Department of Physics, Chemistry and Biology
Final Thesis
Olfactory sensitivity in CD-1 mice for “green” odors
Sathish Kumar Murali
LiTH-IFM- Ex—11/2442--SE
Supervisor: Matthias Laska, Linköpings universitet.
Examiner: Jordi Altimiras, Linköpings universitet.
Department of Physics, Chemistry and Biology
Linköpings universitet
SE-581 83 Linköping, Sweden
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Titel
Title:
Olfactory sensitivity in CD-1 mice for “green” odors.
Författare
Author:
Sathish Kumar Murali.
Sammanfattning
Abstract:
―Green‖ odors comprise a group of eight structurally related aliphatic alkenals and alkenols which are characteristic for the odor of
a wide variety of plant materials. Using an automated olfactometer, the olfactory detection thresholds for ―green‖ odors were
determined in six CD-1 mice and compared with that of spider monkeys and human subjects. Detection threshold values for
alcoholic ‖green‖ odors (cis-3-hexen-1-ol, trans-3-hexen-1-ol, trans-2-hexen-1-ol and 1-hexanol) ranged from 8.1 x 109 to 8.1 x
1011 molecules/cm3 and for aldehydic ‖green‖ odors (cis-3-hexenal, trans-3-hexenal, trans-2-hexenal and n-hexanal) , from 8.1 x
107 to 8.1 x 1011 molecules/ cm3 . Detection threshold values of ―green‖ odor with double bond ranged from 8.1 x 10 7 to 8.1 x 1011
molecules/cm3 and for ―green‖ odor without double bond ranged from 8.1 x 10 8 to 8.1 x 1011 molecules/cm3. Detection threshold
value of cis- configured ―green‖ odors ranged from 8.1 x 108 to 8.1 x 1011 molecules/ cm3 and for trans- configured ―green‖ odors
threshold value ranged from 8.1 x 107 to 8.1 x 1011 molecules/ cm3. Trans-2-hexenal with a double bond at C-2 position in its
molecular structure yielded the lowest detection threshold value when compared the other ―green‖ odors (8.1 x 10 7 to 8.1 x 109
molecules /cm3) which shows not only the presence of double bond plays a major role in detection but the position of the double
bond present. A comparison between the present data and data from the other species showed that CD-1 mice displayed lower
detection thresholds for all ‖green‖ odors than human subjects and spider monkeys except for the cis-3-hexen-1-ol odor. These
findings suggest that the differences in the threshold values between ―green‖ odors are due to the difference in the molecular
structure like the presence of double bond and the position of double bond.
Nyckelord
Keywords:
CD-1 mice, ―green‖ odors, Odor sensitivity and detection threshold values.
Content
1 Abstract
1
2 Introduction
1
3 Materials and methods
3.1 Animals
2
3.2 Stimuli
2
3.3 Behavioral test
4
3.4 Experimental procedure
5
3.4.1 Determination of olfactory detection thresholds
7
3.5 Analysis of data
7
4 Results
4.1 1-Hexanol
8
4.2 n-hexanal
9
4.3 Cis-3-hexen-1-ol
10
4.4 Cis-3-hexenal
11
4.5 Trans-3-hexen-1-ol
12
4.6 Trans-3-hexenal
13
4.7 Trans-2-hexen-1-ol
14
4.8 Trans-2-hexenal
15
4.9 Inter- and intra individual variability
15
4.9.1 Inter individual variability
15
4.9.2 Intra individual variability
16
5 Discussion
5.1 Comparison of olfactory detection thresholds among odorants
17
5.1.2 Alcohols vs Aldehydes
17
5.1.3 Influence of presence or absence of a double bond
18
5.1.4 Position of the double bond
19
5.1.5 The effect of Cis- Trans configuration on detectability
20
5.2 Comparison with other species
21
5.3 Comparison with other odorants tested with CD-1 mice
22
5.4 Future Implications
23
5.5 Conclusion
23
6 Acknowledgements
23
7 Reference
24
1. Abstract:
―Green‖ odors comprise a group of eight structurally related aliphatic alkenals and alkenols
which are characteristic for the odor of a wide variety of plant materials. Using an automated
olfactometer, the olfactory detection thresholds for ―green‖ odors were determined in six CD-1
mice and compared with that of spider monkeys and human subjects. Detection threshold values
for alcoholic ‖green‖ odors (cis-3-hexen-1-ol, trans-3-hexen-1-ol, trans-2-hexen-1-ol and 1hexanol) ranged from 8.1 x 109 to 8.1 x 1011 molecules/cm3 and for aldehydic ‖green‖ odors (cis3-hexenal, trans-3-hexenal, trans-2-hexenal and n-hexanal) , from 8.1 x 107 to 8.1 x 1011
molecules/ cm3 . Detection threshold values of ―green‖ odor with double bond ranged from 8.1 x
107 to 8.1 x 1011 molecules/cm3 and for ―green‖ odor without double bond ranged from 8.1 x 108
to 8.1 x 1011 molecules/cm3. Detection threshold value of cis- configured ―green‖ odors ranged
from 8.1 x 108 to 8.1 x 1011 molecules/ cm3 and for trans- configured ―green‖ odors threshold
value ranged from 8.1 x 107 to 8.1 x 1011 molecules/ cm3. Trans-2-hexenal with a double bond at
C-2 position in its molecular structure yielded the lowest detection threshold value when
compared the other ―green‖ odors (8.1 x 107 to 8.1 x 109 molecules /cm3) which shows not only
the presence of double bond plays a major role in detection but the position of the double bond
present. A comparison between the present data and data from the other species showed that CD1 mice displayed lower detection thresholds for all ‖green‖ odors than human subjects and
spider monkeys except for the cis-3-hexen-1-ol odor. These findings suggest that the differences
in the threshold values between ―green‖ odors are due to the difference in the molecular structure
like the presence of double bond and the position of double bond.
