J Comp Physiol A (2007) 193:955–961 DOI 10.1007/s00359-007-0248-4
ORIGINAL PAPER
Olfactory discrimination ability of CD-1 mice for aliphatic aldehydes as a function of stimulus concentration Matthias Laska · Dipa Joshi · Gordon M. Shepherd
Received: 10 March 2007 / Revised: 13 May 2007 / Accepted: 26 May 2007 / Published online: 20 June 2007 © Springer-Verlag 2007
Abstract Using an operant conditioning paradigm, we tested the ability of CD-1 mice to discriminate between members of a homologous series of aliphatic aldehydes presented at four diVerent concentrations. We found that the mice were clearly capable of discriminating between all odorant pairs when stimuli were presented at concentrations of 1, 0.01, and 0.001 ppm (corresponding to four, two, and one log unit above the highest individual detection threshold) with no signiWcant diVerence in performance between these concentrations. In contrast, the animals generally failed to discriminate above chance level when stimuli were presented at 0.0001 ppm (corresponding to the highest individual detection threshold) although stimuli were clearly detectable. Further, we found a signiWcant negative correlation between discrimination performance and structural similarity of odorants in terms of diVerences in carbon chain length. These Wndings suggest that an increase in stimulus concentration of only one log unit above detection threshold appears to be suYcient for recruitment of additional subpopulations of odorant receptors to allow for qualitative recognition of aliphatic aldehydes. Keywords Olfactory discrimination · Aliphatic aldehydes · Stimulus concentration · CD-1 mice
M. Laska (&) IFM Biology, Linköping University, 581 83 Linköping, Sweden e-mail:
[email protected] D. Joshi · G. M. Shepherd Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA
Introduction Many studies have demonstrated that the neural representations of odorants vary not only as a function of molecular structural features such as carbon chain length (Johnson and Leon 2000a; Xu et al. 2003) or type or position of functional groups (Johnson et al. 2005; Leon and Johnson 2003) but also as a function of stimulus concentration (Fried et al. 2002; Johnson and Leon 2000b; Scott et al. 2006; Stuck et al. 2006). However, at the behavioral level several species have been shown to be capable of recognizing a given odorant over a wide range of concentrations (mouse: Cleland and Narla 2003; spiny lobster: Fine-Levy and Derby 1991; humans: Laing et al. 2003; rat: McBride and Slotnick 2006; honeybees: Pelz et al. 1997). This phenomenon is further complicated by Wndings from human psychophysical studies that reported the perceived quality of some, but not all, odorants to change with stimulus concentration (GrossIsseroV and Lancet 1988; Laing et al. 2003; Wilson and Stevenson 2006). Despite the importance of stimulus concentration for neural coding and thus for the mechanisms underlying recognition and discrimination of odorants surprisingly few studies thus far have systematically assessed the impact of stimulus concentration on odorant recognition at the behavioral level (Neuhaus 1957; Lancet et al. 1993). In the present study we therefore tested the ability of mice to discriminate between members of a homologous series of odorants presented at diVerent equimolar concentrations. We have chosen aliphatic aldehydes as stimuli because a previous fMRI study has shown that they elicit distinguishably diVerent odor maps in the olfactory bulb of mice (Xu et al. 2003). Furthermore, a recent study found aliphatic aldehydes to be prominent components of mouse body odor (Röck et al. 2006) suggesting that these odorants may play
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a role in olfactory social communication. Of additional relevance is the fact that a receptor has been characterized in the mouse speciWc for heptanal (Krautwurst et al. 1998), and threshold data from mice (Laska et al. 2006) and data on discrimination performance from other species for the aldehydes used here are also available (Laska et al. 1999a, b; Laska and Teubner 1999). The aims of the present study are threefold: (1) to provide data on the olfactory discrimination ability of mice for a homologous series of aliphatic aldehydes; (2) to assess whether a correlation between discrimination ability and structural similarity of the odorants under investigation exists; and (3) to assess the impact of stimulus concentration on discrimination ability for these aliphatic aldehydes.
Materials and methods Animals Testing was carried out using ten male CD-1 mice (Mus musculus). The rationale for choosing this 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 for the stimuli used here were obtained in an earlier study using the same animals (Laska et al. 2006) The mice were housed individually in plastic cages in a temperature- and humidity-controlled room and maintained under natural lighting conditions. The mice were 150–170 days old at the beginning of the study. During the experiments, the animals were kept on a water deprivation schedule of 1.5 ml of water per day (Bodyak and Slotnick 1999).
