olfactory sensitivity in cd-1 mice for six l- and d-amino...

Department of Physics, Chemistry and Biology

Final Thesis

Olfactory sensitivity in CD-1 mice

for six L- and D-amino acids

Helena Wallén

LiTH-IFM- Ex--2351--SE

Supervisor: Matthias Laska, Linköpings universitet

Examiner: Per Jensen, Linköpings universitet

Department of Physics, Chemistry and Biology

Linköpings universitet

SE-581 83 Linköping, Sweden

Rapporttyp

Report category

Licentiatavhandling

x Examensarbete

C-uppsats

x D-uppsats

Övrig rapport

_______________

Språk

Language

Svenska/Swedish

x Engelska/English

________________

Titel

Title:

Olfactory sensitivity in CD-1 mice for six L- and D- amino acids.

Författare

Author: Helena Wallén

Sammanfattning Abstract:

The olfactory sensitivity of five male CD-1 mice (Mus musculus) for six amino acids was determined using an operant conditioning paradigm. All

animals significantly distinguished dilutions as low as 0.01 mM L-cysteine, 3.3 mM L-methionine, 10 mM L-proline, 0.03 mM D-cysteine, 0.3

mM D-methionine and 10 mM D-proline from the odorless solvent, with individual animals displaying even lower detection thresholds. Among

the three different L-forms of the amino acids the mice were most sensitive for cysteine and least sensitive for proline, and among the three D-

forms the animals displayed a lower sensitivity for D-proline compared to D-cysteine and D-methionine. A comparison between the present data

and results obtained with other species showed that the CD-1 mice displayed a higher sensitivity than human subjects and spider monkeys with

three (L-Cysteine, D-cysteine and L-proline) of the six amino acids. Results from this report support the idea that the number of functional

olfactory receptor genes is not suitable to predict a species’ olfactory sensitivity.

ISBN

LITH-IFM-EX—05/2351—SE

__________________________________________________

ISRN

__________________________________________________

Serietitel och serienummer ISSN

Title of series, numbering

Handledare

Supervisor: Matthias Laska

Ort

Location: Linköping

Nyckelord Keyword:

Amino acid, Olfaction, CD-1 mice, olfactory receptor genes and sulphur- containing group.

Datum

Date

2010-06-04

URL för elektronisk version

Avdelning, Institution

Division, Department

Avdelningen för biologi

Instutitionen för fysik och mätteknik

Contents

1 Abstract ........................................................................................................................ 1

2 Introduction .................................................................................................................. 1 3 Materials and methods .................................................................................................. 3

3.1 Animals ..................................................................................................................... 3 3.2 Odorants .................................................................................................................... 4

3.3 Behavioral test ........................................................................................................... 5 3.3.1 Begin program .................................................................................................... 6

3.3.2 D2 program......................................................................................................... 6 3.4 Experimental procedure ............................................................................................. 7

3.4.1 Two-odorant discrimination ................................................................................ 7 3.4.2 Determination of olfactory detection thresholds .................................................. 8

3.5 Data analysis ............................................................................................................. 8 4 Results ......................................................................................................................... 9

4.1 L-cysteine .................................................................................................................. 9 4.2 L-methionine ........................................................................................................... 10

4.3 L-proline ................................................................................................................. 11 4.4 D-cysteine ............................................................................................................... 12

4.5 D-methionine........................................................................................................... 13 4.6 D-proline ................................................................................................................. 14

4.7 Inter- and intra-individual variability ....................................................................... 15 4.8 Threshold dilutions .................................................................................................. 15

5 Discussion .................................................................................................................. 17 5.1 Comparison of olfactory detection thresholds among the six amino acids ................ 17

5.2 Comparison with other species ................................................................................ 17 5.3 Comparison of olfactory sensitivity for other odorants tested with CD-1 mice ......... 19

5.4 Odor structure- activity relationships ....................................................................... 21 5.4.1 Chirality............................................................................................................ 21

5.4.2 Receptor property ............................................................................................. 22 6 Acknowledgements .................................................................................................... 23 7 References .................................................................................................................. 24

1

1 Abstract

The olfactory sensitivity of five male CD-1 mice (Mus musculus) for six amino acids was

determined using an operant conditioning paradigm. All animals significantly

distinguished dilutions as low as 0.01 mM L-cysteine, 3.3 mM L-methionine, 10 mM L-

proline, 0.03 mM D-cysteine, 0.3 mM D-methionine and 10 mM D-proline from the

odorless solvent, with individual animals displaying even lower detection thresholds.