Key Words:
CD-1 mice, “green” odors, Odor sensitivity and detection threshold values.
2. Introduction:
The mouse (Mus musculus) is one of the most commonly used model organisms in olfactory
research. A great deal of the current knowledge about the anatomy (Kosaka and Kosaka, 2005;
Zou et al., 2004), physiology (Wachowiak et al., 2004; Xu et al., 2003) development (Burd and
Tolbert, 2000; Mombaerts, 2006) and genetics of olfaction (Godfrey et al., 2004; Zhang and
Firestein, 2002) as well as the mechanisms underlying the neural coding of olfactory information
(Katada et al., 2005; Mori et al., 2006; Zou et al., 2005) has been obtained using the mouse as a
model species. A considerable number of electrophysiological studies (Döving, 1966, Ho et al.,
2003, Leon & Johnson, 2003, Johnson et al., 2004) and imaging studies (Xu et al., 2003, 2005)
on the sense of smell have been also performed in the mouse. Only few studies (Beauchamp and
Yamazaki, 2003; Schaefer et al., 2002; Wysocki et al., 2004; Yamazaki et al., 1994), in contrast,
have so far assessed olfactory performance in the mouse at the organismal level.
Recent genetic studies have demonstrated that mice have ~1194 functional genes coding for
olfactory receptors (Godfrey et al., 2004; Zhang and Firestein, 2002) whereas humans, for
example, express only ~390 olfactory receptor genes (Niimura and Nei, 2006) and spider
monkeys have ~900 functional olfactory receptor genes (Rouquier et al., 2000). These
1
differences in the number of functional olfactory receptor genes between species may be
indicative of between-species differences in olfactory performance.
Only little more than dozen substances have been tested with regard to the olfactory sensitivity
of Mus musculus (Passe and Walker, 1985; Walker and Jennings, 1991; Laska et al., 2006,
2009). To the best of my knowledge, no study with regard to the olfactory sensitivity on ―green‖
odors has been performed previously. ―Green‖ odors comprise a group of eight structurally
related aliphatic alkenals and alkenols which are characteristic for the odor of a wide variety of
plant materials. Thus, they should be of high behavioral relevance for herbivorous species, but
not necessarily for a granivorous species such as the mouse.
Olfactory detection threshold values for the same eight ―green‖ odors have been obtained in
humans (Van Gemert 2003) and spider monkeys earlier (Løtvedt 2011, unpublished). It is
therefore the aim of the present study
1. to determine olfactory detection thresholds in CD-1 mice for ―green‖ odors,
2. to assess the impact of molecular structural features on detectability of the odorants tested,
3. to compare the threshold data obtained here to those of other species tested previously on the
same set of odorants and to evaluate the impact of the number of functional olfactory receptor
genes on olfactory sensitivity.
3 Material & methods
3.1 Animals
Six male CD-1 mice (Mus musculus), 120-150 days old at the beginning of the study, were used
for the behavioral testing. The reason for choosing the CD-1 outbred strain of mice was to use
animals with a variable genetic background that is more similar to wild-type mice than that of
inbred strains. Furthermore, data on olfactory detection thresholds (Joshi et al., 2006, Laska et
al., 2006, 2009) and discrimination capabilities (Laska and Shepherd, 2007; Laska et al., 2008)
were obtained in earlier studies using the same mouse strain. Animals were kept in plastic rodent
cages measuring 41 x 25 x 15.5 cm. As an environmental enrichment for the mice, the cages
were supplied with toilet paper rolls and timber pieces. The animals were kept on a 12/12h lightdark schedule. Animals were supplied with food ad libitum. All animals were kept on a water
deprivation schedule of 1ml of water per day in order to ensure a high motivational status during
the experiments. Each individual received its daily ration (1 ml) of water partly as reinforcement
during the test trials and partly by being hand fed an additional amount of water after the test.
