QUALITY OF LIFE AND MANAGEMENT OF LIVING
RESOURCES PROGRAMME (1998-2002)
FINAL REPORT
Contract number
: QLG3-CT-2001-02004
Project acronym
: DECG
QoL action line
: 1.1.1-9.1 Neuroscience
(State to which key action, generic activity etc the project belongs)
Reporting period for the last progress report:
01/12/03-30/11/04
Reporting period for the final report:
01/12/01-30/11/04)
SECTION I: PROJECT IDENTIFICATION
Contract number:QLG3-CT-2001-02004
(include reference to complementary contracts–e.g. fellowships, INCO)
Title of the project: Dynamics of Extracellular Glutamate
(as in the contract)
Acronym of the project: DECG
(as in the contract)
Type of contract: RTD project
(E.g. RTD project, demonstration project…)
QoL action line:1.1.1.-9.1. Neuroscience
(state to which key action, generic activity etc this contract belongs)
Commencement date: 01.12.01
(DD/MM/YY: normally the first day of the month following the day of the signature of the last contracting party, unless otherwise
stated in the contract)
Duration: 36 months
(in months)
Total project costs: 2,211,724 (two million two hundred and eleven thousand seven
hundred and twenty-four euros)
(in Euro)
EU contribution: 1,640,867 (one million six hundred and forty thousand eight
hundred and sixtyseven euros)
(in Euro)
Project co-ordinator:
 Name (including title): Professor Jon Storm-Mathisen (Department Chair)
 Organisation: Department of Anatomy, IMB, and the Centre for Molecular Biology and
Neuroscience (CMBN), University of Oslo
 Street address: Room 1296, Domus medica, Sognsvannsv 9, 0372 Oslo
 Postal address: P.O. Box 1105 Blindern, N-0317 Oslo, NORWAY
 Telephone: +47 22 85 12 58, + 47 97 19 30 44 (mobile)
 Telefax: +47 22 85 12 78
e-mail: jon.storm-mathisen@medisin.uio.no
website: http://www.cmbn.no/group-storm-mathisen.html
Keywords: glutamate glia neuron
( list up to five keywords that best describe the project)
World wide web address: http://folk.uio.no/jonsm/decg.htm
(Internet address where updated information on the project can be obtained)
Updated list of participants:
(provide same details as for the co-ordinator)
Professor David Attwell
Dept. Physiology, University College London
Gower St. London, WC1E 6BT, UK
Tel: +44 20 7679 7342
Fax: +44 20 7413 8395
e-mail: d.attwell@ucl.ac.uk
Professor Dimitri M. Kullmann
Dept. Clinical and Experimental Epilepsy, Institute of Neurology, UCL
Queen Square, London WC1N 3BG, UK
Tel: +44 20 7837 3611 x 4100
Fax: +44 20 7278 5616
e-mail: d.kullmann@ion.ucl.ac.uk
Professor Andrea Volterra
Institut de Biologie Cellulaire et de Morphologie (IBCM)
Faculté de Medecine, Université de Lausanne
Rue du Bugnon 9, 1005 Lausanne, Switzerland
Tel: +41 21 6925271
Fax: +41 21 6925105 (or 6925255)
e-mail: Andrea.Volterra@unil.ch
Dr. Fabio Grohovaz
Cellular Neurophysiology Unit
2
San Raffael Scientific Instituet Dibit
Via Olgettina 58, Milano, ITALY
Tel: +39 02 2643 4811
Fax: +39 02 2643 4813
e-mail: grohovaz.fabio@hsr.it
Dr. Robert Zorec
Celica, Biomedical Sciences Center
Stegne 21
SI-1000 Ljubljana
Slovenia
Tel: +386 51 31 061
Fax: +386 1 51 123 15
e-mail: robert.zorec@pafi.mf.uni-lj.si
3
SECTION II: PROJECT FINAL REPORT
Table of contents:
1. OVERVIEW OF PROGRESS DURING THE LIFETIME OF THE PROJECT
-
Summary of the main objectives as stated in the contract Technical Annex
-
Overview of the scientific achievements of the project as a whole – significant
scientific achievements and their potential social and economic impact.
-
Update of tables – progress achieved against the planning
2. EXPLOITATION AND DISSEMINATION ACTIVITIES
-
Results achieved towards exploitation
-
Major dissemination activities performed during the period
3. ETHICAL ASPECTS AND SAFETY PROVISIONS
4. CONCLUSIONS
5. ACKNOWLEDGEMENTS
4
1. OVERVIEW OF PROGRESS DURING THE LIFETIME OF THE PROJECT
Main objectives
To achieve the objectives, the consortium has undertaken 16 Work packages (WPs) centred
on 5 Themes:
THEME 1
Innovative tools and approaches (WP1-3)
THEME 2
Sinks of extracellular glutamate (WP4-8)
THEME 3
Sources of extracellular glutamate (WP9-11)
THEME 4
Network aspects of extracellular glutamate (WP12-15)
THEME 5
Integrated view of extracellular glutamate dynamics (WP16)
All of the 5 Themes have been addressed.
Overview of the scientific progress
Tools essential for further work have been provided in the form of antibodies (WP1), knockout mice (WP2), and new optical and electrical recording approaches and glutamate probes
(WP3) under Theme 1 (papers published). The main output of the project is in the form of
scientific papers, 51 of which have appeared or will appear in peer reviewed publications.