Among the three different L-forms of the amino acids the mice were most sensitive for

cysteine and least sensitive for proline and among the three D-forms the animals

displayed a lower sensitivity for D-proline, compared to D-cysteine and D-methionine. A

comparison between the present data and results obtained with other species showed that

the CD-1 mice displayed a higher sensitivity than human subjects and spider monkeys

with three (L-Cysteine, D-cysteine and L-proline) of the six amino acids. Results from

this report support the idea that the number of functional olfactory receptor genes is not

suitable to predict a species’ olfactory sensitivity.

Keyword: Amino acid, Olfaction, CD-1 mice, olfactory receptor genes and sulphur-

containing group

2 Introduction

The mouse is widely used as a model organism in olfactory research. However, only a

few studies, other than regarding body-borne odors, (Beauchamp and Yamazaki, 2003;

Schaefer et al., 2002; Wysocki et al., 2004; Yamazaki et al., 1994), have been conducted

with regard to olfaction at organismal level in the mouse. To my current knowledge, no

study has determined olfactory detection thresholds for amino acids in mice.

In vertebrates the largest gene superfamily is the one containing the olfactory receptor

genes (Zhang and Firestein, 2002). In the olfactory receptor gene family each gene codes

for one olfactory receptor type, while each olfactory receptor itself can bind several

different odorants. This allows the olfactory system to detect a large range of olfactory

stimuli (Gaillard et al., 2004; Buck, 2004). When volatile odor molecules bind to

olfactory receptors, located in the olfactory epithelium in the nose, the signal is

transferred to the olfactory bulb and subsequently to the olfactory sensory area in the

brain (Mori et al., 2006). In 2004, Godfrey et al. identified 913 intact olfactory receptor

genes in the mouse genome located to 51 loci on 17 chromosomes. The number of intact

olfactory receptor genes in humans is 388 (Niimura and Nei, 2006). After a comparison

between human olfactory receptor gene subfamilies with the corresponding ones in the

3

mouse, Godfrey et al. found that the two species recognize many of the same structural

motifs of the odorants; however the mouse could yet be superior in odor discrimination

and sensitivity.

Amino acids constitute the building blocks of proteins that are present in all living

materials on earth. They can evoke specific taste sensations (Schiffman et al., 1981) and

subsequently contribute to the flavor of food (Kirimura et al., 1969). Amino acids have

also been shown to play an important role in chemical communication and foraging in

fish (Knutsen, 1992; Fishelson, 1995). Amino acids can exist as two forms of optical

isomers (L or D), with glycine as the only exception. The amino acids found mainly in

protein are the L form, but the D form can be found in proteins of some sea species as

well as in bacterial cell walls (Campbell and Farrell, 2006; McMurry, 2008). However,

the knowledge about the role of amino acids as olfactory stimuli is limited. Recently

Laska (2010) conducted a study where he examined human olfactory threshold for a set

of six amino acids. Laska concluded that the odor from four of the six amino acids where

detected at lower threshold concentrations compared to the corresponding taste threshold

concentrations. These results indicate that amino acids are perceived both ortho- or

retronasally by the sense of smell as well as by the sense of taste and may thus contribute

to the flavor of food.

Since no study so far has investigated olfactory detection thresholds for amino acids in

mice it is therefore the aim of this project to obtain first data on the olfactory sensitivity

of mice for amino acids, with the following specific aims: 1) to determine olfactory

detection thresholds of the mouse for six amino acids, 2) to compare threshold data

obtained here to those of other species tested previously on the same set of odorants 3) to

evaluate the impact of the number of functional olfactory receptor genes on olfactory

sensitivity and 4) to assess the impact of chirality and other molecular structural features

on detectability of the amino acids.

In order to clarify some of the terms used in this report, I have explained below two (in

this report frequently used) words that could be otherwise confusing: detection; the

ability of a subject to actively detect an odor (to smell it) and discrimination; the ability

of a subject to actively being able to tell the difference between two different odors.

3 Materials and methods

3.1 Animals

Five male CD-1 mice (Mus musculus) were used in the present study. They were between

120-150 days old at the beginning of the experiments. The rationale for choosing the CD-

1 outbred strain was to use animals with a variable genetic background that is more

similar to wild-type mice than that of inbred strains. Further, there is also data available

regarding olfactory detection thresholds (Joshi et al., 2006, Laska et al., 2006, 2009) and

discrimination capabilities (Laska and Shepherd, 2007; Laska et al., 2008) from the same

mouse strain. The animals were kept in standard plastic rodent cages measuring 41 x 25 x

4

15.5 cm in the animal facility at the University Hospital of Linköping. The interior of the

cages included wood shavings, nesting materials and additional environmental

enrichment. The enrichment consisted of two toilet paper rolls and occasionally a ketchup

cup from McDonald`s®. The animals were kept on a 12/12 hour light-dark schedule.