The animals were kept in accordance with the Swedish legislation on animal welfare and all
experiments reported here were approved by the local ethics committee.
3.2 Stimuli
Initially six odorants; amyl acetate, cineol, carvone, eugenol, anethole and limonene were used to
train the animals prior to the critical tests. These odorants have been shown to be easy to
discriminate for mice and do not share any functional group or other apparent similarity in
molecular structure with the odorants used for the critical testing. These odorants were also used
as easy training tasks in-between the critical tests in order to prevent that more challenging tasks
2
lead to extinction or to a decline in the animal’s motivation if the mouse failed to perform above
chance level.
―Green‖ odors comprise a group of structurally related aliphatic alkenals and alkenols which are
characteristic for the odor of a wide variety of plant materials. For the critical tests a set of eight
―green‖ odorants was used: 1-hexanol, n-hexanal, cis-3-hexen-1-ol, cis-3-hexenal, trans-3-hexen1-ol, trans-3-hexenal, trans-2-hexen-1-ol, trans-2-hexenal (Table 1). The rationale for choosing
these odors was that they share certain molecular features such as the aliphatic six-carbon
backbone, and differ from each other in molecular features such as the presence or absence of a
double bond, the position of a double bond, and the type of oxygen-containing functional group
(Table 1) (Figure 1). Additionally, comparative data on olfactory detection thresholds from other
species for these odorants are at hand. All substances were obtained from Sigma-Aldrich (St.
Louis, MO) and had a nominal purity of at least 99%. All odorants were diluted using nearodorless diethyl phthalate (Sigma-Aldrich) as the solvent.
Table 1. Odorants used in the critical tests with their molecular formula, CAS number and
nature.
Odorants
Molecular Formula
CAS #
Chemical class
n-hexanal
C6H12O
66-25-1
Aldehyde
1-hexanol
C6H14O
111-27-3
Alcohol
Cis-3-hexen-1-ol
C6H12O
928-96-1
Alcohol
Cis-3-hexenal
C6H10O
6789-80-6
Aldehyde
Trans-3-hexen-1-ol
C6H12O
928-97-2
Alcohol
Trans-3-hexenal
C6H10O
69112-21-6
Aldehyde
Trans-2-hexen-1-ol
C6H12O
928-95-0
Alcohol
Trans-2-hexenal
C6H10O
6728-26-3
Aldehyde
3
Figure 1: Chemical structures of the odorants used.
3.3 Behavioral test
Olfactory detection thresholds of the mice were determined using an automated olfactometer
(Knosys, Tampa, FL). An operant chamber was connected to the olfactometer via a sampling
port in which the odorants were presented. When inserting the nose into the sampling port
(making a nose poke) the mouse breaks a photo beam triggering the 2 s presentation of either an
odorant used as the rewarded stimulus (S+) or a different odorant used as the unrewarded
stimulus (S–). A final valve regulates the presentation of the odorant and the mouse has to keep
its head in the sampling port for ~0.1 s before the odorant is presented. As soon as the final valve
relaxes the odorant is presented. A correct response to the S+ consists of licking at a waterspout
in the sampling port, water reinforcement is presented by the opening of a reinforcement valve
for 0.04 s presenting a water droplet of 2.6 µl through the waterspout. For an incorrect response
(licking the waterspout in response to the presentation of the S-) no water reinforcement is
presented. The two seconds of odorant presentation are divided into ten 0.2 s intervals and the
mouse has to lick the waterspout during at least seven of these 0.2 s intervals for a response to be
4
registered as correct and thus rewarded. For all the critical tests, five to seven blocks (depending
on the time taken by the individuals to complete the blocks) of 20 trials each using a given
stimulus pair were conducted per animal and day. Each block consisted of 10 S+ and 10 Spresentations in pseudo randomized order.
Figure 2: Mouse managing odor port in operant chamber
Before the critical tests the animals underwent seven days of operant conditioning (Bodyak and
Slotnick, 1999). The begin program is composed of two stages: in the first stage the mouse learns
to insert its snout into the sampling port and is immediately rewarded when licking at the
waterspout even without an odorant present. In the second stage an odorant is presented and the
mouse is rewarded if staying in the sampling port and licking the waterspout for a longer period.
During these two stages the mouse acquires the knowledge to rely on an odorant for the
reinforcement, as well as increases its endurance to keep the head in the sampling port. The D2
program introduces the mouse to a two-odorant discrimination task in which two odorants are
presented in pseudorandomized order. Here the mouse learns to respond to the rewarded S+
odorant and to reject the unrewarded S- odorant. Licking the waterspout in response to the S+
(hit) as well as not licking the waterspout in response to the S- (correct rejection) odorant is
registered as a correct response. Licking the waterspout when presented with the S- odorant
(false alarm) as well as a no-response to the S+ odorant (miss) is registered as an incorrect
response.
