, norepinephrine and glutamate and to establish their participation in the consolidation of
taste recognition memories in mice. Results in our laboratory showed that young transgenic or old Non- transgenic mice presented
good taste recognition memory and strong release of dopamine and glutamate. However, as the transgenic mice get older, they start
showing impaired recognition memory, and interestingly the dopamine release was significantly reduced. Moreover, anatomical
analysis revealed that older, but not younger transgenic mice had abundant Beta-amyloid depositions in the amygdala and insular
cortex, structures strongly related to taste recognition memory. These results suggest that dopamine release during cognitive
challenges (like memory formation) could be used as early signal of cognitive impairment in the transgenic mice.
References
Bermúdez-Rattoni F. Molecular mechanisms of taste-recognition memory. Nature Reviews in Neuroscience, 2004, 5(3), 209-217.
Bermúdez-Rattoni F, Ramírez-Lugo L, Gutiérrez R, Miranda MI. Molecular signals into the insular cortex and amygdala during
aversive gustatory memory formation. Cellular Molecular Neurobiology, 2004, 24(1), 25-36.
De la Cruz V, Rodriguez-Ortiz CJ, Balderas I, Bermudez-Rattoni F. Medial temporal lobe structures participate differentially in
consolidation of safe and aversive taste memories. European Journal Neuroscience, 2008, 28(7), 1377-1381.
Supported by UC-Mexus and CONACYT 60478
15
S12 – Sweet proteins, structure, production and function
Göran Hellekant
Department of Physiology and Pharmacology, Medical School, University of Minnesota, Duluth, MN, USA
Although it had been known for a long time that sweet taste can be caused by many different chemicals, it was long thought
impossible that a protein would be sweet, not to speak of intensely sweet. Therefore the discovery of monellin and thaumatin in 1972
was a sensation [1, 2], although the miraculin had preceded them [3, 4]. However, miraculin is not a “true” sweetener, because it has
no sweet taste in itself. It induces sweet taste only in solutions with pH <7.
For more than 10 years monellin and thaumatin were the only known sweet proteins until mabilin was published [5]. It is however
much less sweet than monellin and thaumatin. After another 10 years, brazzein was characterized [6]. It is more heat stable than any of
the other proteins. Since then the sweet protein family has been extended with curculin and gurmarin. Gurmarin suppresses sweet taste
in rodents [7]. Curculin elicits both sweetness and gives sour substances a sweet taste [8]
Sweet proteins are considerably more potent than carbohydrate sweeteners but have a limited species range. Thus, for example,
brazzein, monellin and thaumatin are sweet only to Old-World primates. Structurally sweet proteins range in size from 22 kD for
miraculin to 5,4 brazzein with little structural similarity. Their sweetness is significantly slower than that of non-protein sweeteners.
The slowest is thaumatin, whose sweetness is not evident until after several seconds of exposure. It is not an understatement that the
attempts to relate their structure with function have been slow. Their intense sweetness and natural origin make them attractive for the
food industry.
1. Morris JA, Cagan RH: Purification of monellin, the sweet principle of Dioscorephyllum cumminsii. Biochim Biophys Acta 1972,
261:114-122.
2. van der Wel H, Loeve K: Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus
daniellii Benth. European journal of biochemistry / FEBS 1972, 31(2):221-225.
3. Brouwer JN, van der Wel H, Francke A, Henning GJ: Miraculin, the sweetness-inducing protein from miracle fruit. Nature 1968,
220(165):373-374.
4. Kurihara K, Beidler LM: Taste-modfying protein from miracle fruit.. Science (New York, NY 1968, 161:1241-1243.
5. Hu Z, He M: Studies on mabinlin, a sweet protein from the seeds of capparis masaikai levl. I. Extraction, purification and certein
characteristics. Acta Botanica Yunnanica 1983, 5(2):207-212.
6. Ming D, Hellekant G: Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS letters
1994, 355(1):106-108.
7. Kamei K, Takano R, Miyasaka A, Imoto T, Hara S: Amino acid sequence of sweet-taste-suppressing peptide (gurmarin) from the
leaves of Gymnema sylv estre. J Biochem 1992, 111:109-112.
8. Kurihara Y: Characteristics of antisweet substances, sweet proteins, and sweetness-inducing proteins. Critical Reviews in Food
Science and Nutrition 1992, 32(3)):231-252.
S13 - Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast pichia pastoris
Loïc Briand
UMR FLAVIC INRA- ENESAD - Université de Bourgogne, Dijon, France
Brazzein is a small (6.5 kDa), heat- and pH-stable sweet protein originating from the fruit of Pentadiplandra brazzeana Baillon, a
plant found in West Africa. Brazzein isolated from its natural source exists in two forms differing in sweetness intensity. The major
form (54 amino acids, ~80%), called pGlu-bra, contains a pyroglutamic acid (pGlu) at is N-terminus, while the minor form (53 amino
acids, ~20%), called des-pGlu-bra, lacks the N-terminal pGlu. It has been reported that des-pGlu-bra is twice as sweet as pGlu-bra.
Heterologous expression of brazzein in bacteria is complicated by the presence of a pyroglutamic acid (pGlu) in the major form of
brazzein. Here we describe the heterologous expression of the two natural major forms of brazzein using the methylotrophic yeast
Pichia pastoris. Proteins were efficiently secreted into the extracellular medium using the alpha-factor preprosequence peptide of
Saccharomyces cerevisiae. After dialysis, recombinant brazzeins were isolated from the yeast culture filtrate using ion-exchange
chromatography. Purified proteins were characterized using MALDI-TOF mass spectrometry and 1D NMR spectroscopy revealing
that both sweet-tasting proteins were properly refolded in agreement with their sweetness properties. We also investigated the
capability to brazzein isoforms to stimulate and interact with hT1R2/hT1R3 taste receptor.
16
S14 - Lactic acid bacteria as hosts for the expression of brazzein
Berlec A (1), Jevnikar Z (2), Tompa G (3), Rogelj I (3), Štrukelj B (1,2)
Department of Biotechnology, Jozef Stefan Institute, Ljubljana, Slovenia (1), Faculty of Pharmacy, University of Ljubljana, Ljubljana,
Slovenia (2), Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia (3)
Lactic acid bacteria (LAB) are gram positive bacteria that have been a part of human diet in fermented dairy, vegetable or meat
products for centuries. They are anaerobic or microaerophilic cocci or bacilli and have common metabolic and physiological
characteristics. They have gained increased research attention as hosts for recombinant protein production due to their recorded safe
usage and beneficial, probiotic health properties. The expression of sweet tasting protein brazzein in LAB would have several
advantages. The need for sweetening the dairy products would be decreased, the need for brazzein isolation and purification would be
removed and functional value of LAB in various alimentary products would be increased.
In the course of our research we have expressed brazein in a model LAB Lactococcus lactis with the use of nisin-controlled expression
system (NICE). Because the quantity of expressed brazzein was low, we put our efforts into optimization of its expression. We
successfully directed brazzein into the growth medium with the use of signal peptide SP-Usp, which is beneficial from the applicative
point of view. This however did not affect the overall yield. Similarly, the use of brazzein gene with optimized codon usage did not
contribute to yield increase.
In further optimization we were systematically changing growth and expression parameters (medium composition, inducer
concentration, optical density at induction, pH) in a series of fermentations under controlled conditions in the fermentor. We
determined the conditions, under which 17-fold increase in brazzein yield was achieved. Large influence of pH regulation and medium
composition was determined. We also established a new method for brazzein quantification based on ELISA, which enabled the
comparison of large number of samples, which were obtained during the optimization.
We also tested the influence of plasmid/strain combination of the NICE system on the level of brazzein expression. With the use of
plasmid pNZ8148 in L. lactis NZ9000 we achieved more than 800-fold increase in brazzein expression relative to initial combination
(plasmid pMSP3545 and strain L. lactis IL1403), which is comparable to the expression in Escherichia coli. The expressed brazzein
had detectable sweet taste of low intensity, which is probably the consequence of improper folding.
Other species of LAB, predominantly of genus Lactobacillus, are currently being tested as potential hosts for brazzein expression by
the use of NICE system.
This work was supported by the Slovenian Research Agency Grant No. P4-0127
S15 – Analyses of new brazzein derivatives
Tiffany Cragin1, , D. Eric Walters2, R. Jin1, Jon N. Rumbley3, and Göran Hellekant1
1Department of Physiology and Pharmacology, Medical School, University of Minnesota Duluth, USA 2Department of Biochemistry
and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science and 3Department of
Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota Duluth, USA
Brazzein is a small protein (54 amino acids) derived from the African plant Pentadiplandra brazzeana [1]. It is potently sweet (potency
= 2,000 x a 2% sucrose solution on a weight basis,) and is extremely heat-stable. We have previously modeled its interaction with the
extracellular domains of the sweet taste receptor using different brazzein mutants. Our model suggests the open conformation of the
T1R2 active site is the preferred brazzein-binding site; a model consistent with 21 of 23 previously designed brazzein mutants. We
have now used our proposed model to design new brazzein single and double mutants and describe here the application of that model
to the design of twelve new highly potent analogs of brazzein tested by a human panel. The model was applied and evaluated using
several kinds of mutations. Tyr39 and Arg43 mutations were expected to decrease sweetness, through loss of important receptor
interactions in the “sweet finger” domain/loop (residues 39 to 45). The double mutant Cys16Ala/Cys37Ala tested the importance of
one of the disulfide bonds in brazzein, adjacent to loop 39 to 45, likely supporting structural integrity and rigidity of this critical
domain. Mutation of Asp40 and Lys42 were expected to have no effect on sweetness, since these residues do not contact the receptor
in the modeled conformation. Mutation of Asp29, Glu41, Asp50, and Tyr54 were expected to increase interaction, either by removing
repulsive interactions or, in the case of Tyr54Trp, by increasing a hydrophobic interaction. The purpose of these new mutants was
two-fold: to test the validity of our binding model and to improve the sweetness of brazzein. Brazzein mutants and wild type brazzein
were dissolved in deionized water at a concentration of 100 µg/ml and adjusted to pH 7.0 with 0.1 N NaOH or HCl. The subjects
tasted 150 µl samples delivered with a 200µl pipette to the anterior part of the tongue. Each sample was presented 3 times and in
quasi-randomized order and on separate occasions. This same sample size and method of application have been used in our previous
studies. The subjects scored the sweetness of taste stimuli with the Labeled Magnitude Scale. The qualitative scale was later converted
to a numerical scale and average sweetness scores were calculated for each stimulus. Eight of the twelve analogs have higher
sweetness potency than wild type brazzein. Our results are consistent with our brazzein-receptor interaction model, which predicts
binding of brazzein to the open form of T1R2 in the T1R2/T1R3 heterodimer.
1. Ming D: Brazzein, a new sweet protein from pentadiplandra brazzeana. Ph.D. Thesis. University of Wisconsin at Madison; 1994.
17
S16 – The complexity of sensory perception in insects: electrophysiological evidences
De Cristofaro A, Anfora G*, Germinara GS§, Ioriatti C*, Rotundo G, Tasin M*, Vitagliano S
Department of Animal, Plant and Environmental Sciences, University of Molise, Via De Sanctis, 86100, Campobasso (CB), Italy. (*)
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all'Adige (TN), Italy. (§)
Department of Agro-Environmental Sciences, Chemistry and Plant Protection, University of Foggia, Vi
Insects perceive a wide range of chemicals using different types of chemoreceptors. The chemical stimulus is transformed in action
potentials that can be recorded by electrophysiological experiments. Using different single cell recording techniques, olfactory cells
sensitive only to a single pheromone component or to a plant volatile have been reported. Receptor neurons sensitive to more than one
pheromone compound, or to both synergists and antagonists, have been also found. Most of these cells respond strongly to one
compound and weakly to a few other chemical related constituents. Cells responding to a sex pheromone component and to high doses
of host-plant volatiles have been already detected on male antennae of various Lepidoptera species as well as neurons responding to
more than one plant volatile compound. Recently, the occurrence of cells sensitive to the sex pheromone main component (E,E)-8,10dodecadien-1-ol (E8E10-12:OH) of Cydia pomonella (L.) and to the host-plant volatile ethyl (E,Z)-2,4-decadienoate (Et-2E,4Z-DD,
pear ester) has been revealed both in male and female. Sampling by single cell recordings the olfactory cell responses of different
species of Lepidoptera [C. pomonella, Cydia fagiglandana (Zeller), Cydia splendana (Hübner), Pammene fasciana (L.), Lobesia
botrana (Dennis & Schiffermüller), Ephestia kuehniella (Zeller)] and Diptera [Bactrocera oleae (Rossi), Ceratitis capitata
(Weidemann), Musca domestica L., Drosophila melanogaster (Meigen), Drosophila simulans Sturtevant], we detected, other than cell
types stimulated only by pheromone (sex or aggregation) or plant/food volatiles, the presence of generalist cells sensitive to similar
doses of substances of both categories, sometimes completely different in their chemical structures. In some cases stimuli
superposition and differential saturation EAG experiments confirmed the presence of peripheral interactions during the simultaneous
olfactory perception of pheromone components and volatile organic compounds. The biological meaning of these results is still not
completely understood, since in insects a separate peripheral perception mechanism and response processing of the pheromone
constituents and plant volatiles have been clearly shown. These evidences could be explained by a low selectivity of some receptor
binding proteins or by the presence of different receptor sites on the same neuron, which also support our hypothesis on the possible
existence of an unknown common sensory channel for pheromone components and plant volatiles.
In addition, the ability of plant compounds to change the biological activity of a pheromone component has been already showed by
different authors. As a practical consequence, it is crucial to pay great attention when setting up mixed blends of pheromone
components and plant volatiles to monitor or control insect pest populations since their combined effect have to be considered a priori
unpredictable. As a practical consequence, it is crucial to pay great attention when setting up mixed blends of pheromone components
and plant volatiles to monitor or control insect pest populations since their combined effect have to be considered a priori
unpredictable.
S17 - The complexity of sensory perception in insects: the study case of codling moth
Anfora G, Tasin M, De Cristofaro A*
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all'Adige (TN), Italy
The ethyl (2E,4Z)-2,4-decadienoate (pear ester), a pear derived volatile, has been reported to act as kairomone for males and females,
both larvae and adults, of the codling moth, Cydia pomonella (L.) (Lepidoptera Tortricidae), the major pest of the apple orchard. The
main component of its sex pheromone (E,E)-8,10-dodecadien-1-ol (codlemone) is widely used in monitoring systems and direct
control methods. Several recent studies have reported different levels of interferences in the activity of the two compounds on codling
moth. We present and discuss this evidence as a study case of the complex and often unpredictable behavioural interactions among
pheromone components and plant volatiles.
Electrophysiological studies (electroantennography and single cell recordings) demonstrated the occurrence of a population of
olfactory receptor neurons sensitive both to codlemone and pear ester in male and female C. pomonella. Although the presence of
generalist olfactory cells sensitive both to pheromone components and to plant volatiles has been many times detected, the biological
meaning of these observations are not still completely understood. However, they can not be merely explained by interferences with
the binding proteins or the membrane receptor sites due to high concentrations of stimuli but more likely by their low selectivity or by
the presence of different receptors sites on the same neuron.
The results of catching experiments carried out in the field using single-compound or blended lures, with respect of the effective
dosages and captures sex ratio, are consistent with interactive effects of the two substances. Field trials have also shown the
attractiveness of pear ester to other Tortricid species, particularly Cydia splendana (Hübner), Cydia fagiglandana (Zeller) and Hedya
nubiferana Haworth, which share with the codling moth the presence of codlemone in the pheromone blend. It also supports the
hypothesis of common sensory channels for the perception of codlemone and pear ester.
In addition to the well known integration of the different neural signals occurring at the central nervous system level, the finding of
complex peripheral interferences in odour perception supply a new basis for a better understanding of the reported ability of plant
compounds to enhance or reduce the biological activity of a pheromone component. These results should also be taken into account
when applying semiochemical-based monitoring or control methods.
18
S18 – The complexity of sensory perception in insect: study of an aphid-plant-fungal system
Ganassi S, Claudio A*, De Cristofaro A§, Andolfi A$, Evidente A$, Sabatini MA
(*) Istituto di Scienze delle Produzioni Alimentari, CNR, Bari, Italy
This study is framed in the context of an innovative approach on the search of natural compounds of fungal origin that interfere with
the insect pest processes of host plant selection. Previous studies revealed that the fungal isolates Trichoderma citrinoviride Bisset
ITEM 4484 and Trichoderma harzianum Rifai ITEM 908 can influence feeding preference of two different species of aphids
restraining specimens from feeding on treated leaves. The species are Schizaphis graminum (Rondani), widespread and dangerous for
various cereals of agricultural interest, and Myzus persicae (Sulzer), a cosmopolitan and polyphagous species. A multidisciplinary
study, aiming to identify the metabolites responsible of the effects on aphid feeding, have been carried out. The purification of the
bioactive metabolites from organic extracts of T. citrinoviride culture carried out with chromatographic techniques combined with
behavioral tests has lead to obtain and characterize six metabolites, which belong to three distinct chemical family: a symmetrical
disubstituted hexa-1,3-dienyl ester of acetic acid, a tetrasubstituted cyclohexane-1,3-diol, and four bisorbicillinoids. Four of the six
metabolites influence aphid feeding preference. These metabolites could constitute the base for the development of new active
principles for crop protection from aphids. Preliminary results of the purification of T. harzianum extract showed that this fungus
produces several different bioactive metabolites some of which could be identical to those isolated from T. citrinoviride. In terms of
the aphid sensory organs involved in the perception of the metabolites, electroantennographic studies showed that neither the volatile
compounds of the behaviourally active suspensions and extracts of fungal cultures nor the identified metabolites were able to stimulate
the antennal olfactory sensilla of both species. Using a Single Cell Recording technique we demonstrated that taste cells located on the
aphid tarsomeres are involved in the perception of fungal metabolites. Single-cell responses to some fungal metabolites are dosedependent and consistent with behavioral results. Studies pointed out a different sensitivity across the winged and wingless morphs of
S. graminum towards the fungal cultures and some of the characterised metabolites. Indeed, some metabolites influence winged but
not wingless morph feeding preference. It may be conceived that the different sensitivity might be due to the different distribution and
number of adequate gustatory receptors. This hypothesis is largely supported both by the lower percentage of taste cells that evoke
spike activity in wingless morphs and by the lack of responses to the tested compounds. SEM and TEM studies will be carried out to
describe the morphological features of the implicated sensorial structures in aphids. In particular, we aim to functionally and
morphologically define the sensory structures located on the tarsi which remain unknown to date.
S19 - ORL420, an example of a broadly-tuned human olfactory receptor
Veithen A, Wilkin F, Philipeau M and Chatelain P
TecnoScent S.A., 802 route de Lennik 1070 Brussels, BELGIUM
The mechanism of odorant molecule perception in vertebrates relies on the expression of olfactory receptors (ORs) in olfactory
neurons. Several hundreds of putative olfactory receptors have been identified in the human genome, and it is commonly admitted that
each of them might interact with more than one ligand, while each odorant molecule might trigger several ORs. However, the
molecular receptive range of a given OR is difficult to predict. Here, we present the deorphanisation and characterisation of the human
ORL420, also known as OR2W1. The receptor was transfected into HEK293 cells expressing olfactory chaperones. To favour its
surface expression and to allow its detection by a specific antibody, the twenty first amino acids of the bovine rhodopsine (rho tag)
were added upstream to the sequence of ORL420. The cells surface expression of the receptor was first demonstrated by
immunofluorescence, using an anti-rho tag specific antibody.
In a first odorant screening using a reporter gene-based functional assay and performed with 80 molecules, 29 of them were identified
as ligands. The activity of ORL420 was confirmed with both a calcium imaging-based functional assay and with an alternative assay
allowing direct measurement of cAMP. The potential influence of the rho-tag was also assessed by comparing the functional responses
of the rho-tagged receptor, of the 5 amino acid F5-tagged receptor and of the non-tagged receptor. In each cases, a similar response to
the reference activator anisyl acetate was observed.
In a subsequent structure activity relationship study, most of the initially found ligands were confirmed and additional activators were
found. The efficacies of most of these agonists are in the range of EC50 values varying from 3 to 300 µM. Surprisingly, no obvious
common structural characteristic was shared by all these activators. Although most of the agonists possess an aromatic ring, some
linear aliphatic ligands are also identified. Different chemical groups such as esters, aldehydes or alcohols are represented among the
ligands of ORL420, with the remarkable exception of carboxylic acids.
Interestingly, treatment of anisyl acetate by an esterase, thus converting anisyl acetate to anisyl alcohol and acetic acid which both still
have an odor, severely hampers the triggering of the receptor. This may be explained by the fact that the two products resulting from
esterase activity have lost the structural requirements that allow binding and that lead to activation of the receptor.
This work is supported by the Brussels region
19
S20 - Birth of an olfactory receptor clade in the teleost lineage
Ashiq Hussain, Luis Saraiva, Yen Yen Kwan, Sigrun Korsching
Institute of Genetics, University of Cologne
Trace amine-associated receptors (TAARs) in mammals recently have been shown to function as olfactory receptors. We have
delineated the taar gene family in jawless, cartilaginous, and bony fish. Taar genes are evolutionary much younger than the related OR
and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes
appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new
clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of
zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark,
mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial,
nonolfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete
loss of the aminergic ligand-binding motif, stringently conserved in the other 2 classes. Thus, class III taar genes seem to have
acquired a novel set of ligands and appear poised to eventually become an olfactory receptor gene family. The dN/dS analysis suggests
both minimal global negative selection and an unparalleled degree of local positive selection as another hallmark of class III genes.
The accelerated evolution of class III teleost taar genes conceivably might mark the birth of another olfactory receptor gene family.
Ligand characterization of selected zebrafish taar genes shows specificity for physiological ligands.
Trace amine-associated receptors (TAARs) in mammals recently have been shown to function as olfactory receptors. We have
delineated the taar gene family in jawless, cartilaginous, and bony fish. Taar genes are evolutionary much younger than the related OR
and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes
appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new
clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of
zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark,
mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial,
nonolfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete
loss of the aminergic ligand-binding motif, stringently conserved in the other 2 classes. Thus, class III taar genes seem to have
acquired a novel set of ligands and appear poised to eventually become an olfactory receptor gene family.
S21 - Localization of olfactory receptors heterologously expressed in S. cerevisiae cells
Sanz G, Persuy MA, Vidic J, Wade F, Longin C, Corcelli A, Lobasso S, Monnerie R, and Pajot E
INRA-NOPA Jouy-en-Josas France, INRA-GPL Jouy-en-Josas France, U. Bari Dept Medical Biochemistry Italy
Our group previously showed that S. cerevisiae yeast cells constitue an efficient means to express heterologous olfactory receptors
upon a galactose induction carried out at the low temperature of 15°C. In the present work, we study the kinetics of a human olfactory
receptor, hOR17-40, and its localization and functional response in sucrose gradient density subfractions, or in subfractions of a
detergent gradient (Optiprep), starting from a membrane fraction preparation. The abundancy of olfactory receptors co-localizing with
various protein markers, such as the yeast endoplasmic reticulum (Dpm1p), Golgi (Vps10p), or lipidic rafts of the plasmic membrane
(Pma1), evolves with time post-induction, as shown on immunoblots. Olfactory receptors were also localized and their amount
comparatively evaluated by immunogold transmission electron microscopy using a gold-labeled secondary antibody, in various
sucrose subfractions. The presence of co-expressed Golf (the Gα subunit of G proteins specific of olfactory sensory neurons) was also
examined in the various fractions.
Lipids analysis revealed various compositions for the sucrose subfractions. In addition, Pma1 marker located in the high sucrose
subfraction (plasmic membrane) proved to be attached to lipidic rafts, since it was found in the detergent-resistant fraction of a
subsequent Optiprep separation. On the contrary, olfactory receptors from any sucrose subfraction, including the plasmic membrane
subfraction, proved to be present only in detergent-sensitive domains, i.e. not attached to rafts. Thus, a given sucrose subfraction may
include both rafts and detergent-sensitive microdomains.
The functional response of olfactory receptors present in the various sucrose or Optiprep subfractions was monitored by Surface
Plasmon Resonance as already reported. Whatever the fraction considered, i.e. independently of the cellular localization as indicated
by the various protein markers (Pma1, Dpm1p, and Vps10p), olfactory receptors exhibited a functional response, and the level of
functional response is not directly correlated to the total amount of olfactory receptors and Golf revealed on the immunoblots. Finally,
this functional response is obtained regardless of lipidic rafts presence, and especially in the detergent-sensitive fraction of the
Optiprep gradient.
Altogether, our results thus demonstrate that localization of olfactory receptors within rafts lipidic microdomains is not an absolute
requirement for their efficient functional response.
This work was partly supported by the French ANR-07-PCVI-0027 contract.
20
S22 - Axon wiring and neural map formation in the mouse olfactory system
Sakano H
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
In the vertebrate nervous system, sensory information is spatially encoded in the brain, forming topographic neural maps that are
fundamental for cognition and higher-order processing of sensory information. Molecular mechanisms of the topographic map
formation have been extensively studied in the visual system. Nearly half a century ago, R. W. Sperry proposed the “chemoaffinity”
hypothesis, in which target cells present chemical cues to guide axons to their destinations. Axonal projection of retinal ganglion cells
is instructed by several pairs of axon guidance molecules that demonstrate graded expression in the retina and tectum.
Olfactory information is also encoded in a topographic map formed on the olfactory bulb (OB), a part of the forebrain. In rodents,
odors are detected with ~1,000 types of odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) in the olfactory
epithelium (OE). Each OSN expresses only one functional OR gene in a mono-allelic manner. Furthermore, OSNs expressing a given
type of OR converge their axons to a specific glomerulus on each glomerular map in the OB. During the olfactory development, OSN
axons are guided to approximate locations in the OB by the combination of dorsal-ventral (D-V) patterning based on anatomical
locations of OSNs in the OE, and anterior-posterior (A-P) patterning, regulated by OR-derived cAMP signals. The glomerular
arrangement along the D-V axis appears to be determined by axon guidance molecules expressed in a graded manner in the OE, e.g.,
Robo-2 and Neuropilin-2. Unlike D-V positioning, A-P positioning of glomeruli is independently of positional information in the OE.
Instead, OR-specific cAMP signals determine the expression levels of Neuropilin-1 (Nrp1) at its soluble ligand, Semaphorin-3A, in
OSN axon termini, forming a gradient of Nrp1 on the OB. Thus, the olfactory system also uses gradients of axon guidance molecules
to form the topographic map.
How then do guidance molecules regulate topographic map formation? Does map formation solely depend on axon-target interaction?
Topographic order emerges in axon bundles, well before they reach the target. Here we report the pre-target sorting of OSN axons and
its role in topographic map formation in the mouse olfactory system1).
1) Imai, T., et al.: Pre-target axon sorting establishes the neural map topography.
Science, in press (2009).
S23 - The OR37 subfamily: establishment of the clustered expression pattern
Strotmann J, Bader A, and Breer H.
Institute of Physiology, University of Hohenheim, Stuttgart, Germany
Odorant receptors (ORs) are encoded by the largest gene family in vertebrate genomes, comprising of more than 1000 members. Each
olfactory sensory neuron (OSN) in the olfactory epithelium (OE) selectively expresses one particular receptor gene from this large
repertoire; moreover, only one of the two alleles is chosen per cell. OSNs which express a distinct OR gene are usually broadly
dispersed throughout the OE; in contrast, cells expressing a member of the OR37 subfamily show a unique pattern: they are located
exclusively in a small central patch of the OE. The regulatory mechanisms which govern OR gene choice of individual OSNs in a
topographically restricted manner are still largely elusive. To obtain more insight into the underlying principles we have employed a
transgenic approach in mice that allowed to permanently label all cells that selected a defined OR gene for expression. For this
purpose a knock-in mouse line was generated in which expression of one member of the OR37 subfamily (OR37C) leads to the coexpression of Cre-recombinase (OR37C-IRES-Cre). By crossing this line to Cre-reporter mouse lines, all cells could be visualized that
transcribed OR37C at any time during development. As expected, labelled OSNs were found in the central patch which is typical for
OR37 expression. Surprisingly, however, numerous additional OSNs were found which were broadly dispersed throughout the OE.
Using in situ hybridization, mRNA for OR37C could only be detected in those cells located in the typical ‘OR37 patch’, suggesting
that all ectopic OSNs had ceased OR37C expression. The question whether these cells may undergo premature apoptosis was
addressed by immunohistochemical analysis for active caspase-3; none of them, however, expressed this pro-apoptotic marker. A
close examination of the ectopically positioned OSNs revealed that they all extended an axon towards the olfactory bulb (OB), and
indeed many glomeruli could be detected which contained a few labelled fibers. The location of these glomeruli in the medial and
lateral domains of the bulb indicated that these represented glomeruli that receive input from OSN populations expressing other ORs
than OR37C. Altogether, these data indicate that OSNs which initially express OR37C outside the typical patch do not continue, but
switch to the expression of a different OR gene, suggesting the involvement of a feedback mechanism downstream of gene choice that
restricts OR37C expression to the central patch.
This work was supported by the Deutsche Forschungsgemeinschaft
21
S24 - Olfaction Targeted
Mombaerts P
Max Planck Institute of Biophysics, Frankfurt, Germany
The mammalian olfactory system is increasingly recognized as an attractive model system for studying the formation of neural circuits
during development. An olfactory sensory neuron expresses one the of ~1000 intact odorant receptor genes in the mouse genome.
Axons of neurons a given odorant receptor gene converge onto a few of the ~1800 glomeruli in the olfactory bulb of the mouse. A
mapping problem is thus posed: ~1000 populations of neurons, each expressing a distinct odorant receptor gene, must be sorted onto
~1800 glomeruli. A productive approach to study axonal coalescence has been targeted mutagenesis of odorant receptor genes, to
express axonal markers in neurons expressing a given odorant receptor gene. These experiments have revealed that the odorant
receptor is a critical determinant of axonal coalescence into glomeruli.
S25 - The ESP peptide family and its role in chemical communication
Kazushige Touhara
The University of Tokyo
Many animal species utilizeexternal chemical cues called pheromones to send social and sexual information.In mice, pheromones are
detected by two olfactory sensory circuits, the mainolfactory pathway and the vomeronasal system, which leads to specificbehavioral
and endocrinological outputs. In the course of studies to seek forpheromones that are recognized by the mouse vomeronasal organ, we
identified amale-specific 7 kDa peptide produced in the extraorbital lacrimal gland andreleased in tear fluid (Kimoto et al. Nature
2005). This peptide, which wenamed exocrine gland-secreting peptide 1 (ESP1), stimulates female vomeronasalsensory neurons in
vivo. ESP1 turnedout to be a member of a previously-unrecognized multigene family that consistedof ~40 homologous genes,
suggesting that the ESP family may provide a basis forvariation in the pattern of information signals between individuals, sex,strains,
or species (Kimoto et al. Current Biol. 2007). ESP1 stimulates femalevomeronasal sensory neurons that express V2R-type pheromone
receptors,resulting in induction of c-Fos expression. The c-Fos-positive neuronsstimulated by ESP1 were shown to express only one
type of V2R, called V2Rp5. Furthermolecular biology experiments confirmed that V2Rp5 was a vomeronasal receptorspecific for
ESP1. The ESP1 signal received by V2Rp5 is sent to the accessoryolfactory bulb, which then projects to the basal forebrain, regions
that areimportant for reproductive and mating behavior in a sex-specific manner. Indeed,we observed the involvement of ESP1 in a
sexualbehavior of female mice, providing evidence that ESP1 is a sex pheromone inmice. The present study provides molecular and
functional link between a sex peptide pheromone and a selective neuralcircuitry leading to a behavioral output via a specific receptor
in the mousevomeronasal system. Mice utilize not only volatile odorants andpheromones, but also non-volatile peptides and proteins
as a chemosensorysignal that plays various biological roles. References: Kimoto, H., Haga, S.,Sato, K., and Touhara, K.: Sex-specific
peptides released from exocrine glandsstimulate vomeronasal sensory neurons in mice. Nature 437, 898-901 (2005); Kimoto,H., Sato,
K., Nodari, F., Haga, S., Holy, T., and Touhara, K.: The mouse ESPfamily: sex and strain differences, and implications in the
vomeronasal sensorysystem. Current Biology 17, 1879-1884 (2007)
Grants: This workwas supported in part by the PROBRAIN, JSPS and MEXT.
22
S26 - Dissecting an olfactory subsystem: GC-D neurons and the necklace glomeruli
Munger SD
Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201 USA
The mammalian nose employs several olfactory subsystems to recognize and transduce diverse chemosensory stimuli. These
subsystems differ in the anatomical position of their sensory cells within the nasal cavity, their targets in the olfactory forebrain, and
the transduction mechanisms they employ, and can also differ in the strategies they use for stimulus coding. One olfactory subsystem
includes a subpopulation of olfactory sensory neurons (OSNs) in the main olfactory epithelium that expresses the receptor guanylyl
cyclase GC-D, the cyclic nucleotide-gated channel subunit CNGA3 and the phosphodiesterase PDE2. GC-D-expressing neurons
recognize the peptide hormones uroguanylin and guanylin as well as natural urine stimuli. These peptides stimulate an excitatory,
cGMP-dependent signaling cascade that requires both GC-D and CNGA3 and which results in increased intracellular Ca 2+ and action
potential firing. Necklace glomeruli are the sole main olfactory bulb (MOB) targets of GC-D neurons, and afferent activity to these
glomeruli depends on both GC-D and CNGA3. However, in stark contrast to the homogeneous sensory innervation of canonical MOB
glomeruli from OSNs expressing the same odorant receptor, each necklace glomerulus receives heterogeneous innervation from at
least two distinct sensory neuron populations: one expressing GC-D and PDE2, the other expressing olfactory marker protein. In the
main olfactory system it is thought that odor identity is encoded by a combinatorial strategy and represented in the MOB by a pattern
of glomerular activation. This combinatorial coding scheme requires functionally homogeneous sensory inputs to individual glomeruli
by OSNs expressing the same odorant receptor and displaying uniform stimulus selectivity; thus, activity in each glomerulus reflects
the stimulation of a single OSN type. The heterogeneous sensory innervation of individual necklace glomeruli by multiple,
functionally-distinct OSN subtypes precludes a similar combinatorial coding strategy in this olfactory subsystem. Instead it suggests
that the necklace glomeruli could serve to integrate multiple chemosensory stimuli. Furthermore, necklace glomeruli exhibit extensive
intrabulbar connections with other MOB glomeruli via juxtaglomerular interneurons. The extensive and unexpected connections
between necklace and non-necklace glomeruli offer the opportunity for olfactory information integrated within the necklace glomeruli
to be processed in the context of other odor signals.
Supported by the NIDCD (DC005633)
S27 - A new receptor family in the vomeronasal organ
Rivière S, Challet L, Fluegge D, Spehr M, Rodriguez I.
University of Geneva
Mammals rely heavily on olfaction to interact adequately with each other and with their environment. They make use of seventransmembrane G-protein-coupled receptors to identify odorants and pheromones. These receptors are present on dendrites of
olfactory sensory neurons located in the main olfactory or vomeronasal sensory epithelia, and pertain to the odorant, trace amineassociated receptor and vomeronasal type 1 (ref. 4) or 2 (refs 5-7) receptor superfamilies. Whether these four sensor classes represent
the complete olfactory molecular repertoire used by mammals to make sense of the outside world is unknown. Here we report the
expression of formyl peptide receptor-related genes by vomeronasal sensory neurons, in multiple mammalian species. Similar to the
four known olfactory receptor gene classes, these genes encode seven-transmembrane proteins, and are characterized by monogenic
transcription and a punctate expression pattern in the sensory neuroepithelium. In vitro expression of mouse formyl peptide receptorlike 1, 3, 4, 6 and 7 provides sensitivity to disease/inflammation-related ligands. Establishing an in situ approach that combines wholemount vomeronasal preparations with dendritic calcium imaging in the intact neuroepithelium, we show neuronal responses to the
same molecules, which therefore represent a new class of vomeronasal agonists. Taken together, these results suggest that formyl
peptide receptor-like proteins have an olfactory function associated with the identification of pathogens, or of pathogenic states.
23
S28 - Studying the functional integration of adult-born neurons in the mouse olfactory bulb
Carleton A
Department of Neurosciences, Faculty of medicine, University of Geneva, 1 rue Michel-Servet, 1211 Genève 4, Switzerland
Genetic and epigenetic mechanisms controlling neuronal birth, migration, differentiation and death are key questions in modern
neuroscience.
While most neurons are born during embryonic and postnatal life, few regions keep producing new neurons. Since the thencontroversial reports of adult neurogenesis in the hippocampus and the olfactory bulb (OB) in the sixties, adult neural stem cells have
been extensively studied. The largest pool of adult neural stem cells is found in the subventricular zone (SVZ) located in the lateral
ventricles walls. Daily, SVZ stem cells produce tens of thousands of neuroblasts that migrate tangentially along the rostral migratory
stream into the OB. These neuroblasts then migrate radially and integrate into the OB network as functional interneurons, acquiring
the morphological, molecular and electrophysiological characteristics of granule and periglomerular cells within a month after their
birth.
However, the detailed sequences of maturation and the factors involved remain largely unknown. In order to address that we took
advantages of the relatively neuronal population homogeneity, the simple anatomical organization of the olfactory bulb, and we
performed laser capture microdissection and DNA microarray analysis and studied the transcritpome evolution during the maturation
of adult born granule cells. In addition we performed in vivo gene transfer using lentiviral vector infection that allows the visualization
of the newborn neurons within living tissue of the adult olfactory bulb. We implemented 2-photon time lapse microscopy. 2-photon
microscopy is an imaging technique that offers nowadays high spatial and temporal resolutions over several hours without any
phototoxicity in living tissue. As a consequence, it is now possible to visualize neurons migrating and developing in the middle of
their natural and relevant environment i.e. the brain networks. We found that it is indeed possible to accurately monitor in 4D the
dynamics of small cellular compartments such as growth cone, dendrite, and cell body during cell maturation.
If we want to understand how internal and/or external cues are affecting neuronal maturation, one will need to quantify changes in cell
morphology, speed… For that one need to delineate the borders (also named segmentation) of specific cells and compute different
parameters (such as area, volume, length, speed, growth cone surface, number of branching points…). For that we used methods and
knowledge developed in the field of computer vision that seemed appropriate to
S29 - Learning-dependent neurogenesis in the olfactory bulb determines remote olfactory memory
Sultan S, Mandairon N, Kermen F, Garcia S, Sacquet J, Didier A
Université Lyon1, Université de Lyon, F-69007 Lyon; Neurosciences Sensorielles, Comportement, Cognition, CNRS UMR 5020, 50,
Avenue Tony Garnier F-69007 Lyon, France
Adult neurogenesis is well described in the mammalian olfactory bulb: stem cells proliferating in the subventricular zone of the
telencephalon provide neuronal progenitors which migrate along the rostral migratory stream to the olfactory bulb (OB) where they
differentiate into bulbar interneurons, mostly granule cells (GCs).GCs are inhibitory interneurons which interact with mitral cells, the
relay cells of the OB to shape the output message of the OB to higher olfactory centers.. OB responses to odorants are highly sensitive
to experience and learning and GCs including adult-born cells could be at play in mediating experience-dependent plasticity of the
bulbar network. Indeed, short-term olfactory memory improvement induced by olfactory enrichment is accompanied by increased
newborn neuron survival in the OB and neurogenesis is modulated by olfactory learning. We used the DNA synthesis marker BrdU to
label newborn cells in the olfactory bulb during an associative learning task. Using a computer-assisted two-hole board apparatus,
adult mice were trained in an olfactory associative discrimination task in which they had to use an olfactory cue (+limonene) to find a
food reward as previously described .This task is hippocampus-independent both for acquisition and retrieval. Briefly, the animals
were first trained to dig for a reward placed in one of the holes of the board in the absence of an odorant. Once all the mice reached
80% of success in finding the reward (8 to 12 trials), they were trained to use an olfactory cue to dig in the hole containing the reward.