In order to maintain a high motivational state throughout the behavioral testing the

animals were deprived from ad lib water access and kept on a daily controlled water

intake totaling 1.5 ml. Each individual received water partly as reinforcement during the

behavioral testing and partly by being hand fed an additional amount of water directly

after the daily testing. The mice had ad lib access to SDS food pellets (CRM rodents).

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.

Figure 1 One of the CD-1 mice used in the study.

3.2 Odorants

For the determination of olfactory detection thresholds a set of six odorants was used:

L-cysteine, D-cysteine, L-methionine, D-methionine, L-proline, and D-proline (Figure 2).

These substances were chosen because information on taste detection thresholds in the

mouse as well as on olfactory and taste detection thresholds from humans and spider

monkeys are at hand allowing me to compare the performance of the mice to that of other

species. Further, the use of the L- and D-forms of a given amino acid allowed me to

additionally assess the impact of chirality on olfactory detectability.

Amino acids contains both an amino group (-NH2) and carboxyl group (-COOH).

Cysteine is a polar amino acid with a sulfur-containing (thiol) group. Methionine has also

a sulfur-containing functional group, a thioether group. In contrast, Proline is lacking a

sulfur-containing group. Proline is also called an imino acid since it lacks a primary

amino group, in stead it contains a secondary amine group (imino; >C=NH) and a

carboxyl group (-C(=O)-OH). Both methionine and proline are non polar amino acids

(Figure 2). (Campbell and Farrell, 2006; McMurry, 2008).

5

L-methionine

OH

O

H2N

SH

D-methionine

OH

O

H2N

SH

D-cysteine

O

OH

H2N

SHH

L-cysteine

O

OH

H2N

SHH

D-proline

OH

O

N H

H

L-proline

OH

O

N H

H

Figure 2 Molecular structure of the six amino acids used.

All substances were obtained from Sigma-Aldrich (St. Louis, MO) and had a nominal

purity of at least 99%. They were diluted using demineralised water.

Additionally, the odorants amyl acetate, cineol, carvone, and limonene were used both

for training the animals prior to the critical tests and also as easy training tasks in

between the critical tests in order to prevent the more challenging conditions leading to

extinction or to a decline in the animal´s motivation. These odorants were diluted using

odourless diethyl phthalate (Sigma-Aldrich) as the solvent. They were presented at a gas

phase concentration of 1 ppm (parts per million).

3.3 Behavioral test

Olfactory detection thresholds were determined using an automated olfactometer

(Knosys, Tampa, FL). The olfactometer consisted of an operant chamber connected to an

odorant-delivery unit controlled by a computer. The odorants were presented in a

continuous air stream via an odor port. A mouse was placed in the operant chamber prior

to the begining of the test. When the mouse puts its nose in the sampling port it breaks a

photo beam that triggers a 2 s presentation of either an odorant used as the rewarded

stimulus (S+) or a different odorant used as the unrewarded stimulus (S-). The

presentation of the odorants is regulated by a final valve and the mouse has to keep its

head in the sampling port for ~0.1 s before the odorant is presented. The final valve

relaxes directly after the 0.1 s, if the mouse successfully stayed in the sampling port,

presenting the odorant. A correct response to the S+ stimulus consists of licking at a

6

waterspout in the sampling port, which opens a reinforcement valve for 0.04 s presenting

a water droplet of 2.5 µl through the waterspout. If the mouse licks at the waterspout

when the S- is presented the trial is regarded as incorrect and no reinforcement will be

presented. The 2 s odorant presentation is divided into ten 0.2 s intervals. With the S+

stimulus the mouse has to lick the waterspout for at least seven of these ten intervals

before the response is regarded as correct and thus rewarded. The opposite is true for S-,

licking fewer than seven of the ten intervals is regarded as correct (Figure 3).

Figure 3 Mouse managing odour port in operant chamber

All mice underwent 3 days of a Begin program and then 10 days of a D2 program

(described further down) prior to the determination of olfactory detection thresholds.

3.3.1 Begin program

The Begin program is divided in two stages. The first stage is shaping, in which the

mouse learns to put its head into the sampling port and lick at a waterspout (licking will

be rewarded every time). In the second stage the mouse learns to build an association

between the presentation of an odorant and licking at the waterspout. Stage two is

subdivided into 6 blocks each containing 20 trials. In the first block the odor is

immediately presented when the mouse puts its head in the sampling port (makes a nose

poke), while in the following five blocks, the mouse has to keep its head in the sampling

port for 0.3, 0.6, 0.9, 1.0 and 1.2 s before the odour is presented. At the same time the

reinforcement volume was step-wise increased (Table 1) so that the mice learned to

continously lick at the waterspout when presented with a S+ stimuli.

Table 1 Reinforcement in % during stage two of the begin program where 2.5 microliters

of water equals a reinforcemnt of 100% .