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and Slotnick 1999) to insert their snout into the odor sampling port of a test chamber. This triggered the 2 s presentation of either an odorant used as the rewarded stimulus (S+) or a diVerent odorant used as the unrewarded stimulus (S¡). Licking at a steel tube providing 2.5 l of water reinforcement in response to presentation of the S+ served as the operant response. Accordingly, not licking in response to presentation of the S¡ was regarded as a correct rejection. 120 such trials (60 S+ and 60 S¡ trials in pseudorandomized order) using the same concentration of a given pair of S+ and S¡ were conducted per animal and condition. With each animal, one aldehyde was assigned as the rewarded stimulus (S+) and the four other aldehydes were used as unrewarded stimuli (S¡). To allow an animal to build a robust association between water reward and a given S+, the critical tests were preceded by 120 training trials using geraniol as S¡ and 120 training trials using 1,8-cineole as S¡. The two substances do not share any functional group or other apparent similarities in their molecular structure with the S+, and were used at the same gas phase concentration as the S+. After these training trials, all animals reliably scored above 95% correct choices, implying that they had learned to correctly assign the reward value of the S+. To prevent serial order eVects from confounding the results, each animal was presented with a diVerent order of the critical tasks. To prevent the more challenging conditions leading to extinction or to a decline in the animal’s motivation, these were always followed by a return to an easy training task for 120 trials. This task consisted of the discrimination between the S+ and 1,8-cineole.
Results Odorants General discrimination ability A set of Wve odorants was used: n-butanal, n-pentanal, n-hexanal, n-heptanal, and n-octanal. The rationale for choosing these substances was to assesss the discrimination performance of the mice for odorants representing members of a homologous series of aliphatic compounds, that is, substances sharing the same functional group but diVering in carbon chain length, allowing us to assess the impact of this structural feature on discriminability. All substances were obtained from Sigma-Aldrich (St Louis, MO) and had a nominal purity of at least 99%. They were diluted using odorless mineral oil (Sigma-Aldrich) as the solvent. Behavioral test Olfactory discriminability was assessed using an automated olfactometer (Knosys, Tampa, FL). Mice were trained using standard operant conditioning procedures (Bodyak
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This experiment aimed at assessing the general ability of CD-1 mice to discriminate between aliphatic aldehydes presented at a gas phase concentration of 1 ppm. This concentration is at least four orders of magnitude above the highest individual detection threshold for the aldehydes used here (Laska et al. 2006). Figure 1 summarizes the performance of the mice in discriminating between the ten odorant pairs presented at a gas phase concentration of 1 ppm. When considering the mean percentage of correct decisions across all 6 blocks of 20 trials performed per animal and task (circles in Fig. 1), the mice performed signiWcantly above chance level in all tasks and thus were clearly able to discriminate between all odorant pairs presented (binomial test, P < 0.001 for all tasks and individuals). Interindividual variability with a given task was low as can be inferred from the small SEs. Mean scores
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Fig. 1 Performance of CD-1 mice in discriminating between members of a homologous series of aliphatic aldehydes presented at a gas phase concentration of 1 ppm. Each data point represents the percentage (means § SE) of correct decisions per odorant pair (a) across the 6 blocks of 20 trials performed per animal and task (circles), (b) in the Wrst block of 20 trials (squares), and (c) in correct rejections of the S¡ in the Wrst block of 20 trials (triangles). 4 n-Butanal, 5 npentanal, 6 n-hexanal, 7 n-heptanal, 8 n-octanal. C1 corresponds to the discrimination of aldehydes, which diVer by only one carbon atom, and C2–C4 to the discrimination of aldehydes, which diVer by two to four carbon atoms, respectively
ranged from 92.5% for the discrimination of n-heptanal and n-octanal (odorant pair 7–8) to 97.0% for the discrimination of n-butanal and n-hexanal (odorant pair 4–6). When considering the percentage of correct decisions in the Wrst block of 20 trials only (squares in Fig. 1), mean scores ranged from 75.0% with odorant pair 7–8 (nheptanal vs. n-octanal) to 92.5% with odorant pair 4–8 (nbutanal vs. n-octanal), indicating marked diVerences between tasks in the speed of learning but generally a fast learning process. When considering the percentage of correct rejections of the unrewarded stimulus in the Wrst block of 20 trials only (triangles in Fig. 1), mean scores ranged from 52.5% for the discrimination of n-heptanal and n-octanal (odorant pair 7–8) to 85.0% for the discrimination of n-butanal and n-octanal (odorant pair 4–8), again indicating marked diVerences between tasks in the speed of learning and, additionally, that rejection of the unrewarded stimulus rather than responding to the rewarded stimulus was the challenging part of the problem. The across-task patterns of performance correlated signiWcantly between all three measures of performance (Spearman, rs ¸ 0.67, P < 0.05 with all three comparisons). Figure 2 shows the mean discrimination performance of the mice as a function of diVerences in carbon chain length. When considering the percentage of correct decisions across all 6 blocks of 20 trials performed per animal and group of tasks (circles in Fig. 2), no signiWcant correlation between structural similarity of odorants and discrimination performance was found (Spearman, rs = 0.29, P > 0.05).