(The papers are listed below, only some of the results can be mentioned in the present
overview.) Under Theme 2, the controversy of the enigmatic glutamate transporter of
glutamatergic nerve endings has been resolved (WP4) (papers in preparation). Unexpected
problems of antibody specificity lead to a series of papers on the immunocytochemical
specificity testing. The roles of glutamate uptake for limiting (i) the extracellular lifetime of
the transmitter, (ii) cross-talk between synapses producing activation of AMPA receptors, (iii)
activation of metebotropic glutamate receptors, and (iv) the occurrence of retrograde
cannabinoid signalling, have been studied (WP6-7) (papers published, submitted and in
preparation). Novel proteins interacting with glutamate transporters and with other
intracellular proteins of the cytoskeleton and cell junctions have been further characterized
(WP5). These and other related proteins are potentially of great importance for regulating the
functioning of glutamate transporters and hence of brain synaptic function. On the same note,
the dynamics of the insertion of glutamate transporters into the plasma membrane have been
studied (WP8) (papers published). The apparently paradoxical role of glia as source (not only
as sink) of extracellular glutamate was studied under Theme 3 (WP9-11). Here, significant
progress has been achieved on the intracellular mechanisms governing the Ca2+-dependent
release of glutamate from glia (papers published and in preparation). Further, a novel
vesicular glutamate transporter, VGLUT3, has been identified and demonstrated in glia and in
neurons thought to be ‘non-glutamatergic’ (PNAS 2002), and the first knock-out of a vesicular
glutamate transporter, VGLUT1, was published (Science 2004). Notably, the presence of a
synaptic-like vesicular compartment for storage and exocytosis of glutamate from glia was
directly demonstrated and its role in the control of synaptic transmission in the hippocampus
studied (paper published in Nature Neurosci 2004, J Biol Chem 2004, and submitted). In
addition, results have been obtained on the network aspects of extracellular glutamate (Theme
4, WP12-15). Thus modulation of glutamate release and action has been demonstrated to
occur via a variety of mechanisms, such as corelease of multiple transmitter amino acids (J
Cell Sci 2004, Mol Cell Sci 2004), endocannabinoid signalling (Nature Neurosci 2005), or
5
synergistic control of intracellular signalling pathways by ionotropic and metabotropic
glutamate receptors, ie a coincidence detector that may contribute to synaptic ‘learning’ (J
Neurosci 2006). Signalling mechanisms involved in Nieman-Pick and in Alzheimer
neurodegeneration have been characterized. An unexpected discovery was the presence of an
unusual type of NMDA receptor on the myelinating processes of oligodendrocytes and their
activation in a model of brain ischemia (publiched in Nature 2005 and widely commented on
in prestigious scientific journals). This mechanism may underlie the vulnerability of
myelinataed nerve fibres in a variety of neurological diseases and offers a novel approach to
therapy. Finally, an integrated view of the dynamics of extracellular glutamate has been
established (Theme 5, WP16) by a combination of imaging with fluorescent tracers,
electrophysiological recordings and computational simulations in several brain regions, viz
hippocampus (Neuroimage 2005), cerebellar cortex and in the calyces of Held in the auditory
system (2 papers published in J Neurosci 2005).
Potential social and economic impact of the scientific achievements: Theme 1: the
availability of antibodies of high quality and the establishing of improved procedures for
ascertaining immunocytochemical specificity are of great importance to the scientific
community. (A large number of papers in the literature have probably reached wrong
conclusions due to non-specific immunoreactivity.) Likewise, new electrical and optical
recording devices and procedures will be useful for other investigators. Themes 2-5: the
knowledge gained forms the basis for further investigations into the normal functioning of the
nervous system and its malfunctioning in disease. This knowledge is essential for innovation
of therapy. Specifically, the demonstration (Theme 2) that excitatory nerve endings, contrary
to common notion, is a significant glutamate sink and may be important for replenishing
transmitter stores, provides a change in focus of the quest for ways to modify glutamate
uptake as a therapeutic intervention. Such modification may exploit the physiological
mechanisms for regulation of glutamate uptake also studied under Theme 2. Theme 3: a major
finding of the project is that astrocytes actively release glutamate by an exocytotoic
mechanism similar to the one operating in nerve endings. This provides an essentially altered
way of understanding the operation of the nervous system and hence for understanding
disease mechanisms and treatment. Theme 4: among several regulatory mechanisms revealed
in the present project as underlying ‘network aspects’ of extracellular glutamate the most
important discovery may be the sensitivity of myelin to glutamate through NMDA receptors.
The unusual subunit composition of these receptors opens an avenue to developing selective
drugs for therapy in neurological conditions such as ischemic damage, neonatal asphyxia (that
causes cerebral palsy), spinal cord injury, and demyelinating disease (multiple sclerosis).
Theme 5: the modelling of extracellular glutamate dynamics and glutamate receptor
occupancy provides a tool for pathophysiological research and for developing such novel
therapeutic strategies.
Progress achieved compared with planned activities
The progress achieved is largely as planned. A few Milestones or Deliverables have been
deleted because results obtained showed they were not practicable or because they have been
covered by papers published by others (as indicated for the individual WPs).
Updated Tables 1-3 are presented on the following pages
6
Table 1.