Final valve time 0 0.3 0.6 0.9 1.0 1.2 1.2

Reinforcement % 80 80 80 100 120 130 140

3.3.2 D2 program

The D2 program introduces the mouse to a two-odorant discrimination task in which two

odorants are presented in pseudo randomized order. In this program the mouse learns to

lick in response to a rewarded odorant (S+) and not to lick in response to a non-rewarded

7

odorant (S-). Licking at the waterspout when presented with a S+ (hit) as well as not

licking at the waterspout when presented with a S- (correct rejection) is regarded as

correct responses. Not licking at the waterspout when presented with a S+ (miss) as well

as licking at the waterspout when presented with a S- (false alarm) is regarded as

incorrect response.

Figure 4 a) Overview of the olfactometer used in the present study. b) Mouse inside the

operant chamber with its head inserted in the sampling port.

3.4 Experimental procedure

3.4.1 Two-odorant discrimination

The D2 program was used both in the training of the animals and also in the critical

threshold determination tests. During the training step four odorants were used and the

test combinations were as follows: amyl acetate (S+) versus cineole (S-), carvone (S+)

versus cineole (S-), carvone (S+) versus anethol (S-), limonene (S+) versus anethol (S-)

and finally limonene (S+) versus eugenol (S-) (Table 2). Each odor pair was presented for

two sessions (a total of ten days) before the critical threshold determination tests took

place. Each session comprised five to six blocks of 20 odorant presentations each.

Table 2 The odor pairs used in the training stage.

S+ S-

Amyl acetat cineol

(-)-carvone cineol First positive transfer

(-)-carvone anethol First negative transfer

(+)-limonene anethol Second positive transfer

(+)-limonene eugenol Second negative transfer

a)

a)

)

b)

8

3.4.2 Determination of olfactory detection thresholds

The L-forms of the amino acids were tested first, in the following order: L-cysteine, L-

methionine and L-proline and the same order was subsequently used for the D-forms. The

substances were diluted with demineralised water to a concentration of 100 mM and then

further diluted in steps of ten (to 10 mM, 1 mM, 0.1 mM, etc.) until the animal failed to

discriminate the odorant from the solvent. After that 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 to determine the detection threshold value more

exactly. The odorants described above were used as S+ and demineralised water was used

as S- (blank). Since it is difficult for the mice to learn not to lick when there is no odorant

present (blank) the animals were presented with the 100 mM dilution versus the blank for

at least three days until the criterion of 75 % correct decisions was achieved in two

consecutive blocks of 20 trials. The same criterion was also used in the critical threshold

determination tests. In order to reduce the possibility that the animal learned the water

cue (the “smell” of water) two blanks were used and each blank was only used for two

consecutive blocks before the blanks were switched. Each amino acid was used for a

maximum of three days before new solutions were prepared.

3.5 Data analysis

For each animal, the percentage of correct choices from 40 decisions, from two

consecutive blocks of 20 trials, 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.

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. All tests were two-tailed, and the alpha level was set at 0.05. This corresponds

to at least 30 out of 40 correct decisions which equal 75 % correct.

9

4 Results

4.1 L-cysteine

Figure 5 shows the performance of the CD-1 mice in discriminating between various

dilutions of L-cysteine and the odorless solvent. Three animals, M3, M4 and M6,

significantly distinguished dilutions as low as 0.003 mM from the solvent. The other two

animals, M2 and M5, significantly distinguished dilutions as low as 0.01 mM from the

solvent.

Figure 5 Performance of the CD-1 mice in discriminating between various dilutions of L-

cysteine and the solvent (demineralised water). Each data point represents the

percentage of correct choices from a total of 40 decisions. The five different symbols

represent data from each of the five animals tested. M2: small circle; M3: box; M4:

triangle; M5: circle and M6: diamond. Filled symbols indicate dilutions that were not

discriminated significantly above chance level (binomial test, P<0.05).

10

4.2 L-methionine


dilutions of L-methionine and the odorless solvent. One animal, M6, significantly

distinguished dilutions as low as 0.03 mM from the solvent. M4 significantly

distinguished dilutions as low as 0.1 mM. M2 and M3 both managed to significantly

distinguish dilutions as low as 1.0 mM from the solvent. M5 significantly distinguished

dilutions as low as 3.3 mM from the solvent.


methionine and the solvent (demineralised water). Each data point represents the





11

4.3 L-proline


dilutions of L-proline and the odorless solvent. All five test animals significantly

distinguished dilutions of 10 mM from the solvent.


proline and the solvent (demineralised water). Each data point represents the percentage

of correct choices from a total of 40 decisions. The five different symbols represent data

from each of the five animals tested. M2: small circle; M3: box; M4: triangle; M5: circle

and M6: diamond. Filled symbols indicate dilutions that were not discriminated

significantly above chance level (binomial test, P<0.05).