5
3.4 Experimental procedure
With no prior knowledge of reinforcement and operant conditioning the mice were individually
taught to make a nose poke and to lick the waterspout for reinforcement. At the beginning of this
operant conditioning the animals had then been on a water deprivation schedule for one week,
receiving 1 ml of water per day. The mice received the daily amount of water in a bulk so that a
thirst was built up over twenty-four hours. The training of the nose poke was achieved with the
first stage of the begin program in which the mouse received water immediately when licking the
waterspout. By alternating the time interval between the mouse making a nose poke and
receiving the reinforcement, this first stage also conditions the animal to keep its head inside the
sampling port and to keep on licking until the reward is presented. After 20 reinforcements the
mouse is introduced to the second stage in which an odorant is presented. In the present study,
amyl acetate was selected as the initial S+. In the second stage the time interval between
breaking the photo beam, eliciting the closure of the final valve and the presentation of the
reinforcement is increased gradually throughout the test (Table 2). With the increased time
interval the mouse learns to lick the waterspout continuously. The amount of reinforcement is
also increasing over time ranging from 80-140%, where 100% corresponds to 2.6 µl of water.
The amount of 2.6 µl was kept throughout the begin program as well as during the critical
testing. However, deviations from this schedule were allowed and individuals with low
motivation were given a slightly higher reinforcement. By increasing the reinforcement valve
opening time to 0.05 s the water droplet increased to 2.8 µl.
Table 2. In the second stage of the begin program the closing time of the final valve is increased
throughout the blocks (of 20 trials each) resulting in a gradual increase of the time between the
breaking of the photo beam and the odor presentation. The amount of reinforcement is also
increased in accordance with the final valve time ranging from 80-140% where 100%
corresponds to 2.6 µl of water.
Block 1
1
2
3
4
5
6
7
Final valve Time
0
0.3
0.6
0.9
1.0
1.2
1.2
% Reinforcement
80
80
80
100
120
130
140
After stage 2, animals were trained to discriminate between two stimuli using the
odorants amyl acetate, carvone and limonene as S+ and cineol, eugenol and anethole as S- as
shown in table 2. Each animal received two days of training with each stimulus combination.
After two days of training with a given stimulus pair, the animals were subjected to a change,
either an positive transfer (Change of S+ and keeping the S- the same) or an negative transfer
(Change of S- and keeping the S+ the same) as shown in table 3. In this way all animals acquired
the knowledge that the stimulus combination will be changed during the course of critical tests.
6
Table 3: Stimulus combinations used prior to the critical tests for training the mice.
S+ Stimulus
S- Stimulus
Type of Transfer
Amyl Acetate
Cineol
Initial Odor Pair
Carvone
Cineol
First Positive Transfer
Carvone
Anethole
First Negative Transfer
Limonene
Anethole
Second Positive Transfer
Limonene
Eugenol
Second Negative Transfer
3.4.1 Determination of olfactory detection thresholds
―Green‖ odors with different concentrations were used as S+ and near-odorless diethyl phthalate
was used as the S- in all critical tests. Starting with a gas phase concentration of 1 ppm, each S+
was presented in 10-fold dilution steps for a total of 40 trials until an animal failed to
significantly discriminate the odorant from the solvent (diethyl phthalate). Subsequently, an
intermediate concentration (0.5 log units between the lowest concentration that was detected
above chance and the first concentration that was not) was tested in order to determine the
threshold value more exactly.
3.5. Data Analysis
For each individual animal, the percentage of correct choices from 40 decisions per dilution step
was calculated. Correct choices consisted both of licking in response to presentation of the S+
and not licking in response to the S–, and errors consisted of animals showing the reverse pattern
of operant responses. An individual was considered to have mastered a given stimulus
concentration when it scored an average of at least 75% correct in two consecutive blocks which
corresponds to 30 correct decisions out of a total of 40 decisions.
Significance levels were determined by calculating binomial z-scores corrected for continuity
from the number of correct and false responses for each individual and condition. Wilcoxonsigned rank test was also performed to find the level of significance. All tests were two-tailed,
and the alpha level was set at 0.01.
7
4 Results
4.1 1-hexanol
Figure 3 shows the performance of the six CD-1 mice in discriminating between various
dilutions of 1-hexanol and the solvent. Five out of six mice (M1, M3, M4, M5 and M6)
significantly distinguished dilutions as low as 1:540,000 from the solvent. One animal (M2)
significantly distinguished dilutions as low as 1:54,000 from the solvent.
Figure 3 Performance of CD-1 mice in discriminating between various dilutions of 1-Hexanol
and the solvent. Each data point represents the percentage of correct choices out of 40 trials. The
six different symbols represent data from each of the six animals tested. M1: Circle; M2: Square;
M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled symbols
indicate dilutions that are not significantly discriminated above chance level (Two- tailed
binomial test, p> 0.01).