Work-pack
No
Work package list – unchanged from Technical Annex
Work package title
WP 1
Antibodies to new transporters,
interacting proteins and ligands
WP 2
Responsible
particip. No
Start
month
End
month
Deliver-able(s)
No
(6) #1
0
36
D 1.1
D 1.2
Production and maintenance of
#2
mice lacking glutamate transporters
12 #2
0
36
D 2.1
WP 3
Innovative approaches to studying
glutamate dynamics
#5
(5) #4
26(29) #5
(3) #6
0
36
D 3.1
D 3.2
D 3.3
WP 4
The glutamate transporter in
glutamatergic nerve terminals
#1
18(36) #1
3(1) #2
0
36
D 4.1
D 4.2 (D 4.3)
WP 5
Roles of proteins interacting with
glutamate transporters
#2
21(7) #2
12(24) #1
0
36
WP 6
Correlation of synaptic cross-talk
with glutamate transporter density
and arrangement in area dentata
#3
8(8) #3
(24) #1
0
24
D 6.1
#2
3(3) #2
(12) #1
0
18
D 7.1
D 7.2
0
36
0
36
WP 7
Role of the glutamate transporters
in maintaining the specificity of
synaptic transmission, studied in
transporter deficient animals
#1
Personmonths1
18(6) #6
3(1) #2
6#1 (3)#5
15(29) #4
15(15) #5
(12) #1
(3)#6
D 5.1
D 5.2
D 5.3
D 8.1
D 8.2
D 8.3
D 9.1
D 9.2
D 9.3
D 9.4
WP 8
Regulation of GLT-1 density in
astrocyte plasmamembrane
#6
WP 9
Intracellular signalling events
responsible for glial glutamate
release
#4
WP 10
Role of exocytosis in glial
glutamate release
#6
36(6) #6
(3) #5
(3)#4
0
36
WP 11
Dynamics of glutamate release:
photoreceptor terminal as a model
#6
18(6) #6
2 #2, (3)#5
0
36
WP 12
Modulation of synaptic activity by
glial glutamate release
#4
0
36
WP 13
Intercellular signalling within
neuron/glia networks
#5
30(32) #4
7(7) #5
(3)#6
15(15) #4
24(25) #5
(3) #6
8(12) #3
(18) #1
0
36
D 13.1
D 13.2
D 13.3
0
24
D 14.1
D 14.2
12
36
12
36
WP 14
WP 15
WP 16
Modulation of glutamate action
by aspartate or GABA coreleased
with glutamate
Alteration of glutamate control in
focal epilepsy
Release, diffusion, uptake, and
actions of glutamate: modelling
#3
12(12)#3
(18) #1
#3
#3
8(24) #3
(3)#1
TOTAL
D 5.4
D 5.5
D 10.1
D 10.2
D 10.3
D 11.1
D 11.2
D 11.3
D 12.1
D 12.2
D 15.1
D 15.2
D 16.1
D 16.2
320 (420)
1 The total number of EU budgeted person-months allocated to each work package is split between participants. The non-EUstaff involved is
indicated in parentheses (person-months).
7
Table 2.
Milestones list [digits before decimal point = WP no] (month planned)
Achieved (new month)
M 1.1: First antibody to transporter variant, and to synthetic stubstrate (6) Achieved
M 1.2: First antibody to a transporter-interacting protein (12) Achieved (20)
M 2.1: Knock-out mice (within 6) Achieved
M 3.1: Imaging of exocytosis (12) Achieved
M 3.2: Imaging of extracellular glutamate (24) Achieved (36)
M 4.1: Provide unequivocal proof for the existence of a glutamate transporter in glutamatergic terminals (6)
Achieved
M 4.2: Determine if the putative transporter is related to GLT by studying mutant mice (12) Achieved
M 4.3: Develop a cloning strategy and clone the transporter (36) [conditional] Cancelled
M 5.1: Achieve slice cultures of hippocampus and cerebellum (6) Achieved (20)
M 5.2: Find the full p83-sequence (12) Achieved
M 5.3: Characterise synaptic currents in slice cultures (18) Cancelled
M 5.4: Characterise transporter and LIM/p83 protein expression in slice cultures (24) Cancelled
M 6.1: Establish whether outer, but not middle, molecular layer synapses show evidence for temperature and
transporter dependent mismatch between AMPA and NMDA receptor-mediated signalling (12) Achieved
M 6.2: Estimate densities of glutamate transporters in middle and outer molecular layers and/or MNTB (18)
Achieved (24)
M 6.3: Ultrastructural distribution of transporters and astrocyte processes in the vicinity of synapses (24)
Achieved ahead of schedule (18)
M 7.1: Characterise synaptic currents mediated by spillover of glutamate in mice expressing or lacking
particular glutamate transporters (6) Achieved
M 7.2: Control experiments on how hippocampal cells’ glutamate receptor and transporter density is affected by
the knockout of one transporter type (18) Achieved ahead of schedule (12)
M 8.1: Elucidate whether incorporation of new glutamate transporters into the plasmalemma is mediated via
regulated exocytosis (24) Achieved
M 9.1: Information on receptor types stimulating regulated glutamate release in astrocytes (12) Achieved
M 9.2: Information on intracellular signal transduction cascades activated (see M 9.1) (24) Achieved
M 9.3: Establish spatiotemporal correlation between Ca2+ signals and glutamate release (30) Achieved
M 10.1: Test the hypothesis that glia posess mechanisms for exocytotic release of glutamate (36) Achieved
ahead of schedule
M 11.1: Describe the calcium dependence of exocytosis of glutamate-containing vesicles in photoreceptor
neurons (36) Achieved
M 12.1: Appropriate experimental model for studying the active role of astrocytes in synaptic functions (18)
Achieved
M 12.2: Information on specific contributions by astrocytes to synaptic transmission and plasticity (36)
Achieved
M 13.1: Reveal relation between Ca2+ and DAG signalling and glutamate release in glia (24) Achieved
M 14.1: Define the responses to non-saturating mixtures of aspartate and glutamate in various ratios, applied to
membrane patches by rapid perfusion techniques (12) Achieved (18) but results negative
M 14.2: Alterations of AMPA and NMDA receptor-mediated signalling with impaired glycolysis (12) Cancelled
(because of outcome of M 14.1)
M 14.3: Relative importance of glutamate and GABA-mediated signalling following seizures (24) Cancelled
(because of publication by R Gutierrez & E. Cherubini)
M 15.1: Determine the effects of epilepsy (induced in rats) on glutamate spillover using in vitro
electrophysiological methods (24) Achieved ahead of schedule (20)
M 15.2: Relationship between spontaneous seizures in the models and extracellular glutamate (30) Not achieved
(because of difficulties in establishing telemetry)
M 15.3: Characterise density and spatial arrangement of glutamate transporters during epileptogenesis (30)
Achieved (in part)
M 15.4: Find the aspartate/glutamate ratio in nerve endings in epileptic and control hippocampus (36) Cancelled
(because of outcome of WP14)
M 16.1: Establish a geometrical representation of the synaptic microenvironment in brain neuropil (24)
Achieved ahead of schedule (18)
M 16.2: Incorporate neuronal transporters, and data on their kinetics and density (30) Achieved
M 16.3: Generate predictions about the occupancy of each of the subclasses of glutamate receptors (AMPA,
kainate, NMDA, metabotropic) at different time points after discharge of a single synapse, after discharge of a
defined random proportion of synapses in a block of tissue, and after trains of discharges (36) Achieved
Table 3.