12

4.4 D-cysteine


dilutions of D-cysteine and the odorless solvent. Two animals, M2 and M3, significantly

distinguished dilutions as low as 0.001 mM from the solvent. Both M5 and M6 managed

to distinguish dilutions as low as 0.003 mM from the solvent. M4 significantly

distinguished dilutions as low as 0.03 mM from the solvent.

Figure 8 Performance of the CD-1 mice in discriminating between various dilutions of

D-cysteine and the solvent (demineralised water). Each data point represents the





13

4.5 D-methionine


dilutions of D-methionine and the odorless solvent. Two animals, M4 and M6,

significantly distinguished dilutions as low as 0.01 mM from the solvent. M3

significantly distinguished dilutions as low as 0.1 mM from the solvent. Both M2 and M5

managed to significantly distinguish dilutions as low as 0.3 mM from the solvent.


D-methionine and the solvent (demineralised water). Each data point represents the





14

4.6 D-proline


dilutions of D-proline and the odorless solvent. One animal, M5, significantly

distinguished dilutions of 3.3 mM from the solvent. The other four animals, M2, M3, M4

and M6, significantly distinguished dilutions as low as 10 mM from the solvent.


D-proline and the solvent (demineralised water). Each data point represents the


represent data from each of the five animals tested M2: small circle; M3: box; M4:



15

4.7 Inter- and intra-individual variability

With the odorants L-cysteine and D-proline the difference in detection thresholds

between the best scoring animal and the poorest scoring animal was a factor of 3. With

the odorants D-cysteine and D-methionine the difference was a factor of 33. With L-

methionine the difference was a factor of 100. With the last amino acid, L-proline, the

difference was a factor of 0.

When examining the individual performance of the mice I found that all individuals were

among the best scoring with at least one odorant and among the poorest with another

odorant.

4.8 Threshold dilutions

Table 3 summarizes the threshold dilutions of the CD-1 mice and also gives

corresponding vapor phase concentrations (Weast, 1987). Conversion from liquid to

vapor phase concentrations is necessary to allow for proper comparisons of performance

across substances since they differ in their respective vapor pressures. Further, these data

allow for a comparison with data obtained in other studies using one of these measures.

In all cases, the liquid dilutions at threshold correspond to vapor phase concentrations of

≤0.1 ppm (parts per million). The best scoring animals were even able to detect a

concentration of ≤0.1 ppb (parts per billion) with four of the six stimuli.

16

Table 3 Olfactory detection threshold values for the six amino acids tested in CD-1

mice, expressed in various measures of vapor phase concentrations.

n

Liquid

dilution

(mM)

Vapor phase concentrations

Molecules/ cm³ ppm

Log

ppm mol/l

Log

mol/l

L-cysteine 2 0.4 1.2 x 10⁹ 0.00004 -4.4 2.0 ×10-¹² -11.7

3 0.13 3.6 x 10⁸ 0.00001 -4.9 6.0 × 10-¹³ -12.2

D-cysteine 1 1.33 3.6 x 10⁹ 0.0001 -3.9 6.0 × 10-¹² -11.2

2 0.13 3.6 x 10⁸ 0.00001 -4.9 6.0 × 10-¹³ -12.2

2 0.04 1.2 x 10⁸ 0.000004 -5.4 2.0 × 10-¹³ -12.7

L-methionine 1 133 3.2 x 10¹¹ 0.01 -1.9 5.3 × 10-¹⁰ -9.3

2 40 9.8 x 10¹⁰ 0.004 -2.4 1.6 × 10-¹⁰ -9.8

1 4.0 9.8 x 10⁹ 0.0004 -3.4 1.6 × 10-¹¹ -10.8

1 1.33 2.9 x 10⁹ 0.0001 -4.0 4.8 × 10-¹² -11.3

D-methionine 2 13.3 2.9 x 10¹¹ 0.01 -2.0 4.8 × 10-¹⁰ -9.3

1 4.0 9.8 x 10⁹ 0.0004 -3.4 1.6 × 10-¹¹ -10.8

2 0.4 9.8 x 10⁸ 0.00004 -4.4 1.6 × 10-¹² -11.8

L-proline 5 400 1.8 x 10¹² 0.07 -1.2 3.0 × 10-⁹ -8.5

D-proline 4 400 1.8 x 10¹² 0.07 -1.2 3.0 × 10-⁹ -8.5

1 133 6.1 x 10¹¹ 0.02 -1.6 1.0 × 10-⁹ -8.9

n indicates number of animals.