Learning took place over five days (4 trials per day) and was evidenced by the decrease in latency (time to find the reward) and the
increase in correct choices (first nose poke in the odorized hole). We found that olfactory learning favors survival of newborn neurons
in odor-specific areas of the olfactory bulb. Furthermore, newborn neurons selected to survive during learning are later on
preferentially recruited upon recall of the task as evidenced by expression of the immediate early gene Zif268. Finally, using
pharmacological manipulation of glutamatergic transmission, we show that learning improvement by the NMDA agonist Dcycloserine is accompanied by increased duration of both survival of newborn neurons and memory of the olfactory task. These results
show that adult born neurons in the olfactory bulb support remote olfactory memory. Olfactory bulb neurogenesis thus sub serves
crucial role in long-term olfactory memory.
24
S30 - Integration and survival of newly-formed neurons in the AOB of adult mice
Paolo Peretto
Dipartimento Biologia Animale, Università Torino
Integration of new neurons into the adult main olfactory bulb (MOB) of rodents occurs throughout life. Newly-formed neurons are
generated in the residual of the embryonic SVZ and migrate through a restricted pathway, the rostral migratory stream, to the granular
and glomerular layers of the MOB where they differentiate into local inhibitory interneurons. Although the functional meaning of this
prolonged neurogenesis is still unclear, several data indicate sensory olfactory experience plays a prominent role in the survival and
differentiation of new neurons. Presence of neuroblasts has also been demonstrated in the posterior dorsal portion of the MOB, namely
the accessory olfactory bulb (AOB). The AOB is part of the vomeronasal system which mediates innate responses elicited by
pheromones. This pathway is sexually dimorphic in different vertebrate species and its development is regulated by steroid hormones.
Functionally, the AOB is subdivided into two sub-regions receiving segregated projections from two distinct sub-populations of
vomeronasal receptor neurons which respond to different types of pheromonal compounds. We have previously shown that 3 weeks
following genesis in the SVZ of adult rats, numerous newborn neuroblasts are still visible in the AOB principally in the granule cell
layer. At this survival time the neurogenesis rate differs in the anterior AOB of the two sexes, suggesting different regulatory
mechanisms in the two functional subdivisions. Nonetheless, to date neither integration of newly generated elements nor activitydependent modulation of neurogenesis has been clearly investigated in this brain region. In order to address these issues, we have
analyzed the AOB of mice by using multiple approaches including: BrdU cell tracer injections, cell transplants, confocal 3-D
reconstructions, analyses of specific markers of AOB interneurons and chronic exposure of females to olfactory/pheromonal stimuli.
The obtained results indicate: i) a pool of SVZ neuroblasts specifically targets the mouse AOB of both sexes equally and acquires
morphological and neurochemical profiles of adult neurons; ii) differences between the dymanics of integration and selection of newly
formed neurons into the main OB and AOB; iii) direct contact with male soiled bedding, but not exposure to the derived volatiles,
significantly enhances survival of new neurons in the whole AOB, but not in the MOB of female mice. Overall, these results indicate
the AOB of mice represents a unique site of adult constitutive neurogenesis regulated by specific internal and external cues.
Supported byCompagnia di San Paolo (Neurotransplant, 2004.2019)
S31 - Electronic Nose Based Applications in Medicine
A.D’Amico
University of Roma Tor Vergata,Dept. of Electronic Engineering via Politecnico,1 00133 Roma, Italy
The application of an electronic nose in medicine is based on a simple and fascinating idea: the health-state of an individual can be
assessed and monitored with a completely non invasive methodology based on the chemical fingerprint of the volatile part of
biological specimen normally released by human beings (such as exhaled breath, urine, sweat, skin transpiration).
Beside the application of classical analytical chemistry instruments, gas sensor arrays (named electronic noses) represent a valid
alternative for volatile compounds fingerprinting. The positive results collected in the numerous experiments performed with the
electronic nose in the last years form a solid basis for the development of practical diagnostic method.
Electronic nose applications in the medical field can be grouped as in-vitro and in-vivo experiments. Among in-vitro studies bacteria
cultures shows to be the most investigated subject (almost the 50% of electronic nose works in medicine) [1,2]. The prevention, which
is of great importance especially for oncologic diseases, is the main goal of in-vivo experiments which are mainly devoted to cancer
studies. The altered metabolism of tumour cells may result in an anomalous composition of emitted volatile compounds profile. The
contribution of lung cancer to the breath composition is the more investigated case probably because this is the most insidious among
tumors. Many investigations in the last 10 years have emphasized the correlations between the anomalous concentration of a limited
set of compounds with various kinds of lung cancers [3-5], breast cancer [6], and melanoma [7].
Besides the studies on cancer numerous papers are found about the potentialities of chemical sensor arrays for the identification of
several diseases [8]: for the study of the evolution of ulcers and wounds [9] or of metabolic alterations such as those supposed to take
place in neurological disorders [10].
This presentation will review and comment some of the most significant applications, in the medical field, of artificial olfaction
systems also trying to evidence critical drawbacks in the practice of gas sensors arrays. In particular the large variability of human
samples which influences the composition of the volatiles giving rise to unsatisfactory reproducibility of the analysis will be
discussed.
References
[1] Sensors and Actuators B Chemical 2006; 115, 1: 5-13.
[2] Journal of Endodontics 2007; 33, 9:1106-1109.
[3] Chest 2003; 123: 2115–2123.
[4] Biosensors Bioelectron. 2003; 18: 1209–1218.
[5] Breast J. 2003; 9: 184–191.
[7] Skin Research and Technology 2007; 14, 2: 226-236.
[8] Nat Rev, Microbiol 2004; 2: 161–166.
[9] J Wound Care 1995; 4: 404–406.
[10] Med Sci Monitor 2005; 11: 366–375.
25
S32 - Detecting wound infection
Krishna C. Persaud, Anna Maria Pisanelli, Arthur Bailey
The University of Manchester
The process of traditional microbiological diagnostics is often lengthy and costly. Multi-sensor array technology has potential to
provide an alternative, faster and less expensive approach to the clinical identification of specific organisms. Our approach is based on
identifying certain compounds in the gases emitted by bacteria in clinical samples. A need exists for rapid detection of bacterial
infection in critically ill patients who may have serious wounds. We have developed a rapid and reliable method for detection of
microbial infection by monitoring the headspace from the infected wounds. We analysed using gas chromatography-mass
spectrometry the headspace volatiles emitted from Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa in
order to identify volatile markers of infection. Wounds become infected when microorganisms from the environment or from the
patient’s body enter the open wound and multiply. The symptoms related to an infection include abnormal flushing of the skin, heat,
pain and tenderness and abnormal odours, such as fruity odours that often indicate the presence of Staphylococcus or foul odours due
to presence of gram negative bacteria. The results obtained from this study allowed us to select suitable sensor technology to build a
mobile system for non invasive wound monitoring using an array of gas and odour sensors. The criteria for selection of the sensors
was determined by the sensitivity and selectivity of the sensors to a limited number of the volatile compounds (VOC) produced by
living bacteria types that are defined as the most frequently found in clinical conditions during treatment of various types of wounds.
As a result we selected a range of metal oxide gas sensors, which were coupled to a sampling system allowing preconcentration of the
low amounts of volatile compounds emitted from clinical samples. For sampling from swabs or dressings from patients a solid phase
microextraction approach was used for preconcentration of the low concentrations of volatile compounds emitted. An instrument was
constructed that incorporated an automated solid phase microextraction desorption system, a hybrid sensor array, electronics, and data
processing to enable the system to be used for clinical validation. The instrument was then validated on samples taken from burns
patients from two hospitals, one based in Lithuania and the other in the UK. The results indicate that over a wide population of
patients it is possible to swab a wound, and sense volatiles that allow discrimination between infection and non-infection.
IST-027859 EU project WOUNDMONITOR
S33 - Odor Communication for Olfactory Display
Jeong-Do Kim1, Hyung-Gi Byun3, Sang-Goog Lee2, Chang-Hoon Park1, Jin-Ho Ahn1 and Jang-Hoon Lee4
Hoseo University, The catholic University of Korea, Kangwon National University
1 Dept. of Electronic Engineering, Hoseo University, Asan, Chungnam, Korea
2 Dept. of Multimedia System Eng., The catholic University of Korea, Bucheon, Gyeonggi
3 School of Electronic, Information and Communication Eng., Kangwon National University, Samcheok, Kangwondo, 245-711,
Korea
4 Dept. of Environmental Engineering, Hoseo University, Asan, Chungnam, Korea
Smell output is one of the most exciting and popular explorative research areas in the field of Human Computer Interaction(HCI). The
olfactory system influences and stimulates human memory and this mechanism may help to reinforce human senses in a positive
manner. However, the lack of an effective scent generator to generate various odors has hampered research a great deal. Recently,
there has been a great increase in interest in how to manufacture a scent generator device that would be able to represent smell output.
Indeed, a commercial device has recently appeared on the market.
Despite the appearance of commercial scent generators, information related to the olfactory system is not adequately provided as
multimedia information when compared to information related to sight and hearing. Appropriate and credible research needs to be
conducted to provide more exact information on how olfactory data can be presented as multimedia. Olfactory information has to
include existing multimedia information.
In order to transform olfactory information into multimedia, we have to develop the following techniques:
- How to derive information from smell
- How to transmit smell information
- How to use a scent generator
The basic reason for creating odors through computer services is to heighten emotions. Previous studies have reported that smells have
the potential to augment emotions and. For this purpose, this paper proposes a method for presentation, transmission, and computer
service.
In this paper we present a method, that may be a way to use smell as multimedia information, for presenting olfactory information.
Information related to smell can be derived from the characteristics of odors such as intensity, persistence, hedonic tone, and odor
description.
In this paper, we try to solve two questions through experimental activity. First, what is the proper intensity that has to be used in a
scent generator? Second, can emotions be heightened by giving off odors during conversations?
We implemented a computer service like movie with scents and an internet service with scents in order to test the usability of the
olfactory presentation method. In order to confirm the augmentation of emotion, we measured the physiological signals by using four
biosensors: EMG, SC, ECG, and RSP.
In our experiment, we confirmed that physiological emotion can be changed by giving off scents in computer services.
26
S34 - Integration olfactory display into a video game for exercise
Changhoon Park, Jin-Ho Ahn, Hyung-Gi Byun, Jeong-Do Kim, Sang-Goog Lee
Hoseo Univ., Kangwon Univ., Catholic Univ. of Korea
Recently, game controllers for physically realistic experiences have become popular such as Dance Pad for Pump It Up, a Korean
arcade game, EyeToy for Sony's Playstation, and Wii Remote and Wii Balance Board for Nintendo's video game consoles. These
special interfaces allow to detect movement and gestures by using sensor and computer vision technology instead of standard
interfaces such as keyboard and mouse.In this paper, we demonstrate a bicycle-based exercise game to enhance the physical ability
and acquire exercise skills. As ordinary physical training is routine and simple, the people are inclined to stop their training plans
prematurely. Thus, exercise-purposed games must provide the enjoyments as well as the physical effects.
It is well-known that pleasant scents induce positive effects for cognitive, psychological, physiological and physical enhancements of
people. In particular, previous publications show that peppermint odor could reduce the workload due to exercise and arouse attention
efficiently. Both jasmine and lavender odors also caused significant decreases in heart rate. Thus, we expect odors synchronizing with
game contents for exercise induce the increment of exercise time and continuous participation in the game.
Consisting of collaborative contents, game interfaces stimulating the reality, and user-dependent services, the proposed bicycle-based
game integrating display can satisfy most requirements for exer-gaming. We evaluate its result by the analysis of bio-feedback signals
of subjects.
S35 - Methods for building computer–controlled, fMRI–compatible, inexpensive olfactometers suitable for event-related
imaging designs
Lundström JN, Gordon AR, Alden EC, Albrech J, Boesveldt S.
Monell Chemical Senses Center, Philadelphia, PA
Many human olfactory experiments call for fast and stable stimulus-rise time as well as exact and stable stimulus-onset time. These
features are particularly important in modern neuroimaging experiments, which frequently employ techniques such as event-related
functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation. Due to their temporal demands, these
techniques require an olfactometer, a piece of equipment that either comes with a high price tag or requires a high degree of technical
expertise to build. Here, we detail the construction of two olfactometers, one 8-channel olfactometer, aimed primarily at behavioral
experiments, and one multi-channel olfactometer (8-64 channels) aimed primarily at event-related fMRI experiments. Both are built
almost exclusively with “off-the-shelf” parts, require little technical knowledge to build, have relatively low price tags ($8.000 and
$14.000, respectively), and are controlled by E-Prime, a turn-key ready and easily-programmed software commonly used in
psychological experiments. Both olfactometers can present either solid or liquid odor sources, and exhibit a fast stimulus-rise time
(53ms and 34ms, respectively, for 90% concentration; measured at the nose) and stimulus-onset time (38ms and 314ms inside scanner
bore; measured at the nose). In both olfactometers, airflow can be regulated separately for each channel, and odors can be delivered
either mono- or birhinally. Fast switching between odor and clean air channels occurs near the nose, and potential tactile cues (puffs)
are eliminated by embedding the flow into a low-flow air stream. Both olfactometers are designed so that they are easy to move and
store in public places. We will provide detailed descriptions of the olfactometers construction as well as its individual parts with their
expected price and potential optional modifications. Moreover, we will be presenting concentration curves, as measured with a
photoionization detector, for various airflows together with corresponding GC analyses of signal intensity drop. Data from three
validation studies will also be presented. We measured olfactory ERP responses using the behavioral olfactometer and BOLD
responses using the fMRI olfactometer using both even-related and block designs. We were able to reliable record olfactory responses
in all studies. Many human olfactory experiments call for fast and stable stimulus-rise time as well as exact and stable stimulus-onset
time. These features are particularly important in modern neuroimaging experiments, which frequently employ techniques such as
event-related functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation.
Supported by Monell Chemical Senses Center and NIDCD (R03DC009869) awarded to JNL
27
S36 - Scientific and nonscientific wine descriptors in wine language
Adrienne Lehrer
University of Arizona
When wine scientists, especially enologists, communicate about the taste and aroma of wine, it is essential that their words have the
same meaning for the whole scientific community. To achieve this goal, they must be carefully trained, and some standard stimuli or
samples must be available to the whole community. Ann Noble and colleagues at the University of California at David have devised a
language for wine aromas with instructions for creating such standards. (I will briefly contrast wine standards with those used in
training experts for ciders and beer.) In evaluating wine, experts (scientists and nonscientists) after tasting a wine, try to identify the
varietal or regional type, and evaluate it with respect to that category. Many of the descriptive words are scalar, e.g., 'full-bodied' or
'light', and what is appropriate for wine type like a Medoc might be very different for a California Zinfandel. A heavy Grenache might
be light for a Syrah. Individuals who evaluate beverages and foods for commercial purposes also need to use language consensually,
and they are carefully trained as well, but for them it is less important that their usage be universal.
Other experts may try to use this language as well, generally without the technical chemical terms. Wine writers and critics use much
of the scientific language, but in addition, they use a colorful, literary language with metaphors, images, and references that the
scientists would abhor, like 'a muscular, big-boned, blockbuster of a wine.' Yet this makes the writing livelier and more interesting to
read. In addition, the wine preferences of these writers are revealed in their descriptions and judgements, and readers have to decide
whether to trust them. Wine merchants and sommeliers are experts of different type, and they have to gear their language to that of
their customers.
Many non-expert wine drinkers understand and use the language of experts, but many, especially novices, do not. Yet some believe
that they should learn this vocabulary, and they enroll in courses and workshops and attend directed wine tastings. The advantage to
them is that they can understand the wine writers, and they can order wine in stores and restaurants better by describing the kinds of
wines they like.
However, often discussions of wine take place in intimate social settings, like dinner parties, where an important function of the
discussion about wine is for social bonding, sharing the experience, amusing other participants, and having a good time. In situations
where people are discussing the wines they are drinking, language is used to point out properties of the wine, and the words used may
not be 'correct' from the point of view of the experts, but if the discourse succeeds in getting other participants to notice something
they did not notice before, the speaker has succeeded.
S37 - Communicating Taste
Bieler LM (a), Diederich C (b)
(a) University of Zurich, (b) University of Basel, Switzerland
The content of this presentation connects to the project Sensory Language and the Semantics of Taste (Sens), an interdisciplinary
project in which the universities of Zurich and Basle, the ETH Zurich and the Zurich University of Applied Sciences are involved.
Linguists, food sensory analysts and cognitive scientists investigate the lexis of taste in the German and English language. The project,
sponsored by the Gebert Rüf Stiftung, conducts basic research at the interface of sensorics and language and is application-oriented,
working together with experts from the food industry to compile an online lexicon of taste. SenS brings together linguists, sensory
analysts and psychologists to provide a broad porfolio of skills in the fields of language, sensorics and food marketing
(www.sensorysemantics.ch). The aim of the project is an online dictionary of German taste terms which can be used for both scientific
and commercial reasons. The focus is not only on the actual semantics (meaning) of various taste terms but also on the pragmatics,
namely how we use language to communicate taste and, most importantly what methods can be used to go about. The question of how
language and taste relate to each other is still very much unresearched.
In this contribution, two methods will be introduced which can be used to demonstrate everyday communication on taste: focus group
discussions and corpus research. How we speak about taste on an everyday basis can differentiates strongly from how taste is
measured and described in sensory science. The meaning of an adjective describing taste e.g. is not fixed but derives in the discussion
and changes according to its respective context. The aim is to highlight both advantages and challenges regarding the two
methodologies and emphasizing how they can contribute to a linguistic and scientific understanding of language and sensory
perception. The method of corpus research can serve as a tool for an inter-linguistic approach. With the help of different corpora we
can analyze usage of taste terms in different languages and thus contribute to the aspect of translation, both interesting for academic
and commercial purposes. Initial results of the “taste terms” ‘crunchy’/’crispy’ and their German aquivalents knusprig/knackig
regarding this qualitative and quantitative method will be presented in a second part of the presentation. These adjectives appear
frequently in food advertisements. Our goal is to observe what senses these words aim to address and how they serve as tools to make
a product seem more appealing (thus respond to the hedonic aspect of product marketing). It is important to see how these terms can
be defined most adequately and what features must be considered in a definition, which can serve the industry. With a linguistic
approach and methodology the word meaning and broad potential of usage can be demonstrated.
28
S38 - Lexicon in the Sense of Smell: Etymology vs. Motivation. A Comparison Between English and French.
Rémi DIGONNET (Assistant, MA, PhD in progress, University of Lyon, France)
University of Lyon 3 - France
The study of olfactory lexicon shall investigate the various lexical processes used for the appearance of such a lexicon in English and
French. From the words smell and odeur, a typology of different lexical processes shall be defined to point either morpho-semantic
neology (odourless), or semantic neology (smell a rat), or morphological neology (electro-olfactogramme = EOG). The first category
includes affixation (prefixation, suffixation, back-formation), composition (compounding, blend) and phonic imitation such as
onomatopoeia. The second category encompasses either change in grammatical category such as conversion or change in meaning
(metaphor, metonymy). The third category gives way to morphological reduction thanks to clipping or initialing. Besides the
consideration of internal matrices, external matrices such as loan words (bouquet) may contribute to the study of an estimated limited
lexicon.
The focus on both etymological and motivational approaches calls into question the definition of the concept of “reality”. Does the
emergence of lexicon come from an objective reality (etymology) or from a certain vision of reality (motivation)? To what extent is
the contemporary lexicon in the sense of smell rooted in etymological foundations? What is the part of motivation in olfactory word
formation? From direct motivation (phonetics) to indirect motivation (morphology and semantics), we may wonder whether
experience influences the emergence of a new lexicon in the sense of smell. Synaesthesia will appear as one of the main focuses in so
far as the different senses (vision, touch, taste, etc.) are often used to express the sense of smell. An attempt to compare motivation in
various languages shall determine different ways of building words.
In a diachronic perspective, we may focus on both opacification and remotivation phenomena. Opacification occurs when the meaning
of a word cannot be inferred from its form anymore. The word is perceived as being opaque, unmotivated. On the contrary, the
principle of remotivation (folk etymology) befalls when a new relationship, which did not exist, is established between the form of a
word and what it refers to. The word is remotivated thanks to new personal/cultural bindings, i.e a new motivation is artificially
reconstructed. Opacification and remotivation can be quite telling about the potential continuum to take place between etymology and
motivation. Indeed, it will be interesting to highlight borderline cases as an attempt to explain genesis of lexicon in the sense of smell.
S39 - Taste and food acceptance: sweet, bitter, salty and umami tastes, a complex relationship
Piero A. Temussi
Department of Chemistry, University of Naples "Federico II", Via Cinthia, 80126 Naples, Italy and National Institute for Medical
Research, The Ridgeway, London NW7 1AA (UK)
Sweet, bitter salty and umami are the main taste modalities linked to food acceptance, with sweet governing the acceptance of valuable
caloric food, bitter presiding on the rejection of toxic compunds and umami leading to proteic food. Both sweet and bitter molecules
are very numerous. The response of nature to the need of sensing many different ligands has been to generate many different bitter
receptors and one versatile receptor for sweet molecules. molecules are recognized by more than 30 GPCRs, named T2Rs. of the
molecular bases of sweet taste is very important not only for its intrinsic biological significance but also for the design of new artificial
sweeteners. Up to few years ago design was complicated by the common belief that different classes of sweet compounds, notably
sweet proteins, might interact with different receptors altogether. The identification and functional of the receptor for sweet taste
shown that there is but one receptor, drastically changing our approach to the development of new sweeteners. The explanation of how
the sweet receptor can bind several different classes of molecules is that rather than multiple receptors there are, apparently, multiple
sites on the single sweet taste receptor. In the present talk, the mechanisms of interaction of small and macromolecular sweet
molecules will be examined, with particular emphasis on sweet proteins. homology modelling, reliable models of all possible hetero
dimers of the human T1R2 and T1R3 sequences with the closed (A) and open (B) conformations of the mGluR1 glutamate receptor,
used as template. The most important result of these studies is the “wedge model”, the first explanation of the taste of sweet proteins.
Before the discovery of taste receptors, it was common belief that sweet and bitter receptors are closely related, but it has been found
that there is a single sweet receptor (T1R2-T1R3) and many unrelated bitter receptors (T2Rs). It is proposed that bitter chiral isomers
of sweet compounds, do not bind to one of the T2Rs but to T1R1-T1R3, the umami receptor, the third modality linked to food
acceptance. This hypothesis is made possible by recent developments in code signalling: although most receptor cells do respond to
only one taste, most presynaptic cells accept signals from taste cells responding to two or more different taste qualities.
29
S40 - Sweet taste: from receptor to perception
Wolfgang Meyerhof, Marcel Winnig, Bernd Bufe
German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
Humans evaluate the quality of their food by the sense of taste. They categorize taste stimuli into the five distinct qualities, sweet,
bitter, umami, sour, and salty with each taste quality thought to fulfill a specific physiologic function. Sweet taste presumably
functions as a sensor for calories in form of carbohydrates. Indeed, many sugars elicit strong sweet taste perception. However, sweet
stimuli are not confined to mono- and disaccharides. They include numerous other chemically diverse substances such as
oligosaccharides, polyols, nitroanilines, sulfonyl amides, aryl ureas, oxims, hydrazides, sulfamates, carboxy benzophenones, dipetide
esters and amides, dihydrochalcones, sulfones, halogenated sugars, terpenoids, L- and D-amino acids, polypeptides, and metal ions.
Sweet taste is elicited through activation of a special taste receptor present in a subset of taste receptor cells of the oral cavity that are
tuned to detect sweet stimuli. The receptor protein is a heterodimer of the subunits, TAS1R2 and TAS1R3, members of class C of G
protein-coupled receptors. Functional expression studies revealed that all tested sweet stimuli activated the TAS1R2-TAS1R3
heterodimer suggesting that the receptor possesses multiple interaction sites in different regions. This finding, genetic data, and
observations in gene-targeted mice suggested that the heterodimer functions as a general sweet taste receptor. However, TAS1R2 and
TAS1R3 homodimers may also contribute to sweet taste sensation induced by high sugar concentrations or by few definite sweet
compounds.
Our objective is to explain sweet taste perception on the basis of the receptor’s pharmacological properties. To this end we combined
human sensory studies, homology modeling, and functional expression. Our results show, in line with published data, that various
ligands interact with different sites located in the large N-terminal ectodomains or in the transmembrane regions of both TAS1R2 and
TAS1R3. Moreover, the data also indicated that the sulfonyl amide sweeteners saccharin and acesulfame K interact at low
concentrations with an orthosteric site, whereas at high concentrations these compounds also interact with an allosteric site. This
eventually leads to a bell shaped activation curve of the receptor that is paralleled by psychometric functions explaining the off-taste of
these compounds at high concentrations. Removal of the sweeteners from the allosteric site causes strong receptor activation in vitro
and the phenomenon of "sweet-water" taste, a sensory after-impression, in vivo. Together, our data suggest that even complex sensory
experiences can be explained on the basis of the sweet taste receptor’s molecular properties.
S41 - Downstream signaling effectors in sweet taste
Kinnamon S, and Chaudhari N
University of Colorado, Denver Colorado USA
Sugars, synthetic sweeteners, and sweet proteins bind to the G protein coupled receptor, T1R2/T1R3. The canonical transduction
pathway for sweet involves Gbg activation of PLCb2, production of the second messengers IP3 and diacylglycerol, release of Ca2+
from intracellular stores via IP3R3, and Ca2+-dependent activation of a monovalent selective cation channel, TRPM5. These events
lead to membrane depolarization and release of ATP as a transmitter. The contribution of these intracellular effectors was documented
by genetic knockout of PLCb2, IP3R3, and TRPM5 in mice. Each severely compromises sweet taste transduction. Although less
investigated, cAMP may also play a significant role in sweet taste. Sweet taste stimuli elevate cAMP levels in taste tissue in mice and
rats, and cAMP depolarizes sweet-sensitive taste cells by blocking a K+ conductance. Several effectors that regulate cAMP levels are
expressed in sweet-sensitive taste cells, including adenylyl cyclases, phosphodiesterases, and the Ga protein, gustducin. Although
gustducin knockout mice are compromised to sweet stimuli in anterior taste fields, gustducin’s role in transduction remains elusive.
By analogy with transducin, gustducin is assumed to activate phosphodiesterase and decrease, rather than increase cAMP levels. We
tested whether basal (unstimulated) cAMP levels may be altered in the gustducin knockout mice, by measuring cAMP levels in taste
buds. Gustducin knockout mice had significantly elevated resting cAMP levels compared to wildtype mice. To determine if the
elevated cAMP impacts transduction, we examined Ca2+ responses to the bitter stimulus, denatonium in Gus-GFP-labeled taste cells
of wildtype and knockout mice. As expected, labeled cells of the knockout mice had significantly depressed Ca2+ responses compared
to the Gus-GFP cells of the wildtype mice. To determine if the depressed response stems from cAMP-dependent Protein Kinase A
(PKA), we we applied the PKA inhibitor, H-89. We found that H-89 dramatically unmasked responses to denatonium in Gusknockout taste cells and even enhanced responses in wildtype GFP-labeled taste cells. We hypothesize that PKA inhibits transduction
by phosphorylating PLC signaling effectors. Our data suggest that a primary role of a-gustducin in circumvallate taste buds is to
maintain cAMP levels low, thereby preventing tonic adaptation to bitter taste stimuli. We are currently examining whether gustducin
plays a similar role in regulating sweet taste transduction in the anterior tongue and palate.
30
S42 - Adventurous TRPs: Chemoreception at the interface of nutritional and biomedical sciences
Appendino G.
DISCAFF - Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale,
Novara (Italy)
The discovery of specific sensory receptors activated by spice constituents has identified modern targets for age-old chemoreception
leads like capsaicin from chili, piperine from black pepper, eugenol from clove, cinnamaldehyde from cinnamon, allicine from garlic
and menthol from mint.1 These receptors are ion-channels of the TRP-type, and their manipulation holds great potential not only in
terms of pharmacological and nutritional exploitation, but also for the study of chemoreception and its physiological implications.2 A
further fascinating feature of several TRPs is their complex and promiscuous gating by endogenous lipids and plant compounds also
acting at cannabinoid receptors (CBs),3 while there is also growing awareness that TRP channels in mammals are essential elements
of all sensory pathways with the exception of sight.4
Research aiming at identifying chemoreception leads from spices and at elucidating the molecular bases of their activity will be
presented, navigating at the germane and blurred interface between nutrition and medicine. In particular, the following issues will be
described as case-histories of research in the realm of TRPs:
1. The identification of the active conformation of anandamide on TRPV1. By synthesizing conformationally constrained analogues of
the polar ethanolamidic head of this endocannabinoid, it was possible to demonstrate that TRPV1 recognizes the anti conformation of
anandamide, while cannabinoid receptors do not show any discrimination between the syn and the anti conformations of this
endolipid.
2. The identification of umbellulone the offensive principle of the headache tree (Umbellularia californica Nutt.), a clarification of the
mechanism by which this compound activates TRPA1, and the surprising and puzzling chemical behavior of this monoterpene ketone.
3. The development of an ultrapotent analogue of capsaicin, and a discussion of the dissection between sensory properties of vanilloids
and their potency as TRPV1 activators.
Taken together, these case-histories will give a glipse of the challenges facing research on TRPs, also highlighting their relevance for
sensory transduction and for chemoreception in general.
1.Appendino, G.; Minassi, A.; Pagani, A.; Ech-Chahad, A. Curr. Pharm. Design 2008, 14, 2-17. 2. Gavva, N. R. Trends Pharmacol.
Sci. 2008, 29, 550-557. 3. Akopian, A. N.; Ruparel, N. B.; Jeske, N. A.; Patwardhan, A.; Kenneth, M.; Hargreaves, M. Trends
Pharmacol. Sci. 2009, 30, 79-84. 4. Demann, N.; Voets, T.; Nilius, B. Curr. Biol. 2008, 18, R880-R889.
S43 - Some like it cool : novel technologies for testing the menthol receptor
Sabrina Corazza, Michela Stucchi,
Axxam SpA
Menthol is a plant-derived cyclic monoterpene alcohol that gives plants of the Mentha genus their characteristic minty smell and
flavour. When menthol is applied to the skin or mouth at low concentrations, menthol elicits a pleasant cool sensation. Menthol is used
in a wide range of products, such as confections, candy, toothpastes, gels and aroma therapy inhalations. Since Hippocrates and Galen,
sporadic reports have described the use of cooling to produce analgesia. Clinical trials show beneficial effects of cooling on chronic
back pain, dental pain, postoperative pain, and muscle injuries. Preparations containing menthol are used topically to relieve neuralgia
in traditional Chinese and European medicine. Mint oil has been reported to alleviate thermally elicited pain and post-hepetic
neuralgia. In 2002, with the cloning of TRPM8, also called CMR1 (Cold and menthol receptor), a receptor was identified which
confers menthol-sensitivity in a temperature dependent manner. The receptor was shown to be functional also when expressed in
heterologous expression system. In addition to menthol, a number of cooling agents, including icilin, eucalyptol, and WS-3, activate
TRPM8 in vitro. Due to the complex regulation of the TRPM8 activity, several aspects have to be considered in developing an assay
suitable for the identification of potent TRPM8 agonists in High-throughput screening (HTS) applications. In Axxam laboratories was
developed a cell based assay for the heterologous expressed TRPM8 receptor. The assay was first validated by testing the response of
the cell line to all the major known activators and blockers of the TRPM8 receptor. The validated assay was then used in a high
throughput screening campaign using a calcium sensitive fluorescence dye approach . The screening was conducted on an high
diversity library, composed of 100,000 synthetic compounds. Identified hits were confirmed and validate on the TRPM8 cell line as
well as on not transfected cells and on a cell line expressing the TRPM8 receptor but using a luminescent read out. At the end of the
process, the validated hits were tested for specificity of action, using cell based assays expressing other TRP channels. The most
promising compounds where finally tested using the classical electrophysiological analysis. The presentation will put the emphasis on
assay development, screening design and hit confirmation strategies, which are essential for the identification of meaningful TRPM8
agonists.
31
S44 - Function of TRP channels and cannabinoid CB1 receptors, and their interactions, in chemoreception
Vincenzo Di Marzo
Institute of Biomolecular Chemistry, National Research Council
Studies on the molecular mode of action of capsaicin, the pungent principle of hot chilli peppers, led to the discovery, in 1997, of a
plasma membrane cation channel, belonging to the large family of the Transient Receptor Potential (TRP) channels, and named TRP
of Vanilloid type-1 (TRPV1). TRPV1 is activated by noxious heat (>42°C) as well as by pH <6.5. Capsaicin, and, with considerably
higher potency, the other plant toxin, resiniferatoxin, are both capable of binding to TRPV1 and to lower the threshold of its activation
by heat down to physiological body temperatures. This causes the gating of cations, namely calcium ions, into the cell, and, since
TRPV1 are abundantly expressed in unmyelinated sensory fibres of C- and Aδ-type, this, in turn, results in neuron depolarisation and
mimics the “hot” feeling otherwise caused by higher temperatures. The discovery of TRPV1 triggered the cloning of other similar
TRPV channels, four of which (TRPV1-4) are also involved in high temperature sensing. Furthermore, other TRP channels, belonging
to the Ankirin-type and Melastatin-type subfamilies, were also shown to act as “thermosensors”. In particular, TRPA1 transduces the
pungent feeling associated with mustard oil isothiocyanates and several other pro-nociceptive compounds (but has been involved also
in “cold” sensing), and TRPM8 is activated by cool temperatures (<25°C), and is responsible for the “cooling” feeling associated with
menthol and some synthetic compounds used as additives in the food, pharmaceutical and cosmetic industries.The observation of the
chemical similarity between capsaicin and anandamide, one of the endogenous agonists of cannabinoid receptors CB1 and CB2, two
G-protein-coupled receptors activated by Cannabis psychoactive principle, Δ9-tetrahydrocannabinol, led, in 1999, to the discovery that
anandamide can also act as an “endovanilloid” (i.e. an endogenous agonist at TRPV1 receptors), and, in 2002, to the finding, in
nervous tissues, of N-arachidonoyl-dopamine (NADA), which also activates both CB1 and TRPV1 receptors. Furthermore, it was
shown that CB1 receptors are very often co-expressed, in both sensory fibres and the brain, with TRPV1, the activity of which they
can modulate in various ways. The “TRP-cannabinoid link” was reinforced by the subsequent finding that anandamide and NADA can
potently antagonize the activation of TRPM8 receptors by both menthol and the synthetic compound, icilin, and that several plant
cannabinoids can variedly modulate the activity of TRPV1, TRPM8 and TRPA1 channels. These findings have several possible
implications for chemoreception and pain transmission, which will be also discussed at the meeting
S45 - The TRPA1 channel: a sensor for oxidative stress and more.
Pierangelo Geppetti
Department of Preclinical and Clinical Pharmacology, University of Florence, Italy
The transient receptor potential (TRP) family of channels is represented with at least 6 members in primary sensory neurons. These
include the vanilloid subtypes 1 (TRPV1), TRPV2, TRPV3 and TRPV4, the cold and menthol receptor TRPM8 and the TRPA1. Much
interest has been directed to the study of the TRPV1 because, capsaicin, has been instrumental to discover the unique role of a subset
of primary sensory neurons to cause nociceptive responses, to activate reflex pathways, including cough, and to produce neurogenic
inflammation. TRPV1 is now regarded as an integrator of diverse sensory modalities. Evidence in experimental animals and in
patients with airway diseases indicates a marked hypersensitivity to cough induced by TRPV1 agonists. Novel data in different models
of neuropathic and inflammatory pain have revealed a remarkable effect of TRPV1 antagonists. Recently, another channel expressed
by sensory nerve terminals, the TRPA1, has been identified as a sensor of oxidative, nitrative and carbonylic stress and to mediate
neurogenic responses to cigarette smoke and other environmental and occupational agents responsible for airway and lung disease. In
addition, TRPA1 has been proposed as a key integrator of interactions between the immune and nervous systems in the airways,
driving asthmatic airway inflammation following inhaled allergen challenge, as it greatly contributes to hyperresponsiveness and
cytokine production evoked by antigen sensitization in mice. The observation that TRPA1 agonists are powerful tussive agents,
suggests TRPA1 as a novel suitable target for novel antitussive medicines. In addition to sensing noxious agents in the respiratory
system TRPA1 is apparently involved in pain trasmission. We recently found that TRPA1 entirely and selectively mediates to
nociceptive behaviour and neurogenic inflammatory responses elicited by products of lipid peroxidation generated by oxidative stress,
including the highly reactive molecule, 4-hydroxy-2-nonenal. Moreover, we demonstrated that, in contrast with the sensitizing, but nt
pain-producing, action of the classical prostaglandin, PGE2, a series of cyclopentenone prostaglandins and isoprostane, including 15d-PGJ2, PGA2 and PGA1and 8-iso-PGA2 are selective stimulants of the TRPA1 and by this mechanism produce marked pain
behavior. All these molecules possess an electrophilic carbon atom that undergoes nucleophilic attack by the cysteine residues of
proteins. TRPA1 gating by α,β-unsaturated aldehydes and other reactive molecules occurs through this unusual mechanism, involving
covalent modification within the cytoplasmic N-terminal domain of the channel. Design and development of TRPA1 antagonists may
represent a new avenue to treat inflammatory and neuropathic pain, where oxidative, nitrative and carbonylic stress play a role.
32
S46 - Context-dependent modulation of activity in the vertebrate olfactory epithelium
Eisthen HL
Dept of Zoology, Michigan State University
Neuronal plasticity enables an animal to respond flexibly and appropriately to its environment, and to alter its responses with respect
to its physiological state. We are examining a form of plasticity in the vertebrate olfactory epithelium that involves modulation by
peptides released from the terminal nerve, an anterior cranial nerve that wraps around the outside of the nasal sensory epithelia in most
vertebrates. In axolotls (Ambystoma mexicanum), a type of aquatic salamander, about half the fibers of the terminal nerve contain the
peptide gonadotropin releasing hormone (GnRH) and other half contain neuropeptide Y (NPY). Electro-olfactogram (EOG)
recordings in axolotls reveal that NPY enhances responses elicited by amino acids, which serve as feeding cues for aquatic vertebrates.
In contrast, GnRH suppresses responses evoked by amino acids, but the response recovers and sometimes rebounds above baseline
when the peptide is washed off. Using whole-cell recordings from olfactory receptor neurons in epithelial slices, we are also
examining the effects of these peptides on voltage-activated currents in axolotls. We find that both peptides increase the magnitude,
but do not alter the kinetics, of the voltage-activated sodium current. Interestingly, these effects are dependent on the animal’s
behavioral context or physiological state: the effects of GnRH are more robust when the animal is in reproductive condition, and NPY
has dramatic effects in food-deprived animals but no effect on animals that have recently been fed. These results suggest that the
terminal nerve functions to alter responses to odorants in a way that is adaptive given the animal’s immediate physiological or
behavioral state. We are currently working to understand the mechanisms by which the effects of peptides change with the animal’s
physiological state. Given that the peptides in our experiments are bath-applied, we suspect that changes in physiological state lead to
changes in the peptide transduction pathways in the olfactory epithelium. To investigate this possibility, we have sequenced the genes
for all three expected GnRH receptor subtypes in axolotls (GnRH R1, R2, and R3), and for two of the three expected NPY receptor
subtypes (Y1 and Y5). PCR data indicate that all five receptor subtypes are expressed in the nose in axolotls. We are now working to
identify the cells that express the different receptors, and to determine whether and how their activity changes with the animal’s
physiological state.
Supported by the Natl Institutes of Health (DC05366) and Natl Science Foundation (IOS 0817785).
S47 - Specialist Drosophila Avoids Costs of Appetitive Olfactory Learning
Teun Dekker1,*, Barbara Randlkofer1, Peter Anderson1, Holger Daniels1, Stephan Knapek2, Bill S. Hansson
1Division of Chemical Ecology, Swedish University of Agricultural Sciences, PO Box 102, Alnarp, Sweden. 2Max Planck Institute
for Neuroscience, Department of … Munich, Germany. 3Max Planck Institute for Chemical Ecology, Department of Evolutionary
Neuroethology, Jena, Germany.