Deliverables list
Deliverable
No
Deliverable title
Target
month
Nature Dissem.
level
1. D 2.1
Mice lacking particular glutamate transporters
6 A -36 A
P
RE
2. D 5.1
Achieve slice cultures of hippocampus and cerebellum
6A
P
RE
3. D 1.1
Antibodies to transporters, interacting proteins and synthetic
substrates
12 A
O
PU
4. D 3.1
Set up of a prism-based TIRM for monitoring exocytosis and plasma
membrane translocation of GFP-tagged molecules
12 A
P
RE
5. D 3.2
Set up of an apparatus to simultaneously i) monitor Cm, ii) measure
[Ca2+]c, iii) evoke photolysis of caged compounds
12 A
P
RE
6. D 3.3
Set up of videoimaging approaches for the measurement of
extracellular glutamate
36 A
P
RE
7. D 4.1
Resolve controversy over the excistence of a nerve terminal glutamate
uptake and clarify its substrate selectivity and distrib
12 A
R
PU
8. D 5.2
Isolate the full sequence of the p83 protein
12 A
P
RE
9. D 7.1
Electrophysiological data on the properties of synapses in transporter
knock-out mice
12 A
P
RE
11. D 8.1
Fusion constructs between GLT-1 and GFPs
12 A
P,R
PU
12. D 9.1
Identification of transmitter receptors promoting calcium- and
prostaglandin-dependent glutamate release from astrocytes
12 A
R
PU
13. D 10.1
14. D 11.1
Calcium dependence of surface area changes in astrocyte
Photolysis of caged calcium and fast capacitance measurements in
photoreceptors
Clarification of the interaction of p83 with GLT-1
12 A
12 A
R
R
PU
PU
18 C
R
PU
16. D 7.2
Report the effect of transporter knockout on excitatory synaptic
transmission, in a full paper
18 A (12)
R
PU
17. D 12.1
Identification of appropriate in situ and/or in vitro models to study the
contributions of astrocytes to synaptic functions
18 A
R
PU
18. D 4.2
Obtain crude estimates of the relative capacity and affinity of nerve
terminal glutamate uptake in tissue slices
24 A (12)
R
PU
19. D 5.4
Characterise synaptic currents, and transporter and LIM/p83 protein
expression, in slice cultures
Correlation between (1) distribution of glutamate transporters and (2)
spillover-mediated signalling at medial and lateral perforant path
synapses.
24 C
P
RE
24 A
R
PU
Expression and properties of the GLT-1-GFP constructs in cultured
astrocytes observed under confocal microscope
Botulinum neurotoxin sensitivity of surface area changes in astrocytes
24 A
R
PU
24 A
R
PU
Ca2+-dependent capacitance changes in photoreceptors compared to
bipolar cells
24 A
R
PU
15. D 5.3
20. D 6.1
21. D 8.2
22. D 10.2
23. D 11.2
A=achieved (new month) C=cancelled
9
24. D 13.1
Identification of signalling processes responsible for intercellular
communication in neuron-astrocyte networks
24 A (36)
R
PU
25. D 14.1
Effect of aspartate on AMPA, kainate and NMDA receptor-mediated
signalling following metabolic perturbations, related to the aspartate
content of presynaptic terminals (Could not establish model)
24 C
R
PU
26. D 14.2
Ratio of GABAergic to glutamatergic mossy fibre signalling
following seizures
Publication on data in normal rats
24 A
(partly)
R
PU
27. D 16.1
Generalisable
geometric
representation
of
the
synaptic
microenvironment: a compartmental model with adjustable
pararameters
30 A (12)
P
RE
28. D 1.2
Further antibodies to new transporters, interacting proteins and
ligands
36 A
O
PU
[D 4.3]
Conditional
Cloning of the transporter (provided discoveries leading to a cloning
strategy)
36 C
R
PU
29. D 5.5
Report on the effects of altering expression of GLT-1-associated
proteins on synaptic transmission in slice cultures
36 C
R
PU
30. D 8.3
Comparison of GLT-1-GFP dynamics in normal astrocytes and
astrocytes from GLT-1 (-/-) and (+/+) mice
36 C
R
PU
31. D 9.2
Spatiotemporal characterization of receptor-induced [Ca2+]i elevations
in astrocytes, and correlation with the dynamics of glutamate release
36 A
R
PU
32. D 9.3
Characterisation at the molecular level of mechanism and
determinants of PGE2 control on Ca2+i elevation and glutamate
release in astrocytes
36 A
R
PU
33. D 9.4
Identification of intracellular glutamate storing compartments in
astrocytes
36 A (12)
R
PU
33. D 10.3
Correlation between plasma membrane surface dynamics and
extracellular glutamate measured optically
36 A
R
PU
34. D 11.3
Ca2+-induced changes in membrane capacitance and optical
measurements of extracellular glutamate
36 A
R
PU
35. D 12.2
Characterisation, in the identified models, of specific contributions of
astrocyte glutamate release to synaptic transmission and plasticity
phenomena
36 A
R
PU
36. D 13.2
Definition of the influence of intercellular communication on
Ca2+/DAG signalling pathways in neuron-astrocyte networks
36 A(24)
R
PU
37. D 13.3
Characterisation of the role of the intercellular signalling network in
the control of glutamate release
36 A
R
PU
38. D 15.1
Glutamate spillover-mediated signalling measured in hippocampal
slices from animals following experimental status epilepticus, related
to the density and distribution of transporters
36 A(24)
R
PU
39. D 15.2
Extracellular glutamate measurements from epileptic animals related
to the density and distribution of transporters
36 C
R
PU
40. D 16.2
Spatio-temporal concentration profiles of glutamate: dependence on
synapse type and transporter function, and consequences for AMPA,
NMDA, kainate and metabotropic receptors
36 A
R
PU
10
2. EXPLOITATION AND DISSEMINATION ACTIVITIES
Progress towards exploitation of the project results
The most important output of the DECG project is the publication of scientific papers in peer
review journals (see under Dissemination activities). The junior members of the research
teams are obtaining scientific training, which represents an important aspect of the
exploitation of the project activities to the benefit of the community. As an example, Runhild
Gammelsæter, who did her work for the PhD under the present project, attained the position
as a CSO in a company, Regenics (http://www.nucleotech.com/corporate/), newly created to
develop commercially reprogramming of cell function. No patents have been filed.