8
4.2 n-Hexanal
Figure 4 shows the performance of the six CD-1 mice in discriminating between various
dilutions of n-Hexanal and the solvent. Two animals (M4 and M5) significantly distinguished
dilutions as low as 1:12,600,000 from the solvent. Four animals (M1, M2, M3 and M6)
significantly distinguished dilutions as low as 1:126,000,000 from the solvent.
Figure 4 Performance of CD-1 mice in discriminating between various dilutions of n-Hexanal
and the solvent. Each data point represents the percentage of correct choices out of 40 trials. The
six different symbols represent data from each of the six animals tested. M1: Circle; M2: Square;
M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled symbols
indicate dilutions that are not significantly discriminated above chance level (Two- tailed
binomial test, p>0.01) .
9
4.3 Cis-3-Hexen-1-ol
Figure 5 shows the performance of the CD-1 mice in discriminating between various dilutions of
cis-3-hexen-1-ol and the solvent. Two animals (M1 and M3) significantly distinguished dilutions
as low as 1:48,000 from the solvent. The four other animals (M2, M4, M5 and M6) significantly
distinguished dilutions as low as 1:480,000 from the solvent.
Figure 5 Performance of CD-1 mice in discriminating between various dilutions of cis-3-hexen1-ol and the solvent. Each data point represents the percentage of correct choices out of 40 trials.
The six different symbols represent data from each of the six animals tested. M1: Circle; M2:
Square; M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled
symbols indicate dilutions that are not significantly discriminated above chance level (Twotailed binomial test, p> 0.01).
10
4.4 Cis-3-Hexenal
Figure 6 shows the performance of the six CD-1 mice in discriminating between various
dilutions of cis-3-hexenal and the solvent. Two animals (M1 and M6) significantly distinguished
dilutions as low as 1:147,000,000 from the solvent. Three animals (M2, M3 and M5)
significantly distinguished dilutions as low as 1:4,900,000 from the solvent. One mouse (M4)
significantly distinguished dilutions as low as 1:4,700,000 from the solvent.
Figure 6 Performance of CD-1 mice in discriminating between various dilutions of cis-3-hexenal
and the solvent. Each data point represents the percentage of correct choices out of 40 trials. The
six different symbols represent data from each of the six animals tested. M1: Circle; M2: Square;
M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled symbols
indicate dilutions that are not significantly discriminated above chance level (Two- tailed
binomial test, p> 0.01).
11
4.5 Trans-3-Hexen-1-ol
Figure 7 shows the performance of the six CD-1 mice in discriminating between various
dilutions of trans-3-hexen-1-ol and the solvent. All animals significantly distinguished dilutions
as low as 1:480,000 from the solvent.
Figure 7 Performance of CD-1 mice in discriminating between various dilutions of trans-3hexen-1-ol and the solvent. Each data point represents the percentage of correct choices out of 40
trials. The six different symbols represent data from each of the six animals tested. M1: Circle;
M2: Square; M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled
symbols indicate dilutions that are not significantly discriminated above chance level (Twotailed binomial test, p> 0.01).
12
4.6 Trans-3-Hexenal
Figure 8 shows the performance of the six CD-1 mice in discriminating between various
dilutions of trans-3-hexenal and the solvent. All animals except M3 significantly distinguished
dilutions as low as 1:1,470,000 where M3 significantly distinguished dilutions as low as
1:147,000 from the solvent.
Figure 8 Performance of CD-1 mice in discriminating between various dilutions of trans-3hexenal and the solvent. Each data point represents the percentage of correct choices out of 40
trials. The six different symbols represent data from each of the six animals tested. M1: Circle;
M2: Square; M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled
symbols indicate dilutions that are not significantly discriminated above chance level (Twotailed binomial test, p> 0.01).
13
4.7 Trans-2-Hexen-1-ol
Figure 9 shows the performance of the six CD-1 mice in discriminating between various
dilutions of trans-2-hexen-1-ol and the solvent. Four animals (M1, M2, M3 and M4) significantly
distinguished dilutions as low as 1:450,000 from the solvent. Two animals (M5 and M6)
significantly distinguished dilutions as low as 1:4,500,000 from the solvent.
Figure 9 Performance of CD-1 mice in discriminating between various dilutions of trans-2hexen-1-ol and the solvent. Each data point represents the percentage of correct choices out of 40
trials. The six different symbols represent data from each of the six animals tested. M1: Circle;
M2: Square; M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled
symbols indicate dilutions that are not significantly discriminated above chance level (Twotailed binomial test, p> 0.01).
14
4.8 Trans-2-Hexenal
Figure 10 shows the performance of the six CD-1 mice in discriminating between various
dilutions of trans-2-hexenal odor and the solvent. One animal (M6) significantly distinguished
dilutions as low as 1:450,000,000 from the solvent. All other animals significantly distinguished
dilutions as low as 1:4,500,000 from the solvent.