Modulation of behavior in response to previous experiences allows an animal to mould its behavior to a changing environment. The
plasticity in behavior permits an animal to respond adequately to stimuli that are indicators of survival and reproduction. For insects
resource availability is often spatially and temporally dynamic, particularly across generations. As insects rely strongly on their sense
of smell, fine-tuning its olfactory circuitry to available hosts through learning is advantageous for survival. Several insect genera and
species, such as the honeybee, are well-known for their remarkable olfactory learning abilities. But even the small fruit fly, D.
melanogaster, is able to form robust associations between an odor and a punitive stimulus. However, its appetitive learning ability
seems to be less astounding. Generally, low levels of short-term appetitive learning have been reported. We hypothesized that longterm appetitive memory may need longer exposure to the odor-reward combination, and would improve when flies can freely roam in
an assay, with much of their behavioral repertoire conserved. In a new paradigm flies exposed to an odor combination of butyl
butyrate and ethyl hexanoate paired with either sucrose or quinine were indeed capable of high levels of learning . Furthermore, the
paradigm resulted in long term memory, as learning persisted over many days in wild-type flies, was normal for radish1 flies, and was
absent in flies fed the protein synthase inhibitor cyclohexamide. Since learning is advantageous over innate behavior in temporally and
spatially dynamic environments, a species’ ability to form new neuronal representations of combinations of external cues may reflect
its ecology. Here we show that specialist fruit fly D. sechellia, which feeds exclusively on morinda fruit, has a severely depressed
appetitive learning ability in our learning paradigm compared to its generalist sibling species D. melanogaster. Ecologically relevant
fruity esters were especially poorly learning, which were the odors particularly well learned by generalist D. melanogaster. A costbenefit analysis in shows that conditioning increased the accuracy and speed of decision making, but reduced survival rates under
starvation conditions. D. sechellia exposed in a similar manner did not show any fitness costs. Anosmic Or83b- mutant flies did not
learn odors, nor did they incur any cost in the assay. The olfactory learning deficit in D. sechellia is ecologically relevant and may
have evolved to avoid the direct fitness costs as well as shifts in odor preferences, which are useless or even detrimental to this
species.
33
S48 – Olfactory plasticity: a key to survival for honey bee queens?
Vanina Vergoz, Kyle T. Beggs, Lisa H. Geddes, Alastair Aiken, Jamie McQuillan and Alison R. Mercer
Department of Zoology, University of Otago, Dunedin New Zealand
Queen mandibular pheromone (QMP) is a multifunctional blend of chemicals produced by honey bee queens. Among its many
functions, QMP helps prevent the development of ovaries in worker bees [1], modulates the behavioural development of workers [2],
influences comb building activities [3], and inhibits the rearing of new queens [4]. QMP is also used to entice workers to feed and
groom the queen and distribute her pheromones throughout the colony [5].
QMP is reported to be strongly attractive to young worker bees. Our data suggest, however, that while some bees are very responsive
to QMP most are not attracted to this pheromone. Indeed, older (foraging age) bees appear to be repelled by QMP. We have been
investigating worker-bee attraction to QMP in order to understand why responses to this critically important pheromone are so
variable. Our data suggest that QMP responsiveness may be affected by homovanillyl alcohol, a key component of QMP itself. HVA
reduces dopamine levels in the brain of the bee and alters cellular responses to this important neuromodulatory amine [6]. We have
recently shown in addition, that HVA activates one of three dopamine receptor subtypes in the bee [Beggs and Mercer, Curr. Biol., in
press). Changes in QMP responsiveness appear to be one of several consequences of HVA’s actions. This is surprizing, but suggests
that QMP-induced olfactory plasticity may provide a key to queen survival, and play a central role also in the survival of the honey
bee colony as a whole.
References:
1. Hoover, S. E., Keeling, C. I., Winston, M. L. & Slessor, K. N. (2003) The effect of queen pheromones on worker honey bee ovary
development. Naturwiss. 90, 477-480.
2. Pankiw, T., Huang, Z., Winston, M. L. & Robinson G. E. (1998) Queen mandibular gland pheromone influences worker honey bee
(Apis mellifera L.) juvenile hormone titres and foraging ontogeny. J. Insect Physiol. 44, 685-692.
3. Ledoux, M. N., Winston, M. L. , Keeling, C. I. , Slessor, K. N. & Le Conte, Y. (2001) . Queen and pheromonal factors influencing
comb construction by simulated honey bee (Apis mellifera L.) swarms. Insectes Soc. 48, 14-20 .
4. Winston, M. L., High, H. A., Colley, S. J., Pankiw, T. & Slessor. K. N. (1991) Effects of various dosages of queen mandibular
gland pheromone on the inhibition of queen rearing in the honey bee (Hymenoptera, Apidae) Ann. Entomol. Soc. Am. 84, 234-238.
5. Slessor, K. N., Winston, M. L. & Le Conte, Y. (2005) Pheromone communication in the honeybee (Apis mellifera L.). J. Chem.
Ecol. 31, 2731-2745.
6. Beggs K. T. et al. (2007) Queen pheromone modulates brain dopamine function in worker honey bees. Proc. Natl. Acad. Sci. USA,
104, 2460-2464
This work was supported through a grant from the Royal Society of New Zealand Marsden Fund
S49 - Mating-dependent plasticity of the olfactory system in an insect
Anton S, Barrozo RB, Jarriault D, and Gadenne C
INRA, UMR Physiologie de l'Insecte: Signalisation et Communication, Route de St Cyr, F-78000 Versailles Cedex, France
In many animals including insects, reproduction involves detection and central processing of relevant chemical cues that lead to
appropriate behaviours. Sex pheromones are generally considered as attractants and play a key role in the encountering of sexual
partners, ultimately leading to copulation. In the contrary to females, for which mating induces drastic behavioral and physiological
changes, males can often remate after a variable time delay. However, males are limited with respect to the number of ejaculates they
can deliver and the time required to restore depleted reserves. In the moth, Agrotis ipsilon, males cease to respond to the femaleproduced sex pheromone after mating. This plasticity is not only seen at the behavioural level, but is accompanied by a change in the
sensitivity of central olfactory neurons (Gadenne et al. 2001). The loss of sensitivity after mating is restored during the next day. This
transient neuronal plasticity serves as an energy-saving strategy by switching off the olfactory system and therefore preventing males
from mating unsuccessfully.
We investigated the neurobiological mechanisms underlying this mating-induced change in behaviour. Neither electroantennogram,
nor extracellular recordings from individual antennal receptor neurons revealed differences in responses to the sex pheromone between
virgin and mated males. Detailed analysis of intracellularly recorded projection neuron responses in the antennal lobe (the primary
olfactory centre of insects) revealed, however, clear differences not only in the response threshold to pheromone, but also in other
measured parameters such as spike frequency and latency of the responses between virgin and mated males. These physiological
changes were restricted to responses to sex pheromone, but were not observed for responses to general odours, such as plant volatiles.
In addition we could show that biogenic amines such as octopamine and serotonin might play a role in the mating-induced changes of
pheromone sensitivity by using pharmacological approaches. Although no oriented flight could be induced when injecting biogenic
amines in mated males, antennal lobe neurons in mated males increased in sensitivity after injection. Both orientation behaviour and
sensitivity of central neurons were reduced in virgin males when an octopamine receptor antagonist was applied.
Reference:
Gadenne C, Dufour MC, Anton S (2001) Transient post-mating inhibition of behavioral and central nervous responses to sex
pheromone in male moths. Proc Royal Soc Lond B 268:1631-1635
34
S50 - Pheromone-plant odour interactions and mating effects in the antennal lobe of Agrotis ipsilon males
Deisig N, Anton S, Gadenne C
INRA, UMR Physiologie de l'insecte: signalisation et communication, Route de Saint-Cyr, 78000 Versailles, France
In Lepidoptera, olfaction is essential for searching sexual partners (in males), host-plants for egg-laying (in females) and food (both
sexes). Males are thus able to detect and code sensory information pertaining to plant odours and sexual pheromones, by means of two
relatively distinct olfactory pathways. In nature, females emit a blend of sexual pheromone when situated on plants (host or non-host)
and it is thus vital for males to extract the information on the females’ pheromone from their olfactory environment. Plant odours
could either facilitate the location of females (synergy) or mask the female pheromone (suppression). We found, by means of in vivo
optical calcium imaging in Agrotis ipsilon males, that plant odours evoke high activation in ordinary glomeruli, while the pheromone
blend strongly activates the macroglomerular complex (MGC). When adding the plant odour to the pheromone blend, this strongly
reduced (suppressed) pheromone-evoked activity in the MGC, whereas plant odour-evoked activity in ordinary glomeruli was not
altered. Previous studies have shown that the physiological state of a male moth can change the pheromone-guided behaviour. For
example, male Agrotis ipsilon do not respond to the female pheromone after mating any more and in parallel, central neurons within
the antennal lobe have been shown to decrease strongly in sensitivity. We now compared the activation of antennal lobe glomeruli in
response to different concentrations of plant odours, the pheromone blend and plant odor/pheromone mixture in virgin and mated 5day old males, using in vivo calcium imaging. Our results show that mating did not have any effect on pheromone-evoked activity in
the MGC and that it did not change the strong suppression of plant odour on pheromone-evoked responses in the MGC. However,
mating induced stronger plant odour-evoked responses in ordinary glomeruli when applied plant odours were presented at high
concentrations. In conclusion, we find strong interactions of pheromone and plant-odours within the male-specific part of the antennal
lobe, independently of the mating status, and further an effect of mating on plant-odour activation which is concentration dependent
within the sexually isomorphic glomeruli of the antennal lobe.
S51 - Increased dopamine after mating impairs olfaction and prevents odor interference with pregnancy
Che Serguera, Viviana Triaca, Jakki Kelly-Barrett, Mumna Al Banchaabouchi & Liliana Minichiello
European Molecular Biology Laboratory, Mouse Biology Unit, Via Ramarini 32, 00015 Monterotondo, Italy
In mammals, exchange of body odors favors the establishment of social and sexual interactions and contributes to the regulation of
reproduction. In rodents, these odorants are mainly contained in urine, affecting neuroendocrine status, ovulation, opposite-sex
tethering and mating. In the nasal cavity of rodents, the the vomeronasal organ (VNO) and the main olfactory epithelium (MOE)
contain two sets of neuronal populations with exclusive chemosensory functions, such as detection of pheromones by the VNO and
detection of odorants by the MOE. Recent evidence suggests, however, that both structures are essential for pheromone-mediated
responses through specific and nonredundant mechanisms, which suggest a higher level of complexity in the integration of chemical
information in rodents. The MOE and the VNO project parallel fibers that innervate the main olfactory bulb (MOB) and the accessory
olfactory bulb (AOB), respectively. One specific difference is the absence from the AOB of a prominent dopaminergic neuronal
population, the juxtaglomerular dopaminergic interneurons, that is characteristic of the MOB. Urine constituents activate the VNOAOB and the MOE-MOB systems, which modulate innate responses such as sexual receptivity (lordosis) and ovulation and adaptive
responses like sex discrimination and mate choice. An effective mating therefore requires both systems to be functional. Because
odors favor fecundity, they might harm pregnancy, a condition that is not compatible with cycling. It is known that the odor of male
mouse urine can induce ovulation or block pregnancy within 3 days post coitus. Females avoid the action of such olfactory stimuli
after embryonic implantation. The mechanisms underlying these changes are unknown. Here we report that shortly after mating, a
surge in dopamine in the mouse MOB impairs the perception of social odors contained in male urine. Treatment of females at 6.5 days
post coitus with a dopamine D2 receptor antagonist restores social odor sensing and favors disruption of pregnancy by inhibition of
prolactin release, when administered in the presence of alien male urine odors. These results show that an active sensory barrier blocks
social olfactory cues detrimental to pregnancy, consistent with the MOB being a major relay through which social odor modulates
reproductive status. The postcoital rise of dopamine in the MOB and consequent decrease of olfactory perception abilities are an
unexpected mechanism by which female mice prevent male odor interference with neuroendocrine luteotrophic processes.
Serguera. C., Kelly-Barrett, J., Triaca, V., Minichiello, L. Pregnancy mediated rise of dopamine impairs olfaction and prevents odor
interference with prolactin release.
Nat Neurosci. 2008, 11: 949-56.
35
1A - Highly specific responses to amine odorants of individual olfactory receptor neurons in situ
Gliem S (1), Schild D (1,2) and Manzini I (1,2)
1 Department of Neurophysiology and Cellular Biophysics, 2 DFG Research Center for Molecular Physiology of the Brain (CMPB),
University of Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
The main olfactory system of larval Xenopus laevis is made up of at least two subsystems consisting of subsets of olfactory receptor
neurons (ORNs) with different transduction mechanisms. One ORN subset lacks the canonical cAMP transduction pathway and
responds to amino acid odorants. The second subset is endowed with the cAMP transduction pathway but as yet suitable odorants are
unknown. Here we report the identification of amines as proper olfactory stimuli for larval Xenopus laevis using functional Ca2+imaging and slice preparations of the olfactory epithelium and olfactory bulb. The response profiles of individual olfactory receptor
neurons to a number of amines were extremely complex and mostly highly specific. To gain information on how aminergic stimuli
may be transduced in the olfactory epithelium we tested on the stimulus forskolin, an activator of the acknowledged cAMP
transduction pathway. We found that the broad majority of amine-sensitive olfactory receptor neurons responded also to forskolin
indicating that cAMP can be considered as one major component of the intracellular signal transduction cascade in amine-responsive
olfactory receptor neurons. Furthermore, we investigated whether trace amine-associated receptors (TAARs) are expressed in the
olfactory organ of larval Xenopus laevis, since TAARs recently have been shown to act as olfactory receptors for amines in the
mammalian olfactory system. However, of the TAAR genes reported for Xenopus only one was found to be expressed in the larval
olfactory organ. The complex responses of individual olfactory receptor neurons to amines appear to require more than a few
receptors, indicating that besides TAARs other receptor families may be involved in the olfactory recognition of amines. The
collective evidence of the present study indicates that amines are proper olfactory stimuli for larval Xenopus laevis and suggests the
existence of highly specific olfactory receptors for these odorants mainly coupled to the cAMP-dependent transduction pathway. Supported by DFG Research Center Molecular Physiology of the Brain (CMPB, Project B1/9) to I.M.
2A - Expression of the adiponectin receptor 1 in the olfactory mucosa of mice
Maria-Isabell Burry, Nicole Hass, Karin Schwarzenbacher, Heinz Breer
Institute of Physiology, University of Hohenheim
Chemosensory information derived from the olfactory mucosa is a major factor for appreciating the palatability of food, thus
influencing food intake and energy homeostasis. However, recent findings indicate that the interaction between the sense of smell and
the energy balance status of the body might be mutual: the sensitivity of the olfactory system seems to be reduced if energy stores are
well-stocked, whereas the sensitivity is especially high under conditions of malnourishment or hunger. These adaptive alterations in
olfactory reactivity have been suggested to be mediated by energy balance hormones. One of the hormones which is, among other
metabolic functions, also involved in the regulation of energy homeostasis is the adipocyte proteohormone adiponectin. Serum
adiponectin concentration is regulated depending on adipose tissue mass, with a reduction of adiponectin levels seen in obesity and
high circulating levels of adiponectin in lean individuals. Thus, high levels of adiponectin are a signal for the physiologically
challenging state of reduced adipose tissue and starvation, respectively, a severe physiological state which might also influence
olfactory performance. In fact, AdipoR1 is expressed in mature sensory neurons of the olfactory mucosa of mice, in a pattern
reminiscent of the olfactory marker protein OMP. Newly generated differentiating neurons in the basal portion of the olfactory mucosa
were not AdipoR1-positive, thus contradicting a potential involvement of AdipoR1 signaling in developmental or differentiation
processes. AdipoR1 expression levels in the olfactory mucosa were observed to gradually increase during late embryogenesis until
adulthood. No local expression of adiponectin was detected in nasal tissues, indicating that serum adiponectin is the ligand for
AdipoR1 in olfactory sensory neurons. Thus, AdipoR1 function in the olfactory epithelium seems to be directly linked to the
nutritional status of the body. Furthermore, expression of AdipoR1 has been found in almost all mature sensory neurons of the main
olfactory epithelium and the septal organ. No AdipoR1 expression was detected in sensory neurons of the vomeronasal organ,
however, indicating that AdipoR1 expression is present distinctly in those chemosensory subsystems which are involved in food
seeking or evaluation of the quality and palatability of ingested food. Taken together, these findings suggest a potential modulatory
role for AdipoR1 in the adjustment of the olfactory system to energy balance requirements.
This work was supported by the Forschungsfonds ZEM Tübingen / Hohenheim.
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3A - Taste signaling elements in the gastrointestinal tract.
Nicole Hass, Karin Schwarzenbacher, Heinz Breer
Institute of Physiology, University of Hohenheim
Depending on the nutritional composition of a meal, gastrointestinal (GI) motor and secretory function as well as satiety signaling are
adjusted to optimise the digestive and absorptive capacity of the system and to maintain energy homeostasis. Potentially harmful
substances that have escaped the check-up of the gustatory system may elicit a protective increase in mucus secretion, altered
gastrointestinal motility or even emesis. Thus, the chemicals present in the GI lumen must be closely monitored in order to trigger
adequate responses and reflexes. Gustducin-expressing brush cells scattered throughout the GI mucosa are considered candidate
sensory cells for accomplishing this task. Gustducin-expressing brush cells are also positive for another key element of gustatory
chemosensory signaling, the ion channel TRPM5, and they even morphologically remind of gustatory receptor cells owing to their
tapering somata and apical tufts of microvilli. Therefore, a chemosensory function is readily conceivable for this peculiar cell type.
Many digestive processes are initiated as soon as the chyme reaches the stomach, in particular those related to the degradation of
proteins and lipids as well as satiety signaling. Furthermore, the stomach represents the venue of the body’s last defense against
potential noxae via emesis before substances are irreversibly ingested. Thus, the gastric mucosa can be thoroughly considered a
chemosensory hotspot of the gastrointestinal tract, and we therefore focused on the potential chemosensory capacity of cells within the
gastric mucosa and their relation to enteroendocrine effector cell types of the stomach that regulate its digestive and homeostatic
functions. We have identified a large cluster of gustducin-positive cells which is located exactly at the boundary between the fundic
and the oxyntic mucosa of the mouse stomach, at the so-called “limiting ridge”. These cells exhibit the typical morphology of brush
cells and also co-express TRPM5. In close association with the candidate chemosensory cluster, two populations of enteroendocrine
cells were found: one population containing the satiety regulating hormone ghrelin, the other population comprising serotoninsecreting enterochromaffin cells. The particular arrangement of gustducin-expressing cells and enteroendocrine cells at the limiting
ridge suggests a direct interplay between these cell types with immediate implications not only for digestive processes in the stomach,
but also for parameters controlling the satiety status.
This work was supported by the BMBF
4A - Grueneberg ganglion – a dual sensory organ?
Joerg Fleischer, Katharina Mamasuew, Heinz Breer
Institute of Physiology, University of Hohenheim, Stuttgart, Germany
The detection of odors and pheromones in mammals is mediated by chemosensory neurons of the main olfactory epithelium (MOE)
and the vomeronasal organ (VNO), which generally express the olfactory marker protein OMP. We have found that OMP is also
expressed in cells of the so-called Grueneberg ganglion (GG), a cluster of neuronal cells in the vestibule of the anterior nasal cavity.
Similar to other nasal sensory cells, GG neurons extend axonal processes which fasciculate to form nerve bundles that project caudally
along the roof of the nasal cavity and through the cribriform plate, finally terminating in the olfactory bulb of the brain. These
observations indicate a chemosensory function of the GG. Chemosensory responsiveness of olfactory neurons is based on the
expression of distinct receptors: odorant receptors in the MOE or pheromone receptors in the VNO, respectively. To scrutinize
whether neurons in the GG may indeed be chemosensory cells, they were subjected to molecular phenotyping by RT-PCR and in situhybridization. It was found that a given vomeronasal receptor type (V2r83) was expressed in the majority of GG neurons which were
concomitantly endowed with the G proteins Go and Gi; both are also present in sensory neurons of the VNO. Expression of odorant
receptors was only observed in very few cells during perinatal stages; a similar number of cells expressed adenylyl cyclase type III and
Golf/s. These findings demonstrate that the GG mainly comprises cells with a VNO-like phenotype. GG neurons lacking expression of
V2r83 are endowed with trace amine associated receptors (TAARs) which have been identified recently as a novel class of olfactory
receptors. Interestingly, expression of V2r83 as well as TAARs predominantly occurs in perinatal stages and is significantly reduced
in adults, suggesting that the GG might be particularly important for neonates.
In search of stimuli activating the GG, we have found that a large, distinct subset of GG neurons is activated by cool ambient
temperatures. These coolness-induced responses are particularly intense in neonates. Attempts to unravel the signaling mechanisms
underlying coolness-induced GG responses revealed that the coolness-sensitive TRP ion channel TRPM8 is not expressed in the GG.
Similar to thermosensory neurons in the nematode worm Caenorhabditis elegans, molecular phenotyping of the coolness-responding
GG neurons demonstrated that they are endowed with elements of a cGMP-mediated signaling pathway, including a distinct guanylyl
cyclase subtype, GC-G, and the phosphodiesterase PDE2A. Ongoing experiments indicate that cGMP signaling may indeed be
involved in responses of GG neurons to cool temperatures. In summary, the expression of olfactory signaling elements and axonal
projections to the olfactory bulb suggest a chemosensory function of GG neurons; their responsiveness to coolness may indicate that
the GG is a dual sensory organ.
This work was supported by the Deutsche Forschungsgemeinschaft.
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5A - Outgrowing olfactory axons contain the Reelin receptor VLDLR and navigate through the reelin-rich cribriform
mesenchyme
Carina Schnaufer, Heinz Breer, Joerg Fleischer
Institute of Physiology, University of Hohenheim, Stuttgart, Germany
Olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) project their axonal processes to the olfactory bulb (OB) where
they synapse with the dendrites of second-order neurons in a neuropil structure called glomerulus. Regarding the formation of the
axonal projection pattern, two processes have to be distinguished: first, the axons have to grow out from the OE and to navigate to the
OB. Second, within the OB, only congeneric olfactory axons coalesce in a given glomerulus. While the convergence and precise
targeting of axons in a glomerulus relies on given factors (such as the odorant receptor subtype) which are characteristic for each
subset of olfactory axons, the projection from the OE to the OB seems to be dependent on rather global cues. Concerning the latter
process, starting at early stages of development, on their trajectory from the OE to the OB, primary axons extend from the OE and
grow dorsally into the overlying frontonasal (cribriform) mesenchyme. The molecular cues guiding these axons through the cribriform
mesenchyme to the OB are largely unknown. Therefore, to identify molecules influencing the axonal trajectory in the murine
cribriform mesenchyme, we performed microarray analyses. Thereby, we focused on extracellular matrix (ECM) proteins present in
this tissue, since the ECM is generally considered to provide important cues for axonal outgrowth and guidance. In these approaches,
Reelin, a large glycoprotein of the ECM, turned out to be an interesting candidate. Reelin was found to be expressed by numerous
cells in the cribriform mesenchyme during the relevant embryonic stages when the first axons navigate from the OE to the OB;
however, no Reelin expression was observed within the OE itself. The Reelin-positive cells in the murine cribriform mesnechym were
closely associated with olfactory axons and apparently lack glial and neuronal markers. In the mesenchyme underlying the OE,
localization of the Reelin protein was not confined to the Reelin-expressing cells but it was also observed to be widely distributed in
the ECM – most prominently in regions traversed by olfactory axons. Importantly, these outgrowing axons were found to be endowed
with the “Very-low-density-lipoprotein-receptor” (VLDLR), which is known as a receptor for Reelin. Interestingly, the expression of
Reelin in the cribriform mesenchyme as well as the presence of its receptor VLDLR in olfactory axons seemed to be confined to
prenatal stages. Finally, Reelin expression was also detectable in the periphery of the OB. These Reelin-positive cells, which are
contacted by VLDLR-positive olfactory axons, also expressed neuronal markers, indicating that they are periglomerular and/or mitral
cells.
In summary, these results suggest that a Reelin/VLDLR signaling pathway might contribute to the formation of olfactory projections
to the OB and the establishment of initial contacts between the incoming axons and neurons in the OB.
This work was supported by the Deutsche Forschungsgemeinschaft
6A - Properties of the Ca2+-dependent chloride current in olfactory receptor neurons (ORNs) of the moth Spodoptera littoralis
Grauso M., Pézier A., Rospars J-P. and Lucas P.
INRA, UMR 1272, Physiologie de l’Insecte - Signalisation et Communication, Route de St Cyr, 78026 Versailles Cedex, France
We are interested in insect olfaction mechanisms with special focus on transduction and modulation of odorant response at the
peripheral level. The response of moth olfactory receptor neurons (ORNs) is accompanied by an increase in intracellular Ca2+
concentration ([Ca2+]i). The molecular target of the Ca2+ rise remains elusive. We therefore studied Ca2+-activated currents with the
whole-cell patch-clamp technique on cultured ORNs of the Noctuid moth Spodoptera littoralis.
Our experiments revealed in ORNs a chloride current of 5.4 ± 1.6 pA/pF at 20 nM [Ca2+]i, which is probably close to the Ca2+
concentration at rest. Chloride current rises in a dose-dependent manner upon perfusion with higher [Ca2+]i until a maximal current
density of 22.2 ± 3.3 pA/pF at 100 µM [Ca2+]i and with a K½ of ca. 3 µM. The chloride current had a linear I-V relationship in
symmetrical intra- vs extracellular chloride concentrations, it was voltage independent, it showed partial inactivation with time and it
had an anion permeability ratio of I : NO3 : Br : Cl : MeS = 1.8 : 1.7 : 1.1 : 1 : 0.6.
Pharmacological studies showed that this chloride current was reversibly inhibited by the general chloride current blockers flufenamic
acid, niflumic acid and NPPB; it is insensitive to PKC inhibitors (Staurosporin, Chelerytrin and H7) and activator (PMA); it is
insensitive to the CaMKII inhibitor, KN-93. This chloride current was cell-volume sensitive because its dependence to calcium was
reduced in extracellular hyperosmotic solution and dramatically increased in extracellular hyposmotic solution with currents reaching
56.8 ± 4.5 pA/pF at 100 µM [Ca2+]i. Thus, the chloride current in S. littoralis ORNs doesn’t share all the properties of classical
neuronal Ca2+-activated chloride channels (CaCC) but seems to behave as a Ca2+-gated chloride channel and a volume-regulated
anion channel (VRAC). This current is most likely involved in the receptor potential generation because in vivo recording of responses
to pheromone stimuli were dependent on the chloride concentration in the sensillar lymph bathing ORN outer dendrites.
With the aim of identifying the molecular basis of the S. littoralis ORN chloride currents we screened by BLAST analysis a S.
littoralis male EST antennal library including 20,000 clones. We used members of all known families of chloride channels and we
found three whole cDNAs belonging to the sub-family of bestrophins. Their sequences share high similarity with the Drosophila Best1
and Best2 genes. RT-PCR demonstrated that they are expressed mainly in peripheral sensory tissues (antennae and proboscis) and in
the ORNs primary culture that we used for patch clamp experiments.
This work was supported by ANR-BBSRC Pherosys and FP7 Neurochem.
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7A - Characterization of clonal cell lines derived from mouse taste buds and their responsivity to gustatory stimuli.
Miyamoto T (1, 2), Nishiyama M, Sako H(3), Tomooka T(3)
(1)Dept. Chem. Biol. Sci. and (2)Div. Mater. Biol. Sci., Grad. Sch. Sci., Japan Women’s Univ., Tokyo, 112-8681, (3)Dept. Biol. Sci.
and Tech., Grad. Sch. Ind. Sci. and Tech., Tokyo Univ. of Sci., Noda-shi, Chiba, 278-8510
A taste bud consists of approximately 100 of cells, and the cells turnover repeatedly due to their relatively short life span. Taste cells
are divided into 4 subtypes morphologically and immunohistochemically. Each subtype of taste bud cells is thought to represent
different properties and functions. The cells that comprise a taste bud have short lifespans, and they continuously renewal and
maintain the taste bud structure. However, the cell lineages and detail of taste bud differentiation are unclear. We have already
established taste bud cell lines, TB cell line from taste papillae of a p53 deficient mouse. To examine the cell lineage and
differentiation in taste buds, we attempted to establish culture methods for taste bud reconstruction with clonal cell lines derived from
murine taste buds (TB cell lines). After enzymatic dissociation and limiting dilution of cells, each cell type was categorized by its
morphological characteristics, resulting the establishment of clones of 11 cell lines. All these cell lines were confirmed to be derived
from the epithelial tissue by immunohistochemical analysis using an epithelial cell marker, cytokeratin 18 and a mesenchymal cell
marker, vimentin, because all the cell lines were immunopositive to cytokeratin 18 but not to vimentin. Immunocytochemical analysis
demonstrated that the cell lines are positive to gustducin and T1R3 as Type II cell markers or NCAM as Type III marker but negative
to GLAST as Type I cell marker. In addition, it was also demonstrated by RT-PCR that TB cell lines express several receptor
molecule for tastants including sour, salty, sweet and bitter tastes. TB cell lines cultured in collagen gels formed cell clusters that had
internal cavity. In addition, at the surface of the clusters, laminin, one of marker protein for basal membrane, was detected
immunohistochemically. These results suggest that TB cell clusters have basal membrane-like structure and recognize apical-basal
polarity. In TB cell clusters, however, all cells expressed T1R3, gustducin and NCAM, although in taste buds in vivo, T1R3- or
gustducin-positive cells are distinct cell types from NCAM-positive cells. This result suggests incomplete differentiation of TB cells in
collagen gels. Experiments using calcium-imaging technique showed some TB cell lines can respond to at least sour taste and ATP.
8A - TMEM16B is a novel Ca2+-activated Cl- channel candidate in vertebrate olfactory transduction
Pifferi S, Dibattista M, Menini A
International School for Advanced Studies, Scuola Internazionale Superiore di Studi Avanzati, SISSA, and Italian Institute of
Technology, SISSA Unit, Trieste, Italy
The process of olfactory transduction in vertebrates involves the activation by calcium of a depolarizing chloride current. The Ca2+activated Cl- current constitutes up to 90% of the total current but, despite its importance, the molecular identity of the Ca2+-activated
Cl- channel is still unknown. Some members of the protein family named transmembrane 16 (TMEM16; also known as anoctamin)
have been recently proposed to function as Ca2+-activated Cl- channels. At least one member of this family, TMEM16B, is present in
the mouse olfactory ciliary proteome (Mayer et al., Proteomics, 2009), and we investigated the functional properties of the mouse
TMEM16B (mTMEM16B) after expression in human embryonic kidney (HEK) 293T cells. Currents were measured with the patchclamp technique both in the whole-cell configuration and in inside-out excised patches. In whole-cell, a current induced by
mTMEM16B was activated by intracellular Ca2+ diffusing from the patch pipette, released from intracellular stores through activation
of a G-protein coupled receptor, or photoreleased from caged Ca2+ inside the cell. Furthermore, in inside-out excised patches, bath
application of different Ca2+ concentrations rapidly activated a current, indicating that mTMEM16B is directly gated by Ca2+. Both
in the whole-cell and in the inside-out configurations, the Ca2+-induced current was anion selective, displayed a Ca2+-dependent
rectification, and was blocked by the chloride channel blocker niflumic acid. In inside-out patches, the Ca2+ concentration for halfmaximal current activation decreased from 4.9 µM at -50 mV to 3.3 µM at +50 mV, while the Hill coefficient was >2. Currents
showed a time-dependent decrease (inactivation) at -50 mV in the presence of a constant high Ca2+ concentration and, moreover, an
irreversible rundown, not observed in whole-cell recordings, indicating that some unknown modulator was lost upon patch excision.
We compared the electrophysiological properties of native Ca2+-activated Cl- currents in mouse olfactory sensory neurons with those
of mTMEM16B-induced currents in HEK cells. We found that dose-response relations for Ca2+ activation, estimated single channel
conductance, rectification properties, Ca2+-dependent inactivation and irreversible rundown are remarkably similar indicating that, at
present, mTMEM16B is the best candidate for being the main molecular component of the native Ca2+-activated Cl- channel in the
cilia of vertebrate olfactory sensory neurons.
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9A - The calcium-activated chloride current in the cilia of intact mouse olfactory sensory neurons
Sagheddu C, Boccaccio A and Menini A.
Neurobiology Sector, International School for Advanced Studies (SISSA) and Italian Institute of Technology, Trieste, Italy
Olfactory transduction takes place in the cilia of olfactory sensory neurons. Odorant molecules bind to odorant receptors on the ciliary
membrane, activating a second messenger enzymatic cascade that produces a cAMP concentration increase. Ciclic nucleotide-gated
channels allow Na+ and Ca2+ influx and Ca2+ ions open Clֿ channels. Olfactory sensory neurons maintain an elevated Clֿ concentration
in the cilia, therefore Clֿ ions will exit, amplifying the odorant response. The Clֿ component represents most of the olfactory
transduction current, nevertheless it is not well characterized and the molecular identity of the Ca2+-activated Clֿ channel is still not
established.
In this study we peformed an electrophysiological characterization of such a chloride conductance in isolated olfactory neurons from
mouse. We used the whole-cell voltage-clamp technique to record currents elicited by selective Ca2+ photorelease in the cilia. In order
to avoid any K+ current contamination a Cs+ pipette solution was used. The current reversal potential was about 0 mV in symmetrical
Clֿ conditions, did not vary when the external Na+ was replaced by choline, while it shifted toward negative values when Clֿ in the
pipette solution was substituted with a non permeant anion. The channel permeability sequence, obtained measuring the current
reversal potential by substituting the external Clֿ with other anions, was: SCN>I>NO3>Br>Cl>MeS.
To investigate the pharmacology of the current we applied some chloride current blockers at the external side, such as Niflumic Acid,
DIDS, NPPB and 9AC.
The properties of Ca-activated Cl currents in the cilia of mouse olfactory sensory neurons will be compared with those measured in
knockout mice for putative channels
10A - Signalling Mechanisms in Mouse Bitter Responsive Taste Cells
Narukawa M. (a, †), Minamisawa E. (a), and Hayashi Y (a)
(a) Graduate School of Agriculture, Kyoto University, (†) Present address: Graduate School of Nutritional and Environmental
Sciences, Global COE Program, University of Shizuoka
Several bitter signal transduction candidates have been previously reported, including the T2Rs, α-gustducin (Ggust), PLCβ2, IP3R3,
and TRPM5. Although many studies have reported on bitter signalling components, no physiological evidence is available regarding
their involvement in signal transduction in taste cells that encounter bitter compounds. The use of double immunostaining in our study
examined the expression pattern of Ggust and IP3R3 in mouse taste cells that responded to bitterness. Furthermore, to examine the
participation of intracellular Ca2+ stores in bitter responses, we examined them under intracellular Ca2+-depleted and extracellular
Ca2+-free conditions. First, we investigated expression patterns of Ggust and IP3R3 in denatonium-responsive taste cells (64 cells).
Among taste cells that responded to 1 mM denatonium, 63% (n = 40/64) and 37% (n = 24/64) were positive and negative,
respectively, for anti-Ggust antibody staining, while, 56% (n = 36/64) and 44% (n = 28/63) of taste cells were positive and negative,
respectively, for anti-IP3R3 antibody staining, respectively. In the taste cells that expressed Ggust, 78% of the cells coexpressed
IP3R3 (n = 31/40). The proportion of taste cells that expressed either Ggust or IP3R3 was 70% (n = 45/64). There were 22% (n =
19/64) that expressed neither Ggust nor IP3R3. Next, to investigate the intracellular Ca2+ increase mechanisms in bitter transduction,
we examined denatonium responses under intracellular Ca2+-depleted and extracellular Ca2+-free conditions. After the taste cells were
stimulated by denatonium, they were exposed to 1 μM thapsigargin for 15 min. The taste cells were then challenged once more with
the denatonium. During thapsigargin exposure, [Ca2+]i gradually increased in most of the taste cells. A total of 83% of the taste cells (n
= 82/99) that had responded to denatonium lost the response after thapsigargin treatment; the remainder retained their responsiveness.
Among the taste cells responsive to denatonium in the presence of 1 mM Ca 2+, 39% (n= 33/84) retained their responsivity after
removing Ca2+ from the extracellular space; the remainder were unresponsive. In this study, the hypothesis that Ggust and IP3R3 are
key components in bitter transduction was demonstrated physiologically. For the intracellular bitter transduction, we propose that the
release of Ca2+ from intracellular stores plays an important role and that IP3-dependent and -independent pathways exist in the
mechanism.
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11A - PKD1L3 and PKD2L1 in human sour taste perception
Stähler F(1), Nitschke S (1), Demgensky S (1), Raguse JD (2), Meyerhof W (1)
(1) Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116,
14558 Nuthetal, Germany; (2) Clinic and Polyclinic for Oral and Maxillofacial Surgery and Plastic Surgery, Charité, Campus Virchow
Hospital, 13353 Berlin, Germany
Sour taste appears to be attractive at low and aversive at high acid concentrations. It is thought to be involved in the maintenance of
acid-base homoeostasis and in warning us against ingestion of irritant concentrations of acids contained in spoiled food or unripe
fruits. The identity of sour receptors and transduction mechanisms is still controversially discussed. Recent investigations identified
two members of the polycystic kidney disease like ion channel family, Pkd2l1 and Pkd1l3, as molecular markers of sour taste receptor
cells in mice. It is assumed that they functionally interact to form a sour taste receptor candidate. In the present report we addressed
the question whether or not PKD2L1 alone or in combination with PKD1L3 may have a role in human sour taste perception. RT-PCR
identified the mRNAs for both polypeptides in circumvallate papillae and non-chemosensory lingual tissue. PKD2L1 mRNA was also
found in fungiform papillae. While we found only one PKD2L1 mRNA species in these tissues several alternative splice variants have
been detected for PKD1L3. In situ hybridization experiments revealed that both, PKD2L1 and PKD1L3 mRNAs are present in ~7% of
the cells of circumvallate taste buds. In fungiform taste bud cells PKD2L1 or PKD1L3 specific RNAs have not been detected.
However, in line with our RT-PCR experiments, we found PKD2L1 mRNA in cells of the lamina propria adjacent to fungiform taste
buds. To further assess the involvement of PKD2L1 and/or PKD1L3 in human sour taste transduction, we performed functional
expression studies in Xenopus oocytes. Two-electrode voltage clamp analysis was used to record PKD-dependent responses after
application of acids. Addition of acetic acid at pH 5.0 to PKD2L1 RNA-injected oocytes clamped at -40 mV induced hyperpolarizing
membrane currents. However, removal of the acidic stimulus resulted in an inward current with off-responsive properties. Bath
application of HCl at the same pH and its removal did not cause membrane current changes. These findings support earlier human
sensory studies showing that organic acids are perceived as sourer than inorganic acids at the same pH. Oocytes expressing PKD1L3
alone or both polypeptides did not exhibit responses suggesting that, in oocytes, PKD2L1 forms a functional channel by its own or
associates with an endogenous partner. Taken together, PKD2L1 appears to be an interesting signaling molecule present in taste tissue.
Its precise role in sour taste transduction, however, needs to be precisely defined in future investigations.
12A - Expression of taste reception-related molecules in circumvallate papilla and gastrointestinal tract of developing rat
Wakisaka S*, Okada H*, Kadono K*, Honma S*, Maeda T#
*Osaka University Graduate School of Dentistry, # Niigata University Graduate School of Medical and Dental Sciences
It is now established that some cells in the gastrointestinal tract (stomach and duodenum) of the adult rat expressed mRNA and protein
for taste reception-related molecules such as α-gustducin, T1R’s and mGluR. However, it is not elucidated when mRNA’s for these
molecules begins to express in the gastrointestinal tract. Developmental studies have shown that mature taste buds, i.e. taste buds with
apparent taste pore, appeared after birth in circumvallate papilla of the rat, and single cells expressing immunoreactivity for αgustducin, a taste-specific G protein, a solitary chemosensory cells, were recognized in the gustatory epithelium, prior to the
appearance of mature taste buds. To date, however, it is unknown when mRNA’s for taste reception-related molecules other than agustducin begin to express in developing circumvallate papilla. In the present study, therefore, we design to examine the
developmental expression of mRNA’s for taste reception-related molecules in oral (circumvallate papilla) and gastrointestinal tract
(stomach, duodenum and rectum) in rat by RT-PCR. Circumvallate papilla, stomach, duodenum and rectum were collected from
developing rat from embryonic day 14 (E14) to postnatal day 3 (PN3) as well as from adult rat, and gene expression of α-gustducin,
T1R1, T1R2, T1R3 and mGluR1 was analyzed by RT-PCR. In the circumvallate papilla, we found that mRNA for α-gustducin, T1R1
and T1R3 began to express from E18, T1R2 from PN1, and mGluR1 from E16. In the stomach and duodenum, mRNA for α-gustducin
began to express from E18, T1R1, T1R2, and mGluR1 from E16. Interestingly, mRNA’s for these molecules were also recognized in
rectum, and developmental expression patter was almost identical to that in stomach and duodenum. In summary, mRNA for bitter
transduction-related molecule (α-gustduin) began to express from E18 from both oral (circumvallate papilla) and gastrointestinal tract.