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Fig. 2 Discrimination performance (means § SE) of CD-1 mice as a function of diVerences in carbon chain length. Each data point represents the percentage (means § SE) of correct decisions (a) across the 6 blocks of 20 trials performed per animal and task (circles), (b) in the Wrst block of 20 trials (squares), and (c) in correct rejections of the S¡ in the Wrst block of 20 trials (triangles). C1 corresponds to the discrimination of aldehydes, which diVer by only one carbon atom, and C2–C4 to the discrimination of aldehydes, which diVer by two to four carbon atoms, respectively
However, when considering the percentage of correct decisions in the Wrst block of 20 trials only (squares in Fig. 2) or the percentage of correct rejections of the unrewarded stimulus in the Wrst block of 20 trials only (triangles in Fig. 2), highly signiWcant negative correlations between discrimination performance and structural similarity of odorants in terms of diVerences in carbon chain length were found (Spearman rs = 0.66, P < 0.001, respectively). These results show that the novelty and diYculty of the task is sensitive to odorant concentration. Figure 3 illustrates the ability to discriminate between the individual odorants. The mean scores across the four tasks that involved a given odorant did not diVer signiWcantly between stimuli (Friedman, P > 0.05). This was true with all three measures of performance, the percentage of correct decisions across all 6 blocks of 20 trials performed per animal and task (circles in Fig. 3), across the Wrst block of 20 trials only (squares in Fig. 3), and across the correct rejections of the unrewarded stimulus in the Wrst block of 20 trials only (triangles in Fig. 3). Discrimination ability as a function of stimulus concentration The results of experiment 1 demonstrate that CD-1 mice are clearly able to discriminate between aliphatic aldehydes
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Fig. 3 Discriminability of the Wve aliphatic aldehydes. Each data point represents the percentage (means § SE) of correct decisions across the four tasks that involved a given odorant (a) across the 6 blocks of 20 trials performed per animal and task (circles), (b) in the Wrst block of 20 trials (squares), and (c) in correct rejections of the S¡ in the Wrst block of 20 trials (triangles). 4 n-Butanal, 5 n-pentanal, 6 n-hexanal, 7 n-heptanal, 8 n-octanal
presented at a gas phase concentration of 1 ppm, that is, at least four orders of magnitude above the highest individual detection threshold, and that there is a signiWcant negative correlation between discrimination performance and structural similarity of the odorants in terms of diVerences in carbon chain length. Experiment 2 aimed at assessing the impact of stimulus concentration on discrimination performance. To this end, we tested the animals’ ability to distinguish between the same odorant pairs as in experiment 1 but presented them at gas phase concentrations of 0.01, 0.001, and 0.0001 ppm, respectively. The two higher concentrations were at least two and one log units above the highest individual detection thresholds for the aldehydes used here, and the lowest concentration represented the highest individual detection threshold of the animals (Laska et al. 2006). Figure 4 shows the performance of the mice in discriminating between the ten odorant pairs presented at four diVerent concentrations. Given are percentages (mean § SE) of correct decisions across all 6 blocks of 20 trials performed per animal and task. At gas phase concentrations of 0.01 ppm (squares in Fig. 4) and 0.001 ppm (triangles in Fig. 4), corresponding to two and one log units above the highest individual detection thresholds, respectively, the mice performed signiWcantly above chance level in all tasks and thus were clearly able to discriminate between all odorant pairs presented (binomial test, P < 0.001 for all tasks