Figure 10 Performance of CD-1 mice in discriminating between various dilutions of trans-2hexenal and the solvent. Each data point represents the percentage of correct choices out of 40
trials. The six different symbols represent data from each of the six animals tested. M1: Circle;
M2: Square; M3: Upside Triangle; M4: Downside Triangle; M5: Diamond; M6: Hexagon. Filled
symbols indicate dilutions that are not significantly discriminated above chance level (Twotailed binomial test, p> 0.01).
4.9 Inter- and intra individual variability
4.9.1 Interindividual variability
With Five of the odorants (cis-3-hexen-1-ol, trans-2-hexen-1-ol, trans-3-hexenal, 1-hexanol and
1-hexanal) the difference in detection thresholds between the best scoring animal and the worst
scoring animal was A factor of 10. With the odorant trans-3-hexen-1-ol the difference was a
15
factor of 0. With the odorant cis-3-hexenal the difference was a factor of 33. With the odorant
trans-2-hexenal the difference was a factor of 100.
4.9.2 Intraindividual variability
When examining the individual performance of the mice, individual M6 was the best scoring
animals with all eight odorants. Individual M2 was the poorest scoring animals and scored
lowest values with five out of eight odorants. M3 and M1 mice shared the lowest threshold value
for the odor cis-3-hexenal. M1 to M5 scored the same threshold value for the odor trans-2hexenal while M6 scored lowest threshold value out of all eight odorants tested. M1, M2 and M3
mice scored their highest threshold values on cis-3-hexen-1-ol, 1-hexanol and trans-3-hexenal
respectively. For the odor trans-3-hexen-1-ol, all mice scored the same threshold value. M6 alone
displayed highest threshold with trans-2-hexenal. M1 and M2 scored the highest threshold value
for the odor cis-3-hexen-1-ol while other mice scored the same threshold value. M2 alone also
scored the highest threshold value for the odor 1-hexanol.
Table 4 summarizes the olfactory detection threshold dilutions of the mice for the eight odorants
and their respective conversion from liquid to gas phase concentrations. Since all odorants have
different vapor pressures, it is necessary to convert liquid to gas phase concentrations in order to
make a proper comparison. Further, these data allows the reader to compare the present threshold
data to those from other studies using one of these measures.
1-Hexanol
n-Hexanal
Cis-3-hexen-1-ol
Cis-3-hexenal
N
liquid dilution
molec./cm3
ppm
5
1:540,000
8.1 x 1010
0.003
-2.52
1.3 x 10-10
1
1:54,000
8.1 x 10
0.03
-1.52
1.3 x 10
-8.87
4
1:126,000,000
8.1 x 108
0.00003 -4.52
1.3 x 10-12
-11.87
2
1:12,600,000
8.1 x 10
0.0003
-3.52
1.3 x 10
-11
-10.87
4
1:480,000
8.1 x 1010
0.003
-2.52
1.3 x 10-10
-9.87
2
1:48,000
8.1 x 1011
0.03
-1.52
1.3 x 10-9
-8.87
3
1:4,900,000
2.7 x 109
0.0001
-4.00
4.5 x 10-12
-11.35
2
1:147,000,000
8.1 x 10
-12
-11.87
1
1:14,700,000
8.1 x 10
-11
-10.87
11
9
16
log ppm mol/l
log mol/l
-9
8
0.00003 -4.52
1.3 x 10
9
0.0003
1.3 x 10
-3.52
-9.87
Trans-3-hexen-1-ol
6
1:480,000
8.1 x 10
10
0.003
-2.52
1.3 x 10
-10
-9.87
Trans-3-hexenal
5
1:1,470,000
8.1 x 1010
0.003
-2.52
1.3 x 10-10
-9.87
1
1:147,000
8.1 x 1011
0.03
-1.52
1.3 x 10-9
-8.87
4
1:1:450,000
8.1 x 1010
0.003
-2.52
1.3 x 10-10
-9.87
2
1:4,500,000
8.1 x 10
9
0.0003
-3.52
1.3 x 10
5
1:4,500,000
8.1 x 10
9
0.0003
-3.52
1
1:450,000,000
8.1 x 107
0.000003 -5.52
Trans-2-hexen-1-ol
Trans-2-hexenal
-11
-10.87
1.3 x 10
-11
-10.87
1.3 x 10-13
-12.87
N= Number of individuals
Table 4 Olfactory detection threshold values in CD-1 mice for eight ―Green‖ odors, expressed in
various measures of vapor phase concentrations.