Sweet receptor mRNA (T1R2/T1R3) and umami receptor mRNA (T1R1/T1R3 and mGluR1) began to express from PN1 and E16,
respectively in circumvallate papilla, and from E16 and E16, respectively in gastrointestinal tract. The present results suggest that
mRNA’s for taste reception-related molecules were expressed from late embryonic stage in oral (circumvallate papilla) – when
apparent matured taste buds are not present – and gastrointestinal tract (stomach, duodenum and rectum).
Supported by grants-in-aid for Scientific Research from JSPS (#19390464 to SW) and Research Grant from Society for Research on
Umami Taste)
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13A - Relationship between salivary zinc and taste sensitivity
Tomassini Barbarossa I *, Padiglia A **, Zonza A *, Atzori E ** and Angioy AM *
* Department of Experimental Biology, General Physiology Section, University of Cagliari, Italy; ** Department of Science Applied
to Biosystems, Biochemistry and Molecular Biology Division University of Cagliari, Italy
Variations of taste sensitivity are widely described and are mainly genetically determined. In the Caucasian population, several
sensitivity levels to the bitter chemical 6-n-propylthiouracyl (PROP) have been demonstrated. This compound contains a chemical
moiety NC=S which is responsible of bitter taste. Sensitive and non sensitive individuals are defined tasters and nontasters,
respectively. Recently, the PROP tasters have been further divided into supertasters and medium tasters by comparing their sensitivity
to PROP against that to NaCl. Sensitivity to PROP, inherited as a mendelian recessive trait, may be correlated with different
expression of salivary proteins involved in taste function like the gustin/carbonic anhydrase (CA)VI. This protein, a zinc-dependent
enzyme present in human parotid saliva, is a trophic factor that promotes growth and development of taste buds through its action on
stem cells.
In this work we examined the relationship between sensitivity to PROP and the free Zinc concentration in saliva. Analysis of the gene
codifying for gustin, and in particular the gene portion responsible for binding sites for Zn ions, are in progress.
In seventy-five subjects (28 men and 47 women), the intensity of taste perception evoked by increasing suprathreshold concentrations
of PROP and NaCl was estimated by using the Labeled Magnitude Scale (LMS). Each subject was assigned to a PROP taster category
by comparing the slopes of the dose-response curves to the two compounds. The salivary free Zinc concentration was determined by
spectrophotometric method. Molecular analysis of the gene codifying for the gustin by PCR are underway to determine differences
among supertasters, medium tasters and nontaster.
Subjects were classified as PROP supertasters (n = 27), medium tasters (n = 28) or nontasters (n = 20). ANOVA revealed a significant
three-way interaction of Taster group × Solution type × Concentration on the intensity ratings (P < 0.00001). One-way ANOVA
showed that the free Zinc concentration in human saliva depends on taster status (P < 0.0044). In addition, Newman-Keuls post hoc
test revealed that free Zinc concentration in saliva was greater in nontasters than in tasters (P = 0.0253).
These data demonstrated that the sensitivity to PROP is inversely related to the concentration of Zinc ions in human saliva. The
different salivary free Zinc levels in supertaster, medium taster and nontaster groups, could be associated to changes in gustin active
site structure that may affect protein activity. Investigations are in progress for verifying a possible role this Zn-dependent protein may
play in PROP detection.
14A - Identification of vesicular nucleotide transporter (VNUT) in taste cells
Ken Iwatsuki (1), Reiko Ichikawa (1), *Miki Hiasa (2), *Yoshinori Moriyama (2), Kunio Torii (1) and Hisayuki Uneyama (1)
1 Institute of Life Sciences, Ajinomoto Co. INC., Kawasaki, Japan, 2 Okayama University Graduate School of Medicine, Density and
Pharmaceutical Sciences, Okayama, Japan
Taste receptor cells are chemosensory epithelial cells that sense distinct taste quality such as umami, sweet, bitter and salty. These
cells could be divided morphologically into light, dark and intermediate (or type I, II, III) cells or could be distinguished from one
another by selective molecular markers. It is now widely accepted that type II cells express taste receptors for amino acids, sugars and
bitter compounds and type III cells express receptors for acids (sour tastants). Several reports have suggested involvement of ATP
together with other neurotransmitters in taste signal transduction, however, there is a paucity of molecular information about how
these molecules are being stored and released to nerves which innervate taste papillae. One possible pathway for ATP release is
through hemichannels as previously shown. However there remains another possibility that ATP and other neurotransmitters are
exocytotically released from presynaptic vesicles. The latter possibility has not been proved though there are ample evidence that type
II and type III taste cells contain components of synaptic vesicles despite that type II cells do not form conventional synapses.
Recently, vesicular nucleotide transporter (VNUT) has been identified and shown to be associated with chromaffin granules in adrenal
gland and with secretory granules in PC12 cells. Similarly, it is likely in neural circuits where VNUT expression is found that stored
nucleotides within synaptic vesicles are released into synaptic cleft by exocytosis after an action potential invaded the presynaptic
neuron. Therefore, it is of our great interest to see whether VNUT is being expressed in taste cells in order to elucidate the mechanism
of how purigenic signal transmission occurs within the taste system. In the present study we have explored VNUT expression in the
taste tissue where ATP is being released by taste stimuli. We found that within tongue epithelium VNUT mRNA is abundant in
circumvallate papillae (taste tissue) but less in surrounding epithelium (non-taste tissue). By using antibodies raised against VNUT we
found that the molecule is selectively expressed in particular type of taste cells. Furthermore, we demonstrated that during taste bud
development VNUT expression was always accompanied by the expression of taste cell markers. Our results, together with previous
studies, strongly suggest that VNUT plays a role in taste cell signal transduction.
42
15A - The extra-oral taste buds in crucian carp
Kvernenes SA, Isachsen, C-J, Døving KB.
IMBV, University of Oslo, Norway
The endocytosis in the receptor cells of taste buds is a prominent feature and its visualization is facilitated by use of the FM dyes
(Døving et al., 2009). Crucian carp have numerous taste buds in the skin of the head, the gular, and over the flank of the body
(Gomahr et al., 1992). The morphology and function of these extra-oral taste buds are poorly understood.
In order to stain and observe the receptor cells of the extra-oral taste buds in crucian carp, we have used the fluorescent dye FM1-43
and its fixable analogue AM1-43. The latter dye stains receptor cells, presumably in all types of taste buds. The differential staining
reveals three different types of taste buds. In one type of taste buds the receptor cells are few (~15) and they are long and slender
(Type I TB). The second type of taste buds (Type II TB) has a similar morphology to Type I TBs, but their receptor cells are stained
with AM1-43, but not with FM1-43. The sole difference between the FM1-43 and its analogue AM1-43 is that AM1-43 has an extra
amino group, which apparently plays a role in staining receptor cells of Type II TB. In the third type of taste buds (Type III TB), the
receptor cells are short and more numerous than those in Type I and II TBs, i.e. between 30 and 50.
We observed the Type I TBs most frequently on the gular region, but they are also present on skin of the upper part of the head. The
Type II TBs were mainly observed on the lips, but as they have receptor cell morphology similar to that of the Type I TBs, they could
also be present on the gular region. The Type III TBs were found on the head, gular, and consistently on the caudal rim of the scale on
the sides of the body. There are about eight TBs per scale.
Detailed investigation of the tip of the receptor cells with confocal microscopy and analysis by the software Imaris®, reveal staining
around a central domain of the cell. This observation is congruent with the demonstration of a column of microtubules in the centre of
taste receptor cells (Hansen & Reutter, 2004 In: Chemosensory systems in fish: Structural, functional and ecological aspects. Kluwer
Academic Publishers, Boston. p 72.)
Crucian carp feed on numerous types of invertebrates living in the bottom mud, e.g. the hypoxia tolerant Tubifex sp. Attempts to
determine which prey animals and possible substances from these animals that induce staining of the receptor cells of the different
types of taste buds have so far been unsuccessful.
16A - Bitter taste transduction involves potassium and calcium conductance in the blowfly Protophormia terraenovae
Masala C, Solari P, Sollai G, Crnjar R, Liscia A
Department of Experimental Biology, Section of General Physiology, University of Cagliari, Cittadella Universitaria, SS 554 Km 4.5,
09042 Monserrato (CA), Italy.
The study on transduction mechanisms underlying bitter stimuli is a particularly interesting challenge for taste researchers. The present
study investigates, in the labellar chemosensilla of the blowfly Protophormia terraenovae, the transduction mechanism by which
saccharin evokes a response from the “deterrent” cell (Liscia et al., 2004), with particular attention to the contribution of K+, Cl- and
Ca2+ currents and the modulatory role of cyclic nucleotides on these currents. To this end, a number of pharmacological probes as
modulators of the above mentioned ion channels were used.
As assessed by extracellular single-sensillum recordings (Hodgson et al., 1955), the addition of both EGTA, an extracellular Ca2+
chelator, and SK&F-96365, an inhibitor of receptor-mediated calcium entry, decreases the response of the “deterrent” cell to
saccharin, thus implying that a Ca2+ current and/or an intracellular Ca2+ cascade may be somehow involved in the depolarization of
the receptor cell. The diminishing effect following the addition of both Mibefradil, a calcium T-type channel blocker, and W-7, an
antagonist of calmodulin that blocks the Ca2+ cascade, strengthens this hypothesis. Also, Nifedipine, a blocker of the “L”-type
calcium channels, elicits a strong inhibitory effect, which is further increased by the addition of EGTA, thus confirming the
involvement of Ca2+ ions. Since K+ current is known to sustain receptor potential in the blowfly (Gödde and Krefting, 1989), we
tested 4-aminopyridine, an inhibitor of K+ channels, and found a robust decrease in spike firing frequency of the “deterrent” cell in a
dose-dependent manner. Conversely, chloride is not involved in the “deterrent” cell response, as suggested by the lack of effect of
NPPB, a chloride channel blocker: also in this case the addition of EGTA strongly inhibited the response. On the other hand, nonpermeable cGMP, which was suggested to be a second messenger in the sugar cascade, is ineffective; as an expected consequence,
neither GDP-β-S nor EHNA (non-specific G-protein and PDE2 inhibitors, respectively) were effective. Surprisingly, the membrane
permeable analogue 8Br-cGMP activated the “deterrent” cell in absence of saccharin; the response was purely additive when the two
compounds were presented as a mixture.
On the whole, our results are consistent with the hypothesis that in the labellar chemosensilla of the blowfly both Ca2+ and K+ ions
are involved in the transduction mechanism of the “deterrent” cell in response to saccharin and suggest that saccharin and 8Br-cGMP
share a common pathway.
43
17A - Variations in Human Tas1R1 and Tas1R3 taste receptors affect taste responses to MSG in functional assays
M. Raliou (1,2,3), M. Grauso (3), C. Schlegel-Lepoupon (3), D. Trotier (1), C. Nespoulous (3), H. Débat3, J.C. Pernollet (3), A.
Faurion (1), J.P. Montmayeur (2) & L. Briand (3).
(1) NBS-NOPA, INRA, Jouy-en-Josas, France; (2) CESG-CNRS, Dijon, France; (3) BOG-NOPA, INRA, Jouy en Josas
Inter-subjects differences in taste sensitivity may reflect differences in taste receptors. For example, genetic factors play a role in the
sensitivity to phenylthiocarbamide (PTC) and its detection is partly related to specific variants in Tas2R38 (Drayna et al, 2003).
Recently, our team (Lugaz et al., 2000) found in a French population, a multimodal distribution of the detection thresholds for
monosodium glutamate (MSG). Besides normal tasters, some subjects presented a decreased sensitivity to the anion (hypo-tasters) and
about 2% of the population was unable to detect the anion (non-tasters). Molecular biology analysis from these subjects (Raliou et al.,
2009a) confirmed the existence of non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region of human
Tas1R1 and Tas1R3 (Kim et al., 2006) and extended their number. In addition we found that the presence of some nsSNP was related
to the sensitivity of our subjects to MSG (Raliou et al., 2009b). For example, nsSNP A110V in the amino-terminal extracellular
domain (ECD) of Tas1R1 and R757C in the intracellular loop 3 of Tas1R3 (as well as P993S in carboxy-terminal intracellular tail in
mGluR1) were statistically significantly associated to the glutamate non-taster trait whereas nsSNP A372T in the ECD of Tas1R1 was
associated to the taster trait (Raliou et al, 2009b).
We co-expressed human Tas1R1 and Tas1R3 variants with G-alpha16i3 in HEK293 heterologous expression system. Calcium
imaging indicated that responses to MSG were decreased in the mutants comparatively to responses obtained in the wild type Tas1R1Tas1R3. These findings support the hypothesis of a role of nsSNPs A110V Tas1R1 and R757C Tas1R3 in decreasing the sensitivity to
umami.
Drayna D, Coon H, Kim UK, Elsner T, Cromer K, Otterud B, Baird L, Peiffer AP, Leppert M (2003) Genetic analysis of a complex
trait in the Utah Genetic Reference Project: a major locus for PTC taste ability on chromosome 7q and a secondary locus on
chromosome 16p. Hum Genet 112:567-572.
Kim UK, Wooding S, Riaz N, Jorde LB, Drayna D (2006) Variation in the Human TAS1R Taste Receptor Genes. Chem Senses.
Lugaz O, Pillias AM, Faurion A (2002) A new specific ageusia: some humans cannot taste L-glutamate. Chem Senses 27:105-115.
Raliou M, Boucher Y, Wiencis A, Bézirard V, Pernollet JC, Trotier D, Faurion A, Montmayeur JP (2009a) Tas1R1-Tas1R3 taste
receptor variants in human fungiform papillae. Neurosci Lett. 451(3):217-21.
Raliou M, Wiencis A, Pillias A-M, Planchais A, Eloit C, Boucher Y, Trotier, Montmayeur JP and Faurion A (2009b) Non
synomymous SNPs in human tas1r1, tas1r3, mGluR1 and individual taste sensitivity to glutamate. AJCN
18A - Vesicular Glutamate transporters localization in the rat lingual papillae
F. Zerari-Mailly b , A. Brauda, J. Azerad a,b, Y. Bouchera b
a. Faculté d'odontologie Paris 7 Denis-Diderot, 5, rue Garancière 75006 ;
L-glutamate is an ubiquitous excitatory neurotransmitter in the vertebrate central nervous system, which plays an important role in
many neuronal processes such as fast synaptic transmission and neuronal plasticity. Vesicular glutamate transports (VGluts) are
widely expressed in neurons of the central nervous system. Three distinct VGluts isoforms have been identified with VGlut 1 and
VGlut 2 accounting for most of the presumed excitatory glutamatergic terminals in the central nervous system, whereas VGlut 3 is
diffusely distributed in the brain, defining a discrete subpopulation of non-glutamatergic neurons. VGluts have also been observed in
peripheral sensory nerves fibres innervating the pulmonary neuro-epithelial body in the lung, in vagal afferents, in muscle spindles in
the triceps surae muscle, in intrafusal nerve fibres of muscle spindles in the masseter muscle and in free nerve endings in the mouse
palatine mucosa. Although there is evidence for glutamate signalling in lingual mucosa, its physiological significance has not been
established. Glutamate has been detected in lingual taste and non-taste epitheliums, non-NMDA glutamate receptors have been
observed in basal processes of the cells of taste buds and the presence of glial glutamate-aspartate transporters (GLAST) has been
demonstrated in the basal cellular layer of the lingual epithelium and in taste buds of circumvallate papillae. However, the question of
which vesicular transporters release glutamate in the rat lingual mucosa is still unresolved. In the present study we examine for the
first time the distribution of VGluts in the rat lingual mucosa using immunohistochemical methods. Examination of axial and
transverse sections of rat lingual mucosa showed a uniform punctate immunoreactivity for VGlut 1 in a subset of nerve fibres located
in the epithelium. In non-taste epithelium, the labelling for VGlut 1 was detected in fibres circulating in the lamina propria and in subepithelial layers, especially in the core of the papillae. A weak VGlut 1 immunoreactivity was detected in all taste papillae of the
tongue, in the core of the papillae and in taste epithelium. In the fungiform and foliate buds, the immunoreactive perigemmal nerve
fibres circulate near supporting cells of the taste buds and between epithelial cells whereas in circumvallate papillae, VGlut 1 labelled
nerve fibres were observed both outside taste buds besides supporting cells and in taste buds circulating near taste cells. Neither VGlut
2 nor VGlut 3 was detected in the lingual epithelium. These findings demonstrate the specific lingual pattern of distribution for VGluts
and suggest that only VGlut 1 takes part in glutamatergic regulation of epithelial and taste cells within the tongue in physiological
conditions.
taste papillae
44
19A - TrpM5 channel in solitary chemosensory cells is necessary for the trigeminal respiratory depression elicited by the bitter
compound denatonium benzoate
M. TIZZANO (1,4), A. VANDENBEUCH (2,4), W.L. SILVER (3), S.C. KINNAMON (2,4), T. E. FINGER (1,4)
(1) Dept. of Cell & Devel. Biol., (2) Dept. of Otolaryngology, (4) Rocky Mountain Taste & Smell Ctr., Univ. of Colorado Denver,
Aurora, CO; (3) Dept. Biology, Wake Forest Univ., Winston-Salem NC
Non-olfactory, trigeminally-mediated chemosensitivity of the airways serves to protect the respiratory tract from damage due to
inhalation of irritants or toxic substances. Activation of nasal trigeminal sensory innervation triggers protective reflexes that include
apnea or sneezing. This modality is mediated not only by direct chemosensitivity of capsaicin-sensitive and acid (ASIC) receptors on
the trigeminal nerve fibers, but also via solitary chemosensory cells (SCCs) distributed in the respiratory epithelium, with the highest
numbers positioned just posterior of the nasal vestibule. These SCCs synapse with peptidergic (substance P & CGRP) trigeminal
sensory fibers, and express many signalling effectors typical of taste transduction including the G-protein alpha-gustducin, PLCβ2,
TrpM5 and some bitter receptors (T2Rs). SCCs respond to a variety of irritating gaseous compounds as well as to certain bitter
substances (Lin et al. 2008; Gulbransen et al 2008). In particular, Gulbransen demonstrated using calcium imaging experiments, that
isolated TRPM5- and gustducin- expressing nasal SCCs respond to the T2R agonist denatonium benzoate via a PLC-coupled
transduction cascade typical of T2Rs in the taste system. Functional studies indicate that denatonium, when applied to the intact
epithelium, can activate the trigeminal nerve and evoke changes in respiratory rate (Finger et al 2003). Here, using a respiratory
monitoring approach, we tested whether the denatonium-mediated respiratory depression depends on the TrpM5 channel expressed by
SCCs but not by the nerve fibers. TrpM5 -null mice show no respiratory responses to nasal stimulation with denatonium benzoate,
although stimulation with capsaicin and acetic acid (capable of stimulating the trigeminal nerve fibers directly) still evokes a strong
respiratory depression. These findings demonstrate the importance of the TrpM5 channel for the funcionality of SCCs and its necessity
to activate trigeminal protective reflexes to irritants detected via SCCs.
20A - Synthesis and biological activity on TRPA1 receptor of perillaketone analogues
Bassoli A.(a), Borgonovo G.(a), Morini G.(b), Calamello C.(a), Di Marzo V.(c), De Petrocellis L.(c)
(a) DISMA Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, via Celoria 2, I-20133 Milano Italy. (b)
University of Gastronomic Sciences, Piazza Vittorio Emanuele, 9, I-12060 Pollenzo - Bra (Cuneo) Italy. (c)Istituto di Chimica
Biomolecolare CNR, Via Campi Flegrei 34, Comprensorio Olivetti, I-80078 Pozzuoli (NA), Italy
Several spices and food plants used in traditional cooking contain interesting bioactive compounds. Among these, we are particularly
interested in chemesthetic compounds, both for their use in gastronomy and for their medical, pharmacological and agrochemicals
applications. Perilla frutescens is an Asiatic food plant largely used in particular Korea and Japan as herbal remedy and culinary plant
in many gastronomic preparations. For its interesting sensory profile, bioactive compounds content and easy cultivation in our climate
this plant is considered to be extremely appealing for new applications in medicine, agriculture and food industry. Perillaketone (PK),
the major metabolite Perilla f. PK type has attracted considerable attention for the potent in vitro activity on TRPA1 channel[1], one
of the member of TRP (transient receptor potential) family ion channels that are activated by several stimuli (cold temperature,
pungent natural compounds, environmental irritant) and involved in pain perception[2]. Basing on this molecular evidence, we
hypothesized that these compounds are likely responsible both of the chemesthetic properties found in the taste and flavour of this
plant. The literature suggests that some TRPA1-activating compounds are electrophiles able to react through a Michael addition with
nucleophilic thiol residues of cysteine on the receptor. In fact many of the known agonists of TRPA1 have a molecular structure
containing electrophilic groups such as the isothiocyanates contained in horseradish and many brassicaceae, or unsaturated aldehydes
as acrolein or cinnamaldehyde. PK, that has the structure of a furylketone, represent therefore a new lead for the rational study of the
role of structure-activity relationships with the instruments of drug-design, where the target is the identification of the important
pharmacophores in generating the biological activity. This furylketone is susceptible to many possible targeted modifications of
pharmacophores; some analogues of perillaketone are been therefore designed and prepared by synthesis. The modifications on the PK
lead compound include the introduction of a C=C double bond in the side chain, reduction of the carbonyl group to alcohol, or
introduction of different functional groups which are known to be usually associated to chemesthetic activity such as the amide group
in various position of the side chain on the furanic ring. All the new compounds have been submitted to in vitro test with the cloned
TRPA1 receptor to evaluate the biological activity and the response used to derive information for structure-activity relationship.
[1] Bassoli A.; Borgonovo G.; Caimi S.; Scaglioni L.; Morini G.; Schiano Moriello A.; Di Marzo V.; De Petrocellis L., Journal of
Bioorganic & Medicinal Chemistry,17, 1636-1639, 2009. [2] Clapham D.E., Nature, 426, 517-524, 2003.
we thank the Italian Ministry for Foreigner Affairs (MAE) for the project of exchange Italy-Korea
45
21A - Anatomical and functional relationships between trigeminal dental primary afferents and lingual gustatory afferents
within the NTS
Braud A, Zerari-Mailly F, Felizardo R, Boucher Y
Faculté d'odontologie Paris 7 Denis-Diderot, 5 rue Garancière 75006 & CRicm UMRS 795
A recent clinical study indicated that dental deafferentation resulted in altered gustatory perceptions (Boucher et al. 2006). The aim of
this study was then to understand functional and anatomical relations between alveolar inferior nerve (NAI) and gustatory afferents
innervating the tongue within the Nucleus of the Solitary Tract (NTS.(1) We first investigated the effects of electrical stimulation of
the IAN (inferior alveolar nerve) on tastants (mix of NaCl, citric acid, sucrose) evoked-gustatory responses of electrophysiologically
recorded single gustatory neurons of the NTS in pentobarbital-anaesthetized rats. Electrical stimulation of the IAN elicited a
significant reduction of the amplitude of gustatory responses suggesting a modulation of gustatory neurons by trigeminal afferents. (2)
We then studied the anatomical relations of primary afferent of the IAN and gustatory neurons of the NTS. We used BDA as a retro
and anterograde tracer of primary afferent fibres of the IAN and Fos-immunoreactivity (Fos-IR)in neurons of the NTS following
electrical lingual stimulation in pentobarbital-anesthetized rats with section of ipsilateral lingual trigeminal afferents. We observed that
some primary afferents of the IAN project to the NTS and present boutons "en passant" near Fos-IR neurons in the dorsal and central
subdivisions of the rostral part of the NTS. Taken together our results document anatomical and functional interactions between
trigeminal dental and gustatory afferents in the brainstem.
22A - Effects of flavour prenatal exposure on taste related behavior during adolescence in rats
Márquez, R*, Gámiz, F**, De la Casa, G* and Gallo, M**
* Department of Experimental Psychology. University of Seville. Spain. ** Department of Experimental Psychology and Physiology
of Behavior. Institute of Neurosciences. University of Granada. Spain.
Both appetitive and aversive foetal flavour learning has been demonstrated in rats. Long-term postnatal consequences of flavour
prenatal exposure have been reported during the lactating period, including increased preferences and reinforcing properties. We have
investigated if the effects of prenatal flavour exposure can be extended to the adolescence period (PN35-PN43).
Eight pregnant Wistar rats were exposed during the last 10 gestational days to a solution of either cider vinegar (2%) or decaffeinated
coffee (0,5%) for 5 hours daily. On postnatal day 2 (PN2) litters were culled to 8 pups, leaving the maximum number possible of
males. After weaning, only male pups were tested. On PN27 they were water deprivated and habituated to drink water twice a day for
4 days. Afterwards they were assigned to two groups: prenatal-preexposed and prenatal-non-preexposed, being the flavour (vinegar or
coffee) appropriately balanced in both of them. The experimental procedure was aimed to test taste neophobia and second-order
conditioning, among other taste related behaviours by using either the prenatal exposed or non-exposed flavour depending on the
behavioural group.
Taste neophobia was tested by recording the flavoured solution intake during three consecutive days during the morning drinking
session. The potential reinforcing properties of the prenatal exposed flavour was assessed using a second-order conditioning procedure
in which either the prenatally preexposed or the non-preexposed flavour was presented in a simultaneous compound with vanilla
solution (2%) during four consecutive days during the morning drinking session being recorded the intake of vanilla solution presented
alone during the following two testing days
No effect of prenatal exposure was evident in taste neophobia. However the adolescent rats showed a peculiar neophobic response
compared with the adult rats. While adult rats exhibit a significantly higher intake of coffee compared with vinegar solution,
adolescent rats showed similar intake of both solutions. Prenatal flavour preexposure induced a significant effect on the amount of the
added flavour solution ingested after being presented in compound. The prenatally exposed group drank significantly lower amounts
of vanilla solution during the test sessions than the non-exposed group.
The results demonstrate a long-term effect of prenatal flavour preexposure lasting until the adolescence period. More research is
needed in order to clarify an interpretation. in terms of taste discrimination changes and/or a modification of the pup preference for
mother-related stimuli, that can be associated with the peculiar behavioural features of the adolescence period.
Research grants HUM 02763 (Junta de Andalucía. Spain), SEJ2006/1489 (MICINN. Spain) and PSIC2008-03933 (MICINN. Spain),
all supported by FEDER funding.
46
23A - Behavioral assessment of the effect of a long-chain polyunsaturated fatty acid supplemented diet in aged rats.
Díaz, A*, Ramírez, M **, Manrique, T*, Barranco, A**, Rueda, R** and Gallo, M*
*Department of Experimental Psychology and Physiology of Behavior. Institute of Neurosciences. University of Granada. Spain. **
Discovery Technology R&D, Abbott Nutrition, Granada-Spain.
Well-known changes in the hippocampal function have been related to a lower performance of aged rats in various memory tasks,
which depend on the hippocampal system in adult rats. Although normal aging does not induce loss of hippocampal neurons, the
variety of neurophysiological and biochemical alterations in the hippocampal function during aging may account for the failure to
support certain types of memories. Among the memory tasks proven to be sensitive to aging –induced brain decay are the Morris
hidden platform water maze and the novel object recognition procedure. It is well known that n-3 long-chain polyunsaturated fatty
acids (LCP) play an important role in brain function. We have investigated the behavioral effects of a LCP supplemented diet in aged
rats (29 months old) using both in novel object recognition memory and spatial memory in the Morris hidden platform water maze.
Twenty-eight male 24 months-old Wistar rats were assigned to two groups: control group (C; n=12) and supplemented group (N;
n=16). The diets were given for 5 months. Animals were trained in novel object recognition memory and Morris hidden platform
tasks. In the first task, the animals explored two identical objects in a chamber during 15 minutes and the recognition memory was
assessed in a second testing session taking place 24 hours later with the familiar and novel object. In the Morris task, animals received
8 blocks of training, applied in daily sessions during 8 consecutive days and 3 probe trials (immediate, 24 hours later and 4 days later).
Recognition memory impairment in the group C was evident, showing there were no differences in the exploration time devoted to the
familiar and novel objetcs, whereas group N exhibited significantly longer exploration time of the novel object than the familiar. In the
Morris task, animals were assessed the latency to reach the hidden platform. Although both groups showed spatial learning (reduced
latencies and distances to reach the platform) group C exhibited longer latency and path length than group N.
The results show a significant enhancement of cognitive functions by the dietary supplement in aged rats. The beneficial effect of the
supplemented diet is clear in long-term (24 hours) object recognition memory and spatial learning acquisition, being both tasks
previously associated with a proper hippocampal function.
Supported by Abbott Laboratories S.A. (contract # 2847-00). Abbott Laboratories contributes as a promoting observer entity to the
research project HUM 02763 (Junta de Andalucía. Spain).
24A - Hunger and Satiety Modulate Abnormal Brain Response to Taste in Restrained Eaters
Claire Murphy and Nobuko Kemmotsu
San Diego State University and the University of California, San Diego
Globally, obesity has increased to epidemic proportions, with significant health consequences. The abundance of highly palatable,
high calorie food may contribute to obesity, yet not everyone exposed to a nutrient-abundant "obesogenic" environment becomes
obese. Persons who exercise cognitive restraint over eating behavior may provide insights into underlying mechanisms of obesity.
"Restrained eaters" attempt to control weight by cognitive restraint, actively regulating the quantity and quality of food intake.
Physiological state (i.e., hunger and satiety) affects the processing of appetitive stimuli and associated brain activation in normal
individuals by changing the reward value and relative salience of food-related stimuli. Both orbitofrontal cortex and the amygdala play
important roles in modulating the allocation of neuronal resources based on physiological state in normal individuals. Restrained
eaters attempt to over-ride the influence of physiological state with cognitive restraint; however, whether their brain response to
gustatory stimuli is altered by hunger and satiety has been unknown. In this study we manipulated both physiological state and
nutritional value of sweet taste stimuli, and used functional magnetic resonance imaging (fMRI) to investigate brain response to
gustatory stimuli in restrained and non-restrained eaters. We aimed to investigate whether individuals with cognitively restrained
eating style show altered cortical response to caloric and non-caloric sweet (pleasant) taste stimuli, and to investigate whether the
physiological states of hunger and satiety have differential impacts on brain response in non-restrained and restrained eaters.
Participants were "restrained eaters" defined by Factor 1 (cognitive restraint) items of the Three Factor Eating Questionnaire (TFEQ;
Stunkard & Messick, 1985), and an equal number of "non-restrained eaters." All were female. Activation on fMRI was investigated
when the participants were hungry and satiated with a nutritional preload, in response to saccharin and sucrose presented in the mouth
while participants were scanned at 3T. Restrained eaters showed decreased activation in response to saccharin when satiated compared
to when they were hungry, the same pattern observed in the non-restrained eaters in response to sucrose. A greater understanding of
plasticity in brain response under the conditions of hunger and satiety has significant potential for clinical applications in obesity, as
well as broader implications for the economics of obesity and public policy.
Supported by NIH grant R01AG04085 to CM. We thank L. Haase, E. Green, A. Jacobson, B. Cerf-Ducastel,
functional MRI
47
25A – Chemical alarm signals are not inducing morphology changes in crucian carp
Stabell O.B. (*), Faeravaag A.C. (*), and Tuvikene A (§)
Dept. of Natural Sciences, University of Agder, Kristiansand, Norway (*), and Centre for Limnology, Estonian Agricultural
University, Rannu, Estonia (§)
Inducible defences released by chemical cues are widespread among aquatic organisms. Crucian carp (Carassius carassius) is known
to develop a deeper body in the presence of chemical cues from predators, like northern pike and perch. The morphological changes
are measurable after 50 days of exposure to odours from predators, and deeper bodies make the prey fish less vulnerable to gapelimited predators. The chemical cues in question are released by predators that have eaten crucian carp, but are also found present in
the filtrate of homogenised skin from sacrificed crucian carp. Accordingly, the classical alarm signals present in the epidermis have
been suspected as the active components for inducing a body depth increase. Such signals may be released continuously by predators
that have been chemically labelled with alarm signals from crucian carp following ingestion of prey. We improved the method for
extracting chemical alarm signals from crucian carp by collecting the supernatant after centrifugation of skin homogenates. With that
method we removed the minute particles that normally make a filtered sample get turbid. The supernatant was subsequently diluted
and frozen into ice-cubes according to standard procedure. The presence of alarm signals in the supernatant was confirmed by
presenting melted ice-cubes in behaviour tests. This was carried out at the start of the laboratory experiments, and for the remaining
time of the experiments ice-cubes were added three times a week. The body-depth-promoting properties of alarm signals were tested
in two different laboratory experiments and one field experiment. Despite growth and weight increase of crucian carp in all three
experiments, no morphological changes were obtained in alarm-signal exposed fish compared to control fish. Accordingly, we
conclude that the classical water-soluble alarm signals are not the active compounds inducing a body depth increase in crucian carp.
We suspect that the sources of chemical signals controlling morphology changes are found in the particles removed during
centrifugation, i.e. in the precipitate. The tissue content of these particles may normally be metabolized by bacteria, either in the
intestine of predators or in the water, resulting in water soluble cues. Such latent alarm cues, inducing morphology changes in the
purpose of prey defences, have previously been found in other aquatic organisms, but have hitherto not been demonstrated in fishes.
Further studies are now under way to clarify the true origin of chemical cues inducing morphology changes in crucian carp.
26A - Olfactory coding neurons of the black bullhead catfish Ameiurus melas
Valentini T.* and Dolenšek J.**
*Department of Biology, University of Ljubljana, Slovenia and **Institute of Physiology, University of Maribor, Slovenia
Electrophysiological identification of olfactory coding neurons in catfish was illusive until recently. Responses of 555 spontaneously
active olfactory receptor neurons (ORNs) and of 142 ORNs which were inactive before stimulation with amino acids will be compiled
to identify coding activity in the olfactory system of the black bullhead catfish. The activities of the spontaneously active neurons
responding to amino acids were recorded in freshwater (FW) whereas the responses to amino acids in the spontaneously inactive
ORNs were made visible in highly purified water (HPW). In FW the investigators approached the electrode to the spontaneously
active ORN until action potentials >200µV were detected and then their responses to amino acids were monitored. Most of the
spontaneously active ORNs responded to amino acids with suppression. In most cases the suppressive responses were not dosedependent (56 of 72 ORNs) and were not stimulus specific; the repeatability of ORNs response was very low. Out of 555 neurons only
16 neurons responded to amino acids with dose-dependent suppression, which had to be determined on ordinal rather than interval
scales. Very few (N=8) spontaneously active ORNs responded to amino acids with a dose-dependent excitation. In HPW small
shunting of ORNs signals increased their detection probability and HPW did not affect viability of the epithelium. Excitatory
responses of ORNs inactive prior to stimulation were thus observed. All the spontaneously inactive ORNs responded to amino acids
dose-dependently (N=27), their responses were robust, repeated identical stimulations released identical responses. Based on the
electrophysiological response repeatability and its strict dose-dependence we propose that most of the ORNs that code amino acids are
inactive before stimulation. In HPW electrodes placed in the middle of the lamellar receptive fields with the greatest ORNs densities
frequently detected multiunit responses to amino acids. Detection of single unit activities was possible in 142 cases. The black
bullhead catfish ORNs are in most cases highly specialized neurons responding best to a single naturally occuring amino acid. Such as
spontaneously active ORNs the spontaneously inactive neurons are located over the entire receptive fields of the olfactory lamellae.
The suppressive responses of the spontaneously active neurons do not need to be stimulus specific. The background activity of the
spontaneously active ORNs is a noise in the olfactory system that needs to be suppressed during stimulation, their suppressed activity
increases the stimulus to noise ratio in the olfactory system. We hypothesize that the ORNs that are excited by odorants in a dosedependent manner code for stimulus quality, whereas ORNs that respond to odorants with suppression improve the signal to noise
ratio of the odorant response.
We acknowledge ARRS Slovenia for funding this study
48
1B - The purinergic system of the olfactory epithelium: effects on sustentacular cells and olfactory epithelium progenitors
Manzini I(1,2), Hassenklöver T(1,2), Kurtanska S(1), Junek S(1), Bartoszek I(1) and Schild D(1,2)
(1) Department of Neurophysiology and Cellular Biophysics, University of Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
(2) DFG Research Center Molecular Physiology of the Brain, Göttingen, Germany
Extracellular nucleotides are important signaling molecules that mediate various biological effects via cell-surface receptors termed
purinergic receptors. Here we employed various labeling techniques, immunohistochemistry, electron microscopy, functional calcium
imaging and a BrdU incorporation assay to investigate the effect of extracellular nucleotides on the different cell types of the olfactory
epithelium (OE) of larval Xenopus laevis. We show that purinergic receptors are expressed in sustentacular cells (SCs) and basal cells
(BCs), but not in olfactory receptor neurons. Nucleotides evoked distinct increases in the intracellular calcium concentration [Ca 2+]i in
both, SCs and BCs. In SCs, nucleotides elicited intracellular calcium waves running from their apical part to their endfeet in the basal
part of the OE. Accordingly, SCs could carry information from the apical part of the OE to the basal cell layer. On the other hand, we
found that nucleotide-induced Ca2+ signaling in BCs, the stem cells of the OE, had an influence on their proliferation rate. This, in
turn, strongly suggests that purinergic signaling plays a role in the regulation of the cell turnover in the OE. The characteristic
increases in [Ca2+]i in these two cell types could be evoked in both, the presence and absence of extracellular calcium. In contrast, a
depletion of the intracellular calcium stores with cyclopiazonic acid, a specific inhibitor of the sarcoplasmic-endoplasmic reticulum
Ca2+ ATPase, abolished the [Ca2+]i responses. This strongly indicates that SCs and BCs express only metabotropic purinergic
receptors. Furthermore, we pharmacologically defined the receptor subtype(s) involved in the nucleotide-induced responses of the two
cell types. The determined order of potency of purinergic agonists and antagonists suggests that multiple P2Y receptor subtypes are
involved in the nucleotide-induced responses in the OE. While SCs probably express a P2Y2/ P2Y4-like receptor, BCs most likely
express various P2Y receptor subtypes. Taken together, our findings certainly contribute to a more thorough comprehension of the
tissue OE. We found that the OE has a complex purinergic system. Nucleotides released from OE cells, most probably damaged or
dying olfactory receptor neurons, could initiate Ca2+ waves in neighbouring SCs, which in turn could carry this information to the
basal cell layer. The elevated [Ca2+]i in the endfeet of SCs could then trigger nucleotide release in the basal cell layer, thereby
stimulating cell proliferation.
Supported by DFG Research Center Molecular Physiology of the Brain (CMPB, Project B1/9) to I.M.
2B - Inhibition of olfactory cytochrome P450-dependent activities modifies the electro-olfactogram responses to quinoline and
coumarin in rats
Thiebaud N*, Veloso Da Silva S**, Jakob I**, Sicard G**, Artur Y*, Heydel JM* and Le Bon AM*
* UMR 1129 FLAVIC, INRA-Université de Bourgogne-ENESAD, 17 rue Sully, F-21000 Dijon, France ** UMR 5170 CESG,
CNRS-Université de Bourgogne-INRA, 15 rue Hugues Picardet, F-21000 Dijon, France
Mammalian olfactory epithelium expresses a large number of xenobiotic metabolizing enzymes such as cytochrome P450-dependent
monooxygenases (CYPs) or transferases. In general, these enzymes catalyze biotransformation of exogenous compounds in order to
facilitate their excretion. The exact role of these enzymes in the olfactory epithelium remains unclear. Besides their role in the
protection against inhaled toxic compounds, these enzymes could also be involved in peri-receptor events. Indeed, since odorants
reach the olfactory epithelium continuously, a rapid inactivation and/or clearance of odorants are needed to maintain the sensitivity of
the olfactory system. Xenobiotic metabolizing enzymes could catalyze biotransformation of odorants resulting in their inactivation.
Such a role has been demonstrated in insects (review in Vogt R. G., 2005).