17

5 Discussion

5.1 Comparison of olfactory detection thresholds among the six amino acids

By comparing the olfactory detection thresholds of the six amino acids one can see a

pattern regarding the sensitivity displayed by the CD-1 mice. Among the three different

L-forms of the amino acids the mice were able to discriminate the odorant L-cysteine

from the solvent at the lowest vapor phase concentrations. Further, among the L-forms,

the animals showed least sensitivity for L-proline, placing L-methionine in between L-

cysteine and L-proline regarding odor sensitivity. The same pattern was observed with

the D-forms. With D-proline the best scoring animal was able to correctly discriminate a

higher vapor phase concentration of D-proline than D-cysteine and D-methionine,

indicating that the animals displayed a lower sensitivity for D-proline compared to D-

cysteine and D-methionine.

When comparing the threshold values for the individual animals for the L- and D- form

of a given amino acid, one can see that with methionine all five animals had lower

threshold values for D-methionine compared to L-methionine. With cysteine, however,

only three individual animals had lower threshold values for D-cysteine compared to L-

cysteine. One animal had the same threshold value for both L- and D- cysteine. The fifth

animal had a lower threshold value for L-cysteine compared to D-cysteine. With proline

four animals had the same threshold values for both the L- and D- form. One animal had

a lower threshold value for D-proline compared to L-proline.

When examining the threshold range among the amino acids one can see an overlapping

pattern of the threshold sensitivity between the L- and D- forms of the amino acids. The

odor sensitivity ranged from 109 to 10

8 (molecules/cm

3, Table 3) with L- and D-cysteine

and with proline the fluctuation was zero for L-proline and between 1012

and 1011

(molecules/cm3) for D-proline. The largest observed fluctuation was with D-methionine

where the vapor phase concentrations varied between 1011

and 108 compared to L-

methionine (between 1011

and 109). The mice seem to display a larger variation in

olfactory sensitivity for methionine compared to cysteine and proline.

5.2 Comparison with other species

Figure 11 shows a comparison of mean olfactory detection thresholds among spider

monkeys (Ateles geoffroyi) (Engström, 2010), human subjects (Homo sapiens) (Laska,

2010) and CD-1 mice for the six amino acids. One can observe that for both L- and D-

proline humans are less sensitive (that is: display higher thresholds) than both CD-1 mice

and spider monkeys. CD-1 mice displayed a higher sensitivity (that is: lower threshold)

than spider monkeys for L-proline. However for D-proline the pattern is reverse, with

spider monkeys showing a higher sensitivity than CD-1 mice (Figure 11). Spider

monkeys were least sensitive for the four amino acids, L-cysteine, D-cysteine, L-

methionine and D-methionine, compared with CD-1 mice and humans. For both L- and

D-methionine humans showed the highest sensitivity among the three species. For both

L- and D-cysteine the CD-1 mice displayed a higher sensitivity than both humans and

18

spider monkeys. The CD-1 mice displayed the highest sensitivity for three (L-Cysteine,

D-cysteine and L-proline) of the six amino acids tested compared to humans with two (L-

methionine and D-methionine) out of six and spider monkeys with one (D-proline) out of

six. Spider monkeys, in turn, showed the least sensitivity for four (L-cysteine, D-cysteine,

L-methionine and D-methionine) out of six amino acids compared to humans with two

(L-proline and D-proline) out of six and CD-1 mice who did not show least sensitivity for

any of the amino acids tested.

Figure 11 Mean olfactory detection thresholds for spider monkeys (n=3) (Engström,

2010), human (n=20) (Laska, 2010) and CD-1 mice (n=5) for six amino acids.

One possible determining factor of olfactory sensitivity is the number of functional

olfactory receptors genes. Niimura and Nei (2006) stated that the number of functional

olfactory receptor genes in mice is around 1000 compared to approximately 400 in

humans. The high number of olfactory receptor genes in the mice genome compared to

the human genome could explain why mice display a higher overall sensitivity for the

amino acids tested in this report (Niimura and Nei, 2006). Another explanation pointed

out by Shepherd (2004) could be that higher brain mechanisms may give humans an

advantage in olfactory capabilities not expected from the small number of functional

olfactory receptor genes. Since humans, in fact, have a higher olfactory sensitivity for

certain odorants than both dogs and rats this explanation seems plausible (Laska et al.,

2000; Shepherd, 2004). However, there are an increasing number of studies which

19

propose that it is not possible to predict the olfactory sensitivity of a species based on the

number of functional olfactory receptor genes (Laska, et al., 2005; Shepherd, 2004). The

number of functional olfactory receptor genes in spider monkeys is approximately 900

which seem to oppose the fact that spider monkeys displayed the least overall sensitivity

for the amino acids tested compared to humans or mice, since a higher number of

functional olfactory receptor genes should indicate better olfactory ability according to

Niimura and Nei (2006). It is therefore not possible in the present study to draw a

conclusion on species differences in olfactory sensitivity based on the number of

functional olfactory receptor genes as some authors suggest (Rouquier et al., 2000; Gilad

et al., 2004).

