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
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
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
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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 cysteine, 3.3 mM 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
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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
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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 S H D-methionine OH O H2N S H D-cysteine O OH H2N SH H L-cysteine O OH H2N SH H 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
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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
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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) )
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3.4.2 Determination of olfactory detection thresholds
The forms of the amino acids were tested first, in the following order: 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.
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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
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4.2 L-methionine
Figure 6 shows the performance of the CD-1 mice in discriminating between various 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.
Figure 6 Performance of the CD-1 mice in discriminating between various dilutions of
L-methionine 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
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4.3 L-proline
Figure 7 shows the performance of the CD-1 mice in discriminating between various dilutions of L-proline and the odorless solvent. All five test animals significantly distinguished dilutions of 10 mM from the solvent.
Figure 7 Performance of the CD-1 mice in discriminating between various dilutions of
L-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).
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4.4 D-cysteine
Figure 8 shows the performance of the CD-1 mice in discriminating between various 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 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
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4.5 D-methionine
Figure 9 shows the performance of the CD-1 mice in discriminating between various 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.
Figure 9 Performance of the CD-1 mice in discriminating between various dilutions of
D-methionine 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
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4.6 D-proline
Figure 10 shows the performance of the CD-1 mice in discriminating between various 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.
Figure 10 Performance of the CD-1 mice in discriminating between various dilutions of
D-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
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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.
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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 dilution Liquid (mM)
Vapor phase concentrations
Molecules/ cm³ ppm ppm Log mol/l mol/l Log
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
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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 proline, placing 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 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- L-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 108 (molecules/cm3, 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 C1 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, cysteine, D-cysteine, L-methionine and D-L-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
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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-(L-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
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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 1011 molecules/cm3, 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 1011 molecules/cm3) as displayed by the mice with the amino acids tested in the present study.
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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 1012 molecules/ cm3 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 1010 molecules/ cm3) than in the other three classes.
Figure 12 Comparison between threshold data expressed in molecules/ cm3 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).
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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 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 S H D-methionine OH O H2N S H D-cysteine O OH H2N SH H L-cysteine O OH H2N SH H 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
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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
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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.
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