The present study was designed to test the hypothesis that CYPs are involved in odorant metabolism, and therefore have an impact on
the olfactory signal in rats. CYPs catalyze a variety of reactions including hydroxylation. For this purpose, we started by exploring the
consequences of CYP inhibition on electro-olfactogram (EOG) recordings. A CYP suicide inhibitor, 1-aminobenzotriazole (ABT),
was applied on the olfactory mucosa. Then responses of odorants were recorded using a submerged EOG technique, and were
compared with responses obtained before application of the CYP inhibitor. Three odorant molecules have been tested: quinoline and
coumarin, which are known to be metabolized by CYPs, and isoamyl acetate, whose metabolism is known to not be CYP-dependent.
Application of ABT provoked a rise of the maximal signal amplitudes elicited by both coumarin and quinoline. We also observed that
CYP inhibition resulted in an extension of the recovery time of olfactory epithelium sensibility. Conversely, when isoamyl acetate was
tested, neither the signal amplitude nor the recovery time were affected, which confirms the specificity of the ABT treatment. In a
separate study, we showed that hydroxylated derivatives of quinoline and coumarin elicited lower EOG signals when compared to
parent molecules. When taking together, these results support evidence that CYPs are involved in the biotransformation of certain
odorants into hydroxylated metabolites, which lead to a decrease of the olfactory input. Our findings are consistent with the hypothesis
that xenobiotic metabolizing enzymes may act at the peri-receptor level to maintain the sensitivity of the olfactory system in
mammals.
49
3B - Retinoic acid-regulated gene expression and survival of vomeronasal neurons during postnatal development
Hörnberg M, Öztokatli H, Gussing F, Berghard A, and Bohm S.
Department of Molecular Biology, Umeå University, Umeå, SE90187, Sweden
Signaling by heterotrimeric G protein-coupled vomeronasal receptors in vomeronasal sensory neurons (VSNs) mediates detection of
chemosensory signals that influence sexual, aggressive and social behaviors in the mouse. In addition G protein-coupled vomeronasal
receptor signaling mediates cell survival. This is exemplified by the fact that approximately half of the VSNs die when the
vomeronasal receptors cannot couple to a corresponding heterotrimeric G protein. VSNs are divided into two major groups, apical or
basal, which express unique sets of vomeronasal receptors (V1Rs and V2Rs), and heterotrimeric G protein- subunits (Galphai2 and
Galphao). We show that cells of the vomeronasal nerve express the retinoic acid (RA) synthesizing enzyme retinal dehydrogenase 2
(RALDH-2). RA is a vitamin A metabolite that regulates neural patterning, neural differentiation and axon outgrowth during
development. An increasing body of evidence indicates that RA continues to play an important role in the postnatal and adult brain,
i.e. sustaining learning and memory capabilities by regulating synaptic plasticity processes. Analyses of transgenic mice with VSNs
expressing a dominant-negative RA receptor indicate that basal VSNs differ from apical VSNs with regard to a transient wave of RAregulated and caspase-3-mediated cell death during the first postnatal week. The fact that both neural activity and RA regulate
neuronal survival raises the central question of how these mechanisms are connected at the molecular level. Analyses of G-protein
subunit deficient mice indicate that RA and vomeronasal receptor signaling combine to regulate expression of proteins that has been
implicated in odorant receptor- dependent formation of precise axonal projections of olfactory sensory neurons. Collectively the
results indicate that apical and basal VSNs are intrinsically different in their dependence on locally produced RA for cell survival
during a neural activity-regulated phase of sensory map development. The results also reveal a novel connection between presynaptic
RA receptor signaling and neural activity-dependent events that together regulate neuronal survival and maintenance of synaptic
contacts.
This work was supported by grants from Swedish Natural Science Research Council (B5101-1250/2001 and
4B - Odorant receptors in olfactory cilia and axons
Strotmann J, Eberle J, Levai O, and Breer H.
Institute of Physiology, University of Hohenheim, Stuttgart, Germany
Odorant receptors (ORs) are supposed to act not only as molecular sensors for odorants but also as cell recognition molecules guiding
the axons of olfactory sensory neurons to their appropriate glomerulus in the olfactory bulb. This concept implies that OR proteins are
located in sensory cilia and in the axons. To approach this critical issue, antibodies were generated against two peptides, one derived
from olfactory receptor mOR256-17, one derived from the ‘mOR37’ subfamily. By means of immunohistochemistry and doublelabelling studies using transgenic mouse lines it was demonstrated that the newly generated antibodies specifically recognized the
receptor proteins. Western Blot analyses of isolated cilia preparations revealed a single immunoreactive band for each OR. To
scrutinize the hypothesis that OR proteins may also be present in the axonal processes and the nerve terminals, serial sections through
the olfactory bulb were probed with the antibodies. Two glomeruli in each bulb were stained by anti-mOR256-17, one positioned in
the medial, one in the lateral hemisphere. Fiber bundles approaching the glomeruli through the outer nerve layer also displayed intense
immunofluorescence. In immunoblotting experiments using microdissected OB tissue anti-mOR256-17 recognized a band with a
molecular mass different from that in cilia. Altogether, these data demonstrate that OR proteins are indeed present in the cilia and the
axons of olfactory sensory neurons and indicate that OR versions with different molecular features may be present in these two
compartments
This work was supported by the Deutsche Forschungsgemeinschaft
50
5B - Uptake of odorant binding proteins in the olfactory epithelium
Brose H, Breer H, and Strotmann J.
Institute of Physiology, University of Hohenheim, 70593 Stuttgart, Germany
The detection of odorants is mediated by distinct chemosensory neurons in the main olfactory epithelium (MOE) of the nose. It is
supposed that the hydrophobic odorous compounds are dissolved in the nasal mucus by means of specialized globular proteins, the
odorant binding proteins (OBPs) in order to reach the chemosensory cells. To assure the responsiveness to odors of each inhalation,
odorant molecules have to be removed rather quickly from the immediate vicinity of the sensory neurons. Therefore, it has been
hypothesized that mechanisms exist which remove OBP/odorant complexes from the ciliary environment. To scrutinize this concept,
recombinant mouse OBP1a was fluorescently labelled, loaded with odorous compounds and applied to the MOE. It was found that
within less than a minute, labelled OBP disappeared from the surface and appeared in the sustentacular cells, glial-like cells which
form the apical border of the MOE. This uptake occurred only when the OBP was loaded with appropriate compounds. A candidate
system for mediating an uptake of OBP/odorant complexes into sustentacular cells represents the scavenger receptors which are
involved in internalizing lipocalin/hydrophobic ligand complexes. RT-PCR and in situ hybridisation studies revealed that only the low
density lipoprotein receptor Lrp2/megalin was specifically expressed in the sustentacular cells. Immunohistochemical analyses
localized megalin to the microvilli of sustentacular cells. Immunoreactivity was visible throughout the olfactory epithelium; whereas
the respiratory epithelium was devoid of megalin. To analyze whether megalin is capable of internalizing OBP/odorant complexes, in
vitro uptake studies were performed using a megalin expressing cell line. It was found that internalization of OBP/odorant complexes
occurred within short period of time and internalized OBP occurred within lysosomes of the cells. Also in the in vitro system, uptake
of OBP1a/odorant complexes only took place when OBP1a was loaded with appropriate odorous compounds. A pre-incubation of the
cells with the “receptor associated protein” (RAP), an inhibitor of Megalin-function, blocked the uptake process; RAP also blocked
internalization of OBPs into sustentacular cells of the olfactory epithelium. These data support the notion, that the uptake of
OBP/odorant complexes into sustentacular cells mediated by megalin represents an important mechanism for a local elimination of
odorants.
This work was supported by the Deutsche Forschungsgemeinschaft
6B - The Calmodulin-binding protein Neurogranin identifies a subpopulation of mature granule interneurons in the adult
mouse olfactory bulb
Gribaudo S, Bovetti S, Garzotto D, Fasolo A and De Marchis S
Dept of Animal & Human Biology, University of Torino, Torino (Italy)
Neurogranin (Ng) is a brain specific molecule involved in the modulation of Ca2+ signalling in neurons. It is highly expressed in
several adult telencephalic brain regions, including cerebral cortex and hippocampus. In this study, we investigated Ng expression and
localization in the adult mouse olfactory bulb (OB), the first relay station in odor information processing and integration. OB neuronal
circuits are highly plastic, both at the synaptic and cell renewal level. OB plasticity plays a central role in olfactory learning and
memory functions (Mori et al., 1999; Laaris et al.,
2007), however the molecular mechanisms underlying these processes are still largely unknown. Identifying new plasticityrelated molecular properties of OB neurons may lead to better understanding of olfactory information processing. As a postsynaptic protein, Ng is thought to be implicated in various forms of activity-dependent synaptic plasticity, regulating the availability of
free Calmodulin (CaM) for activation of Ca2+/CaM dependent signal transduction. By RT-PCR and Western blot analyses we found
significant levels of both Ng mRNA and protein in OB tissue extracts. Our immunohistochemical analysis indicates that Ng localizes
in the soma and dendrites of a defined subpopulation of mature granule cells enriched in the deep portion of the granule cell layer
(GCL). These cells project Ng-positive processes through the mitral cell layer and into the external plexiform layer. Genetic label for
the glutamic acid decarboxylase 67KDa (GAD67) showed that the large majority of Ng-expressing cells belong to the GABAergic
phenotype. Consistent with the preferential distribution of Ng-immunopositive cells in the GCL, we found high level of co-expression
with the Ca+/CaM-dependent kinase IV (CaMKIV), a downstream protein of CaM signalling cascade enriched in deep granule cells,
whereas no co-localization was observed between Ng and the calcium binding protein calretinin, mostly distributed in the superficial
GCL. Finally, we demonstrate that adult neurogenesis contributes to the Ng-expressing population, with more new-generated Ngpositive cells integrated in the deep GCL. Together, these results provide a new specific neurochemical marker to identify a
subpopulation of olfactory granule cells. Moreover, considering the role of OB granule inhibitory interneurons in the modulation and
integration of mitral/tufted cells activity, our data suggest possible functional implications for Ng in OB plasticity mechanisms.
51
7B - Rat olfactory epithelium RNA profiles are age related
Rimbault M, Robin S and Galibert F
Institut de Génétique et Développement de Rennes (IGDR), CNRS UMR 6061, Faculté de médecine 2 av. Leon Bernard, 35000
Rennes, France
Olfactory receptor (OR) genes were first identified in rat olfactory epithelium by Buck and Axel in 1991. The receptors they encode
play an essential role in olfaction, constituting a key element at the origin of a cascade of biochemical reactions leading to odorant
perception and recognition. Gene cloning and in silico mining of a number of mammalian genome sequences have identified between
600 OR genes in the human genome and up to 1500 OR genes in the rat genome. Indeed, these genes constitute by far the largest gene
family in mammalian genomes. A substantial percentage of OR genes, from 50% in humans to around 20% in rodents and dogs have
been identified as pseudogenes. Only a small number of OR proteins have been deorphanized and the role of the vast majority of these
proteins in olfaction remains uncertain. Microarray analyses have added a further layer of complexity by showing that pseudogenes
can be transcribed and that the transcription of some OR genes is not restricted to the nasal mucosa, but can also be detected in several
other tissues. OR are expressed on the surface of the cilia of the olfactory sensory neurons (OSN) lining the neuroepithelium in the
nasal cavity. Each OSN expresses one OR and from one allele only. Studies based on mRNA sequencing and microarray hybridization
have shown that only a subset of the mouse OR genes repertoire is expressed to detectable levels in the olfactory epithelium. Here we
analyzed the evolution of the gene expression pattern in Brown Norway rat olfactory epithelium at birth, adulthood and old ages, by
microarray hybridization and RTqPCR. RNA have been extracted independently from right and left olfactory epithelia dissected from
8 adult rats (6 weeks old) and 4 “old” rats kept in the animal house for 22 months and from 24 newborn rats (2 to 5 days) from 5
different litters. However for the newborn rats, for technical reasons, only one side of the epithelium was dissected. Following
extraction, the RNA integrity were assessed (RIN > 8.8), then RNA were labeled with Cyanine 3 and hybridized onto whole rat
genome Agilent microarrays. Array data, analyzed with GeneSpring GX software (Agilent Technologies), lead us to conclude that : a)
a large number of transcripts corresponding to 2/3 of the probes are detected at all ages. Nevertheless the level of expression of the
genes expressed by all groups of ages were not identical and a clear clustering according the age groups was obtained b) adult and old
rats express OR genes at a similar level, but higher than the level of expression achieved at birth, as confirmed with RTqPCR c)
However a number of OR genes, not expressed or poorly expressed in adulthood, were observed to be more or specifically expressed
at young age. Implication of the observation of a specific set of OR genes over expressed at birth, at a time where the animals are deaf
and blind, will be discussed in light of behavior, mother-pup relationship and nipple recognition.
8B - Determination of a number of olfactory receptor/ligand pairs of importance for explosives and human odor detection
Robin S*, Rimbault M*, Benbernou N and Galibert F
Institut de Génétique et Développement de Rennes (IGDR), CNRS UMR 6061, Faculté de médecine 2 av. Leon Bernard, 35000
Rennes, France
Olfactory receptors (OR) constitute the largest group of G-protein-coupled receptor families described to date. They play a key role in
the recognition of thousands of odorant molecules by the olfactory sensory neurons (OSN) in the olfactory epithelium. In response to
odorants, OSNs transmit signals to the brain, resulting in odour perception. Mammals have as many as 500 to 1500 different olfactory
receptors and we have shown that the dog OR genes are highly polymorphic with many alleles coding for slightly different proteins. It
has been shown that a particular OR can bind different chemical compounds and that one chemical compound can be recognized by
several different OR. This whole process is defined as a combinatorial code allowing several hundred OR to recognize thousands of
different odorants. However among the many thousands of possible OR/ligand pairs less than one hundred has been worked out. This
is essentially due to the difficulties enticed with the deorphanization of OR, in particular if the goal is to identified the receptors that
recognize a given odorant.
Detection of explosives or illicit substances such as drugs is heavily depending upon the interaction between a dog and its handler
even nowadays. With in mind that dog breeding selection, as well as the development of electronic devices based on bio-sensors could
greatly benefit from the optimal OR identification, we developed a strategy aimed at identifying a number of OR/ligand pairs of
interest.
Here we will report on such a strategy in which we associated the exposure of an animal to an odorant with a punishment reward
paradigm. As for practical and ethical reasons we could not work with dogs, the tests have been developed and used with Brown
Norway rats. In these circumstances, rats submitted to repetitive flushes of odorant(s) associate the two events. In order to avoid the
punishment they increase their detection sensitivity of the odorant(s) by increasing the level of expression of the cognate receptors, the
expression level of which is determined by microarray hybridization of olfactory epithelium RNA onto whole rat genome Agilent
microarrays.
We further showed that the olfactory receptors expressed at a higher level following repetitive exposure of the animal to an odorant do
indeed bind this odorant when express at the surface of in vitro transformed HEK293 cells.
Here we report the identification of a number of rat OR binding to chemical odorants that characterize human odor and explosives. We
also determined by sequence comparison analysis (reciprocal BLAST) their canine OR ortholog.
* these two authors had equal contribution.
52
9B - Characterization of olfactory receptors expression in BON cells
Sanz G, Legenre E, Dewaele A, Pajot E
INRA, UMR1197 Neurobiologie de l'Olfaction et de la Prise Alimentaire, Domaine de Vilvert, F-78350 Jouy-en-Josas, France;
Université Paris-Sud 11, UMR 1197, F-91400 Orsay, France; IFR 144 Neuro-Sud Paris, France
BON cells are a cell line derived from human enterochromaffin cells. They were previously described to express various olfactory
receptors (ORs), but it is unclear whether a single cell expresses several types of ORs, as reported for spermatogenic cells, or a single
type of OR as for olfactory sensory neurons. The goal of the present work was first to answer this question and secondly to explore
whether BON cells could be used for functional studies of heterologously expressed ORs.
BON cells were kindly provided by Kirk Ives (Department of Surgery UTMB Galveston, TX 77551). As the cell population appears
heterogeneous in term of morphology, we first picked scattered clonal BON cells. Then, we carried out RT-PCR on mRNAs from
clones of BON cells. RT-PCR products were subsequently cloned in the pGEMT vector and sequenced. Clonal BON cells appear to
express more than one OR and most of the ORs identified are expressed by each clone of BON cells analysed. So, while the various
clones of BON cells do not display the same morphology, nor the same growth rate, they seem to express a common set of olfactory
receptors. To confirm that a single cell really expresses several olfactory receptors, single-cell RT-PCR was also performed.
Since BON cells endogeneously express ORs, we suspected they could also express heterologous ORs, which is still a challenge for
most ORs in mammalian cells. To do, we tried to heterologously express two human ORs (OR1G1 and OR17-40) in BON cells. We
demonstrated by confocal immunofulorescence microscopy that BON cells efficiently expressed both olfactory receptors at the plasma
membrane level. Furthermore, we conducted calcium imaging with BON cells heterologously expressing the OR1G1 receptor and
confirmed the activation of OR1G1 by some of its known ligands. For this purpose, we used a clone of BON cells that does not
respond to the OR1G1 ligands tested when mock-transfected. While other ORs shoud be tested for functional expression by BON
cells, these first results suggest that BON cells constitute an interesting means to carry out functional studies of heterologously
expressed ORs.
10B - Modulation of rat olfactory mucosa gene expression profile by nutritional status: a preliminary microarray approach
Badonnel K, Durieux D, Salesse R, Caillol M and Baly C
INRA, UMR1197 Neurobiologie de l'Olfaction et de la Prise Alimentaire, Domaine de Vilvert, F-78350 Jouy en Josas, France;
Université Paris-Sud 11, UMR 1197, F-91400, Orsay, France ; IFR 144 Neuro-Sud Paris, France
Various studies indicate that physiological factors are able to modulate the odour perception in mammalian species. For example,
numerous common hormones and their receptors are found expressed in the olfactory system of various species. Among them are
some peptides involved in the control of food intake, such as leptin, insulin or orexins, whose roles have been individually deciphered
in the olfactory system at several levels of sensory inputs, from the olfactory sensory neuron to the bulb neuronal network. If their
individual functional role on olfactory signal modulation is highly complex, the interplay between these systems is still poorly
understood in the whole animal context.
In the present study, we investigated the effects of a global variation of endocrine factors on olfactory mucosa plasticity by
manipulating the nutritional status of rats. We investigated the effect of a 48 hr food deprivation on the olfactory mucosa gene
expression profile by high-throughput screening on a rat whole genome microarray.
As a preliminary experiment, several metabolic and olfactory-based behavioral parameters were measured on 4 month-old Wistar rats
either fed ad libitum or food-deprived. Total RNAs extracted from Wistar rats either normally fed (n=4) or 48 hr food-deprived (n=4)
were then labeled and hybridized on DNA chips using Cogenics facilities on Agilent 4x44K microarrays, which display the whole rat
genome information. Among the 448 genes displaying both a p-value of <0.05 (t-test) and a 2 fold change between food-deprived and
normally fed status, 380 genes are potentially down-regulated, whereas 68 are up-regulated by fasting. Patterns on a subset of genes
were validated using quantitative real-time reverse transcription polymerase chain reaction. Clustered analyses of affected genes as
well as our preliminary interpretation of the results are presented, indicating that several classes of genes are affected by the nutritional
deprivation. A peculiar attention to genes corresponding to olfactory receptors, which are present on the microarray, is also given.
Therefore, our findings support the conclusion that the olfactory mucosa is a sensor of the nutritional status by modulating several
genes related or not to olfactory functions. This short-term adaptation of the transcriptional pattern of the olfactory mucosa cells might
help individuals to adjust their sensory inputs to the variations of internal status.
53
11B - The RFamide peptides influence feeding, locomotion, and odor response
Ruthann Nichols
Biological Chemistry Department, University of Michigan Ann Arbor
The family of peptides related by the C terminus RFamide contains many members and is present throughout the animal kingdom in
vertebrates and invertebrates, from human to fly. This superfamily of peptides can be further divided into subfamilies based on the Cterminal structure -XRFamide. Numerous RFamide peptides are present in a single species. Multiple precursors encode polyproteins
from which the RFamide peptides are processed to produce mature bioactive molecules.
The C terminal RFamide is strictly conserved although the N-terminal extension is variable in length and amino acid
composition. Structure-activity studies indicate the C terminus is critical for binding and the N-terminal extension plays a role in
conferring activity. Typically, RFamide peptides signal through G protein-coupled proteins; however, other transduction pathways are
known to be used by RFamide peptides.
The RFamide peptides are typical of other bioactive peptides in that they are present in the central nervous system and the
gastrointestinal tract. In addition, they affect multiple physiological processes suggesting they are involved in numerous biological
functions. The first RFamide peptide identified is the tetrapeptide FMRFamide from mollusc by Price and Greenberg [1977] as a
cardioexcitatory peptide from mollusc. Subsequently members of this family of peptides were discovered to affect feeding,
locomotion, odor response and the gastrointestinal and reproductive systems as well as cardiovascular parameters.
Five different genes encode multiple, structurally distinct RFamide precursors in Drosophila melanogaster. The names of the genes in
the order they were identified are FMRFamide, drosulfakinin, dromyosuppressin, neuropeptide F and short neuropeptide F. The
symbols used to identify the gene products are FMRFamide, DSK, DMS, NPF, and sNPF. Vertebrates contain homologous gene
products of the D. melanogaster RFamide-containing peptides.
The D. melanogaster larva is a developmental stage in which there is tremendous growth; it vigorously feeds and locomotion or
movement is essential to gather food, and to avoid predators, and a noxious or possibly toxic environment. Our work is designed to
contribute to the understanding of the role of RFamide peptides to coordinate reception of external cues to regulate the response of
physiological parameters in the whole organism.
We investigated the response of D. melanogaster larvae to odor cues in the presence of increased DMS and DSK peptides. We also
measured locomotion in the presence of increased DMS and DSK peptides. We collected data from our biological measurements that
are in keeping with the effects of RFamides in odor response and locomotion in other animals. In addition, the cellular distribution of
DMS and DSK are in agreement with a role for these conserved peptides in receiving and conveying external cues to impact
physiological functions consistent with the activities observed.
12B - Molecular and cellular basis of thermoreception of the malaria mosquito Anopheles gambiae
Qiu YT, Wang G*, Lu T*, Kwon H-W*, Pitts J, van Loon JJA, Takken W & Zwiebel LJ*
Laboratory of Entomology, Wageningen University, The Netherlands; * Department of Biological Science and Pharmacology,
Vanderbilt University, Nashville, USA
The malaria vector Anopheles gambiae and other blood-feeding mosquitoes rely on temperature cues in addition to olfactory stimuli
for host location. Heat is emitted by mammalian hosts, serves as a universal attractant to many mosquito species and synergizes with
host odor to increase the efficiency of host-seeking behaviors. Furthermore, recent behavioral studies have indicated that heat alone
has no effect on the landing response of female An. gambiae, but significantly synergizes the attraction to the host odor.
Despite some knowledge of the physiology of antennal thermoreceptive neurons, little is known about genes that are involved in
peripheral temperature detection in mosquitoes. In Drosophila, a family of 13 transient receptor potential genes encode sixtransmembrane nonselective cation channels, several of which have been identified as central nervous system (CNS) thermoreceptors.
Of these, dTRPA1 is activated by warm temperatures, with a threshold of ~27ºC, and regulates thermotaxis. Furthermore, dTRPA1
has recently been shown to be activated indirectly through pathways coupled to phospholipase C to discriminate between the optimal
temperature of 18ºC and slightly higher temperatures (19–24ºC). Not surprisingly, additional studies in Drosophila have revealed
distinct TRP channels that are activated by cold temperature.
Here we examine the molecular basis for peripheral heat sensitivity in adult female An. gambiae. By using single sensillum recording,
we studied the response of neurons in the small coeloconic sensilla to the change of temperature. One neuron in the small coeloconic
sensilla at the distal-most region of the 13th antennal segment responded to an increase of temperature. One of the neurons in these
small coeloconic sensilla also increased firing frequency when the relative humidity increased. Interestingly, small coeloconic sensilla
located in a more central region of the 13th segment contain one neuron that increased its spike frequency when the temperature
dropped to a value lower than room temperature, whereas no neuron responding to the increase of temperature was found in these
sensilla.
We have screened the An. gambiae homologs of Drosophila TRPA channels for antennal expression and have identified AgTRPA1 as
a consequence of its specific localization to the distal antennal small SCs. Furthermore, we have functionally characterized AgTRPA1
heterologously expressed in Xenopus oocytes to demonstrate that it encodes a functional thermoreceptor. In these studies, AgTRPA1
was activated by temperature increases from 25 to 37ºC, consistent with the hypothesis that An. gambiae relies on antennally
expressed AgTRPA1 to detect increasing temperature gradients derived from host body heat during crucial close-range, host-seeking
behaviors.
54
13B – Olfactory function in a viviparous insect
Poddighe S *, Dekker T **, Scala A ***, and Angioy AM *
(*) Department of Experimental Biology, University of Cagliari, Italy; (**) Department of Crop Science, Division of Chemical
Ecology, Swedish University of Agricultural Science, Alnarp, Sweden; (***) Department of Animal Biology, Section of Parasitology
and Parasitic Disease, University of Sassari, Italy
Oestrus ovis L. (Diptera: Oestridae), the sheep’s nasal bot fly, is a myiasis-causing insect species that is widely distributed in the world
(Touré, 1994). Gravid females larviposit first instar larvae into the nostrils of sheep and goats. From the first to the third stage, larvae
develop in frontal sinuses of the animal. Third stage larvae are sneezed from animal nostrils and pupate in the soil. The obligate
myiasis can severely affect animal production, and represents a high risk factor for the human health causing internal infections and
injuries in the mouth, the nose, the eyes and the ears (Pampiglione et al., 1997). Results of a field study indicated that the
larvipositional behaviour of O. ovis bot flies was mainly dependent on climatic conditions and on visual function (Cepeda-Palacios
and Scholl, 2000).
We conducted the present investigation to analyze the fly’s ability to detect odours that may play a role in attraction of the fly to the
host.
Third stage larvae were collected from heads of freshly slaughtered sheep in Sardinia. They were maintained in a sterile substrate to
pupate at constant conditions of temperature (24° C), humidity (50-60 % R. H.) and photoperiod (12-h light and:12-h dark). Pupal
development and fly emergence were recorded. The adult insect is lacking in the alimentary apparatus. Adults are equipped with
small-sized spheroid antennae with aristae. Scanning electron microscopy showed that the proximal subsegment of the antennal
flagellum carries trichoid and basiconic olfactory sensilla, as well as grooved peg formations. These structures are distributed all over
the anterior surface of the flagellar subsegment, while only basiconic and grooved peg sensilla are located on the posterior side.
Confocal microscopy on the Central Nervous System pointed out the presence of well defined antennal lobes with a glomerular
organization. By delivering pulses of odour vapours on the antennae of 2-days-old flies, electroantennographic recordings were
obtained in response to several classes of chemical coumpounds.
Our findings give the first evidence on an olfactory function in the both fly, O. ovis. The biological importance of olfactory perception
in guiding attraction and larvipositional behaviour of the insect will be the object of future studies.
Cepeda-Palacios R., Scholl P. J. (2000) Vet. Parasitol., 91, 93-105
Pampiglione S., Giannetto, S., Virga, A. (1997) Parassitologia, 39, 415–418
Tourè S. M. (1994) Rev. Sci. Tech. Off. Int. Epiz. 13, 1053-1073.
14B - Novel natural ligands for OSNs in the coeloconic sensilla of Drosophila
Irene Ibba*,#, Teun Dekker#, Francesca Cadeddu*, Bill Hansson¤, AnnaMaria Angioy*
*Department of Experimental Biology, Section of General Physiology,University of Cagliari, Italy. ¤Max Planck Institute for
Chemical Ecology, Department of Evolutionary Neuroethology, Jena,Germany. #Division of Chemical Ecology, Swedish University
of Agricultural Sciences, Alnarp, Sweden.
In Insects olfactory stimuli are detected and discriminated by olfactory sensory neurons (OSNs) housed in sensory hairs, called
sensilla, which are located on the antennae as well as the maxillary palps. In the Drosophila antenna, OSNs are housed in three major
morphological types of sensilla: basiconica, tricoidea and coeloconica. Most of the OSNs express two evolutionarily related insectspecific chemosensory receptor families, the odorant receptors (Ors) and gustatory receptors (Grs). Recently it has been found (
Benton et al. 2009) that some OSNs housed in coeloconic sensilla express a novel family of chemosensory receptors, called ionotropic
receptors (Irs). Yao and colleagues demonstrated that there are 4 coeloconic sensilla types: ac1, ac2, ac3, ac4 (Yao et al. 2005).
Among these, only ac3B neurons, that express coreceptor Or83b, respond to the 80% of the odours, specifically to alcohols. Or83b is
expressed in 80% of all OSNs ( Larsson et al. 2004), and acts together with other Ors to generate responses to odorants. The other
coeloconic sensilla showed only few and weak responses to a set of 45 odours. Here we investigated which natural odour ligands
present in fruit elicits response in coeloconic sensilla. We performed headspace collections from ecologically relevant sources such as
different fruits at different stage of ripeness, yeast and vinegar. Through gas chromatographic-electroantennographic recordings (GCEAD) from flies with a targeted mutation of Or83b, we found novel natural chemicals to which OSNs housed in coeloconic sensilla of
Drosophila respond to. Through the GC-MS (mass spectrometry) technique, we were able to identify the compounds that elicited
response in the ORNs, e.g. 2methyl propanoic acid; 2methyl butanoic acid; 3-(methylthio)propanoic acid ethyl ester. In order to
identify which OSN from coeloconic class was responding to these natural compounds, we performed gas chromatography coupled
with single sensillum recording (GC-SS) in Or83b- as well as wild type Drosophila melanogaster. We found some OSNs that
responded strongly to individual compounds within the extracts. Synthetic samples of these compounds will be used to establish dose
response-curves as well as their effect on Drosophila melanogaster behavior.
Benton R., Vannice K. S., Gomez-Diaz C., and Vosshall L. B. Cell 136, 149–162, 2009.
Larsson, M.C., Domingos, A.I., Jones, W.D., Chiappe, M.E., Amrein, H., and Vosshall, L.B. Neuron 43, 703–714 2004.
Yao, C.A., Ignell, R., and Carlson, J.R. J. Neurosci. 25, 8359–8367 2005
Acknowledgments: MIUR: International Inter-University Cooperation
55
15B - Functional study of gustatory receptors in Drosophila
Faucher C, and Chyb S.
CSIRO Entomology, Canberra, Australia
The fly Drosophila melanogaster detects taste compounds by contacting a substrate with sensory hairs located on gustatory organs,
such as proboscis, legs, wings, and ovipositor. Each sensory hair houses two to four gustatory receptor neurons that send projections to
the brain. Recently, a family of 60 putative gustatory receptor (Gr) genes has been identified, consisting of 68 predicted seventransmembrane-domain proteins. They are thought to be involved in the transduction of the chemical information (detection of a taste
molecule) into a neuronal signal (train of action potentials). However, for the majority of these receptors, their ligand specificity and
role in taste discrimination remains to be elucidated.
Here, we describe the functional characterisation of two gustatory receptors heterologously expressed in an insect cell line using
calcium imaging. Gr5a has been previously shown to encode a receptor tuned to the sugar trehalose (Chyb et al., 2003), while Gr64f
seems to function as a co-receptor of Gr64a and Gr5a (Jiao et al., 2008).
We investigated the ligand specificity of Gr5a and Gr64f when expressed individually or in combination. Cell expression vectors
containing one of these two gustatory receptors have been generated and transiently expressed in the Sf9 cell line. Their sensitivity to
tastants has been assessed by monitoring the rise of cytosolic calcium using fluo-4 as a calcium indicator. For homogeneity of the data,
values were related to the maximal fluorescence emitted by the cells by addition of ionomycin, a calcium ionophore that affects the
permeability of the plasma membrane and thus leads to maximal entry of external calcium into the cell. This method is based on that
developed by Kiely et al., 2007. The results of this study will be discussed in light of the present knowledge of GR specificity in vivo.
References:
Chyb S., Dahanukar A., Wickens A., and Carlson J.R. (2003) Drosophila Gr5a encodes a taste receptor tuned to trehalose. Proc. Natl
Acad Sci USA 100 (suppl. 2): 14526 – 14530.
Kiely A., Authier A., Kralicek A.V., Warr C.G., and Newcomb R.D. (2007) Functional analysis of a Drosophila melanogaster
olfactory receptor expressed in Sf9 cells. Journal of Neuroscience Methods. 159; 189 – 194.
Jiao Y., Moon S.j., Wang X., Ren Q., and Montell C. (2008) Gr64f is required in combination with other gustatory receptors for sugar
detection in Drosophila. Current Biology. 18; 1797 – 1801.
16B - Implication of a putative UGT in Drosophila male pheromonal discrimination
Fraichard S, (1), Legendre A (2), Artur Y (2), Ferveur JF (1) and Heydel JM. (2)
(1) UMR CNRS 5548 DCCI, 21000 Dijon, France - (2) UMR INRA 1129 FLAVIC, 21000 Dijon, France
Xenobiotic Metabolizing Enzymes ( P450, UGT...) take in charge exogenous molecules to eliminate them from the organism. There
are several evidences of specific Xenobiotic Metabolizing Enzymes activity in the chemosensory organs but still very few
experimental evidence yet support their functional role in chemosensation. In this study, we have investigated the effect of Xenobiotic
Metabolizing Enzymes on pheromonal discrimination in Drosophila melanogaster.
We used a Drosophila mutant strain containing a P-element inserted in a putative UDP-Glycosyl transferase (UGT) gene and we
observed that under red light, in which flies are virtually blind, mutant males do not preferentially court females as control male do.
Moreover, quantitative RT-PCR (Q-PCR) measurement revealed an increased expression of the UGT in mutant flies in comparison to
control flies.
We targeted several RNA interference transgenes (RNAi) to affect the expression of the UGT-coding gene in various tissues of the fly.
Surprisingly, when most adult peripheral sensory neurons were targeted, transgenic RNAi males showed enhanced sex discrimination
under red light, in comparison to control male genotypes. To refine our results, we targeted specific subsets of peripheral
chemosensory neurons potentially involved in pheromonal perception and more specifically taste and olfactory neurons. We first used
the Gr66a-GAL4 driver, known to be expressed in a group of taste neurons involved in the detection of an aversive male pheromone.
The discriminatory response of transgenic males was not changed in comparison to control males. This result would indicate that the
mechanism, which involved the UGT in sex pheromones discrimination, does not take place in Gr66a neurons. Subsequently, we used
the Or83b-GAL4 transgene to target UGT in most olfactory peripheral neurons. In that case, transgenic males better discriminate sex
pheromones than control males. These results showed that the overexpression (P-element) and the underexpression (RNAi) of the
UGT gene induced reciprocal effects on male ability to discriminate sex pheromone (respectively decreased and improved). To
confirm, we measured their combined effects both on discriminatory behavior and mRNA expression. Males combining UGT P
mutation with RNAi transgene (with OR83b-GAL4 driver), strongly rescued their discriminatory ability and the mRNA level of UGT
gene was very similar to that shown by control flies.
Our data indicate that the overexpression of the UGT gene induces a loss of pheromonal discrimination whereas its underexpression in
sensory organs leads to a higher discrimination. This suggests that the genetic misexpression of a putative UGT can modulate in two
ways (improvement or impairment) pheromonal perception and/or discrimination in Drosophila males.
56
17B - Experience-dependent plasticity in the olfactory system of a moth
Kauer I, Minoli S, Colson V, Marion-Poll F, Anton S
INRA, UMR Physiologie de l'Insecte: Signalisation et Communication, Route de St Cyr, F-78000 Versailles Cedex, France
Male moths innately have a high sensitivity to female-produced sex pheromones. In the noctuid moth Spodoptera littoralis, however,
behavioural responses to the pheromone can be further increased by brief pre-exposure to the sex pheromone (Anderson et al., 2003;
2007). This increase in behavioural sensitivity is observed in two time windows after exposure: a short term effect within 15 minutes
and a long term effect lasting up to two days. In parallel, an increase in sensitivity of neurons within the primary olfactory centre, the
antennal lobe, was observed 27 h after pre-exposure with pheromone. As a first step to test if the observed pre-exposure effect is a
form of general sensitization or rather a phenomenon of selective attention, we tested effects of pre-exposure with gustatory stimuli on
the behavioural and central nervous sensitivity to sex pheromones.
We used a pre-exposure procedure with a sucrose stimulus applied to the antenna, eliciting a proboscis extension reflex in the male
moths. As a first step, sucrose- and quinine-exposed moths were subsequently tested with different concentrations of sucrose solution.
Sucrose- and quinine-exposed males showed lower proboscis extension response thresholds to diluted sucrose solutions than naïve
moths, indicating an increased sensitivity. In addition, sucrose-exposed males, tested on a locomotion compensator during stimulation
with the female-produced sex pheromone, responded to lower doses of the pheromone than naïve moths, not differently from
pheromone-exposed males. To study the neurobiological mechanisms underlying the observed behavioural plasticity, we investigated
the sensitivity of antennal gustatory receptor neurons by means of extracellular recording techniques as a function of pre-exposure
with sucrose or quinine. In addition we studied the sensitivity of central olfactory neurons within the antennal lobe in sucrose-exposed
males as compared to naïve and pheromone-exposed males with intracellular recording techniques. Our results indicate that preexposure to a variety of relevant stimuli might lead to a general sensitization of different sensory systems rather than eliciting selective
attention.
References:
Anderson P, Sadek MM, Hansson BS. 2003. Pre-exposure modulates attraction to sex pheromone in a moth. Chem Senses. 28:285–
291
Anderson P, Hansson BS, Nilsson U, Han Q, Sjöholm M, Skals, N, Anton, S (2007) Increased behavioural and neuronal sensitivity to
sex pheromone after brief odour experience in a moth. Chem Senses 32:483-491
18B - A unique arrangement of male specific glomeruli in Helicoverpa assulta as compared to related species and hybrid
offsprings
Zhao XC1, Dong J2, Wang CZ3, Berg BG
1Dept. of Psych/Neurosi Unit, Norwegian University of Science and Tecnology, Norway, 2Forestry College, Henan Univerity of
Science and Technology, Peoples R China, 3Institute of Zoology, Cinese Academy of Science, Peoples R China
The noctuid moths Heliothinae comprise more than 80 species, distributed in all five continents. These organisms have been widely
used for studying the neural pathways mediating chemosensory signal information, in particular signal information underlying
reproductive behavior. Especially fascinating is the fact that sympatric species of the heliothine group communicate through distinct
chemical signals not only within but also across the species. A certain pheromone substance produced by a particular female may act
as a behavioral antagonist on the male of sympatric species. The male has evolved receptor neurons tuned to pheromones, responsible
for attraction and sexual behaviour, and in addition neurons tuned to interspecific signals, responsible for interruption of attraction.
The female-produced pheromones are detected by sensory neurons housed within long hairs on the male antennae. The sensory axons
project to the male specific macroglomerular complex (MGC) that is situated dorsally in the antennal lobe. The Oriental tobacco
budworm, Helicoverpa assulta, is unique among the species studied so far by using a pheromone blend containing a principal
compound different from that used in the blend of other species. By tracing of physiologically characterized projection neurons we
have investigated the functional organization of the three MGC units in H. assulta. In accordance with previous findings in other
heliothine species, the MGC is chemotopically organized. Interestingly, the inter-specific differences between the pheromone blends
are reflected in the MGC of the various species. Thus, in H. assulta, the large MGC unit, the so-called cumulus, has a functional
tuning different from that of the other species. Also the MGC representations of the two remaining insect-produced substances
detected by the H. assulta male, one produced by the conspecific female and one by a sympatric female, are intriguing from a
comparative point of view. Particularly fascinating is to compare with the closely related species Helicoverpa armigera that utilizes
the same principal pheromone components as H. assulta, but in the opposite ratio. In both species the MGC consists of three units,
however differently arranged. Since H. assulta and H. armigera are able to mate and get offspring, we have investigated, by synapsin
immunostaining and confocal microscopy, the anatomical organization of the MGC in the hybrids. Based on the assumption that H.
assulta is the most original of the two, the results are interesting in an evolutionary perspective.