Another possible explanation for these differences in olfactory sensitivity could be

differences in the diet of the species. Fürst and Stehle (2004) stated that methionine is an

essential amino acid in the human diet whereas cysteine and proline are not. Among the

three species tested humans showed the highest sensitivity for both D-and L methionine

compared to the other two species. This could explain why humans were less sensitive

with cysteine and proline and more sensitive with methionine. Aside from that, humans

are in between CD-1 mice and spider monkeys regarding their sensitivity for the amino

acids tested which could be because proteins and corresponding free amino acids are a

main part of the human diet. Mice, on the other hand, are granivores in nature, but they

can adapt rapidly to new environmental settings. Mice are smaller creatures, with a low

body mass, and could therefore be more rapidly affected by starvation than larger animals

such as humans. It could therefore be more important for mice to be able to find food and

therefore have to depend on their olfactory ability in a broader spectrum (Niimura and

Nei, 2006). That could explain why CD-1 mice display a high sensitivity for several of

the amino acids in this report. The fact that spider monkeys show the least sensitivity for

several of the amino acids could also be supported by this idea. Spider monkeys eat

fruits, but can occasionally consume flowers, insects and leaves, a diet containing

comparatively few protein and thus few free amino acids. It could therefore be less

important for spider monkeys to be able to detect these amino acids at a low

concentration.

5.3 Comparison of olfactory sensitivity for other odorants tested with CD-1 mice

Olfactory detection threshold values for other odorants have also been determined in CD-

1 mice. The same cohort of mice used in this current study has also been tested with a set

of seven aromatic aldehydes (Larsson, 2010). When comparing vapor phase

concentrations one can see in figure 12 that the odorants tested in the study by Larsson

have a vapor phase concentration at threshold ranging from 103 to 10

11 molecules/cm

3, in

contrast with the present study showing a vapor phase concentration ranging from 108 to

1012

molecules/ cm3 (Table 3). This could be an indication that the amino acids tested in

this report are detected at lower concentrations than the substances of Larsson’s study.

However, if we eliminate the substance that the animal showed the lowest sensitivity for

in Larsson’s (2010) study (bourgeonal) the other remaining substances have a similar

range in vapor phase concentration (108 to 10

11 molecules/cm

3) as displayed by the mice

with the amino acids tested in the present study.

20

Further, in Figure 12 one can see a comparison between CD-1 mouse threshold data for

aliphatic aldehydes, alkylpyrazines, aromatic aldehydes and amino acids (Laska et al.,

2009; Laska et al., 2006; Larsson, 2010). One can see that the threshold values from the

chemical classes vary roughly between 107 and 10

12 molecules/ cm

3 with the exception

from the aromatic aldehydes, where one of the chemicals is detected at around 103

molecules/ cm3. One can also see that the variation in the class of aliphatic aldehydes is

smaller (between 108 and 10

10 molecules/ cm

3) than in the other three classes.

Figure 12 Comparison between threshold data expressed in molecules/ cm

3 for aliphatic

aldehydes (n=3), alkylpyrazines (n=4), aromatic aldehydes (n=5) and amino acids (n=5)

obtained with CD-1 mice. The symbols indicate average threshold discrimination results

across individual animals ( Laska et al., 2009; Laska et al., 2006; Larsson, 2010).

21

5.4 Odor structure- activity relationships

5.4.1 Chirality

Both cysteine and methionine have sulfur-containing functional groups that are perceived

to have a foul odor, which is characteristic for putrefaction processes (Figure 13). Being

able to distinguish food that is rotten is important and could explain why the mice display

a lower olfactory detection threshold for both cysteine and methionine compared to

proline. This idea is supported by studies conducted with catfish (Caprio, 1977), the

hammerhead shark (Tricas et al., 2009) and the zebrafish (Michel and Lubomudrov,

1995) in which electrophysiological recordings showed that L- cysteine and L-

methionine stimulated olfactory receptors more effectively than L-proline. This idea is

also in line with results from a study evaluating the olfactory sensitivity among humans

for six amino acids (Laska, 2010). The results indicated that humans can detect the odors

of L- and D-cysteine and L- and D-methionine at significant lower vapor phase

concentrations compared to both forms of proline. However, it cannot be determined

whether the differences in olfactory detection depend on the presence or absence of a

sulphur-containing group. The amino group could also be a factor determining

detectability, because in proline the amino group is secondary (as an imino group)

whereas in both cysteine and methionine the group is primary. This could also be a

plausible explanation for the observed differences in detectability among the amino acids.

L-methionine

OH

O

H2N

SH

D-methionine

OH

O

H2N

SH

D-cysteine

O

OH

H2N

SHH

L-cysteine

O

OH

H2N

SHH

D-proline

OH

O

N H

H

L-proline

OH

O

N H

H

Figure 13 Chemical structure of the amino acids. The sulphur-containing group is

marked by a circle and the imino group is marked by a star.