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Fig. 4 Performance of CD-1 mice in discriminating between members of a homologous series of aliphatic aldehydes presented at a gas phase concentration of 1 (circles), 0.01 (squares), 0.001 (triangles), and 0.0001 ppm (diamonds), respectively. Each data point represents the percentage (means § SE) of correct decisions per odorant pair across the 6 blocks of 20 trials performed per animal and task. 4 n-Butanal, 5 n-pentanal, 6 n-hexanal, 7 n-heptanal, 8 n-octanal. C1 corresponds to the discrimination of aldehydes, which diVer by only one carbon atom, and C2–C4 to the discrimination of aldehydes, which diVer by two to four carbon atoms, respectively
and individuals). Mean scores ranged from 95.3 to 99.0% for the discrimination of aldehydes at a concentration of 0.01 ppm, and from 88.0 to 98.0% at 0.001 ppm, and thus were in the same range as the scores obtained at a concentration of 1 ppm (circles in Fig. 4). The across-task patterns of performance correlated signiWcantly between all three concentrations (Spearman, rs ¸ 0.69, P < 0.05 with all three comparisons). At a gas phase concentration of 0.0001 ppm (diamonds in Fig. 4), corresponding to the highest individual detection threshold, between one (with odorant pair 4–7) and three (with odorant pairs 5–6, 6–7, and 5–7) out of the four mice tested per task failed to score above 75.0% correct and thus did not signiWcantly discriminate between a given odorant pair (binomial test, P > 0.05). The across-task pattern of performance at this lowest concentration did not correlate signiWcantly with those of the three higher concentrations tested (Spearman, rs · 0.58, P > 0.05 with all three comparisons). Figure 5 shows the mean discrimination performance of the mice as a function of diVerences in carbon chain length at four diVerent stimulus concentrations. Given are percentages (mean § SE) of correct rejections of the unrewarded stimulus in the Wrst block of 20 trials. At gas phase concentrations of 1 ppm (circles in Fig. 5), 0.01 (squares in Fig. 5), and 0.001 ppm (triangles in Fig. 5), highly signiWcant negative correlations between discrimination performance and structural similarity of odorants in terms of diVerences in carbon chain length were found (Spearman, rs ¸ 0.60, P < 0.001, with all three concentrations). At a gas phase concentration of 0.0001 ppm (diamonds
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Fig. 5 Discrimination performance of CD-1 mice as a function of diVerences in carbon chain length and stimulus concentration. Each data point represents the percentage (means § SE) of correct rejections of the S¡ in block 1 presented at a gas phase concentration of 1 (circles), 0.01 (squares), 0.001 (triangles), and 0.0001 ppm (diamonds), respectively. C1 corresponds to the discrimination of aldehydes, which diVer by only one carbon atom, and C2–C4 to the discrimination of aldehydes, which diVer by two to four carbon atoms, respectively
in Fig. 5), no such correlation was found (Spearman, rs = 0.12, P > 0.05). Similar to the Wndings at a gas phase concentration of 1 ppm (see Fig. 3), the discriminability of the individual odorants across the four tasks that involved a given odorant did not diVer signiWcantly between stimuli when presented at gas phase concentrations of 0.01, 0.001 and 0.0001 ppm, respectively (Friedman, P > 0.05 with all three concentrations). This was true with all three measures of performance, the percentage of correct decisions across all 6 blocks of 20 trials performed per animal and task, across the Wrst block of 20 trials only, and across the correct rejections of the unrewarded stimulus in the Wrst block of 20 trials only.
Discussion The results of the present study demonstrate (a) that CD-1 mice have a well-developed discrimination ability for aliphatic aldehydes, (b) a signiWcant negative correlation between discrimination performance and structural similarity of odorants in terms of diVerences in carbon chain length, and (c) that discrimination performance with aliphatic aldehydes already reaches a plateau when stimuli are presented at a factor of 10 above detection threshold.