57
19B - Visualization of inhibitory interneurons in the olfactory pathways of the moth
Hoel Fjaerli H, Halvorsen JM, Zhao XC, Berg BG
Dept. of Psych/Neurosci Unit, Norwegian University of Science and Technology, Norway
It is well known that GABA is the major inhibitory neurotransmitter in the central nervous system of most living organisms. The
primary olfactory processing center of the insect brain, the antennal lobe, is well suited for studying the neurochemical organization of
a functional neural network. This system includes only four types of neurons: terminals of sensory neurons, projection neurons which
carry signal information to higher integration regions in the protocerbrum, local interneurons which communicate within the antennal
lobe, and centrifugal neurons which modulate the signal information by sending projections from the protocerebrum into the antennal
lobe. The synaptic connectivity pattern made up by these elements is thoroughly investigated in several species. The main portion of
the antennal-lobe local interneurons is presumed to be GABAergic. The inhibitory input from local interneurons is believed to induce
synchronization of projection neuron activity. In the noctuid moth Heliothis virescens about 330 GABA-immunoreactive somata have
been counted, all located in the lateral cell cluster of the antennal lobe. However, a minor category of these belong to GABAergic
projection neurons as comprehensive immune-reactivity has been found in two antenno-cerebral pathways, the middle and the dorsal
antenna-cerebral tract. Particularly, the middle antenna-cerebral tract which targets the lateral protocerebrum merely has shown a
considerable amount of GABAergic fibers. The function of the inhibitory output-neurons is not yet understood. We have traced the
antennocerebral tracts by applying a fluorescent dye in the protocerebral target areas of antennal-lobe projection neurons and then
combined these back-fill stains with GABA immuno-staining. The double-labelled preparations have been scanned in a confocal
microscope and analyzed by visualization software programs. The results indicate the number and the location of somata belonging to
the inhibitory projection neurons. Widespread occurrence of GABA in the antennal lobe has previously been demonstrated by dense
immunoreactivity throughout all glomeruli. In the present study we also performed another kind of double-labelling; preparations with
single antennal-lobe projection neurons and local interneurons physiologically and morphologically identified through the intercellular
recording and staining technique, were labelled with antiserum against GABA. Thus, the results present physiological and
morphological characteristics of inhibitory antennal-lobe interneurons.
20B - Pheromones and biodiversity - monitoring change in a changing world
Larsson MC (1), Svensson GP (2), Ryrholm N (3)
1) Swedish University of Agricultural Sciences 2) Lund University 3) University of Gävle
Monitoring the abundance of species in space and time is crucial to determine the conservation status of potentially threatened species.
Insects and other invertebrates deserve special attention in this regard as the largest and most diverse group of organisms, and with a
disproportionately high number of threatened species. Insects also constitute excellent potential indicators of environmental and
climate change, as many species have high dispersal ability and respond quickly to change. Assessing their threat status can be a
formidable task, however, due to taxonomic diversity and the difficulty of obtaining population estimates and accurate geographical
distributions.
Within the umbrella project PheroBio – pheromone monitoring of biodiversity – we are developing methods to facilitate monitoring of
rare and threatened species that are otherwise largely inaccessible with other methods. Insect sex pheromones have been used
extensively in integrated pest management but not yet for monitoring of rare and threatened insects, despite the growing concern for
biodiversity and conservation. The legendary attractiveness of sex pheromones makes them an ideal tool for studying many insect
species occurring at very low population densities, including newly established invasive species. We present two model systems that
are likely the first to exploit pheromones in the context of conservation and biodiversity:
1) The sex pheromone-kairomone system of the threatened saproxylic scarab beetle Osmoderma eremita and its predator, the click
beetle Elater ferrugineus, which breed in hollow trees in fragmented oak stands in pastoral landscapes.
2) Monitoring a whole range of tineid and clearwing moths in fragmented forest landscapes, using only a few single compounds from
their phylogenetically restricted pheromone systems. Tineid moths breed in dead wood and/or associated fungi, making them
potentially valuable indicators of mature forest with high levels of biodiversity.
Our investigations illustrate the versatility of pheromones for precise monitoring in space and time and include estimates of absolute
population size by capture-recapture studies; estimates of presence/absence and population density on landscape and regional scales;
monitoring of population fluctuations over several years, and studies of dispersal frequency and dispersal distance. We have shown
that previous recapture models of O. eremita likely lead to severe underestimations of population size, and that patterns of
disappearance for tineid moths appear to be very different from those of saproxylic beetles, which are studied more frequently.
58
21B – Olfactory responses of Hyalesthes Obsoletus to host plants volatile compounds
P. Riolo (1), R. L. Minuz (1), G. Anfora (2), M. V. Rossi Stacconi (3), N. Isidoro1 & R. Romani (3)
(1) Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Ancona; (2) IASMA
Research and Innovation Centre, Fondazione E. Mach (FEM), S. Michele all'Adige (TN); (3) Dipartimento di Scienze Agrarie e
Ambientali - Entomologia, Università degli Studi di Perugia
Hyalesthes obsoletus Signoret (Hemiptera: Auchenorrhyncha: Cixiidae) is a palearctic planthopper, known as the natural vector of
stolbur phytoplasma (16Sr-XIIA), that causes the grapevine yellow disease known as Bois Noir (BN). BN is the most widespread
grapevine yellow disease in Europe and Mediterranean basin, and is the major limiting factor of viticulture.
Genetic, physical, metabolic and sensory capacities of the insect are the constraints of its host plant choice; in the case of H. obsoletus
the host plant choice is made at the adult stage. Its plant preference changes depending on the geographic region. The primary hosts of
H. obsoletus are: Convolvolus arvensis L. (bindweed) and Ranunculus spp. (crowfoot) in north Europe (Maixner et al., 1995; Sforza et
al., 1998 e 1999), while in Italy Urtica dioica L. (nettle) is the herbaceous plant on which adults lay eggs.
Behavioural, morphological and electrophysiological studies were carried out in order to deepen the knowledge on the role of volatile
organic compounds in H. obsoletus host plant recognition.
The behavioural assays were made using a Y-olfactometer, and the host plants tested were: Urtica dioica, Convolvolus arvensis,
Calystegia sepium, Vitex agnus-castus and Vitis vinifera. For every trial (stimulus vs blank) 60 males and 60 females were tested
singularly. The results showed a significant attraction of females to U. dioica and of males to V. agnus-castus.
Ultrastructural studies of the antennae showed the presence of at least two typologies of olfactory sensilla, at the level of the pedicel.
The first type is represented by complex structures in literature known as “plaque organs”, innervated by several tens of sensory
neurons. The second type is formed by trichoid sensilla, each one innervated by two sensory neurons.
Volatile compounds from host plants were collected, and the extracts analysed by coupling gas-cromatography to both
electroantennography (GC-EAD) and mass-spectrometry (GC-MS). The extracted volatile compounds were identified, and some of
them were shown to elicit significant electrophysiological responses on male and female antennae. The identification of such
molecules and the study of behavioural mechanisms involved in host recognition are of fundamental importance to develop a new
possible strategy of monitoring and control of H. obsoletus.
This work was funded by the Italian MIUR, PRIN 2007 “Mechanisms of host-plant location and host-plant preferences in two
leafhoppers (Hemiptera: Auchenorrhyncha), vectors of grapevine yellow diseases”
22B - Olfactory coding in trichoid sensilla of Anopheles gambiae s.s.
Remco A. Suer, Yu Tong Qiu, Joop J.A. van Loon and W. Takken
Laboratory of Entomology, Wageningen University, 6700 EH, P.O.Box 8031, Wageningen, The Netherlands
Anopheles gambiae is one of the major malaria vectors in sub Saharan Africa. The high vectorial capacity of this mosquito species is
mainly because females mosquitoes are highly antropophilic (prefer human blood), endophagic (feed indoors), endophilic (rest
indoors) and susceptible to the infection of Plasmodium falciparum (the most fatal form of malaria). Female Anopheles gambiae use
olfactory cues to seek for sexual mates, blood-hosts, sugar sources or breeding sites. Understanding olfactory coding in Anopheles
gambiae will eventually help us to design better means for the disruption of odour-mediated behaviour, a possible strategy to diminish
malaria transmission.
The olfactory system of a mosquito consists of three pairs of olfactory organs; the antennae, maxillary palps and the proboscis. The
antennae of a female An. gambiae mosquito contain the largest number of the olfactory receptor neurons (ORN) (1500–1600 ORNs
per antenna). The antennal ORN’s are innervated mainly in two morphologically distinct types of sensilla: trichoid sensilla and
grooved-peg sensilla. Using single sensillum recording technique, previous research has characterized the response spectra of
grooved-peg sensilla and two of the five morphological subtypes of the trichoid sensilla using a panel of 44 chemical compounds
known to be components of either human odours or breeding site-related volatiles. In the present study we investigated the responses
of ORN’s contained in two of the remaining morphological subtypes of trichoid sensilla using an extended panel of 130 chemical
compounds.
Using single sensillum recording we characterised the function of the olfactory receptor neurons innervating trichoid sensilla subtype
B and D. Based on an odour panel consisting of 130 chemical compounds, including known and potential infochemicals for female
An. gambiae, five functional types for trichoid sensilla subtype B and four functional types for trichoid sensilla subtype D were
identified. Most ORNs we studied were generalistic, responding to heterocyclics, alcohols and aromatics. All four functional types of
trichoid sensilla D responded to more than 20 compounds. With the exception of one specific functional type that responded to only
one odourant, the response spectra of ORNs in B-subtype trichoid sensilla were more broadly tuned than ORNs innervating D-subtype
trichoid, responding to more than 50 odours.
Next to these characterization studies the responses of ORNs to binary blends were also studied to better understand the mechanisms
of odour coding to odour mixtures in the peripheral olfactory system.
59
23B - Odorant Capture: Heterogeneity of Cuticular Lipid Coatings on Moth Trichoid Sensilla
Thomas C. Baker
Center for Chemical Ecology, Department of Entomology, Penn State University, University Park, Pennsylvania, USA 16802
The lipid coatings on trichoid sensilla of male moths may be involved in both the selective adsorption of pheromone molecules from
the air, as well as focusing these molecules onto sensillar pores. If the lipids on trichoid sensilla were better at adsorbing pheromonetype molecules than other surfaces of the antennae, pheromone molecules would be selectively focused onto the trichoid hairs.
Secondly, different regions of the sensilla could be coated heterogeneously with different forms or orientations of lipids to expedite the
movement of adsorbed pheromone molecules down the pores to the binding proteins and then to the receptor neurons in the sensillum
lumen. We found that in Helicoverpa zea moths, there were in fact significant differences in the lipids on male antennae compared to
those of females (1) and these could be attributed to the trichoid sensilla that exist in huge numbers on male antennae but not on
female antennae. Thus at the macro-level we found evidence for differential coating of male trichoid sensillar surfaces that might
selectively adsorb pheromone molecules.
We then used atomic force microscopy (AFM) on the sensilla to determine their individual nano-terrains, followed by chemical force
microscopy (CFM) to contact the surfaces and probe for differences in chemical bonding forces that could indicate differences in lipid
coatings on different local sensillar regions. Using a hydrophilic CFM silica tip, we found that H. zea trichoid sensilla exhibited
heterogeneity in their lipid coatings, with the multitudes of ridges on each sensillum possessing more hydrophobic coatings than the
pore-containing areas. These results suggest that there is a lipid-based nano-focusing of the aldehyde pheromone molecules into the
pores from non-pore-containing areas of the sensilla. As expected, when the CFM tip was coated with a monolayer of C18
hydrocarbon to eliminate its hydrophilicity, the chemical force differences between the ridges and planar areas containing pores
disappeared. A second species, Utethesia ornatrix, which uses hydrocarbon pheromone components and not aldehydes, exhibited no
lipid heterogeneity on its trichoid sensillar surfaces when the hydrophilic CFM tip was used. Moreover, the pores on U. ornatrix
sensilla were always located at the bases of the ridges, unlike those of H. zea, which are nearly always located in the middle of the flat,
inter-ridge planar areas. These findings suggest that the mechanisms of adsorption of the aldehyde- versus the hydrocarbon-based
pheromone components on the sensilla of these two species are different. It is possible that the pheromone molecules solubilize in this
matrix and do not merely surface-diffuse down the pores to reach the lumen of the sensillum.
1. J. Insect Physiol. 54: 1385-1391.
24B - Representation and discrimination of pheromones and plant odours in the antennal lobe of codling moth males and
females
Trona F., Ignell R., and Witzgall P.
1 IASMA Research and Innovation Center, Fondazione E. Mach - Genomics and Crop Biology Area, via E.Mach 1, 38010 S.Michele
a/A (TN), Italy 2 SLU, Chemical Ecology Group, 23053 Alnarp, Sweden
In the codling moth, Cydia pomonella (Lepidoptera: Tortricidae), olfactory receptors neurons (ORNs) on male and female antenna
were shown to respond to both the main pheromone component, codlemone (E,E)-8,10-dodecadienol) and to pear ester (ethyl (E,Z)2,4-decadienoate), a plant volatile attractant, suggesting that the response to both compounds is mediated through a common sensory
channel (De Cristofaro et al. 2004, Ansebo et al. 2005). Plant volatiles attract both males and females by upwind flight, and synergize
also the male response to female-produced sex pheromone, indicating a close evolutionary relationship between olfactory
communication with pheromones and plant volatiles. The goal of this study was to improve understanding of how information from
social and environmental cues is processed. To this purpose we studied the responses of males and females of C. pomonella to
pheromones, plant volatiles and odour blends in the antennal lobe (AL), the primary olfactory center of the insect brain. A 3dimensional atlas was constructed from mass-stained ALs of both sexes. Using intracellular recordings and staining technique, a
morphological and physiological characterization of projection neurons (PNs) and local interneurons (LNs) was performed, testing
behavioural active olfactory receptor ligands (13 plants volatiles, 4 pheromone compounds). The interaction between compound
blends was investigated in order to evaluate (1) the response properties of PNs that innervate the ordinary glomeruli to mixtures of
plant odours, (2) the responses of the PNs in the macroglomerular complex to pheromonal blends (3) the effects of the main
pheromone component on plant odour discrimination. In the 3-D reconstructions, landmark glomeruli were identified. That allowed us
to locate in each sex the arborisations of 30 stained neurons. The physiological responses of 50 neurons per sex were analysed. This
poster illustrates the complex excitatory and inhibitory response patterns characterizing the PNs and LNs. It also shows mixture
interactions in PN responses to different blends, both in terms of synergism and suppression.
Ansebo L., Ignell R., Lofqvist J., Hansson B.S. (2005) Responses to sex pheromone and plant odours by olfactory receptor neurons
housed in sensilla auricillica of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae), Journal of Insect Physiology, 51, 10661074.
De Cristofaro A., Ioriatti C., Pasqualini E., Anfora G., Germinara G.S., Villa M., Rotundo G. (2004) Electrophysiological responses of
Cydia pomonella to codlemone and pear ester ethyl (E,Z)-2,4-decadienoate: peripheral interactions in their perception and evidences
for cells responding to both compounds. Bulletin of Insectology, 57, 137-141.
60
1C - Memory and discrimination in three senses: olfaction, vision and kinesthesis
Møller P(1), Piper D(2), Hartvig D(1), Mojet J(3) Köster EP(1,3)
(1)University of Copenhagen, Frederiksberg, Denmark; (2)Symrise Germany, Holzminden, Germany;(3) Centre for Innovative
Consumer Studies, Wageningen University and Research Centre, the Netherlands
Background Since vision and olfaction are senses with different "ecological roles" it should not be assumed that their functional
organization is similar. Here we investigated if memories of incidentally and intentionally learned olfactory and visual stimuli differ
w.r.t. modality, mode of learning, gender and age. We also added a weight memory task since the weight of an object cannot be named
and this type of memory thus is immune to verbalization. To be able to compare memory in the visual, olfactory and kinesthetic
modalities we also measured visual, olfactory and weight discriminability between the stimuli used as targets and distractors in the
memory tests. Method One hundred and seventeen young and 114 elderly Ss participated in the experiment which had 2 sessions on 2
separate days. Stimuli consisted of 12 odorants and 12 images of objects emanating the odorants used. Half of each set of 12 stimuli
were pleasant and the other half less pleasant. Young and elderly Ss were divided into 2 groups who learned the visual and olfactory
stimuli either incidentally or intentionally. Each of the groups were split into 4 groups in which the olfactory and visual stimuli were
either congruent (e.g. pleasant smell of peach and pleasant image of peach skin) or incongruent in pleasantness. In the first task Ss
evaluated pleasantness of 6 odours and 6 images. In the memory test which followed after 5 min, the stimuli from the first task served
as targets and the other 6 odours and 6 images served as distractors. Finally, the incidentally learned memory for the weight of the
bottles used to present the olfactory stimuli was measured. On day 2 Ss performed 4 tasks: discrimination of odours and images used
as targets and distractors in the memory test, a congruency test where each odour was presented with the 2 corresponding images and
the task was to pair the odour with one of the images and vice versa for images, a weight discrimination test and a hedonic test of all
24 olfactory and visual stimuli.Results There is a double dissociation between memory and discrimination for vision and olfaction. Ss
discriminate the visual stimuli better, but don't remember them as well as they remember the olfactory stimuli. This holds for young as
well as elderly subjects. Furthermore, incidentally learned visual and olfactory stimuli are remembered equally well as when they are
learned intentionally. Congruent (visual and olfactory) stimuli are not remembered better than incongruent stimuli, but for both
modalities and learning conditions young Ss have better memories than old Ss (ANOVA, p<0.001). For weight memory we find no
differences between the 2 age groups. Conclusion The results of this experiment strongly suggest that visual and olfactory memories
do not share the same functional organization. Furthermore, the results on weight memory suggest that only when stimuli can be
verbalized do young Ss have an advantage over elderly Ss.
2C - A human pheromone affects central nervous face processing
Frey MCM, Weyers P, Pauli P, and Mühlberger A
University of Wuerzburg, Department of Psychology I
Pheromones found in animals as communicative substances are also discussed to exist in humans. Several studies showed that the
steroidal substance androstadienone (AND) has impact on the human brain. It influences the activation of brain regions underlying
face recognition, as well as emotional and visual processing. Therefore, AND might modulate the early automatic evaluation of
emotional facial stimuli. In this study, we used event related brain potentials (ERPs) to investigate influences of AND on central
nervous processing of female and male faces with happy angry and neutral expression in the female brain. The early posterior
negativity (EPN), a ERP component which is suggested to reflect the early, unconscious emotional processing, was examined.
Usually, and as found in our control group, angry compared to neutral male faces, are processed preferentially in the brain to detect
threatening situations rapidly. Interestingly, we found that the pheromone affected the early automatic evaluation of angry males, but
not of happy male faces or angry and happy female faces. AND therefore affected the EPN, an ERP produced by temporo-occipital
brain regions which are linked to early visual processing. This is in line with mentioned brain imaging studies. One could assume that
AND eliminates the facilitated processing of anger, and therefore, angry male faces might not be marked or perceived as important. In
other words, with AND the female brain might not interpret the anger on a men’s face as relevant, and therefore, women smelling
AND might not perceive angry male faces as threatening.
Considering AND as putative male pheromone and men as potential mate partners, AND might serve a communicative function
between men and women. Thus, conveying safety (e.g., not to tag an angry male face as threatening) to women it might weaken their
avoidance tendency to a potentially threatening interaction partner and therefore facilitate sexual approach.
This interpretation is consistent with the idea of AND as a sexual signal that communicates information about male mate quality.
Cornwell et al. (2004) suggested that mate choice is influenced by different sensory input, i.e. visual as well as olfactory signals. They
found positive correlations between the preference for male facial masculinity and the odor of AND, which has been interpreted as
indirect evidence that AND as well as facial features might signal “traits such as testosterone level, dominance or `good genes`” (p.
638) (Cornwell et al., 2004).
We conclude that angry male faces lose their inherent significance for women who smell the putative male pheromone AND. This
suggests that AND acts particularly in intersexual situations by signaling security to women as potential mate partners in order to
facilitate sexual approach.
61
3C - Odour-evoked Autobiographical Memory: Age and gender differences along the life span.
Zucco GM, Aiello L, and Turuani L.
University of Padua, Department of General Psychology
Odours are powerful in bringing back old and vivid memories bearing emotional content (c.f., Richardson & Zucco, 1989; Chu &
Downes, 2000, 2002; Herz & Schooler, 2002; Maylor, Carter & Allott, 2002; Zucco, 2003; 2007; Willander & Larsson, 2006, 2007;).
This inherent hedonic property of olfactory stimuli make this sensory modality particularly suitable for studying autobiographical
memory.
In the present work, teenagers (1st exp.), young adults (2nd exp.) and elderly (3th exp.) were asked to smell 10 familiar odorants and
to report for each of them whether they evoked personal autobiographical memories or referential memories (i.e. names and objects).
The participants were then required to link these memories to triplets of words using the progressive elaboration method of the Loci
mnemonic (c.f., Higbee, 1988). The odorants were presented again and RTs and the number of correct remembered items were scored.
The analyses showed a different pattern of finding according to age and sex. A different trend in the number of odour evoked
autobiographical and non autobiographical memories along the life span was also observed. Results are discussed in terms of
developmental differences, sexual roles and odour-emotion links.
References
Chu S, Downes JJ. 2000. Odour-evoked autobiographical memories: psychological investigation of proustian phenomena. Chemical
Senses. 25: 111-116.
Chu, S., Downes, J.J. 2002. Proust nose best: Odours are better cues of autobiographical memory. Memory and Cognition, 30, 511518.
Herz R, Schooler JW. 2002. A naturalistic study of autobiographical memories evoked by olfactory and visual cues: testing the
proustian hypothesis. American Journal of Psychology. 115: 21-32.
Higbee KL. 1988. Your memory: how it works and how to improve it. Englewood Cliffs, Prentice Hall.
Maylor EA, Carter SM, Allott EL. 2002. Preserved olfactory cuing of autobiographical memories in old age. Journal of Gerontology.
57B: 41-46.
Richardson J, Zucco GM. 1989. Cognition and Olfaction: a review. Psychological Bulletin. 105, 352-360.
Willander J, Larsson M. 2006. Smell your way back to childhood: autobiographical odor memory. Psychonomic Bulletin and Review.
13: 240-244.
Willander J, Larsson M. 2007. Olfaction and emotion: the case of autobiographical memory. Memory and Cognition. 35: 1659-1663.
Zucco GM. 2003. Anomalies in cognition: Olfactory memory. European Psychologist. 8: 77-86.
Zucco GM. 2007. Odour memory: The unique nature of a memory system. In: Holz P,
Pluemacher M, editors. Speaking of colours and odours. Benjamin Press: Amsterdam.
4C - Do children remember the sensory properties of food as adults do?
Laureati M (1), Pagliarini E (1), Mojet J (2) and Köster EP (2)
(1) Department of Food Science and Microbiology, University of Milan, Italy (2) Centre for Innovative Consumer Studies (CICS),
Wageningen University & Research, The Netherlands
Food memory is a kind of implicit memory, i.e. the sensory properties of a food are learned incidentally, without any explicit attention
or awareness.
Only recently systematic attention has been paid to the study of the mechanisms involved in food memory. This is quite surprising
since memory plays a fundamental role in food preference and food choice. Some authors studied memory for texture (Mojet and
Köster 2002, 2005), odour (Møller et al. 2004) and taste (Köster et al., 2004) by means of the implicit paradigm proposed and
validated by Mojet and Köster (2002). Nevertheless, few studies deal with the effect of aging on food memory performance, and only
one (Laureati et al., 2008) compared incidental learning and memory for food considering children, adults and elderly people.
The aim of the present study was to compare incidental learning and memory in children and adults for three different food products
by applying the above-mentioned implicit paradigm.
Two-hundred-ninety children (aged 7-10 yrs) and 78 adults (aged 18-45 yrs) received a snack composed of 3 target products (a biscuit,
a fruit juice and a fruit puree) under incidental learning conditions. 24 hrs later, participants were confronted with a series of samples
consisting of the targets and distractors slightly modified in sweet taste and were unexpectedly asked to perform an “absolute
memory” test (“Did you eat this sample yesterday?”), a “relative memory” test (“Is the present sample less/equal/more pleasant/sweet
than the one you ate yesterday?”), a liking test (“How much do you like this sample?”) and a perception test (“How sweet is this
sample?”).
Age was found to affect liking ratings: children scored higher than adults did for all three food products. Nevertheless, the two age
cohorts showed a similar food preference trend. Actually, both children and adults preferred low sugar concentrations in biscuits and
high sugar concentrations in fruit puree although fruit puree was liked at all the three concentration levels.
Results showed an overall memory effect, indicating that subjects learned incidentally and remembered the food eaten the previous
day. Children had a memory performance comparable to adults in the absolute memory test. For both age groups recognition depended
on rejection of the distractors rather than on the recognition of the target.
For both children and adults relative memory was found to be somewhat superior to absolute memory. Furthermore, relative memory
results showed that memory for the three different foods was related to different sensory aspects: texture instead of sweetness played a
fundamental role in biscuits, while for fruit juice and puree the key sensory attributes were sweetness and sourness.
62
5C - Beyond the inheritance of olfactory preferences: How cognitive factors can implicitly modulate preferences for odours
Coppin G 1, Delplanque S 1, Cayeux I 2, Porcherot C 2, Margot C 2, Velazco MI 2, Sander D 1,3
1Swiss Center for Affective Sciences, University of Geneva, rue des Battoirs 7, 1205 Geneva, Switzerland 2Firmenich, SA, route des
Jeunes 1, 1227 Geneva, Switzerland 3Department of Psychology, FPSE, University of Geneva, 40 Bld du Pont d’Arve, 1205 Geneva,
Switzerland
The extent to which chemosensory preferences could be modulated by cognitive factors and context is a classical debate in psychology
and is still highly debated in cognitive and affective sciences. In fact, although some evidence suggested genetically fixed and
physicochemically based olfactory preferences, many authors have insisted on the high flexibility of olfactory preferences acquisition.
In particular, a growing beam of experimental results showed that olfactory preferences can be strongly modulated by learning and
other cognitive factors. Interestingly, in other sensory modalities, results from a classical paradigm known as the “free-choice
paradigm” revealed a modulation of preferences by a simple choice. Indeed, in this paradigm, it has been shown that, after having
made a difficult choice between two similarly rated alternatives, participants rated the chosen option more desirable and the nonchosen (i.e., rejected) option less desirable than during the first rating. We adapted this paradigm in the olfactory modality in order to
investigate whether the simple act of choosing one odour among two similarly liked odours could likewise induce preference changes
for these two odours. Moreover, we were also interested in investigating the level of processing (implicit/explicit) needed for such
post-choice preference modulation to occur. At the end of the experiment, participants had to complete a memory taskused to
investigate whether post-choice evaluations of the odorants changed as a function of the choice being explicitly remembered or
forgotten. Our results showed that olfactory preferences can be shaped by a simple choice; indeed, as predicted, we demonstrated that
after choosing among two similarly pleasant odours, the preference for the chosen odour increases while the preference for the rejected
odour decreases. Importantly, we also demonstrated that choice did not need to be explicitly remembered for post-choice preference
changes to occur. Consequently, our results revealed a key modulatory role of decision-making and choices on the molding of
chemosensory preferences, consistent with the growing body of researches showing that olfactory preferences are strongly modulated
by cognitive and contextual factors. Importantly, such post-choice preference changes can occur without explicit memory of the
choice, which bring interesting insights into preference construction models, as it invites to take into account explicit as well as
implicit processes.
6C - Short and long-term behavioural and physiological effects of a postnatal olfactory deprivation
Gilbert C, Giroud S, Padzys GS, Martrette J-M, Boivin S, and Trabalon M
IPHC-DEPE, UMR 7178, CNRS-UdS, Nancy-Université
Among mammals, olfactory cues play an important role in the development of numerous behaviours. In particular in rodents, the
olfactory channel is the most important sensorial avenue for a number of functions including conspecific recognition, sexual and
maternal behaviour, feeding behaviour… However, little is known on the physiological and behavioural long-term effects of an early
olfactory deprivation.
We induced on young rats (Rattus norvegicus) a bilateral nasal obstruction (NO) from post-natal day 8 to day 14. Considering shortterm effects, NO rats aged 9-18 days returned less to their nest than control rats and at 21 days, they showed a modified exploratory
behaviour. At 3, 9 and 15 months old, we compared anxiety-related behaviours, using plus-maze and openfield tests, in control and
NO groups. We moreover tested olfactory recognition linked to sexual behaviours for 6 months old rats. NO rats appeared to be more
anxious than the control group, this anxiety varying with their age. While NO rats where significantly more anxious at 3-months old
than control rats, anxiety-related behaviours of 9 and 15 months old rats were not significantly different. Interestingly, NO male rats
also showed an impaired olfactory recognition of sexually receptive females: they failed in choosing a receptive female vs. a nonreceptive female. This thus could possibly impact on their reproductive success. Moreover, an early and reversible bilateral nasal
obstruction induced a significant decrease in olfactory bulb development: NO male rats showed a 36%, and NO female rats a 30%
decrease in their olfactory bulb mass compared to control individuals. Furthermore, corticosterone plasma levels of NO rats were
significantly different, measured both in young and adult rats, suggesting a chronic maladaptation to stress.
Similarly to recent studies focusing on long-term impacts of postnatal stressors, this work reveals the importance of early olfactory
experiences in the long-term behavioural and physiological development of individuals.
63
7C - Relationships between odour properties as a function of hedonic category
Jelena Djordjevic, Allana Goodman, and Mats J. Olsson
Montreal Neurological Institute, McGill University, Montreal, Canada, and Karolinska Institutet, Stockholm, Sweden
Relationships between different odour properties have been studied extensively; they are complex and vary with a number of factors.
However, it is unclear whether different patterns of these relationships characterize pleasant and unpleasant odours. Some limitations
of previous research included limited samples of odourants and failure to study pleasant and unpleasant odors separately. We
examined whether the relationships between four odour properties (intensity, pleasantness, familiarity, and irritability, for a total of six
pair-wise relationships) depend on odour hedonics, and whether these relationships can be predicted by rated odour
pleasantness. Thirty healthy participants, all university students with a mean age of 20.4 years rated a set of 24 odours (half pleasant,
half unpleasant) for four odour properties. Each odour was presented in three concentrations (weak, intermediate, and strong) for a
total of 72 odours, and each participant received them in a different randomized order. Stimuli were presented birhinally, and the
interstimulus interval was 30 seconds. Ratings were completed using four visual analogue rating scales, one corresponding to each
rated property. Three of the six analyzed relationships were strongly predicted by odour hedonics, and these were relationships
between intensity and pleasantness, pleasantness and familiarity, and intensity and familiarity. In all cases, these correlations were
positive for pleasant odours, while correlations were either just weakly positive (pleasantness-familiarity and intensity-familiarity) or
negative (intensity-pleasantness) for unpleasant odours. In addition, mean correlations between pleasantness-irritability were stronger
for unpleasant than for pleasant odours, even though they were negative in the case of both hedonic categories. Finally, mean
correlations between intensity-irritability showed a statistical tendency to be stronger for unpleasant than pleasant odours, although
these were positive for both odour categories. The relationship between irritability and familiarity was the only one that was totally
unaffected by odour pleasantness. Taken together, pleasant and unpleasant odours show different patterns of relationships between
odour properties, and some of these relationships can be strongly predicted by odour hedonics. These findings suggest that pleasant
and unpleasant odours represent fundamentally different odour categories and corroborate previous claims that pleasantness is a
primary dimension in olfaction.
RGPIN 335938-08 awarded to JD by NSERC
8C - Crossmodal associations between vision and olfaction: Evidence from eye movements
Alix Seigneuric, Karine Durand, Tao Jiang, Jean-Yves Baudouin, Benoist Schaal
Centre Européen des Sciences du Goût, UMR-CNRS 5170
Cross-modal linkage between the olfactory and visual senses is still largely under-explored. In this study, we investigated cross-modal
olfactory-visual integration by testing whether and how visual processing of objects was affected by the presence of olfactory cues. To
this aim, we explored the influence of prior learned associations between an odor (e.g. odor of orange) and a visual stimulus naturally
associated with that odor (picture of orange) on the guidance of the eyes through a complex scene. Participants were asked to freely
explore a photograph containing an odor-related visual cue embedded among other objects while being exposed to the corresponding
odor (subjects were unaware of the presence of the odor). Eye movements were recorded to analyse the order and distribution of
fixations on each object of the scene. Our data show that the odor-related visual cue was explored sooner and for a shorter time in the
presence of the congruent odor. First, the mean fixation duration for each exploration was shorter for the area corresponding to the
odor-congruent object compared to the same area when the object was not congruent to the odor. Second, the latency between the
onset of the test and the observer’s first fixation on a given object was shorter when that object and the odor were congruent than when
they were not. These findings suggest that cross-modal olfactory-visual integration occurs in an automatic manner and influences
identification and localization of the possible odor source. We predicted that the effects of the odor on gaze control were in the
direction of a reduction 1) of the fixation duration on the odor-related object, and 2) in the time elapsed until the first fixation on the
odor-related object. Our results provide support for both effects. We found that mean fixation duration was shorter when gaze was in
the area of the object related to the odor. This effect suggests facilitation of identification due to the presence of an odor congruent
with an object. The second major finding of this study concerns the effect of crossmodal integration between odor and sight on
oculomotor attentional capture. Our data indicate that the object associated with the concurrent odor is prioritized in gaze fixation.
This effect can be interpreted in terms of oculomotor capture due to the detection of a potential link between the odor and a visible
object resulting in gaze and attention attraction. This result is in line with those obtained in crossmodal processing based on visualauditory interactions, and may be related to the hypothesis of the localization function of crossmodal integration.
64
9C - The Effect of Odor Image on Identification of City Gas Odor
Tomoko Matsubasa and Yasushiro Gomi
Technology Research Institute, TOKYO GAS CO.,LTD., Yokohama , Japan
Special offensive odor is usually added to city gas to warn gas leaks. Recently, gas consumers have fewer experiences of smelling the
gas odor in daily life because the safety devices installed in gas appliances have been well developed. It resulted in the decrease in
percentage of consumers who recognize the gas odor, especially among younger generations, and this trend is believed to continue in
future.
The properties of gas odorants have been investigated, but there are few researches on olfactory aspects of gas odor. In this study,
experiments were carried out to clarify the characteristics of gas odor and the way how people distinguish the gas odor from other
offensive odors in daily life.
In the experiments, 48 subjects (43 females and 5 males, 41±8 years old) participated. 13 offensive odors in squeeze bottles were
presented to each subject. The odors were chosen from offensive odors in everyday life, odors similar to city gas and the city gas odor
itself. The perceived odor intensities were controlled between “easy detectable” and “strong”.
At the very beginning of the experiment, before experiencing the actual odor, all the subjects were asked to rate their “image of gas
odor” in their mind by using 15 pairs of polar adjectives that describe different sensory experiences. In the next step, the subjects
sniffed each odor to rate intensity, hedonicity, familiarity and similarity to their “image of gas odor”. A Yes/No question asking “ Do
you make an emergency call to your gas company when you smell this odor in your everyday life? “ was added to figure out the
tendencies of the people’s realistic behavior. After that, the subjects once again evaluated the odor by using the same adjectives.
The cluster analysis of the similarity ratings and “the image of gas odor” of each odors represented that the subjects were classified
into two groups. One group can distinguish the gas odor and the other odors very similar to it（Group1）, and the other group cannot
distinguish them (Group2).
To examine the characteristics of both groups, principal component analysis was carried out for each group by using the adjectives.
Two common factors, “danger” and “sharpness”, merged from the analysis in both groups, and the sensory maps of odors were
obtained for each group.
In the map of Group1, "the actual gas odor" and "the image of gas odor" were positioned closer to each other compared with the
Group2 map, and located at dangerous-side decidedly. On the other hand in the map of Group2, "the image of gas odor" was located at
rather safer-side while "the gas odor" was still positioned at dangerous-side as seen in Group1 map.
It was suggested that the Group2 could not identify the gas odor due to their less recognition as “dangerous odor”.
10C - Influence of information-manipulation of the same odor stimulus on cardiovascular response
Kobayashi T, Akiyama Y, Nagano Y, Saito S, and Kobayakawa T
Bunkyo Gakuin University, National Institute of Advanced Industrial Science and Technology
In ECRO 2004 and 2006, we introduced intermittent presentation method of short-duration odor to evaluate the cognitive effects on
odor adaptation / habituation and evaluated these cognitive effects using electrophysiological techniques such as electroencephalograph (EEG) and magneto-encephalograph (MEG). So far we have obtained results showing that the intermittent
presentation method is suitable to detect both cognitive and physiological effects of information-manipulation on the same odor
stimulus through minimizing adaptation to the odor. Autonomy system responses such as cardiovascular responses following odor
presentation with similar experimental conditions, however, have not been investigated. Thus, in this study, the effects of positive,
neutral, or negative information on various cardiovascular responses, as well as subjective responses such as odor perceived intensity
and pleasantness, were examined. Sixty participants including 30 males and 30 females (Mean=20.20, SD=1.80) took part in this
study. The odor used was anise seed oil, unfamiliar odor to the Japanese people. The odor was presented for 8 sec with 17-sec interstimulus interval (1 trial) through DIY olfactometer with a similar changeover mechanism as Kobal-olfactometer, and an experimental
session for one participant consisted of 25 trials. The participants were given positive, neutral, or negative information on the identical
odor prior to the experimental session. Dependent variables in this study were cardiovascular responses (systolic blood pressure: SBP;
diastolic blood pressure: DBP; cardiac output: CO; peripheral resistance: TPR; heart rate: HR) measured by Finometer, and subjective
variables as odor pleasantness, odor perceived intensity, and odor quality. As results, the effect of odor information was significant in
part of cardiovascular responses. To be precise, those who were given negative information on anise seed oil (the negative group)
perceived the odor more unpleasant as compared with participants given positive information (the positive group), and the systolic
blood pressure (SBP) and the diastolic blood pressure (DBP) of the negative group were also significantly higher than those of the
positive group. In addition, the effects of information on the subjective variables such as pleasantness were significant, which is
consistent with our previous findings. These results suggest that a preconception on the odor manipulated by the odor information
influenced participants’ autonomy system responses, perception, and cognition, resulting in significant effects on cardiovascular
responses as well as odor pleasantness.
65
11C - Does fetal and neonatal exposure to odour variety affect later response to novelty in mice?
Hébert B, Lara E, Schaal B, and Patris B
Centre Européen des Sciences du Goût, CNRS, Université de Bourgogne-Inra, Dijon, France
Vertebrate embryos and neonates are competent to encode odour information, and to later use it in guiding neonatal, juvenile, and,
sometimes, adult behaviour. These data generally derive from experiments in which single odorants were administered. Here, we
consider whether the perinatal exposure to multiple odorants administered sequentially via mother’s fluids (amniotic liquid, milk and
saliva) will affect later behaviour. Mouse perinates were exposed to contrasted chemosensory regimen through the mother’s ingestion
of odorized water from days E15 through P21. The effects of 3 treatments were considered: 1/ olfactory monotony (MON; females
accessing a benzaldehyde solution 5 days/week; n=46); 2/ olfactory variety (VAR; females accessing each other day solutions of
benzaldehyde, vaniline, lyral, limonene, cineol; n=45); 3/ Control (CON; females receiving non-odorized water; n=46). At weaning
(P21), all groups received a standard regimen. Treatment influence was assessed in measuring exploration behaviour in contexts
involving chemosensory or visual novelty. On P23, all mice underwent an open field test (2min), and then an episode of confrontation
with a novel object (3min) deposited in the centre of the open-field. On P27, half of each group underwent a 24-hr 2-bottle choice test
pairing water and an unfamiliar odorant (anethole), the other half undergoing a 5min paired-odour choice test, in a square open-field
divided in 4 areas: 2 neutrals and 2 odorized respectively with a familiar (benzaldehyde) and an unfamiliar (anethole) odorant.
Perinatal odour exposure influenced the behaviour of the mice at weaning. In the open field test, CON mice spent 1) significantly less
time than MON and VAR mice (which were not differentiable) in the central area before to introduce the object and 2) significantly
more time in the coins areas in the object’s presence. In the 2-bottle choice test, MON and VAR mice indistinctly drank significantly
more of the solution odorized with the unfamiliar odorant than CON mice. Finally, in the paired-odour choice test, VAR young mice
stayed significantly longer in the odorized areas than MON young mice, and spent more time closer to the unfamiliar odorant,
significantly during the second part of the test. In sum, this study confirmed that the perinatal manipulation of the odour environment
positively influences latter responses to new odorants. It further reveals that perinatal exposure to odour variability has contrasted
effects on pup responsiveness in contexts bearing chemosensory and visual novelty.
