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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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Licentiatavhandling

x Examensarbete

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Svenska/Swedish

x Engelska/English

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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 10

9 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 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 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.

ISBN

LITH-IFM-A-EX-11/2442-SE

__________________________________________________

ISRN

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Serietitel och serienummer ISSN

Title of series, numbering

Handledare

Supervisor: Matthias Laska

Ort

Location: Linköping

Nyckelord

Keywords:

CD-1 mice, ―green‖ odors, Odor sensitivity and detection threshold values.

Datum

Date

20110603

URL för elektronisk version

Avdelning, Institution

Division, Department

Avdelningen för biologi

Instutitionen för fysik och mätteknik

Avdelningen för biologi

Instutitionen för fysik och mätteknik

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

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 1-

hexanol) ranged from 8.1 x 109 to 8.1 x 10

11 molecules/cm

3 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 10

11

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 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 107

to 8.1 x 109 molecules /cm

3) 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.

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

http://chemse.oxfordjournals.org/content/29/2/101.full#ref-33

2

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 light-

dark 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

3

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-hexen-

1-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 near-

odorless 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

4

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

5

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 S-

presentations 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.

6

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.

7

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. Wilcoxon-

signed 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.

8

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).

9

4.2 n-Hexanal


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





binomial test, p>0.01) .

10

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-hexen-

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 (Two-

tailed binomial test, p> 0.01).

11

4.4 Cis-3-Hexenal


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)


Figure 6 Performance of CD-1 mice in discriminating between various dilutions of cis-3-hexenal





binomial test, p> 0.01).

12

4.5 Trans-3-Hexen-1-ol


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-3-

hexen-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



13

4.6 Trans-3-Hexenal


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.


hexenal and the solvent. Each data point represents the percentage of correct choices out of 40





14

4.7 Trans-2-Hexen-1-ol


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)



hexen-1-ol and the solvent. Each data point represents the percentage of correct choices out of 40





15

4.8 Trans-2-Hexenal


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.


hexenal and the solvent. Each data point represents the percentage of correct choices out of 40





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

16

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-2-

hexenal 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.

N liquid dilution molec./cm3 ppm log ppm mol/l log mol/l

1-Hexanol 5 1:540,000 8.1 x 1010

0.003 -2.52 1.3 x 10-10

-9.87

1 1:54,000 8.1 x 1011

0.03 -1.52 1.3 x 10-9

-8.87

n-Hexanal 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 109 0.0003 -3.52 1.3 x 10

-11 -10.87

Cis-3-hexen-1-ol 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

Cis-3-hexenal 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 108 0.00003 -4.52 1.3 x 10

-12 -11.87

1 1:14,700,000 8.1 x 109 0.0003 -3.52 1.3 x 10

-11 -10.87

17

Trans-3-hexen-1-ol 6 1:480,000 8.1 x 1010

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

Trans-2-hexen-1-ol 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 109 0.0003 -3.52 1.3 x 10

-11 -10.87

Trans-2-hexenal 5 1:4,500,000 8.1 x 109 0.0003 -3.52 1.3 x 10

-11 -10.87

1 1:450,000,000 8.1 x 107

0.000003 -5.52 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 1-

hexanol 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 10

11 molecules/ cm

3 while the threshold values of aldehyde ―green‖ odors

range from 8.1 x 107 to 8.1 x 10

11 molecules/ cm

3. Statistical analysis shows that the aldehyde

18

―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 n-

hexanal). 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/cm

3)

.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).

19

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-2-

hexenal 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-2-

hexenal, 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.

20

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: Cis-

configuration ―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

8.1 x 1011 molecules/ cm

3. The detection threshold values of trans-configuration ―green‖ odors

fall in the range of 8.1 x 107 to 8.1 x 10

11 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

21

threshold values in humans for ―green‖ odors range from 2.4 x 109 to 6.6 x 10

13 molecules/ cm

3

(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 / cm

3. 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

22

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, n-

pentanal, 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 10

11 molecules/ cm

3, 2.5 x 10

8 to 7.5 x 10

10 molecules/

cm3, 1.2 x 10

8 to 1.8 x 10

12 molecules/ cm

3 and 1 x 10

8 to 7.5 x 10

12 molecules/ cm

3,

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/ cm

3. From figure 15, it is also evident that the

range in the threshold values of ―green‖ odors (108 to 10

12 molecules/ cm

3) 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

23

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 cis-

3-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.

24

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