It is known that some optical isomers display different odor qualities and also differ in

their odor intensity. Laska and Teubner (1999b) investigated the ability of human

subjects to distinguish between a set of 10 enantiomeric odor pairs. The results showed

that the test group as an entity could only discriminate three of the 10 odor pairs. There

was also a great variance between the individual test subjects in their discrimination

22

performances. The results could indicate that the presence of both enantiomeric forms in

the species odorous nature is important for one species odor discrimination ability for

these substances. It is also possible that prolonged or repeated exposure to the

enantiomers might lead to lower detection threshold values compared to short term or

rare exposure. Further, the discrimination ability of human subjects for enantiomeric odor

pairs could be substance-specific which support the idea that not all volatile enantiomers

have enantioselective molecular odor receptors.

Further, Joshi et al. (2006) assessed olfactory sensitivity of both mice and spider

monkeys regarding enantiomers of carvone and limonene. They also concluded that “the

effect of chirality on detectability of the enantiomers to be substance specific”. One

possible explanation is that the occurrence of the two chiral molecules in the environment

of the species may differ significantly between substances (Knudsen et al., 1993;

Kubeczka, 2002) which could lead to differences in expression patterns of chiral-specific

olfactory receptors previous mentioned. Therefore oxygen moiety, differences in carbon

chain length and the occurrence of chiral molecules in the environment of a species

probably play a key role in receptor binding and subsequently olfactory discrimination

ability. However, no conclusions can be drawn in this report regarding the impact of

chirality on detectability of the amino acids tested; therefore further studies have to be

performed on mice that regard the topic of chirality.

5.4.2 Receptor property

Several studies have found that amino acids constitute highly potent food-related

olfactory stimuli for fishes. Electrophysiological thresholds for amino acids in fishes vary

from micromolar to nanomolar concentrations (Sorensen and Caprio, 1998; Sutterlin and

Sutterlin, 1971; Suzuki and Tucker, 1971). Biochemical (Bruch and Rulli, 1988; Cagan

and Zeiger, 1978; Kalinoski et al., 1987; Rehnberg and Schreck, 1986) and

electrophysiological (Caprio and Byrd Jr., 1984; Caprio et al., 1989; Kang and Caprio,

1991; Ohno et al., 1984; Sveinsson and Hara, 1990) studies indicate the presence of

multiple olfactory receptor sites for amino acids. Further, studies in catfish indicate that

the olfactory receptor neurons either could have an inhibited or activated response to

amino acids (Kang and Caprio, 1995; Kang and Caprio, 1997). Support to this idea came

from Nikonov and Caprio (2007), who showed that amino acids indeed bind to receptors

in two different ways that can cause either an activation or an inhibition. The interaction

involves a three point binding of an amino acid to its binding pocket, where the

negatively charged carboxyl group and the positively charged amino group both bind to a

proximal binding pocket within the receptor and the amino acid side chain binds to a

distal binding pocket. This results in activation. However, another amino acid could

potentially also bind to the same receptor but since the amino acids differ in molecular

structure the other amino acid is likely to only bind with two of these three points in the

receptor, leading to inhibition. Further, amino acids are thought to be detected by four

types of olfactory receptors in freshwater fish (Caprio and Byrd, 1984; Friedrich and

Korsching, 1997). The four types of amino acid receptors are specific for amino acids

displaying basic, acidic, short and neutral and long and neutral side chains, respectively.

However, whether these binding properties also apply to receptor binding in mammals is

23

unknown. Therefore further studies have to be conducted on amino acids and olfactory

receptor binding regarding mammals before any more conclusions can be drawn.

Based on the findings in this report the next logical step would be to conduct a study

regarding the binding between amino acids and the olfactory receptors in mammals. It

would also be interesting to compare the olfactory sensitivity in fishes for L- and D-

forms of cysteine and L- and D- forms of methionine with the vapor phase concentrations

obtained from other species, in order to learn more about the intriguing olfactory

perception capability that amino acids hold.

Conclusion

Based on the results from this study I can conclude that mice are able to detect the six

amino acids tested. The mice also displayed the lowest olfactory threshold for three of the

amino acids when compared to human subjects and spider monkeys. I can also conclude

that a species’ number of olfactory receptor genes is not a good predictor for the species’

olfactory sensitivity.

6 Acknowledgements

I want to thank supervisor Matthias Laska for his patience and excellent advises that have

helped me tremendously in the making of this report. I also want to thank the animal

caretakers at the animal facility at the Universital Hospital of Linköping for their help

during my time there. Last my thank goes to the mice used in the experiments who made

this experiment possible.

24

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