12C - Impact of pre-exposure on the perception of odorant mixtures in humans
Sinding C. (1,2), Coureaud G. (1), Bervialle B. (1,2), Schaal B. (1), Thomas-Danguin T. (2)
(1) Centre des Sciences du Goût, Equipe d’Ethologie et de Psychobiologie Sensorielle, UMR 5170 CNRS(/)UB(/)INRA, Dijon,
France; (2) FLAVIC, Equipe Perception de la Flaveur, UMR 1129 ENESAD(/)INRA(/)UB, Dijon, France
A mixture of odorants can be perceived as an addition of each odour component (elemental perception) or as a new odour (configural).
Recent data suggest that the configural perception of an odour mixture may be modulated by perceptual learning. Indeed, a preexposure to single odorants of a blending mixture (i.e. initially configurally perceived) entailed a decrease of the mixture specific
odour typicality (Le Berre et al., 2008). It has also been shown that pre-exposure to two odorants of a binary mixture increased their
perceived similarity (Stevenson et al., 2005). However, it was not known whether the exposure to a blending mixture can enhance the
configural processing strategy.
We investigated such effect of perceptual learning using a binary blending mixture of ethyl isobutyrate (EI: strawberry odour) and
ethyl maltol (EM: toffee odour). When mixed, these odorants generate the perception of a pineapple-like odour. The aim of our study
was to determine whether a pre-exposure to the EI+EM mixture promotes its configural perception (i.e., strengthens the perception of
the pineapple odour). Twenty participants (39,7 ± 14,8 years) have been pre-exposed, during 2 sessions spaced by a week, to EI+EM,
when 20 others to EI and EM separately. Two other groups constituted the controls (n = 40): one pre-exposed to a non-blending binary
mixture (initially elementally perceived) and the other to its single components. The day of testing, each participant performed a
typicality rating task with blending and non-blending mixtures and their components.
If we confirm the decrease of mixture pineapple-like typicality when participants have been pre-exposed to single components, we do
not show any effect when subjects have been pre-exposed to the blending mixture. Therefore, we suggest that the elemental perception
is favoured by perceptual learning of the blending mixture components, underlying that the mixture was rated as less typical of the
pineapple odour since the perception of strawberry and toffee odours was improved. This impact of pre-exposure to the components
did not affect perception of the non-blending mixture. We concluded that perceptual learning can affect mixtures’ perception
depending on whether the mixture is subjected to blend or not.
Le Berre, E., T. Thomas-Danguin, et al. (2008). "Perceptual Processing Strategy and Exposure Influence the Perception of Odor
Mixtures." Chem. Senses 33(2): 193-199. Stevenson, R. J. (2001). "Associative Learning and Odor Quality Perception: How Sniffing
an Odor Mixture Can Alter the Smell of Its Parts." Learning and Motivation 32(2): 154-177.
Supported by grants from the Burgundy region and FEDER, and by IFR 92.
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13C - Characterization of industrial and agricultural odors in field studies using semantic differential scaling
Sucker K*, Both R**, Müller F**, Hangartner M***, Winneke G****
*BGFA - Research Institute of Occupational Medicine, German Social Accident Insurance; **North Rhine-Westphalia State Agency
for Nature, Environment and Consumer Protection; ***UMTEC - Institute for Environmental and Process Engineering; ****Medical
Institute of Environmental Hygiene at Heinrich-Heine-University Düsseldorf
Several studies have confirmed exposure-annoyance associations for environmental odors from industrial and agricultural premises.
Furthermore, hedonic tone (pleasantness-unpleasantness) has been shown to have a strong impact on community annoyance and
therefore was recommended to be taken into account in the regulation of environmental odors. In order to establish a feasible and
objective manner to assess hedonic tone within field measurements, semantic differential scaling was used.
Using a list of 29 semantic differential scale adjectives subjects have to judge concrete plant odors as well as their “mental concepts”
of stench and fragrance without having any concrete odor on hand. In order to analyze profile ratings, each of the 29 ratings of every
subject is weighted (i.e. multiplied) with a given factor score. Then each of the 29 ratings are averaged above all subjects.
In two German field studies, trained observers rated six industrial (sweets, rusk, textile, seed-oil, fat, cast iron) and three livestock
odors (poultry, pig, cattle). Thus, 248 odor profiles and 186 profiles of the “concepts” stench and fragrance were collected. The
correlation between the weighted and averaged data of each odor profile with the profiles for the concepts stench and fragrance
showed that sweets and rusk odors can be considered pleasant, whereas fat, cast iron, poultry and pig odors are unpleasant, and textile,
seed-oil and cattle are neither clearly pleasant nor unpleasant.
In a Swiss study untrained subjects gave 109 profiles of the concepts stench and fragrance and 79 profiles of odors from waste water
treatment, wood gluing, barrel cleaning processes, and biscuit production using the same list of 29 semantic differential scale
adjectives. Correlations showed that only odors from biscuit production can be considered pleasant, all other others are unpleasant and
odors from wood gluing are neither clearly pleasant nor unpleasant.
Semantic differential (SD) scaling proved to be a viable method to classify hedonic tone of industrial and agricultural odors within
field measurements. Stable profiles for the concepts stench and fragrance can be achieved with at least eight subjects, irrespective of
age or gender. To establish stable odor profiles for real odors from industrial or agricultural premises at least 16 subjects are needed.
According to the German Guideline on Odor in Ambient Air (GOAA) plant odors can be classified as “clearly pleasant odor” if the
coefficients of the correlation between plant odor and the concept fragrance exceeds 0.5 and between plant odor and the concept
stench exceeds -0.5.
14C - ‘Health sniffers’: Ability, use and perceived importance of smell in a group of health professionals.
Amy N.B. Johnston*, Jana Fleischmann and Alan Mackay-Sim.
Clinical Neuroscience, Eskitis Institute of Cell and Molecular Therapies, Griffith University, Brisbane, QLD AUSTRALIA.
The ability to smell is an important component in the arsenal of healthcare professionals diagnostic tool kit. Patient assessment and
subsequent health investigation and treatment often depends on effective use of sensory systems in those most immediately
responsible for their care; most commonly nurses. Moreover, our previous research suggest that olfaction is one sensory system
which, in otherwise healthy people, appears not to decline with age [1]. Odours from breath, tissue and other clinical samples often
contribute to clinical diagnosis and the instigation of treatment regimens. Despite this, we have yet to find a published study that
examines smelling ability in any singificant professional group or in students who are training in a specific profession, irrespective of
how significant smelling is to that profession. Thus we explored the sense of smell in experienced nurses and student nurses. We
compared their ability to that of the normative Australian population [2]. This study applied the sniffin’ sticks test of olfactory ability
[3], coupled with simple questionnaire data, to a group of nursing students (n=45) and experienced nurses (n=30). These data were
compared to demographically similar Australian data in non-health professionals using the current Australian olfactory data base held
at GU. Nurses reported much greater attention to and importance of their sense of smell than either of the other groups. However,
there were no significant differences (p>0.05) between the actual or the perceived ability to smell by nursing students, nurses and the
normative Australian population for odour identification, odour discrimination or threshold for odour detection. This study is a
significant beginning to our understanding of the use and importance of olfaction in the work-place. It enables us to begin to build an
understanding of how attention to olfactory cues (often indicating disease) may develop during clinical experience and thus to
understand how we can best prepare students in healthcare and medical associated professions for their future ‘information-rich’
clinical olfactory environment.
[1] Mackay-Sim et al., Chemical Senses 2006, 23, 763-71.
[2] Mackay-Sim et al., Journal of Clinical Neuroscience 2004, 11, 874-879.[3] Hummel et al., Eur Arch Otorhinolaryngol, 2007, 264,
237-43.
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15C - Valence of odor mixtures
Olsson MJ (1), Brodin M (1), and Moeller P (2)
(1) Karolinska Institutet, Stockholm, Sweden; ( 2) University of Copenhagen, Copenhagen, Denmark
Studies on odor mixture quality indicate that the quality is well predicted by the relationship between intensities of unmixed
components. A few studies on valence, or hedonics, of mixtures suggest that valence is similarly related to intensities of unmixed
components. However, since the relation between valence and perceived intensity differs between pleasant and unpleasant odors, such
that pleasant odors have an inverted U-relationship to odor intensity and unpleasant odors have a negative correlation with odor
intensity, the valence of odor mixtures are harder to predict than the quality. Here, we investigate the relationship between valence of
mixtures and the valence and intensities of unmixed components using data from two types of mixture procedures. One set of data is
based on a substitutional mixture procedure for which odor intensities are held constant across mixture series. The odorants used, amyl
acetate, n-butanol, pyridine, and binary mixtures thereof, were presented in two equi-intense concentration series resulting in a total of
42 unique stimuli. The other set of data is based on an additive mixture procedure that allows mixtures to vary in odor intensity. The
unmixed stimuli were 4 concentrations of amyl acetate, butanol and pyridine, respectively, and blank presentations. All pair-wise
combinations of these concentrations were presented; in total 75 unique stimuli. Models for the prediction of mixture valence were
tested: (1) The Arithmetic Average Model state that the valence of mixtures is just the average of their components’ valence; (2) The
Intensity-Weighted Average Model use the relative intensity of components to weight the average; (3) the Squared-Intensity Weighted
Averaging Model is similar to the previous but gives much higher weight to the stronger component’s valence; (4) The “Sin Model”
(Haddas et al., 2008) yields similar predictions to that of the previous. Thus, models developed so far are different versions of
averaging models predicting that the valence of a mixture is intermediate to the valences of its components. These models do not take
into account the effect of intensity on valence, per se, that we expect to see when modeling mixture valence. In this work in progress
we will analyze the differences between empirical data and models with the aim to define the conceptual components needed for a
more complete model of how the valence of odors are integrated when mixed.
This work is funded by the Swedish Research Council
16C - Speed of processing olfactory information is reduced in old adults
Poncelet Johan (1), Rouby Catherine (1), Abriat Anne (2) and Bensafi Moustafa (1)
(1) Université de Lyon, CNRS UMR 5020 & (2) L’Oréal DPLI - L’Oréal Recherche, France.
Olfactory function is influenced by age at various levels, from sensitivity to odor identification and to hedonic perception of smells. A
central theory as regards cognitive information processing in the elderly is that aging process is associated with a decrease in the speed
of performing many cognitive operations. This reduction in speed may lead to impairments in cognitive functioning (Salthouse, 1996).
To test whether such theory holds for the olfactory system, we compared two groups of adults: older adults (mean age = 59.79 years
+/-2.99) and young adults (mean age = 21.85 years +/- 3.37). Testing was performed in an experimental room designed specifically for
olfactory experiments. Odorants were presented in 15 ml flasks (opening diameter: 1.7 cm; height: 5.8 cm; filled to 5 ml) and were
absorbed on a scentless polypropylene fabric (3x67 cm; 3 M, Valley, NE, USA) to optimize evaporation and air/oil partitioning.
Participants were asked to sniff the following 25 odorants with various degree of pleasantness: Acetophenone, Allyl Caproate, Amyl
PhenylAcetate, Benzyl Acetate, Carvone-l, 1-Decanol, Dodecanal, Diphenyl oxide, Ethyl Butyrate, Eugenol, Geraniol, Guaiacol,
Heptanal, Heptanol1, Hexanoic Acid, Hexanol3, Ionone-beta, Isoamyl Acetate, MethylAnthranilate, Phenyl Ethanol , Amyl Butyrate,
Valeric Acid, Trimethyl Amine, Santalol, Thioglycolic Acid. All subjects sniffed each vial in random order and were asked to decide
as quickly as possible whether the odor was pleasant or unpleasant. Hedonic judgments and response times were recorded for each
trial and each subject. After each odorant presentation, they were asked to rate again compound pleasantness as well as intensity on a
9-point scale (from 1: ‘‘not at all pleasant’’ or “not at all intense” to 9: ‘‘extremely pleasant’’ or “extremely intense”). Odorants were
presented every 45 sec.
Results revealed that older adults were significantly slower in responding to odor stimuli as compared to younger adults (old adults:
2083ms; young adults: 1799ms; p<.0001). As a general rule, slow processing is a sign of cognitive performance deterioration. One
hypothesis to explain the cognitive deficits for olfaction with aging therefore could be that the products of early (perceptual)
olfactory processing may be no longer available from memory when later (cognitive) processing is completed as a result of slow
processing. This would explain the decrease in odor identification or recognition in older people.
Salthouse (1996). The processing-speed theory of adult age differences in cognition. Psychological Review, 103, 403-428.
This research was granted by the CNRS and L'OREAL France.
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17C - Inheritance of pheromone neuroanatomy and physiology in European corn borer
Zsolt Kárpáti, Bill S. Hansson, Teun Dekker
1 Div. of Chem. Ecol., Swedish Univ. of Agric. Sci., PO Box 44, SE-230 53, Sweden
The European corn borer Ostrinia nubilalis (Lepidoptera: Pyralidae) is an excellent model to study the evolution of olfaction in insects.
In the nature two strains of the species occur. The strains are morphologically indistinguishable. Two pheromone strains produce and
respond to opposite ratios of the two pheromone components, Z11- and E11-tetradecenyl-acetate (Z11-14Ac, E11-14Ac). The Z-strain
uses a ratio of 97:3 of Z11-14Ac : E11-14Ac, whereas the E-strain uses a ratio of 1:99 Z:E. The two strains can hybridize in the
laboratory. F1 hybrid (ZxE and ExZ) females produce an intermediate pheromone blend (65:35 E:Z) and the males respond to blends
containing 3-65% E isomer. In nature mating between strains occurs at a low frequency in sympatry. The genetic factors, responsible
for the major differences between the strains, exhibit simple Mendelian inheritance. Female sex pheromone production and the
peripheral pheromone detection of the males are controlled by a single autosomal factor. On the other hand, the male behavioral
response to the pheromone is controlled by a sex-linked gene.
In our previous study we demonstrated that the pheromone preference is mediated by an antennal factor, which reverses the functional
topology in the first olfactory neuropil, the antennal lobe (AL). In males a specific region, the macroglomerular complex (MGC) exist
in the AL which responsible for the pheromone perception. The MGC of the hybrids consisted of two major compartments, a medial
compartment folded around a lateral one. The question thus is if the functional topology in the MGC we previously found, is inherited
by a sex-linked or autosomal factor. Extensive intracellular recording and immunocytochemistry were used to establish the structure
and function of the male MGCs and AL projection neurons. Physiologically characterized and stained neurons were reconstructed
using confocal microscopy of alfa-synapsin overview stained ALs. The intracellular staining of the projection neurons demonstrates
that EZ and ZE hybrids have an E-type functional topology. However, the volumetric measurement of the two MGC compartment
shows that the size of E and Z glomeruli is intermediate between the two strains. Volumetric measurement and pheromone specific
projection neuron stainings of paternal backcrosses follow a pattern predicted under sex-linkage. No differences were found in
frequency and types of projection neurons encountered between the parental strains, their hybrids, and backcrosses.
18C - Amygdalo-cortical interaction during odor fear acquisition session: Neurochemical and electrophysiological investigation
Hegoburu C (1), Sevelinges Y (1), Gervais R (1), Parrot S (2), Mouly AM (1)
(1) Neurosciences Sensorielles, Comportement, Cognition, UMR 5020, CNRS-Université Lyon 1, France (2) NeuroChem Dpt,
Université Lyon 1, France
Although amygdala seems to be essential to the formation and storage of fear memories, it might store only some aspects of the
aversive event and facilitate the storage of other more explicit aspects of memory in cortical areas. The question of the accurate
chronology of this assumed interaction has received little investigation. In a first experiment, using high temporal resolution (1min
sampling) intracerebral microdialysis, we investigated the temporal dynamics of glutamate and GABA variations in parallel in
amygdala (BLA) and posterior piriform cortex (pPCx) on freely moving rats during odor fear acquisition session. Long-Evans rats
received either six odor(CS)-shock(US) pairings (Odor-Shock group), six presentations of shock alone (Shock group) or six
presentations of odor alone (Odor group). In the Odor-Shock we observed a differential involvement of BLA and pPCx during the
course of successive pairings. Indeed odor-shock learning was accompanied by a transient increase in glutamate and GABA
concentrations in BLA following the first odor-shock pairing, after which amino acid concentrations returned to baseline levels or
showed a slight progressive decrease. In contrast in pPCx, transient increases occurred after most of the odor-shock pairings, and were
also observed after the stopping of the pairings, at the predicted time of occurrence of an additional trial. Furthermore, we observed
that for the first pairing, the increase in BLA occurred earlier than the increase in pPCx. These data suggest that the amygdala is
engaged during the early phase of acquisition and drives the activation of the olfactory cortex which then supports alone the late
phases of acquisition. We propose the projection pathway between amygdala and olfactory cortex could play a crucial role in
sustaining the observed dynamics. Further experiments are therefore currently carried out to study this pathway. In a current
experiment, we are investigating the consequences of an artificial increase of glutamate (consecutive to the blockade of glutamate
reuptake) in the amygdala, on the variations of glutamate and GABA in the olfactory cortex. In parallel, we are recording the field
potential signals induced in the olfactory cortex in response to electrical stimulation of the amygdala, and investigating whether this
pathway can be experimentally potentiated. These experiments should increase our understanding of the interactions between cortical
and limbic structures during odor fear conditioning.
Funding from Université Lyon 1 (BQR to RG) and Centre National de la Recherche Scientifique (CNRS)
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19C - How nasal airflow shapes odor representation at the olfactory bulb level
Courtiol E, Thevenet M, Messaoudi B, Garcia S and Buonviso N
Neurosciences sensorielles, Comportement, Cognition
Olfactory sense and respiration are intimately related: odorant molecules reach receptor cells in the olfactory epithelium in a periodic
way, with inspiration. A strong respiration-shaped activation has been evidenced by imaging methods in the glomerular layer (Spors et
al., 2006). Mitral cell activity is also paced by respiration (Buonviso et al., 2006). Such respiratory-modulated activation has been
observed even without any odorant stimulation (Adrian, 1942) so that a possible mechanical effect of air on receptor cells has been
hypothesized. It has been recently evidenced that olfactory receptor cells are sensitive to the intranasal air pressure (Grosmaitre et al.,
2007).
We wanted to know whether nasal airflow, by its mechanical stimulation, can influence olfactory bulb activity so that the
representation of an odor at the bulbar level would be different according to respiratory dynamics.
For this purpose, we used a double canulation protocol on anesthetized rats in order to make nasal airflow sampling independent from
animal respiration. Thanks to this technique, we were able to test different parameters of the nasal airflow as the frequency or the
strength of inhalation flow. Both extracellular unitary activity of mitral cells and local field potential (LFP) were recorded during
different conditions of respiratory dynamics.
Preliminary results show that mitral cell spontaneous- and odor-evoked activities varied with respiratory dynamics. Airflow tended to
synchronize mitral cell activity with respiratory cycle in 27% of cells. But such air sensitivity did not confer those cells any particular
odor response properties. LFP oscillations were also modified by inspiration strength: a low inhalation flow more often evoked beta
oscillations (15-35Hz) while a high inhalation flow induced more often gamma oscillations (35-90Hz). Amplitude, frequency and
duration of beta and gamma oscillatory episodes were modified according to inhalation flow. Moreover, spikes/LFP relationships were
modified by inspiration strength. Results concerning the effect of respiration frequency onto bulbar activity are in progress.
All these results show that intranasal air dynamics plays a critical role in shaping odor representation at the bulbar level. Since sniffing
behavior in the freely moving rodent has been shown to be highly dynamic and to vary with behavioral context (Wesson et al., 2008),
it is likely that representation of an odor at the bulbar level continuously changes according to the animal
20C - Neuromodulators in the antennal lobe of the heliothine moth
Berg BG, Zhao XC
Dept of Psych/Neurosci Unit, Norwegian University of Science and Tecnology, Norway
In insects, odor molecules are detected by a large number of olfactory sensory neurons located on the antennae. As in vertebrates, the
sensory axons project directly to the primary olfactory center of the brain. This first order synaptic neuropil, called the antennal lobe,
is characterized by an array of spherical structures termed glomeruli. Three categories of central interneurons make up these prominent
structures, namely local interneurons that reside within the antennal lobe, projection neurons that connect the antennal lobe to higher
integration centers in the protocerebrum, and centrifugal neurons that send axonal projections from other parts of the central nervous
system into the antennal lobe. One centrifugal neuron morphologically identified in several insect species is the so-called serotoninimmunoreactive (SI) antennal-lobe neuron, originally described in the sphinx moth Manduca sexta (Kent et al. 1987, J Neurobiol). We
have recently characterized, by intracellular recording and staining combined with immunocytochemistry, this particular neuron in the
central olfactory pathway of the male moth Helicoverpa assulta. The SI neuron that had branching profiles similar to those found in
the sphinx moth, displayed two distinctly different types of action potentials — one with a small amplitude showing increased
frequencies when odors were blown over the antenna and the other with a large, long-duration amplitude showing no observable
responses. The presence of two spike categories may be of particular significance for the modulatory capacity of this widespread
neuron.In addition to serotonin, a number of neuropeptides are assumed to act as modulators in the brain olfactory center of insects. In
the heliothine moth, one small population of antennal-lobe local interneurons immunoreactive against tachykinin showed a strikingly
different staining pattern from that of local neurons immunostained by three other neuropeptide antisera (anti-FMRFamide, antiallatotropin, and anti-allatostatin). Furthermore, double labeling experiments showed no co-localization between tachykinin and the
other neuropetides. A physiological role of tachykinin in the fly antennal lobe was recently reported, indicating that individuals
lacking this neuropeptide had reduced olfactory sensitivity in behavioral assays (Winther et al. 2006, Mol Cell Neurosci).
Interestingly, the somata of the tachykinin-immunoreactive neuron population in the heliothine moth, comprising less than ten, did not
contain GABA.
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21C - Relationships between intracellular activities, oscillatory local field potential and respiration in olfactory bulb of freely
breathing anesthetized rat
Briffaud V., Fourcaud-Trocmé N., Cenier T., Garcia S., Buonviso N. and Amat C.
université de lyon; université Claude Bernard lyon1; CNRS UMR5020 Neurosciences sensorielles, comportement cognition
In mammals, the global olfactory bulb (OB) activity is characterized by oscillatory activity in 3 frequency bands during odor
stimulation. Indeed, local field potential (LFP) recordings display a slow high amplitude oscillation (1- 5 Hz) related to respiration.
Coexisting with this slow modulation, two high frequency band rhythmic activities alternate during a respiratory cycle: a beta
oscillation (10 – 35 Hz) occurring particularly during exhalation and a gamma oscillation (40–80 Hz) appearing only at inhalation /
exhalation transition point. At the same time, a respiratory modulation is also present at two levels of cellular activity. First, a few
studies have shown that, during odor stimulation, an intracellular membrane potential slow oscillation appeared coupled to respiration.
Second, two main spiking patterns have been described as synchronized with the respiratory cycle: the excitatory synchronized pattern
(S+) which shows an increase of firing activity principally at inhalation / exhalation transition point and the inhibitory synchronized
pattern (S-) which displays a strong decrease at this same time of the respiratory cycle. Finally, in our team, Cenier and al. (2009)
recently showed a temporal relationship, on the one hand, between LFP's gamma oscillation and S+ pattern and, on the other hand
between LFP's beta oscillation and S- pattern, suggesting the existence of gamma- and beta-neuronal assemblies which alternatively
appear during the respiratory cycle. From all these data, we hypothesize that OB functioning could be supported by 2 different activity
states (one activated and one less activated) and that some signs of these different states could be found both at the network level and
at the intracellular levels. We supposed that these "activated state" and "less activated state" might occur during inhalation and
exhalation respectively. Our aim here is 1) to demonstrate that these 2 states are observable at intracellular and network levels, and 2)
to examine the relationships existing between network and intracellular activity during both states. For this purpose, we recorded
simultaneously intracellular activity and LFP in OB of freely breathing anesthetized rat during odor stimulation. First, we
characterized membrane potential slow oscillations and their relationship with respiratory cycle. We showed that this intracellular
slow oscillation, following animal respiration, can take a positive (up state) or negative (down state) form. Next, we examined the
different relationships existing between 1/ the two forms of membrane potential slow oscillation (up and down) and action potential
respiratory patterns (S+ and S-), and 2/ the 2 forms of membrane potential slow oscillations and gamma and beta LFP oscillations.
Finally, we wanted to characterize what happen, at global and intracellular levels, during the two different activity states related to the
respiratory phases.
22C - Reading Vital Information from a Pattern-Independent Olfactory Neural Code
Haddad R., and Sobel N.
Weizmann Institute
The structure of the olfactory system, where about 1000 different receptor types each responds to a subset of odor characteristics, has
dictated research focused on pattern coding. According to this framework each odor is uniquely represented by the specific pattern of
activity across a population of neurons it activated. Although the pattern of activated receptors has been shown to discriminate one
odorant from another, a pattern-dependent code for meaningful olfactory information has yet to be found. Furthermore, several
findings imply that the olfactory system uses more than pattern coding alone. For example, olfactory discrimination persisted despite
extensive anatomical and genetic lesions to the spatial pattern substrate. Olfactory discrimination also persisted despite
pharmacological inactivation of temporal pattern components. Finally, whereas both spatial and temporal odor-induced patterns
develop over several hundreds of milliseconds, olfactory discrimination can be made within 200 milliseconds or less. These results
imply that important olfactory information may be carried by global rather than pattern-dependent neural activity. To address this, we
analyzed a large set of previously published olfactory neural response data from different olfactory neurons of seven different species
ranging from flies to humans.
For all species tested, we found two pattern-independent neural axes that captured more than half of the variance in neural
activity. These same cross-species axes predicted both olfactory behavior and perception. The first axis was highly correlated with
the total sum of odor-induced neural activity, and reflected the animal behavior of approach or withdrawal. The second axis was
orthogonal to the first, and reflected odorant toxicity. In other words, using a simple pattern-independent neural code we could predict
two key olfactory decisions: whether to approach or withdraw, and then whether to eat or not.
We suggest that that neural code of olfaction, and possibly other neural systems, relies on multiple levels of coding: The first level we
uncovered here is a simple, fast, robust, and biophysically plausible set of axes that subserve fundamental behavioral decisions. This
level of processing may underlie several previously unexplained olfactory phenomena such as its aforementioned processing speed
and robustness to lesion. Higher levels of processing may rely on complex spatiotemporal pattern coding subserving more demanding
olfactory tasks.
Supported by ERC FP7 Ideas 200850
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23C - Beyond olfactory value to olfactory decision-making
E T Rolls, F Grabenhorst, G Deco
Oxford Centre for Computational Neuroscience, Oxford, England.
Decision-making about affective value may occur after the reward value of a stimulus is represented, and may involve different brain
areas to those involved in decision-making about the physical properties of stimuli, such as intensity. In an fMRI study, we delivered
two odors separated by a delay, with instructions on different trials to decide which odor was more pleasant, or more intense, or to rate
the pleasantness and intensity of the second odor without making a decision. The fMRI signals in the medial prefrontal cortex area 10,
and in regions to which it projects including the anterior cingulate cortex and insula, were higher when decisions were being made
compared to ratings, implicating these regions in decision-making. Decision-making about affective value was related to larger signals
in the dorsal part of medial area 10 and the agranular insula, whereas decisions about intensity were related to larger activations in the
dorsolateral prefrontal cortex, ventral premotor cortex, and anterior insula. For comparison, the mid-orbitofrontal cortex had
activations related not to decision-making, but to subjective pleasantness ratings, providing a continuous representation of affective
value. In contrast, areas such as medial area 10, and the anterior cingulate cortex, are implicated in reaching a decision in which a
binary outcome is produced. How the brain takes decisions and how decision confidence is computed are topics of great current
interest. To provide a fundamental basis for understanding decision-making and decision confidence, we describe and analyze a
neuronal spiking attractor-based model of decision-making that makes predictions about synaptic and neuronal activity, the fMRI
BOLD response, behavioral choice, and decision certainty and confidence. The spiking network model predicts probabilistic decisionmaking with faster and larger neuronal responses on easy versus difficult choices, and these in turn predict larger BOLD responses.
The certainty of the probabilistic choice is reflected in the firing rates of the decision-making neurons. In an fMRI study, we confirm
these predictions by showing that brain areas such as medial prefrontal cortex area 10 have graded responses as a function of the
easiness of olfactory choices. This provides a unifying approach for how spiking-related noise in the brain affects choice, certainty,
confidence, synaptic and neuronal activity, and fMRI signals.
Rolls,E.T., Grabenhorst,F. and Parris,B. (2009) Neural systems underlying decisions about affective
24C - Manganese-enhanced fMRI in olfaction: optimisation of Mn dose with minimal deleterious effects upon odour induced
behaviour in rats
Lehallier B, Ben Moussa A, Ben Hassen W, Coureaud G *, Rampin O #, Schaal B *, Maurin Y #, Bonny JM
UR370 QuaPA, INRA, F-63122 Saint Genès Champanelle, # CNRS-CESG, Dijon, * UMR 1197 NOPA, INRA, Université Paris Sud,
France.
In vivo functional magnetic resonance imaging is a method of choice to identify brain structures activated by odours. BOLD (blood
oxygenation level–dependent) contrast, used for most human studies, yields short lasting signals imposing the stimulus to be delivered
during magnetic resonance images acquisition. Hence, investigations on animal models required anaesthesia which depresses central
activity, altering neural responses to odour. The use of manganese, as a functionnal contrast agent, appears to be an adequate strategy.
Manganese ion enters neurons through voltage-gated calcium channels and can be transported anterogradely along axons and can
cross synapses. This method gives rise to a long lasting signal and allows uncoupling stimulation from magnetic resonance images
acquisition. Manganese is injected in nostrils, and odour stimulations are subsequently applied on awake animals. Animals are then
anesthetized and magnetic resonance imaging is performed a posteriori for long periods to improve signal-to-noise ratio and, hence,
spatial resolution. Yet, an important drawback of this technique is the toxic effect of manganese which may alter locomotor behaviour
and/or olfactory perception. The purpose of our study was to find the maximal manganese dose providing a good contrast in functional
magnetic resonance imaging studies while simultaneously preserving a normal behaviour in animals.
Five groups of three adult rats received increasing doses of manganese in both nostrils (0 µmol, 0.3 µmol, 1 µmol, 3 µmol, 8 µmol) 25
minutes before being tested for behavioural performances in an open-field. The open-field arena was equipped with a sniffing port
through which a familiar food odour was delivered for 5 minutes every 30 minutes. Animals were videorecorded during 120 minutes
in the arena. Latency, frequency and duration of locomotor activity and sniffing behaviour were quantified.
Our results show that, within the dose range studied, locomotor activity without odour stimulation was not affected by manganese,
while frequency and duration of odour-induced behaviours were dose-dependently decreased. These results allow determining the
optimal manganese dose sparing olfactory perception while simultaneously ensuring a good contrast in functional magnetic resonance
imaging studies. Toxic effects of manganese on olfactory ability have thus to be considered before functional interpretation of the
activation maps obtained by manganese enhanced magnetic resonance imaging.
72
25C - Event related potentials in response to odor mixtures and single components
Brodin M (1), Olsson MJ (1), and Hummel T (2)
(1) Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; (2) Smell and Taste Clinic, Department of
Otorhinolaryngology, University of Dresden Medical School, Dresden, Germany
When gradually changing an olfactory stimulus from odorant A to odorant B over mixtures of A and B, the perception of the quality
tend to gradually change accordingly with a maximal probability of reporting both A and B around the middle of this physical
continuum. Similarly, if we teach participants to recognize a mixture of A and B as odor X, and a mixture of B and C as odor Y, and
then morphing these odorants over a pure odorant B, we can investigate how the perceived quality changes. The results showed that
participants were as likely to report both A and B in response to a balanced mixture of A and B, as they were to report both ‘‘X’’ and
‘‘Y’’ in response to stimulus B. (Olsson et al in prep). This implies that at a conscious level we do not differentiate between single and
mixed odorants. However, in a PET study by Boyle et al (2009) they showed that the brain distinguished between single odorants and
binary mixtures. In this study we investigate the difference between these two types of mixtures using olfactometry and EEG. Can we
differentiate between the two types of mixtures suing event related potentials? We are currently testing approximately 24 participants
for each type of mixture. The odorants used in the experiment are eugenol (A), l- carvone (B) and citral (C
C26 - Newborn rabbits perceive odour mixtures elementally or configurally according to their experience
Coureaud G. (1), Sinding C. (1,2), Crepeaux G. (1,2), Schaal B. (1), Thomas-Danguin T. (2)
Centre des Sciences du Goût, Ethology and Sensory Psychobiology Group, UMR 5170 CNRS/UB/INRA, Dijon, France; (2)
FLAVIC, Equipe Perception de la Flaveur, UMR 1129 ENESAD/INRA/UB, Dijon, France
In their everyday environment, and as soon as the olfactory system becomes functional, organisms are exposed to complex odour
mixtures more than to single odorants. However, the questions of how and when animals, including humans, process odour mixtures
either as the addition of their elements, or as a whole independent of the odour of each constituent, have still largely to be elucidated.
Recently, we showed that after a one-trial (pheromonal) conditioning to an AB mixture, known to blend in humans, 2-day-old
newborn rabbits behaviourally respond 24h later to the mixture and to its components (elemental perception). However, after the
conditioning to one component, they do not respond to the mixture (configural perception). This suggests that the perceptual blending
of the AB mixture is incomplete, which led newborn rabbits to process AB both in an elemental and in a configural way (Coureaud et
al., Physiol Behav. 2008, J. Exp. Biol. in press). Here, we set out to examine the effect of experience on the neonatal perception of
odour mixture, and more particularly to investigate this putative effect on the AB mixture processing and perception of newborn
rabbits. To that goal, 58 pups were conditioned to odorant A either 1, 3 or 9 times between postnatal days 1 and 3, before to be tested
for their behavioural response to odorant A, odorant B and the AB mixture on day 4. The hypothesis was that the increasing preexposure to A could improve their ability to detect A in the mixture and thus induce their behavioural response to AB. As
hypothesised, the results showed that while rabbit pups did not respond to AB after a single conditioning to A (5%), 61.1% and 42.8%
of the pups responded to AB respectively after 3 and 9 previous conditionings to A. Conversely, 57 pups were conditioned to the AB
mixture either 1, 3 or 9 times, before to be tested for their response to the mixture and to its components. The hypothesis was that the
repeated pre-exposure to the mixture might promote the selective configural processing of AB, and moderate its elemental processing.
After 3 conditionings to AB, pups both responded to AB, A and B as they did after a single conditioning. In contrast, after 9
conditioning episodes, only 35.3% and 29.4% of pups responded respectively to A and B, while 94.1% still responded to AB. Taken
together, these results suggest that the neonatal perception of an odour mixture, spontaneously elemental, configural or both, may be in
part dependent of the previous experiences that newborns have had with the mixture or with its components.
Supported by grants from the Burgundy Region and FEDER, and by IFR 92.
European rabbit (Oryctolagus cuniculus)
73
C27 - HCN channels in the olfactory bulb
Aho AM (1), Fried HU (1), Müller F (2) and Kaupp UB (1)
(1) Center of Advanced European Studies and Research, Abteilung Molekulare Neurosensorik, Ludwig-Erhard-Allee 2, 53175 Bonn,
Germany ; (2) Forschungszentrum Jülich, Institut für Strukturbiologie und Biophysik, ISB-1, 52425 Jülich, Germany
In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain
regions. Oscillations are crucial for the processing of olfactory information in the bulbar networks. Oscillatory activity is found both
on the network level as well as in single neurons. In many neurons, the so-called hyperpolarisation-activated and cyclic nucleotidegated (HCN) channels generate and control oscillations of the membrane potential. These channels are best known for pacing the
beating of the heart; for this reason, they have been designated „pacemaker channels“. Four genes encoding HCN channel isoforms
have been cloned from mammals (HCN1-HCN4). HCN channels are tetramers composed of four HCN subunits. The functional
properties of homomeric channels comprising only one HCN isoform have been determined in detail by heterologous expression: For
example, the HCN isoforms differ in their activation kinetics, the voltage of half-maximal activation V1/2, and in the extent of cAMP
modulation. Although it has been shown that heteromeric HCN channels can assemble in heterologous expression systems, the
existence of heteromeric channels has only rarely been investigated in the native tissue. Heteromerisation could be a means to increase
the functional diversity of HCN channels, thereby endowing a cell with an individual, customized repertoire of channels.
Here, we describe the expression pattern of the four HCN isoforms in the olfactory bulb of mice using a novel and comprehensive set
of antibodies. We show that all four HCN isoforms are abundantly and differentially expressed in the olfactory bulb. HCN channels
can be detected in most juxtaglomerular cell populations identified by commonly used marker antibodies, implying that most cells in
the glomerular layer require HCN channel function. The combination of stainings with marker and HCN antibodies revealed at least
17 different populations of juxtaglomerular cells. Unexpectedly, the four HCN isoforms give rise to a wealth of co-expression
patterns, suggesting the occurrence of heteromeric HCN channels. Especially HCN3 does not occur by itself, but is always coexpressed with at least one of the other HCN isoforms, whereas HCN1, HCN2 and HCN4 do occur singly. Co-immunoprecipitation
experiments demonstrate that all four HCN isoforms can assemble into heteromeric channels in the olfactory bulb. Because the
properties of heteromeric HCN channels are still unknown, further studies are necessary to finally elucidate their physiological
function. Our results suggest that homomeric and heteromeric HCN channels play an important role in the processing of olfactory
information, possibly by controlling oscillations of the membrane potential.
1D – Social and hormonal status influence the emission of urinary molecules in the mouse
Bondi’ M (1), Da Dalt L (2), Redaelli M (1), Gabai G (2), Zagotto G (3), Cavaggioni A (1), Mucignat(1) Department of Human Anatomy and Physiology,
In male mice, several behavioural traits and physiological characteristics were in the past attributed to social and/or hormonal status.
Analysis of the literature showed that most of these reported associations were obtained in studies that compared male mice with
castrated mice, assuming the latter as a model for non-dominant, submissive mice. We aimed at obtaining a more precise correlation,
by analyzing in adult male mice (Mus musculus) their dominance behaviour, hormonal levels and quantity of molecules emitted with
urine, potentially involved in pheromonal communication. Mice were kept either isolated (n=6) or 6 per cage (n=18) since weaning.
At 2 months of age, the grouped mice were tested for dominance within each cage. The day after, all mice were put singly in a small
cage. Their first micturition was collected, the number of urine drops and fecal pellet released were counted. Immediately afterwards,
mice were sacrificed, their blood and urine residual volume collected and frozen. The internal organs were weighed. GC/MS and
GC/FID were performed on urine to identify and quantify volatile molecules. Major Urinary Protein (MUP) and creatinine were
quantified spectrophotometrically in urine. In plasma, testosterone, dehydroepiandrosterone and corticosterone were detected by
radioimmunoassay. Isolated mice voided a lower volume of urine, with a higher number of urine droplets. The spleen and adrenal
glands were smaller in isolated mice, that also had a larger thymus. No difference was present in the other organs, in the corticosterone
levels and in creatinine. Testosterone levels tended to be higher in isolated and were positively correlated with MUP, 2-sec-butyl-4,5dihydrothiazole and brevicomin excreted in urine. 2-sec-butyl-4,5-dihydrothiazole and 4-ethylphenol were significantly more in the
urine of isolated males. Isolated mice showed a peculiar micturition pattern, with a great number of small drops, possibly intended to
mark the territory, while grouped mice voided a larger volume in a single spot. While behavioural data on dominance were variably
related to hormonal levels, several molecules involved in pheromonal communication showed a positive correlation with testosterone
levels.
This work was supported by the University of Padova
74
2D - The importance of volatile and involatile urine components in stimulating female attraction to individual male mice
Cheetham SA, Beynon RJ and Hurst JL.