Major dissemination activities during the period
The major output of the project is scientific papers. Fifty-one research papers have been
published or will be published in publications with peer review system (see list below). We
have achieved a significant proportion of papers in high key journals. Similarly, presentations
have been made at conferences and workshops, but these have not been recorded
systematically. A web site (http://folk.uio.no/jonsm/decg.htm) has been created, to present the
DECG and published results emanating from the project.
On the occasion of the publication of the Nature Neuroscience paper on glial exocytosis of
glutamate (collaboration of Partners #4 and #1), the collaborating institutions made press
releases, that were diffused by the media at the international level (notably, in several
European countries). Following this, lay public articles and interviews appeared on lay-public
scientific journals, for instance “Science & Vie” and “Biofutur” in France. Similarly, the
Nature paper on NMDA receptors in oligodendrocytes (collaboration of partners #2 and #1)
was widely commented on in the lay press in several European countries (including England,
Iceland and Norway), on TV, in national biomedical journals, and editorially in high profile
scientific journals (Science STKE, Neuron, Nature Reviews in Neuoscience, Trends in
Molecular Medicine and Bioessays).
As an important part of the Technological Implementation Plan (TIP), as set out in the
Technical Annex to the DECG contract, a meeting was organized 3-5 September 2004 at
Losby Gods close to Oslo. The main program of the Losby meeting was a series of lectures
and discussions of the theme “Homeostasis at brain synapses – options for drug targets”.
The meeting invited researchers, physicians and representatives from the pharmaceutical
industry to listen to presentations by DECG participants and selected invited speakers. The
proceedings of this symposium have been published electronically:
Bergersen LH, Storm-Mathisen J (editors) (2005) Homeostasis at brain synapses – options
for drug targets. Proceedings of a symposium organized in conjunction with the EU
sponsored research project “Dynamics of Extracellular Glutamate” DECG and the RCN
sponsored CoE “Centre for Molecular Biology and Neuroscience” CMBN. Losby Gods, Oslo,
Norway, September 3-5, 2004. ISBN 82-995010-2-4
http://folk.uio.no/jonsm/Homeostasis-at-brain-synapses-Losby.pdf
11
List of publications directly emanating from the project
Refereed papers. Abstracts are not included
1
Allen NJ, Attwell D (2004) The effect of simulated ischaemia on spontaneous GABA
release in area CA1 of the juvenile rat hippocampus. J Physiol 561:485-498
2
Allen NJ, Karadottir R, Attwell D (2004) Reversal or reduction of glutamate and GABA
transport in CNS pathology and therapy. Pflugers Arch 449:132-142
3
Allen NJ, Karadottir R, Attwell D (2005) A preferential role for glycolysis in preventing
the anoxic depolarization of rat hippocampal area CA1 pyramidal cells. J Neurosci
25:848-859.
4
Allen NJ, Rossi DJ, Attwell D (2004) Sequential release of GABA by exocytosis and
reversed uptake leads to neuronal swelling in simulated ischemia of hippocampal slices. J
Neurosci 24:3837-3849
5
Attwell D, Iadecola C (2002) The neural basis of functional brain imaging signals. Trends
Neurosci 25:621-625
6
Bergersen L, Ruiz A, Bjaalie JG, Kullmann DM, Gundersen V (2003) GABA and
GABAA receptors at hippocampal mossy fibre synapses. Eur J Neurosci 18:931-941
[Joint publication #1 & #3]
7
Bezzi P, Gravaghi C, Grohovaz F, Volterra A (2006) Prostaglandins mediate a component
of astrocyte calcium elevations responsible for receptor-stimulated glutamate release. In
preparation [Joint publication #4 & #5]
8
Bezzi P*, Gundersen V*, Galbete JL, Seifert G, Steinhauser C, Pilati E, Volterra A (2004)
Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of
glutamate. Nat Neurosci 7:613-620 *These authors contributed equally
[Joint publication #1 & #4]
9
Billups D, Attwell D (2003) Active release of glycine or D-serine saturates the glycine site
of NMDA receptors at the cerebellar mossy fibre to granule cell synapse. Eur J Neurosci
18:2975-2980
10 Boulland JL, Qureshi T, Seal RP, Rafiki A, Gundersen V, Bergersen LH, Fremeau RT,
Jr., Edwards RH, Storm-Mathisen J, Chaudhry FA (2004) Expression of the vesicular
glutamate transporters during development indicates the widespread corelease of multiple
neurotransmitters. J Comp Neurol 480:264-280
11 Cavelier P, Attwell D (2005) Tonic release of glutamate by a DIDS-sensitive mechanism
in rat hippocampal slices. J Physiol 564:397-410
12 Cavelier P, Hamann M, Rossi D, Mobbs P, Attwell D (2005) Tonic excitation and
inhibition of neurons: ambient transmitter sources and computational consequences. Prog
Biophys Mol Biol 87:3-16
13 Chiulli N, Codazzi F, Di Cesare A, Gravaghi C, Zacchetti D, Grohovaz F (2006) Effect of
sphingosylphosphocholine on astrocytes: a possible mechanism mediating neurotoxicity
in Niemann-Pick type A disease. Submitted
12
14 Codazzi F, Di Cesare A, Chiulli N, Albanese A, Meyer T, Zacchetti D, Grohovaz F
(2006) Synergistic control of protein kinase Cgamma activity by ionotropic and
metabotropic glutamate receptor inputs in hippocampal neurons. J Neurosci 26(13):340411
15 De Pietri Tonelli D, Mihailovich M, Di Cesare A, Codazzi F, Grohovaz F, Zacchetti D
(2004) Translational regulation of BACE-1 expression in neuronal and non-neuronal cells.