5. Discussion
5.1 Comparison of olfactory detection thresholds among odorants
The results of the current study show that the detection of ―green‖ odors by CD-1 mice varies
with the structure of the stimuli. Small changes in the molecular structure of odorants like the
presence or absence of double bonds and the position of the double bond can lead to marked
changes in detectability. Among the odorants tested trans-2-hexenal yielded the lowest threshold
value. N- hexanal and cis-3-hexenal yielded the second lowest threshold value which are closely
followed by trans-2-hexen-1-ol. Trans-3-hexen-1-ol, trans-2-hexenal, cis-3-hexen-1-ol and 1hexanol yielded the highest threshold value.
5.1.2 Alcohols vs Aldehydes
Figure 11 shows the comparison of olfactory detection threshold values between two classes of
―green‖ odors: alcohols and aldehydes. Threshold values of ―green‖ odors with alcohol
functional group (cis-3-hexen-1-ol, trans-3-hexen-1-ol, trans-2-hexen-1-ol and 1-hexanol) and
―green‖ odors with aldehyde functional group (cis-3-hexenal, trans-3-hexenal, trans-2-hexenal
and n-hexanal) have a notable difference. Threshold values of alcoholic ―green‖ odors range
from 8.1 x 109 to 8.1 x 1011 molecules/ cm3 while the threshold values of aldehyde ―green‖ odors
range from 8.1 x 107 to 8.1 x 1011 molecules/ cm3. Statistical analysis shows that the aldehyde
17
―green‖ odors scored significantly lower thresholds than alcohol ―green‖ odors (Wilcoxon signed
rank test; p< 0.01).
Figure 11 Comparison of olfactory detection threshold values for ―green‖ odors with an alcohol
functional group (cis-3-hexen-1-ol, trans-3-hexen-1-ol, trans-2-hexen-1-ol and 1-hexanol) and
―green‖ odors with an aldehyde functional group (cis-3-hexenal, trans-3-hexenal, trans-2-hexenal
and n-hexanal). Each symbol indicates the threshold values of individual animals.
5.1.3 Influence of presence or absence of a double bond
Figure 12 shows a comparison of olfactory detection threshold values of two classes of ―green‖
odors: odors with double bond (cis-3-hexenal, trans-3-hexenal, trans-2-hexenal, cis-3-hexen-1-ol,
trans-2-hexen-1-ol and trans-3-hexen-1-ol) and odors with no double bond (1-hexanol and nhexanal). The Odorants trans-3-hexenal, cis-3-hexen-1-ol and 1-hexanol share the highest
threshold value among the eight odorants tested and range from 8.1 x 1010 to 8.1 x 1011
molecules/cm3. The threshold values for n-hexanal and cis-3-hexenal fall within the same range
(8.1 x 108 to 8.1 x 109 molecules/cm3) .The only double bond odorant which scored the lowest
threshold value when compared to single bond odorants is trans-2-hexenal (8.1 x 107 to 8.1 x 109
molecules/cm3). Statistical analysis shows there is no statistical difference between the threshold
values of ―green‖ odors with double bond and without double bond (Wilcoxon signed rank test;
p> 0.01).
18
Figure 12 Comparison of olfactory detection threshold values of ―green‖ odors with double
bond (cis-3-hexenal, trans-3-hexenal, trans-2-hexenal, cis-3-hexen-1-ol, trans-2-hexen-1-ol and
trans-3-hexen-1-ol) and ―green‖ odors without double bond(1-hexanol and n-hexanal) in their
carbon backbone. Each symbol indicates the threshold values of individual animals.
5.1.4 Position of the double bond
Trans-2-hexenal yielded the lowest detection threshold value ranging from 8.1 x 107 to 8.1 x 109
molecules /cm3. n-hexanal and cis-3-hexenal scored the second lowest threshold value ranging
from 8.1 x 108 to 8.1 x 109 molecules /cm3 . The presence of a double bond in the C-2 position is
shared between only two of the odorants; trans-2-hexenal and trans-2-hexen-1-ol. Trans-2hexenal has an aldehyde functional group which makes it the odor with the lowest threshold
value of the eight odors tested. Even though cis-3-hexenal has a double bond like trans-2hexenal, due to the position of double in its molecular structure it has the second lowest
threshold value.
As can be seen from figure 13, Trans-2-hexenal has one double bond in the C-2 position which
is located nearer to the functional group, while cis-3-hexenal also has a double bond which is
located at C-3 position, far from the functional group when compared to trans-2-hexenal (Figure
1). Thus we can draw a conclusion that not only the presence of a double bond plays a role in
detection but also its position.
19
5.1.5 The effect of Cis- Trans configuration on detectability
Figure 13 shows a comparison of detection threshold values of two classes of ―green‖ odors: Cisconfiguration ―green‖ odors (Cis-3-hexen-1-ol and cis-3-hexenal) and Trans-configuration
―green‖ odors (Trans-2-hexen-1-ol, trans-3-hexen-1-ol, trans-2-hexenal and trans-3-hexenal).