The University of Liverpool
Scent plays a critical role in mediating reproductive interactions and sexual attraction in many mammals. Here, we assess the
interaction between volatile (airborne) and involatile components of urine scents in female mate choice among wild house mice (Mus
musculus domesticus). Male mice deposit numerous urine scent marks around their territories, continually refreshing their scent and
countermarking any scent marks from competitor males. These urine scents contain both sex- and individual-specific volatile and
involatile components. Females are more attracted to urine from male over female mice, but this attraction occurs only after nasal
contact with involatile scent components. Further, female attraction is only to the specific individual male’s scent and does not
generalise to other genetically distinct wild males. On contact, females learn the individual airborne scent profile of that male’s urine
and are subsequently attracted to the airborne urinary odour alone. Urine contact also stimulates greater attraction to the individual
male himself compared to a male with unfamiliar scent. This is a specific response to male scents; no such attraction is shown towards
familiar female scents. The freshness of a male’s urine further influences his relative attractiveness, suggesting that urinary volatile
components are also important for sexual attraction. After contact with scents from two males deposited 24h apart, females are more
attracted to the owner of the freshest marks. Nonetheless, females are still more attracted to the owner of familiar 24h aged scents than
to unfamiliar males, whether they contact the aged scent on its own or when the aged scent has been countermarked by fresh scent
from another male. Thus, nasal contact with involatile components in male urine scent marks appears to be critical for stimulating
subsequent female attraction to the individual male and his scent. However, the freshness of the scent contacted also influences the
relative attractiveness of individual males. Both involatile and volatile components of male urine scent marks thus play important roles
in stimulating female attraction to individual male mice.
3D – Combinatorial and coordinated expression of the mouse Major Urinary Proteins (MUPs) may reflect a modular principle
of the olfactory coding
Novikov SN (1), Ermakova II (1), Fedorova EM (1), and Mylnikov SV (2).
(1) I.P. Pavlov Institute of Physiology, Russian Academy of Sciences; (2) Department of Genetics and Breeding, Saint Petersburg
State University, Saint Petersburg, Russia.
Major urinary proteins (MUPs) provide an individual olfactory recognition in Mus musculus L. (Hurst, 2009). They can bind volatile
pheromones and/or by themselves convey essential information about genotype, sex, social rank, reproductive state and age of the
donors (Cheetham et al., 2008; Logan et al., 2008; Mudge et al., 2008; Novikov et al., 2008). However, fine molecular mechanisms of
the MUPs’ participation in the formation of olfactory “fingerprints” remain to be elucidated. We performed detailed studies of MUPs’
profile by PAGE electrophoresis of urine samples from male and female mice of CBA/LacY and C57BL/6JY laboratory strains under
different physiological conditions. The results revealed strong positive correlation of MUPs’ expression patterns between juveniles
and adults of both sexes. Additionally, we showed that while patterns of MUPs expression in castrated males resemble those of
females, they rapidly returned to control ones after testosterone therapy (Novikov et al., 2009). The obtained data indicate that
genotype-specific «adult proportion» profile of different MUP fractions appreares very soon after weaning and resembles a «bar
code». Our results suggest that combinatorial and coordinated patterns of MUPs co-expression are similar in two common,
genealogically unrelated mice strains CBA/LacY and C57BL/6JY and that they could reflect a modular principle of the olfactory
coding in Mus musculus L.. Taking into account the data which demonstrated direct activation of the vomeronasal neurons by MUPs
(Chamero et al., 2007), and that the expression pattern of the pheromone receptors V2Rs is combinatorial and probably
complementary (Silvotti et al., 2007; He et al., 2008), our data provide valuable insights into fine molecular mechanisms of the
olfactory coding based on specific sets (modules) of MUPs combinations.
Supported by Russian Foundation for Basic Research (projects 02... and 04-04-63050 ).
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4D - Temporal changes in deposited urine scent marks
Platt JE, Armstrong SD, Cheetham SA, Robertson DHL, Hurst JL and Beynon RJ
The University of Liverpool
Mice purposely deposit urine, the main source of chemosignals in mice, around their environments as scent marks. Receiving mice can
utilise information from scent marks in subsequent social and mate choices. However, an indeterminate time can lapse between
deposition of a urine scent mark and assessment by a receiver animal, with potential for considerable change in the qualities of the
scent mark. First, evaporation will increase the effective concentration of involatile components. Secondly, volatile components will
be lost from the scent mark, accelerated by the drying process. Finally, interactions between urinary constituents may change the
qualities of the scent mark. There is a clear evolutionary advantage to the receiver to be able to assess the time since deposition. Yet,
to allow the receiver to identify the scent mark donor, some components of the scent mark must constitute an invariant ownership
signal. Thus, different molecular species within a urine scent mark must deliver complex temporal mapping of the signal. The major
urinary proteins (MUPs) account for over 99% of the protein content in mouse urine. MUPs provide an involatile, genetically encoded
signal of individuality, which conspecifics utilise for recognition of kin and to assess and drive maintenance of heterozygosity. As
such, MUPs should remain unchanged throughout the functional lifetime of the signal. However, mouse urine contains potent
endopeptidases that could modify the MUP-mediated signal. We have therefore investigated the ability of urinary endopeptidases or
exopeptidases to modify MUPs and show that MUPs are remarkably refractory to proteolytic degradation. By contrast, peptide ligands
of the Major Histocompatibility Complex (MHC) class 1 molecules are also proposed to serve as chemosignals of individuality within
mouse urine. Although such peptides are Vomeronasal Sensory Neuron (VSN) ligands, thought to mediate pregnancy block, their
presence in mouse urine is controversial. Indeed, as MHC class 1 peptide ligands may arise from invading microbes, it may be of
benefit to the scent owner to destroy such peptides, preventing advertisement of infection status to conspecifics. We show data
supporting the hypothesis that MHC peptide ligands are unable to persist within the proteolytic environment of mouse urine. MUPs
bind volatile semiochemicals and the multiphasic release of volatiles from dried urine is controlled by the proportions and the relative
ligand affinities of the complement of MUPs in the scent mark. The time domain of volatile signaling could be controlled by
manipulation of the MUP complement, however, volatile release kinetics from a range of wild mouse urine scent marks are not greatly
different, notwithstanding the difference in MUP profiles. This suggests that scent mark advertisement is manifest over a time frame
that is matched by territory visits by the scent owner.
5D – Neurogenesis in the AOB of adult mice: role of pheromonal perception on survival of newborn neurons
Oboti L(1), Schellino R(1), Fasolo A(1,2,3), Peretto P(1,2,3)
(1) Department of Animal and Human Biology, (2) Neuroscience Institute of Torino (NIT), (3) National Institute of NeuroscienceItaly (INN)
In the olfactory bulb (OB) of adult mammals inhibitory interneurons are continuously replaced by neuroblasts generated in the adult
subventricular-zone (SVZ). The survival of these newly generated cells highly depends on the olfactory input. This implies the
presence, in the OB, of relevant structural remodelling in response to sensory experience. As we previously reported, adult SVZderived neuroblasts directed to the OB also reach the accessory olfactory bulb (AOB). Their survival strictly depends on sensory
activity involving the vomeronasal system (VNS). To better characterize AOB neurogenesis modulation by pheromonal perception, as
well as the sensory cues/structures involved in this regulatory process, we have used different in-vivo approaches on female mice.
These included phenotypical characterization of AOB newborn cells, fate mapping of BrdU-labelled cells after exposure to malesoiled bedding at different survival times, analysis of the spatial distribution of BrdU-labelled cells in the AOB sub-regions and
analysis of c-fos expression in newborn cells after exposure to male-soiled bedding. Moreover, neurogenesis in the AOB of females
has been quantified after exposure to male-soiled bedding or to its isolated pheromonal components. Finally we have analyzed the
behavioural responses related to the perception of pheromones contained in male-soiled bedding. Here we show that a sensitive period
exists for the activity-dependent survival of AOB newborn neurons. In addition, exposure of females to male-soiled bedding enhances
long-term survival of interneurons in the AOB but not in other neurogenic regions such as the OB and the dentate gyrus of the
hippocampus. Importantly this effect is observed solely when direct access to the source of pheromones is allowed. After such sensory
experience, female mice learn to associate the unconditioned attractive properties of non-volatile pheromonal stimuli to the airborne
scents derived from the same individual. Thus survival of AOB newborn cells seems to be regulated by an experience-specific
mechanism related to the peculiar function of the VNS in the perception of pheromones.
Supported by Compagnia di San Paolo (Neurotransplant Project 2004.2019)
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6D - Dissecting the complexity of major urinary protein signals in rodents
Hurst JL, Cheetham SA, Davidson AJ, Armstrong SD, Garratt MG and Beynon RJ
Department of Veterinary Preclinical Science, University of Liverpool
A scent mark, deposited in the environment, is usually encountered in the absence of the donor. It follows that the scent mark must
encode several layers of information, including information on both the identity and status of the donor. The major urinary proteins
(MUPs) of mice and rats are encoded by a highly polymorphic multi-gene complex that provides persistent species, sex and
individual-specific genetic identity signals in urinary scent marks. The individual-specific expression pattern of MUPs is used by
house mice (Mus musculus) to identify individual scent owners and to recognize close kin. MUPs are particularly important in mate
choice, allowing females to identify individual male territory owners, to avoid inbreeding with close kin, and to assess the genetic
heterozygosity of potential mates. Phenotypic expression of MUPs depends not only on genotype but also on hormone levels, with
growth hormone, testosterone and thyroxine controlling the expression of different sets of MUP isoforms; different MUP classes also
show functional divergence. However, it is not yet known whether MUP signals provide information on physiological status as well as
identity, or how such complex information may be encoded. Here, we examine the effects of genetic background, development, social
experience and reproductive status on MUP expression in both wild house mice and laboratory strains. Individual and sex-specific
differences in MUP expression are more complex among wild mice than is apparent from laboratory strains. However, genetic
background has surprisingly little effect on MUP phenotype within sexes, confirming MUP pattern sharing as a simple marker of
kinship. A rapid increase in both MUP output and scent marking rate among males competing to defend territories enhances the
intensity of their individual competitive signals. Female output varies over the estrous cycle, but high variation in output between
females together with lack of variation in the relative expression of different isoforms suggest that this is unlikely to provide a useful
signal of oestrus state. However, more substantial changes evinced by age and reproduction suggest that MUPs provide other
information on female status. Qualitative MUP patterns thus provide persistent signals of genetic identity in scent marks, while more
subtle quantitative changes both in total MUP output and relative expression of different MUP isoforms has capacity to provide
additional information on current age, reproductive and social status.
Funded by BBSRC
7D - Temporal contrast of salt delivery in mouth increases salt perception
Johanneke L.H.C. Busch, Carole Tournier, Janine E. Knoop(*), Gonnie Kooyman and Gerrit Smit
Unilever R&D Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands. (*) current address TI Food and
Nutrition, located at NIZO food research B.V., P.O. Box 20, 6710 BA Ede, The Netherlands
The impact of salt delivery in mouth on salt perception was investigated. It was hypothesised that fast concentration changes in the
delivery to the receptor can reduce sensory adaptation, leading to an increased taste perception. Salt solutions were delivered via
sipping from cups (at low frequency) and via a gustometer method operating in continuous mode (at low and high frequency). High
frequency sampling could only be reached with the gustometer. Saltiness ratings were scored by a panel over time during various
stimulation conditions involving relative changes in NaCl concentration of 20 and 38 %. Changes in salt delivery profile had similar
effect on saltiness perception when delivered either by the sip-wise method or with the gustometer. The impact of concentration
variations at higher frequency was investigated with the gustometer method. Five second boosts and two second pulses were delivered
during three sequential 10 second intervals, while the delivered total salt content was the same for all conditions. Two second pulses
were found to increase saltiness perception, but only when the pulses were delivered during the first seconds of stimulation. Results
suggest that the frequency, timing and concentration differences of salt stimuli can affect saltiness.
77
8D - Correlation of In Vitro and In Vivo Activities of New Umami Tasting Neomenthyl Derivatives
Ley JP, Stähler F, Batram C, Backes M, Paetz S, Looft J, Meyerhof W, Krammer G
(*) Symrise GmbH & Co. KG, Flavor & Nutrition, Research & Innovation, Mühlenfeldstr. 1, 37603 Holzminden Germany; (§)
German Institute of Human Nutrition, Department of Molecular Genetics, Arthur-Scheunert-Allee 114-116, 14558 PotsdamRehbrücke, Germany
Mammals are able to perceive five different taste qualities, i.e., salty, sour, sweet, bitter and umami. The latter three qualities are based
on transduction mechanisms involving G protein-coupled receptors. In humans, bitter taste perception is mediated by ~ 25 members of
the taste 2 receptor (TAS2R) family. In marked contrast, sweet and umami taste is elicited by taste 1 receptors (TAS1Rs), which are G
protein-coupled receptors of class C. TAS1Rs assemble into heterodimers to form functional receptor molecules, with hTAS1R2hTASR3 being a sweet taste receptor and hTAS1R1-hTAS1R3 being a umami taste receptor (1).
For bitter and sweet tastes numerous agonists are known. The most important sweet tasting molecules are sugars, especially glucose,
fructose and sucrose which provide nutritional value to the organism. However, many non-nutritive strongly sweet tasting molecules
of different structural classes are known such as stevioside, saccharin, and aspartame. In contrast, only few organic acids or salts,
including monosodium glutamate were known as umami tastants until recently (2). As a hallmark, umami taste and activation of
TAS1R1-TAS1R3 can substantially be enhanced by the simultaneous presence of ribonucleotides, IMP, GMP, and AMP (1, 2).
Recent reports identified new, non-amino acid based strong agonists of TAS1R1-TAS1R3 that elicited umami taste in sensory
experiments (3, 4). During our sensory analysis of potential cooling compounds derived from the menthane skeleton in human subjects
we have identified a new class of very strong umami tasting compounds, the 2-isopropyl-5-methyl-bicyclo[4.1.0]heptan-7carboxamides (5) and the neomenthylamides of cyclopropanoic acid (6). In this report, we determined the sensory profile of a set of
these new umami molecules as well as for structural analogs which show no umami taste at all. In order to elucidate the molecular
basis of the umami taste of the active compounds we tested them and their inactive structural analogs in a heterologous expression
system based on the hTASR1-hTASR3 receptor dimer. Our experiments indicate for the investigated neomenthyl derivatives, a strong
correlation between the sensory quality umami and the agonist potency in the receptor assay.
References
(1) Chandrashekar, J., M. A. Hoon, et al. (2006) Nature 444(7117): 288-294
(2) Winkel, C., A. d. Klerk, et al. (2008) Chemistry & Biodiversity 5(6): 1195-1203
(3) Dewis, M.L. et al., US 2004 0,202,760, US 2007 134,389
(4) Tachdijan, C., et al., WO 2005 041,684; WO 2006 084,186
(5) Looft, J. et al., WO 2008 046,895
(6) Looft, J. et al., EP 1,989,944
9D - Gas chromatography/sniffing port analysis for screening of impact odorants in maple syrups
J. Fortin, H. Sabik and N. Martin
1 Food Research and Development Centre, Agriculture and Agri-Food Canada, St-Hyacinthe, Quebec J2S 8E3, Canada
The maple industry is exclusive to North America and is centred mainly in Canada. The value of maple syrup is assessed using a
grading system that is based essentially on light transmittance classes. This project was conducted to improve knowledge of the
composition of maple syrup, to identify the compounds responsible for the characteristic flavour of maple syrup, and to measure their
sensory impact.
Previous work led to consolidation into groups. Sensory analysis was performed using the Quantitative Descriptive Analysis method
(QDA®) on two years of data on maple syrup production in Canada. Based on the results of the cluster analysis, the syrups were
divided into four groups characterizing the flavour of maple syrup. Representative samples of these predominant types of maple
syrups were chosen for gas chromatography/sniffing port analysis.
A headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-flame ionization detector (FID) and
sniffing port (GC-O) was applied to identify aromatic active components of maple syrups. The acquisition time was adjusted to 16
minutes to counter the effects of subject fatigue. Fifteen subjects were selected to compensate for the considerable interindividual
variations observed in the collection of such data. They were encouraged to use their idiosyncratic way of sniffing. Due to the low
encoding capacity of human subjects, the task of participants was limited to indicating their perceptions. A four-point scale of intensity
was used for that purpose, and the participants were free to identify the odours collected.
Four aromagrams resulted from the olfactory analysis performed by 15 trained individuals on 12 samples representing the
predominant types of syrup. The aromagrams were distinctive for the number and intensity of active olfactory zones, as determined
using the nasal impact frequency method. Two of the four zones observed corresponded to furans and pyrazines, which were identified
by HS-SPME-GC/MS under the same separation conditions. Among the 204 compounds found by HS-SPME-GC/MS, 63 flavors
were identified as food flavourings in databases. The following chemical families were the most important: 8 aldehydes, 15 furan
derivatives, 10 ketones and 15 pyrazines.The type of compounds and their frequency of appearance in each type of syrup are known.
Additional work is currently being done to determine which of these components are active in maple syrup. A study of the four
targeted areas of elution will be performed by slowing the ramp temperature on the chromatographic column.
78
10D - Taste and relativity in handbooks of physiology
Sylvain Farge
CRTT (Centre de recherche en terminologie et traduction), University of Lyon,Lyon, France
Based on former conclusion about the difference in conceptualisation of taste in English, French and German, this study will deal
withe the question of the relevance of this finding in a concrete use of language: how is taste presented in handbooks of physiology in
German, French and English? Do the differences in the conception of taste perceivable in a semantic study still appear in specialised
texts or do German-, English- and French speaking specialists presend taste according to the linguistic and cultural structures they
retrieved from their mother tongue?
In fact, the lexicological analysis (by S.Farge and A. Depierre) showed that tast, as objective a sense as it may seem at first sight, is in
fact conceptualised differently in various languages. As a matter of fact, in English, taste is an experience, almost an experiment,
giving way to a broader knowledge of the tastant. In French, taste is a quality intrisic to the tastant, which is sort of picked upby a
taster as an undeniably existing quality of an object of the world. In German, taste is a mutual experience linking the taster and the
tastant, in which the taste does not exist in the mere tastant nor in the mere taster but rather at the intersection between both of them in
the perception.
The problem addressed in that frame is that of the influence language plays on the way people think. Is our perception of the world
necessarily intertwined with linguistic structures or are these factors partly or still totally independent from each other? Are the
semantic structures mentioned above, which are the result of a lexicological analysis, relevant to the way native speakers of English,
German or French perceive and experience taste in everyday life? In that case, to what extent could language still be a relevant factor
for the taste scientist to account for?
The issue is as essential for natural scientists as is the question of their relationship to their research object: can taste (or smell...) be
analysed as an objective and universal object or may the relativity in the conception of this object be questioned in order to situate the
scientific approach? In either case, this study pleads for a tighter collaboration between natural scientists and human scientists and is
part of a broader project aiming at supporting a real collaboration between the various domains of scientific inquiry concerned by
taste.
11D - Smell and taste of Perilla frutescens described by sensory and instrumental evaluation
Laureati M.(1), Pagliarini E. (1), Buratti S. (1), A. Bassoli (2)
(1) Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (Distam) Università degli Studi di Milano, (2) Dipartimento di
Scienze Molecolari Agroalimentari (Disma) Università degli Studi di Milano
Perilla frutescens (L.) Britt. (Lamiaceae), also known as “wild coleus”, “Chinese basil” and “Perilla mint”, is an annual short-day
plant widely used in therapeutics as well as in food preparations in Asian countries. It is classified into Perilla frutescens (L.) Britt.
Var. acuta Kudo forma viridis Makino (green Perilla) and Perilla frutescens (L.) Britt. Var. acuta Kudo (red Perilla). Green- and redleaved Perilla are also broadly classified as smooth and crisp varieties depending on margin leaf.
Perilla leaves and stems are reported to have anti-microbial, anti-HIV, anti-tumour and anti-allergic properties. It is traditionally used
in Chinese medicine to treat various diseases including depression, anxiety, cough and to promote intestinal propulsion. Aside its
pharmacological properties, Perilla leaves are strongly aromatic with a mint-like flavour and, therefore, suitable for food preparations.
Despite its interesting properties this plant is unknown in Western countries. In particular, the studies that systematically investigate
the properties of taste, smell and trigeminal sensations of such a plant are scanty.
The purpose of the present preliminary study was to describe the sensory characteristics of Perilla frutescens by means of sensory
evaluation involving a panel of trained assessors and by instrumental sensory devices such as the electronic nose and electronic
tongue. The relationship between the measures obtained by human senses and instrumental sensory analysis was also investigated.
Eight trained assessors evaluated by means of the Sensory profile method (UNI ISO 13299, 2003) the following three Perilla
frutescens cultivars: two varieties of Perilla frutescens (L.) Britt. var. crispa (one crisp red-leaved Perilla and one crisp green-leaved
Perilla), and one smooth green-leaved Korean variety of Perilla.
These preliminary data appear interesting to perform a rapid and reliable characterization of Perilla samples. Principal Component
Analysis evidenced that the three cultivars were well separated in the multidimensional space. The Korean variety of Perilla was
perceived as the least bitter and was associated to a high intensity of Cooling sensation. The two crisp varieties were also different
from a sensory point of view. The green-leaved Perilla was the most aromatic sample being described by high intensity of Grassy and
Floreal odor and, accordingly, by electronic nose sensors, while the red-leaved Perilla was perceived as the least astringent and
pungent.
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12D - Decrease in conditioned flavour preference after post-training CS-exposure seems to be due to conditioned inhibition
rather than to extinction: US devaluation effect failure and evidence of retardation
Felisa Gonzalez and David Garcia-Burgos
Department of Experimental Psychology and Physiology of Behavior, University of Granada, 18071 Granada, Spain
In a previous study, Garcia-Burgos et al. (2008) evaluated in several experiments whether the decrease in conditioned preference
observed in hungry rats after post-training CS-exposure was in fact due to extinction. The training for the experimental group
consisted in giving thirsty rats a compound solution made of an odour, 1% (v/v) almond, and 20% (w/v) sucrose mixed in tap water
during four conditioning sessions (AB+). After assessing the level of conditioned preference using a two-bottle test (odour vs. water),
rats were food deprived and the odour was presented outside the compound several times (A-). A decrease in conditioned preference
for almond was then observed in the experimental group. This general pattern was found in each of the experiments aimed to check for
several extinction-related phenomena: Spontaneous recovery, reinstatement, renewal—using motivational states as contexts—and
rapid reacquisition. They found neither of these usually robust phenomena and concluded that other process different from extinction
was responsible for the decrease in preference: Conditioned inhibition. The reason to propose conditioned inhibition as the cause of
the decrease was that rats experienced the odour outside the compound for the first time when they were food deprived, so it could
become a signal of calories-intake reduction. The experiments presented here provide further support for this hypothesis. In
Experiment 1 we found the US devaluation effect—the decrease in conditioned preference after the devaluation of the taste of sucrose
by pairing it with i.p. LiCl injections—only if the CS has not been further exposed after training. The effect disappeared if the CS was
presented repeatedly alone after training. The US devaluation effect is found after extinction, both in classical and instrumental
conditioning, showing in the former case that the original CS-US association is intact. However, it would be hardly found after
conditioned inhibition, as the CS does not recover the representation of the US. In Experiment 2, we used the retardation test to assess
the inhibitory properties of the CS after post-training exposure. Reacquisition of the conditioned preference was retarded in the
experimental group compared to the control group. Taken together these results provide compelling evidence against the interpretation
of the decrease in preference under these conditions in terms of extinction, and suggest that during post-training CS-exposure
inhibitory CS-US learning occurs.
Garcia-Burgos et al. (2008). Failure in restoring an “extinguished” conditioned flavour preference based on calories. Paper presented
at the XX Congress of the Spanish Society for Comparative Psychology. Bilbao, Spain (September).
Grants #SEJ2006-13851 (MEC, Spain) and #HUM-02763 (J. Andalucía, Spain). FEDER funds.
13D - Odour-Taste interactions: Sensory modulation or perceptual integration.
Godinot N., Pelletier C., Labbe D., Martin N.
Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland
Flavours are considered as an integration of simultaneous perceptions including taste and odour. It has been demonstrated that taste
can be enhanced by odour, and such enhancement could be correlated with increased activations in integrative brain areas. Although
the cognitive origin of such effects is now generally admitted, the primary sensory areas could be involved through top-down
mechanisms, with recruitment not only during taste detection but also via selective attention to taste(1), attention that could be
triggered by specific aromas. This suggests that odour-taste interactions are supported by sensory enhancement through top-down
facilitation mechanisms such as in audio-visual and visio-tactile interaction.
The most reported odour–taste interaction concern sweetness (2,3), and despite a strong health related drive, only two articles report
similar interactions for saltiness (4,5). Studies on odour–taste interaction were conducted using a classical sequential monadic design,
in which subject have to rate the magnitude of perceptions evoked by a given stimulus in comparison with remembered perception
retrieved from long term memory (everyday life, preceding stimuli or set of references). In these cases the remembered magnitudes
may be impacted by context and differ from actual sensory input. We assessed for the first time odour induced taste modulations using
a direct comparative method, which allow comparison within short time interval of magnitude of perceptions, relying therefore on
short term memory, close to the sensory memory and supporting contrast detection. Using a panel of 12 subjects we measured
magnitudes of perceived saltiness, sweetness, and in mouth aroma from a set of stimulus either using a comparative or a sequential
monadic profiling design. Stimulus comprised pure salty (NaCl) or sweet (sucrose) solutions in water with or without aroma (Bacon or
Strawberry). No odour induced modulation of saltiness is evidenced with either method. However, we show that the odour induced
sweetness enhancement found using a sequential monadic design is lost when allowing subjects to directly compare stimulus. Based
on our results, we are bringing forward the hypothesis that the impact of aroma on taste is the due to integration of both sensations in
high level processes, and reflected in their long term storage, but are probably not involving a direct facilitation in primary sensory
area by top-down mechanisms.
1/Veldhuizen MG et al (2007) Chem. Senses, 32:569-581.
2/Stevenson RJ et al. (1999) Chem. Senses, 24: 627-635.
3/Labbe D et al. (2007) Chem. Senses, 32:205-214.
4/Djordjevic J et al. (2004). Exp. Brain Res., 159:405-408.
5/Lawrence G et al. (2009) J. Food Qual. Pref., 20:241-248.
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14D - Between Ions Discrimination Capacities Of The Gustatory System: The Tastes Of Drinking Water
Puget S(1),(2), Cure Y(1), Béno N(1), Piriou P(3), Guichard E(1) and Thomas-Danguin T(1)
(1) INRA, ENESAD, Université de Bourgogne, UMR 1129 FLAVIC, Dijon, France; (2) Lyonnaise des Eaux, Paris, France; (3)
CIRSEE, Suez Environnement, Le Pecq, France
It has been established for a long time that cations can be detected by the gustatory system. If the role of anions remains poorly
understood, water by itself had been shown to be a taste stimulus (Zotterman, 1956) through hypo-osmotic sensing (Gilbertson, 2002).
Drinking water constitutes the complex mixture of ions dissolved in water and its ionic composition depends on its hydrogeographical origin. Therefore, it is likely that drinking water have a variety of tastes. Indeed, recent investigations demonstrated that
water taste depends on the quantity of dissolved minerals namely water osmolarity (Teillet et al., 2007). However, it is not known
whether variations in ionic content, beyond water osmolarity, could impact on water taste.
In the present study we tested the hypothesis stating that the taste system has the capability to discriminate between various ions ratios
dissolved in water. In a first experiment, 75 untrained subjects had to discriminate water solutions following a triangular testing
procedure. Twenty four water samples were designed to cover 8 ionic patterns at 3 osmolarity levels reconcilables with drinking water
(2, 6 and 10 mmol.l-1). The statistical analysis of discrimination proportions revealed that subjects could discriminate between some
but not all ionic patterns. Moreover discrimination ability was also found to increase with increasing osmolarity. In a second
experiment, bottled mineral water varying in their mineral composition and total dissolved solids contents were compared through
triangular tests performed by 115 untrained subjects. The results showed that the capacity of gustatory system to discriminate between
solutions was based on ionic pattern variations rather than osmolarity differences.
When taken together, the results of both experiments indicated that the gustatory system has the ability to discriminate between water
samples including various ratios of ions. Variations of Na+ and Ca2+ content were especially well discriminated. Moreover, the taste
system was found to be sensitive to osmolarity differences depending on the ionic composition. In addition to the dissolved ions
discrimination ability of the gustatory system evidenced by these results, our findings could also be of interest for water suppliers.
Understanding water ionic perception may help either the water tastes diversity exploitation or the drinking water formulation
following desalinization for example.
Teillet E, Urbano C, Cordelle S, Schlich P. (2007) 7th Pangborn Sensory Science Symposium.
Zotterman, Y. (1956) Acta Physiol. Scand. 37:60-70.
Gilbertson, T A. (2002) Chem. Senses. 27:383-94.
We thank Lyonnaise des Eaux, ANRT (CIFRE n°372/2006) and Burgundy region for financial supports.
15D - Food habit and taste perception: a North and South Vietnam comparative study.
Patris B, Do Vihn B, Nguyen D*, and Valentin D
Dijon-Dresden European laboratory on Taste and Smell, LEA CNRS-TUD 549; Centre Européen des Sciences du Goût, UMR 5170
CNRS-UB-INRA, Dijon, France. ^* HoChiMinh-City University of Technology, HoChiMinh,Vietnam.
We focussed here on cultural habits and taste perception. In particular, we studied the impact of food habits on taste perception,
identification, and appreciation. Previous work suggested that human populations leaving in extreme condition, poor in basic
nutriments present a higher sensitivity for sweet and nutritive molecules. We are interested in evaluating whether such an influence of
environment on gustatory sensitivity can be generalized to other situations. In this study we have compared sensitivity, identification
and hedonic appreciation to saccharine, sodium chloride, quinine chlorhydrate and tartaric acid in a North (Hanoi) and a South (Ho
Chi Minh) Vietnamese population. A survey carried out by the Centre for International Economics indicates that North Vietnamese
population consumes less sugar and sweet food products than South Vietnamese population. This difference in food habit might have
an impact on gustatory sensitivity and food preferences. Seventy North and seventy South Vietnamese participants were invited to
taste twelve taste strips impregnated with three increasing solution of saccharine, sodium chloride, quinine chlorhydrate and tartaric
acid. For each taste strips participants had to evaluate 1) whether they perceived something, and if they did, 2) to indicate the
perceived intensity, 3) to give their hedonic appreciation and 4) to identify the taste sensation. Results on saccharine revealed that
North Vietnamese participants tended to perceived a sensation with the lowest concentrations more often than South Vietnamese
participants. Concerning the salty taste strips, North Vietnamese participants perceived the two higher concentrations as significantly
less intense than South Vietnamese participants and tended to give a higher hedonic score to the intermediate one. Concerning the
bitter taste strips, North Vietnamese participants gave a significantly higher hedonic score for the two lower concentrations than South
Vietnamese participants and tended to prefer also the higher one, without clear trend on perceived intensity. Finally, it appeared that
North Vietnamese participants tended to prefer the lower and the higher concentrated sour taste strips than South Vietnamese
participants. These results confirm that participants coming from the same country but with specific food habits could differ on their
taste perception. It appears also that the difference on sweet food consumption seems not correlated with the sugar perception but
perhaps more with the bitter sensation rejection.
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16D - Infant preferences for food odours over the first 2 years of life.
Monnery-Patris S (1), Rigal N (2), Marlier L (3) Schaal B (4) and Issanchou (1)
(1) INRA, UMR 1129 FLAVIC, Dijon, France; (2) Université Paris 10, Département de Psychologie, Nanterre, France; (3)
Laboratoire d'Imagerie et de Neurociences Cognitives, CNRS-UDS UMR7191, Strasbourg, France; (4) Centre Européen des Sciences
du Goût, CNRS UMR 5170, Dijon, France.
The OPALINE study aims to determine the influence of chemosensory experience on infants’ food likes and dislikes over the first 2
years of life. One specific aim of this study is to assess infants’ hedonic reactions to food odours. Here, we report data on responses to
pleasant/unpleasant food odours in 7, 12 and 22 month-old infants. We also examine the link between infants’ responses towards a set
of odours and their actual food behaviour (as reported by mothers). Infants were videotaped when they were about 7, 12 and 22
months in age-appropriate tasks. They were given 2 series of 6 differently scented or unscented objects to explore. The stimuli were
chosen to represent a priori liked and disliked odour qualities. They included 4 odours of often-rejected foods (e.g. green pepper), 4
odours of often-liked foods (e.g. strawberry), and 4 blanks. Four orders of stimulus presentation were used, but for a given infant, the
order was held constant across age. For each stimulus, the videotapes were coded to determine the time of mouthing interpreted as
reflecting infants’ interest for the stimuli. Acceptance of each odour was defined as the mouthing time for this odour relative to the
sum of mouthing time for this odour and for the blanks. In addition, infants’ usual reactions to food at 22 months old were evaluated
through a questionnaire completed by the mother. The results indicate gender-independent individual variation in the acceptance of the
food odours. For each age group, a main effect of odour quality, and an odour quality by presentation order interaction, came out. On
average, a priori pleasant food odours were more accepted than a priori unpleasant odours in 7 and 22 months. Further, unpleasant
food odours were more rejected when presented at the end than at the beginning of the test (in all age groups). Correlation analyses
revealed that hedonic responses are stable only for the unpleasant odours between 12 and 22 months. Finally, positive correlations
were noted between infants’ responses to unpleasant food odours and neophobia scores as reported by mothers. This study provides
new data on infants’ hedonic response to a set of food odours and it corroborates the stable rejection of certain odours that are a priori
unpleasant (e.g. butyric acid). However, it also shows that infants are sensitive to order effects in their hedonic responses to odours,
indicating that response profiles need to be discussed in relative terms. Finally, this study provides some insights about the relationship
between hedonic responses to food odours and food behaviours in young children.
17D - The influence of culture on the structure of odour-elicited emotions
Ferdenzi C (1), Grandjean D (1,5), Roberts SC (2), Schirmer A (3), Delplanque S (1), Chrea C (1), Cayeux I (4), Margot C (4),
Velazco MI (4), Sander D (1,5), Scherer KR (1,5)
(1)Swiss Center for Affective Sciences, University of Geneva, Switzerland; (2)School of Biological Sciences, University of Liverpool,
U.K.; (3)Department of Psychology, Faculty of Arts and Social Sciences, National University of Singapore; (4)Firmenich, SA,
Geneva, Switzerland; (5)Department of Psychology, FPSE, University of Geneva, Switzerland.
Based on a large range of terms used in the literature on emotion and using a wide variety of odorants, a verbal scale has been
developed with 529 Swiss participants to investigate the semantic representation of odour-elicited emotions: the Geneva Emotion and
Odour Scale (GEOS; Chrea et al. 2009 Chemical Senses 34: 49–62). Factor analyses revealed that the structure of the GEOS scale
differed from classical taxonomies of emotion such as posited by discrete (basic emotions) or bi/tri-dimensional emotion theories
(valence, arousal, dominance). Indeed, the subjective affective feelings induced by odours were structured in six dimensions related to
well-being, sensuality, disgust, energy, relaxation and sensory pleasure. A comparison between GEOS and the basic emotions and
dimensional models, based on the ratings of eight odorants by 111 participants, showed that GEOS (1) was the most relevant scale for
the olfactory domain, i.e. received the highest mean ratings, (2) was characterized by higher inter-subject agreement, i.e. higher intraclass Correlation Coefficient, and (3) had a better ability to discriminate between varied odorants (cluster analysis). However, the
structure of verbal labels used to describe odour-elicited emotions is likely to vary according to culture, possibly because of
differences in verbalization of emotion or olfactory practices. Therefore, the development of the GEOS scale was replicated in another
European country, England, and in an Asian country, Singapore. The same 480 emotion terms used in Switzerland were translated in
English and were rated for their relevance to odours by 148 and 105 participants in Liverpool and Singapore respectively. The eighty
most pertinent terms in each country were then selected and presented to a second group of participants (N = 38 in Liverpool and N =
41 in Singapore) who were asked to smell a series of 24 odorants and rate the extent to which the selected feelings were triggered by
these odours. Exploratory factor analyses provided a first structure of odour-elicited emotions in each country. Several dimensions
overlapped between the three countries, especially the dimension of disgust and the dimension of sensuality / desire. However,
specificities emerged in each country, such as odour-elicited feelings related to spirituality in Singapore. After retaining a smaller set
of relevant affective terms, a final study using larger samples of odorants and participants aimed to validate these preliminary models.
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18D - The Identification and Discrimination of umami taste in German and Norwegian population
Singh B(1,2), Schuster B (1), Seo H(1)
1-Institute of Oral Biology, Faculty of Dentistry, University of Oslo, 2- Smell and taste clinic, Otorhinolaryngology, University of
Dresden medical school, Dresden
Several G-protein coupled receptors have been proposed as taste receptors for glutamate but the role of these receptors in umami taste
transduction is still controversial. The presence of multiple receptors for umami signalling might result into inter-individual
differences in the healthy population.In this study we aim to investigate the inter-individual differences for sensivity to monosodium
glutamate in German and Norwegian population. Further, we compared the distribution of tasters, hypotasters and non-tasters in the
two populations. This study was performed in 201 healthy German subjects and 150 healthy Norwegian subjects. A series of three
successive psychophysical tests was performed on the subjects. Based on the test results, subjects were divided into three groups: nontasters who were unable to perceive monosodium glutamate, hypotasters who perceived monosodium glutamate at relatively high
concentrations and tasters who perceived monosodium glutamate at relatively low concentrations. This study revealed 4.0% of the
German participants to be non-tasters while in the Norwegian population 4.7% of participants were non-tasters. The number of
hypotasters was higher in the norwegian population than in the German population; 12% of the participants in the Norwegian
population and 3.5% of the participants in the German population showed hypotaster status. No significant difference was found with
respect to age and sex in the non-taster group in either of the culturtes. One of the possible reasons behind the inter-individual
differences for sensivity to monosodium glutamate in the healthy subjects might be the multi-receptor hypothesis which suggests
several receptor sites involved in umami taste transduction.
19D - Olfactory dysfunction in allergic fungal sinusitis
Philpott CM*; Lai L*; Zheng G*; Homayoon B*; Clark A**; Javer AR*
(*)St Paul’s Sinus Centre, St Paul’s Hospital, Vancouver, British Columbia, Canada; (**)School of Medicine, Health Policy and
Practice, University of East Anglia, Norwich, UK.
Background: Allergic fungal rhinosinusitis is a chronic disease which requires sustained medical therapy following endoscopic sinus
surgery. However patients appear to complain of olfactory dysfunction in spite of apparent control of their disease based on
endoscopic assessment of their sinus cavities. This persistent complaint is perceived as a significant detriment to their quality of life.
Objective: This study aims to correlate subjective reporting of olfactory function with endoscopic staging and performance on the
Sniffin’ Sticks test in patients with allergic fungal rhinosinusitis. It also aims to assess the impact of this disease on their quality of life.
Methods: 60 patients with allergic fungal sinusitis seen in a tertiary rhinology clinic were recruited to undergo olfactory testing
following routine endoscopic follow up1. Patients were included if they met the modified diagnostic criteria for allergic fungal
rhinosinusitis (immunocompetence, polyps, eosinophilic mucin, CT findings and positive fungal stain)2. The Sniffin’ Sticks3 test was
used to derive their TDI score and a visual analogue score was used for their perceived olfactory ability. Patients were also asked to
complete a short form 36 questionnaire for quality of life scores.
Results: 31 male and 28 female patients with allergic fungal rhinosinusitis underwent olfactory testing over the course of 2 months; 50
of these completed all parts of the assessment. The age range of the patients was 25 to 71 (mean 52). The mean TDI score was 17
showing a poor level of function in this group. There was a significant correlation between patients’ performance on the Sniffin’
Sticks and endoscopic staging and with their reported olfactory ability (p<0.001). There was also a positive correlation between TDI
score and SF36 scores (p<0.01).
Conclusion: All patients with significant sinonasal inflammatory disease should receive evaluation with olfactory testing and be
treated on their merit in order to lessen the impact on their quality of life.
1. Philpott C, Javer A. A novel endoscopic staging system for allergic fungal sinusitis. Rhinology World. Philadelphia, USA 2009.
2. Bent JP, 3rd, Kuhn FA. Diagnosis of allergic fungal sinusitis. Otolaryngol Head Neck Surg. 1994 Nov;111(5):580-8.
3. Hummel T, Sekinger B, Wolf SR, Pauli E, Kobal G. 'Sniffin' sticks': olfactory performance assessed by the combined testing of
odor identification, odor discrimination and olfactory threshold. Chem
Senses. 1997;22(1):39-52.
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