Nucleic Acids Res 32:1808-1817
16 Di Cesare A, Del Piccolo P, Codazzi F, Zacchetti D, Grohovaz F (2006) EP2 receptor
promotes calcium responses in astrocytes via activation of the adenylate cyclase pathway.
In preparation
17 Domercq M, Pietropoli A, Jourdain P, Brambilla L, Pilati E, Kollias G, Matute C, Bezzi P
and Volterra A. (2006) An astrocyte control of excitatory transmission in hippocampus
via purinoreceptors. Submitted
18 Evanko DS, Zhang Q, Zorec R, Haydon PG (2004) Defining pathways of loss and
secretion of chemical messengers from astrocytes. Glia 47:233-240
19 Fremeau RT Jr, Burman J, Qureshi T, Tran CH, Proctor J, Johnson J, Zhang H, Sulzer D,
Copenhagen DR, Storm-Mathisen J, Reimer RJ, Chaudhry FA, Edwards RH (2002) The
identification of vesicular glutamate transporter 3 suggests novel modes of signaling by
glutamate. Proc Natl Acad Sci USA 99:14488-14493
20 Fremeau RT Jr, Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm-Mathisen J,
Chaudhry FA, Nicoll RA, Edwards RH (2004) Vesicular glutamate transporters 1 and 2
target to functionally distinct synaptic release sites. Science 304(5678):1815-1819
21 Furness DN, Dehnes Y, Qureshi A, Rossi D, Hamann M, Grutle N, Gundersen V,
Holmseth S, Lehre KP, Ullensvang K, Attwell D, Danbolt NC (2006) A quantitative
assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes. In
preparation [Joint publication #1 & #2]
22 Gammelsaeter R, Frøyland M, Aragón C, Danbolt NC, Fortin D, Storm-Mathisen J,
Davanger S, Gundersen V (2004) Glycine, GABA and their transporters in pancreatic
neuroendocrine cells: evidence for a transmitter paracrine interplay. J Cell Sci 117(Pt
17):3749-3758.
23 Gundersen V, Holten AT, Storm-Mathisen J (2004) GABAergic synapses in hippocampus
exocytose aspartate on to NMDA receptors: quantitative immunogold evidence for cotransmission. Mol Cell Neurosci 26:156-165.
24 Hamann M, Rossi DJ, Mohr C, Andrade AL, Attwell D (2005) The electrical response of
cerebellar Purkinje neurons to simulated ischaemia. Brain 128:2408-2420.
25 Holmseth S, Dehnes Y, Bjornsen LP, Boulland JL, Furness DN, Bergles D, Danbolt NC
(2005) Specificity of antibodies: unexpected cross-reactivity of antibodies directed against
the excitatory amino acid transporter 3 (EAAT3). Neuroscience 136: 649-660.
26 Holmseth S, Dehnes Y, Furness DN, Plachez C, Bjornsen LP, Mylonakou NM, Bergles D,
Danbolt NC, Lehre KP (2006) The concentration of the excitatory amino acid transporter
3 protein (EAAT3) in young adult Wistar rat hippocampus is 100-fold lower than that of
EAAT2. In preparation
13
27 Holmseth S, Lehre KP, Danbolt NC (2006) Specificity controls for
immunocytochemistry. Anat Embryol (Berl) Jan 25;:1-10 [Epub ahead of print 2006 Jan
25]
28 Holmseth S, Martin VV, Lehre KP, Bergles D, Danbolt NC (2006) Specificity controls for
immunocytochemistry: Pre-absorbing antibodies with the antigen does not test whether
the antibodies bind to the same antigen in tissue sections. In preparation
29 Jourdain P*, Bergersen LH*, Bhaukaurally K, Bezzi P, Domercq M, Matute C, Tonello F,
Gundersen V, Volterra A (2006) Glutamate exocytosis from astrocytes controls synaptic
strength. Submitted *These authors contributed equally [Joint publication #1 & #4]
30 Káradóttir R, Cavelier P, Bergersen LH, Attwell D (2005) NMDA receptors are expressed
in oligodendrocytes and activated in ischemia. Nature 438:1162-1166
[Joint publication #1 & #3]
31 Kreft M, Krizaj D, Grilc S, Zorec R (2003) Properties of exocytotic response in vertebrate
photoreceptors. J Neurophysiol 90:218-225
32 Kreft M, Milisav I, Potokar M, Zorec R (2004) Automated high through-put
colocalization analysis of multichannel confocal images. Comput Methods Programs
Biomed 74:63-67
33 Kreft M, Stenovec M, Rupnik M, Grilc S, Krzan M, Potokar M, Pangrsic T, Haydon PG,
Zorec R (2004) Properties of Ca(2+)-dependent exocytosis in cultured astrocytes. Glia
46:437-445
34 Krzan M, Stenovec M, Kreft M, Pangrsic T, Grilc S, Haydon PG, Zorec R (2003)
Calcium-dependent exocytosis of atrial natriuretic peptide from astrocytes. J Neurosci
23:1580-1583
35 Lehre KP, Rusakov DA (2002) Asymmetry of glia near central synapses favors
presynaptically directed glutamate escape. Biophys J 83:125-134
[Joint publication #1 & #3]
36 Marcaggi P, Attwell D (2004) Role of glial amino acid transporters in synaptic
transmission and brain energetics. Glia 47:217-225
37 Marcaggi P, Attwell D (2005) Endocannabinoid signaling depends on the spatial pattern
of synapse activation. Nat Neurosci 8:776-781
38 Marcaggi P, Billups D, Attwell D (2003) The role of glial glutamate transporters in
maintaining the independent operation of juvenile mouse cerebellar parallel fibre
synapses. J Physiol 552:89-107.