The detection threshold values of cis-configuration ―green‖ odors fall in the range of 8.1 x 109 to
3
8.1 x 1011 molecules/ cm . The detection threshold values of trans-configuration ―green‖ odors
fall in the range of 8.1 x 107 to 8.1 x 1011 molecules/ cm3. Statistical analysis shows that there is no
significant difference between the threshold values of cis-configuration and trans-configuration
―green‖ odors (Wilcoxon signed rank test; p> 0.01).
Figure 13 Comparison of olfactory detection threshold values of cis-configuration ―green‖ odors
and trans-configuration ―green‖ odors. Each symbol indicates the threshold values of individual
animals.
5.2 Comparison with other species
Figure 14 shows a comparison of olfactory detection threshold value of six CD-1 mice, highest
and lowest threshold values of human subjects (Van Gemert 2003) and three spider monkeys
(Løtvedt 2011) for ―green‖ odors. Olfactory detection threshold values in spider monkeys for
―green‖ odors range from 4.8 x 1011 to 2.2 1013 molecules/ cm3 (Løtvedt 2011). Olfactory
20
threshold values in humans for ―green‖ odors range from 2.4 x 109 to 6.6 x 1013 molecules/ cm3
(Van Gemert 2003). It is evident from the figure that spider monkeys are less sensitive towards
all the ―green‖ odors when compared to CD-1 mice and humans. CD-1 mice are more sensitive
towards all the odors than humans and spider monkeys except for cis-3-hexen-1-ol. Humans are
more sensitive than mice for cis-3-hexen-1-ol for which their detection threshold value was 2.40
x 109 molecules / cm3. For the odor trans-3-hexen-1-ol, the range in the detection threshold value
is smaller between CD-1 mice, spider monkeys and humans, while the range in the detection
threshold values is much higher for n-hexanal, cis-3-hexenal and trans-2-hexenal odors between
these species.
Figure 14 Comparison of olfactory detection threshold values of six CD-1 mice (Circles),
highest and lowest detection threshold values of humans (Triangles) and three spider mokeys
(Squares). Six threshold values per odorant for CD-1 mice, three threshold values per odorant for
spider monkeys and highest and lowest threshold values for humans are represented in the table.
5.3 Comparison with other odorants tested with CD-1 mice
Figure 15 shows a comparison between olfactory detection threshold values of different odorant
classes which have been determined using CD-1 mice. The detection threshold values found here
21
for ―green‖ odors are in the same range as those found in previous studies with other classes of
odorants.
The detection threshold values obtained from CD-1 mice for aliphatic aldehydes (n-butanal, npentanal, n-hexanal, n-heptanal, n-octanal and n-nonanal) ranged from 10 x 108 to 10 x 1010
molecules/ cm3 (Laska et al., 2006). The threshold values obtained for alphatic aldehydes fall
within the range of the values obtained from the present study using ―green‖ odors (10 x 107 to
10 x 1011 molecules/ cm3). Similarly, the threshold values for alkyl pyrazines ( Laska et al.,
2009), terpenoids ( Joshi et al., 2006), amino acids (Wallen, 2010) and aromatic aldehydes
(Larsson, 2010) were 2.5 x 107 to 7.5 x 1011 molecules/ cm3, 2.5 x 108 to 7.5 x 1010 molecules/
cm3, 1.2 x 108 to 1.8 x 1012 molecules/ cm3 and 1 x 108 to 7.5 x 1012 molecules/ cm3,
respectively. It is evident from the figure that the threshold values of all odorants approximately
ranged between 107 and 1012 molecules/cm3 with one aromatic aldehyde (Bourgeonal) as an
exception which is detected at 103 molecules/ cm3. From figure 15, it is also evident that the
range in the threshold values of ―green‖ odors (108 to 1012 molecules/ cm3) is almost similar to
that of other classes of odorants except aliphatic aldehydes which have a smaller range (108 to
1010 molecules/ cm3).
Figure 15. Comparison of olfactory detection threshold values for different odorant classes
obtained using CD-1 mice. Each symbol represents the average threshold values obtained from
22
different odorants using CD-1 mice. (Laska et al., 2006; Laska et al., 2009; Joshi et al., 2006;
Wallén, 2010).
5.4 Future Implications
Future studies on olfactory detection thresholds with regard to the presence or absence of a
double bond and position of double bond can lead to a generalized concept whether these
changes in the molecular structure have an effect on discrimination performance in CD-1 mice.
5.5 Conclusion
As shown in the results, CD-1 mice have low detection thresholds for ―green‖ odors except cis3-hexen-1-ol when compared to human subjects and spider monkeys. The mice were more
sensitive for alcohol ―green‖ odors than the aldehyde ―green‖ odors.
6. Acknowledgement
I would like to thank my supervisor Professor Matthias Laska for his excellent supervision and
guidance throughout this thesis. I also want to thank the animal caretakers in the animal facility
at University hospital of Linkoping for their patience and kindness for helping me during my
entire thesis period.
23
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