39 Marie H, Billups D, Bedford FK, Dumoulin A, Goyal RK, Longmore GD, Moss SJ,
Attwell D (2002) The amino terminus of the glial glutamate transporter GLT-1 interacts
with the LIM protein Ajuba. Mol Cell Neurosci 19:152-164
40 Marie H, Pratt SJ, Betson M, Epple H, Kittler JT, Meek L, Moss SJ, Troyanovsky S,
Attwell D, Longmore GD, Braga VM (2003) The LIM protein Ajuba is recruited to
cadherin-dependent cell junctions through an association with alpha-catenin. J Biol Chem
278:1220-1228
14
41 Micheletti M, Brioschi A, Fesce R, Grohovaz F (2005) A novel pattern of fast calcium
oscillations points to calcium and electrical activity cross-talk in rat chromaffin cells. Cell
Mol Life Sci 62:95-104
42 Potokar M, Kreft M, Pangrsic T, Zorec R (2005) Vesicle mobility studied in cultured
astrocytes. Biochem Biophys Res Commun Apr 8;329(2):678-83.
43 Renden R, Taschenberger H, Puente N, Rusakov DA, Duvoisin R, Wang LY, Lehre KP,
von Gersdorff H (2005) Glutamate transporter studies reveal the pruning of metabotropic
glutamate receptors and absence of AMPA receptor desensitization at mature calyx of
Held synapses . J Neurosci 25: 8482-8497 [Joint publication #1 & #3]
44 Rusakov DA, Lehre KP (2002) Perisynaptic asymmetry of glia: new insights into
glutamate signalling. Trends Neurosci 25:492-494 [Joint publication #1 & #3]
45 Rusakov DA, Saitow F, Lehre KP, Konishi S (2005) Modulation of presynaptic Ca2+
entry by AMPA receptors at individual GABAergic synapses in the cerebellum. J
Neurosci 25:4930-4940 [Joint publication #1 & #3]
46 Savtchenko LP, Rusakov DA (2005) Extracellular diffusivity determines contribution of
high-versus low-affinity receptors to neural signaling. Neuroimage 25:101-111
47 Scimemi A, Fine A, Kullmann DM, Rusakov DA (2004) NR2B-containing receptors
mediate cross talk among hippocampal synapses. J Neurosci 24:4767-4777
48 Scimemi A, Schorge S, Kullmann DM, Walker MC (2006) Epileptogenesis is associated
with enhanced glutamatergic transmission in the perforant path. J Neurophysiol 95:12131220
49 Stenovec M, Kreft M, Grilc S, Pangršic T, Zorec R (2005) EAAT2 at the astrocyte plasma
membrane and Ca2+ -regulated exocytosis. Submitted
50 Volterra A, Steinhauser C (2004) Glial modulation of synaptic transmission in the
hippocampus. Glia 47:249-257
51 Zhang Q, Pangrsic T, Kreft M, Krzan M, Li N, Sul JY, Halassa M, Van BE, Zorec R,
Haydon PG (2004) Fusion-related release of glutamate from astrocytes. J Biol Chem
279:12724-12733
3. ETHICAL ASPECTS AND SAFETY PROVISIONS
No ethical or safety issues have arisen during the period.
15
4. CONCLUSIONS
Tools for further work have been provided in the form of antibodies, KO mice, and new
optical and electrical recording approaches. Results have been published or will be published
in the form of 51 refereed scientific papers, conference presentations, communications to the
media and through the project’s web site. A significant proportion of the papers are in high
impact journals, such as Nature, Nat Neurosci, Science, PNAS, J Neurosci, Neuroimage,
TINS, J Biol Chem. The proceedings of a conference, “Homeostasis at brain synapses –
options for drug targets”, based on the activities of the project have been published and are
available as a pdf on the web.
The controversy of the enigmatic glutamate transporter of glutamatergic nerve endings has
been resolved. The roles of glutamate uptake for limiting the extracellular lifetime of the
transmitter and the cross-talk between synapses have been studied. Novel proteins interacting
with glutamate transporters and with other intracellular proteins of the cytoskeleton and cell
junctions have been investigated. The dynamics of the insertion of glutamate transporters into
the plasma membrane have been addressed. The apparently paradoxic role of glia as source
(not only as sink) of extracellular glutamate has been established, and mechanisms governing
it have been unravelled. A novel vesicular glutamate transporter, VGLUT3, has been
identified and demonstrated in glia and in neurons thought to be ‘non-glutamatergic’, and the
first KO of a vesicular glutamate transporter was published. Corelease of multiple transmitter
amino acids was demonstrated, and novel modulatory mechanisms were revealed, such as
modulation of GABA release through presynaptic AMPA receptors, via synergistic control of
intracellular cascades by ionotropic and metabotropic glutamate receptors, or through
endocannabinoid signalling. Enhanced release of glutamate was demonstrated in a rat model
of epilepsy. A mechanism (viz an unusual type of NMDA receptor) rendering myelinated
nerve fibres vulnerable in various neuropathologic conditions was discovered. Numeric
models have been presented for the dynamics of extracellular glutamate and the control of
receptor occupancy in different brain sites (hippocampus, cerebellum, calyces of Held in the
auditory pathway).
The outcome of the project was as expected and according to the plans set out in the
Technical Annex. Several young scientists have obtained training through the project. The
results provide new insight into how the nervous system works. Through this new insight the
results have important socio-economic implications as they offer new approaches and tools to
tackle diseases with major impact on human health.
5. ACKNOWLEDGEMENTS
The co-ordinator and the partners would like to thank the EC for supporting our research.
A major part of the resources required to carry out the EU sponsored project Dynamics of
Extracellular Glutamate (DECG) QLG3-CT-2001-02004 came from other sources, as shown
by the figures for manpower in Table 1 (Technical Annex), ie 320 personmonths funded by
the EU grant, compared to a total of 740 personmonths. Correspondingly, a major proportion
of the resources for consumables etc came from non-EU funds. Thanks are due to the
institutions of the individual partners #1 - #6 and to national and international granting bodies.
16
SECTION III: SCHEMATIC DESCRIPTION OF THE PROJECT
Overall objectives of the project:
If allowed to accumulate in the extracellular space, glutamate may act as a powerful toxin and may stimulate
glutamate receptors to the extent that cellular suicide processes get triggered. This phenomenon, known as
excitotoxicity, develops with some latency, implying a possibility for therapeutic intervention. The ambitious
end objective of the DECG is to provide a comprehensive numeric model that describes the extracellular
glutamate concentration and ensuing receptor activation. Glutamate reaching postsynaptic as well as
presynaptic and extrasynaptic receptors, the latter on neurons as well as on glial cells, needs to be taken into
account. Major sources and sinks of extracellular glutamate as well as several regulatory mechanisms are
known, but their relative roles must be clarified and unknowns identified. Major factors that need further
research include the evoked release of glutamate from glial cells, the still unidentified glutamate transporter of
excitatory nerve endings, the roles of proteins interacting with glutamate transporters, and the question of
modulation of glutamate action by co-released aspartate and GABA. In addition to unravelling these and other
factors, and using them as inputs to the model, we will test the performance of the model in relation to disease
by experiments on simulated ischemia and in epileptic rats.
Experimental approach and working method:
The overall methodology ranges from morphology via electrophysiology to molecular biology. The participants
are masters of complementary aspects within these disciplines. The expertise of the individual participants (#)
include inter alia the following:
#1
Neuroanatomy, electronmicroscopy, immunocytochemistry (including quantitative postembedding
immunogold), antibodies, protein biochemistry, molecular biology
#2
Patch-clamping, conductance assay of glutamate transporters, functional fluorescence imaging,
computer-modelling, cell/tissue culture, gene knock-out, molecular biology
#3
Patch-clamping, fast drug application, computer-modelling, epilepsy models
#4
Cell culture, intracellular signalling, oxidative stress, molecular biology, TIRF microscopy
#5
Confocal/2-photon microscopy, TIRF (evanescent wave) microscopy, imaging, quick-freeze/drying
#6
Capacitance recording, flash photolysis, exocytosis
Achievements and results (51 refereed papers produced):
Tools for further work have been provided in the form of antibodies, KO mice, and new optical and electrical
recording approaches. The controversy of the enigmatic glutamate transporter of glutamatergic nerve endings
has been resolved. The roles of glutamate uptake for limiting the extracellular lifetime of the transmitter and the
cross-talk between synapses have been studied. Novel proteins interacting with glutamate transporters and with
other intracellular proteins of the cytoskeleton and cell junctions have been investigated. The dynamics of the
insertion of glutamate transporters into the plasma membrane have been addressed. The apparently paradoxic
role of glia as source (not only as sink) of extracellular glutamate has been established, and mechanisms
governing it have been unravelled. A novel vesicular glutamate transporter, VGLUT3, has been identified and
demonstrated in glia and in neurons thought to be ‘non-glutamatergic’ (PNAS 2002), and the first KO of a
vesicular glutamate transporter was published (Science 2004). Corelease of multiple transmitter amino acids
was demonstrated, and novel modulatory mechanisms were revealed, such as modulation of GABA release
through presynaptic AMPA receptors, via synergistic control of intracellular cascades by ionotropic and
metabotropic glutamate receptors, or through endocannabinoid signalling (Nature Neurosci 2005). Enhanced
release of glutamate was demonstrated in a rat model of epilepsy. A mechanism (viz an unusual type of NMDA
receptor) rendering myelinated nerve fibres vulnerable in various neuropathologic conditions was discovered.
Numeric models have been presented for the dynamics of extracellular glutamate and the control of receptor
occupancy in different brain sites (hippocampus Neuroimage 2005, cerebellum, calyx of Held).
The five most relevant publications emanating from the project (additional high impact papers in box above):
Bezzi* P, Gundersen* V, Galbete JL, Seifert G, Steinhauser C, Pilati E, Volterra A (2004) Astrocytes contain a
vesicular compartment that is competent for regulated exocytosis of glutamate. Nature Neurosci 7:613-20
*These authors contributed equally [Joint publication partners #1 & #4]
Zhang Q, Pangrsic T, Kreft M, Krzan M, Li N, Sul JY, Halassa M, Van BE, Zorec R, Haydon PG (2004)
Fusion-related release of glutamate from astrocytes. J Biol Chem 279:12724-33 [Partner #6]
Káradóttir R, Cavelier P, Bergersen LH, Attwell D (2005) NMDA receptors are expressed in oligodendrocytes
and activated in ischemia. Nature, 438:1162-66 [Joint publication partners #1 & #2]
Rusakov DA, Saitow F, Lehre KP, Konishi S (2005) Modulation of presynaptic Ca 2+ entry by AMPA receptors
at individual GABAergic synapses in the cerebellum. J Neurosci 25:4930-40 [Joint publication #1 & #3]
Codazzi F, Di Cesare A, Chiulli N, Albanese A, Meyer T, Zacchetti D, Grohovaz F (2006) Synergistic control
of protein kinase Cgamma activity by ionotropic and metabotropic glutamate receptor inputs in hippocampal
neurons. J Neurosci 26:3404-11 [Partner #5]
17