Our Wnding of a well-developed discrimination ability for aliphatic aldehydes is in line with the few reports so far that assessed discrimination performance in mice for monomolecular odorants using psychophysical procedures. Employing the same method as in the present study, Laska and Shepherd (2007) demonstrated Mus musculus to be capable of distinguishing between 15 enantiomeric odor pairs, although to diVerent degrees. Similarly, Bodyak and Slotnick (1999) reported that mice successfully discriminated between two esters (butyl acetate vs. amyl acetate), two terpenes (citral vs. cineol), and two benzenes (toluene vs. benzene), and Abraham et al. (2004) found two esters (amyl acetate vs. ethyl butyrate) and two terpenes (cineol vs. eugenol) to be discriminable for mice. Future studies should elucidate whether the ability to perceive small diVerences in the molecular structure of odorants is a general feature of the mouse olfactory system or whether— similar to other species—this ability is substance classspeciWc (Laska et al. 1999a, b; Laska and Teubner 1999). Our Wnding of a signiWcant negative correlation between discrimination performance and structural similarity of aliphatic aldehydes in terms of diVerences in carbon chain length is in agreement with previous studies that showed human subjects (Laska and Teubner 1999) and squirrel monkeys (Laska et al. 1999b) to display the same regular connection with these odorants. It should be noted, however, that whereas squirrel monkeys—like the mice tested in the present study—were able to discriminate signiWcantly between all aldehyde pairs presented to them, human subjects, both individually and as a group, failed to perform above chance level in distinguishing between several odor pairs involving aldehydes that diVered by only one carbon atom. A possible explanation for this diVerence between species in their ability to distinguish between direct neighbors in the homologous series of aliphatic aldehydes may be that humans have only t350 functional genes coding for olfactory receptors (Glusman et al. 2001) whereas both mice (Godfrey et al. 2004) and squirrel monkeys (Rouquier et al. 2000) have t900 such functional genes. Whereas this marked diVerence in the size of the repertoire of olfactory receptor types is not necessarily predictive of betweenspecies diVerences in olfactory sensitivity (Joshi et al. 2006; Laska et al. 2005, 2006) recent studies suggest that it may correlate with olfactory discrimination performance (Laska and Shepherd 2007). However, honeybees have also been shown capable of discriminating between all members of a homologous series of aliphatic aldehydes (Laska et al. 1999a) although they possess only t160 functional olfactory receptor genes (Robertson and Wanner 2006). This suggests that not only the number of functional olfactory receptor genes but also the behavioral relevance of odor stimuli may aVect a species’ discrimination performance as aliphatic aldehydes are
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constituents of the odor of a variety of bee-pollinated Xowers (Knudsen et al. 1993). Our Wnding that discrimination performance with aliphatic aldehydes already reaches a plateau when stimuli are presented at a factor of 10 above detection threshold is in line with an earlier report that found performance of mice to rise from chance level to near saturation within one log unit when determining detection thresholds, that is, discriminating between an aldehyde and a blank stimulus (Laska et al. 2006). It is commonly agreed that detection thresholds (deWned as the lowest concentration at which a given odorant can be detected or discriminated from a blank stimulus) can be distinguished from recognition thresholds (deWned as the lowest concentration at which a given odorant can be assigned a recognizable quality or discriminated from another preceptible odorant) with the latter being higher than the former (Doty and Laing 2003; Wilson and Stephenson 2006). Few studies have directly assessed the concentration diVerence between these two thresholds. Hudson et al. (2006) found the human recognition threshold for coVee and orange odors (in terms of correctly identifying the quality of these stimuli) to be a factor of 8–16 above their respective detection thresholds and thus in the same range as found in the present study. Similarly, Wright and Smith (2004) reported the thresholds for detection and discrimination of monomolecular odorants to diVer in the honeybee. Interestingly, they found that discrimination performance markedly increased with concentration of structurally dissimilar odorants (1-hexanol, geraniol, 2octanone) but much less so with structurally similar odorants (1-hexanol, 1-heptanol, 1-octanol). Unfortunately, the authors did not determine proper detection threshold values for their stimuli so that the diVerence in concentration between detection threshold and plateau performance of discrimination cannot be quantiWed. The authors interpret their Wndings as mirroring the recruitment of additional subpopulations of olfactory receptors with increasing concentration. More direct evidence for recruitment of receptors as concentration increases has been obtained in functional imaging studies. Using high-resolution calcium imaging, Fried et al. (2002) found that for a range of aldehydes presented at low concentrations, approximately 10–20 glomeruli in the dorsal area of the mouse olfactory bulb were activated, with relatively little overlap between aldehydes. At higher concentrations, corresponding to two log units above signal threshold (only log unit steps were tested), about 80 glomeruli were activated with much more overlap between aldehydes. Employing the [C14]2-deoxyglucose method, Johnson and Leon (2000b) reported that for two odorants that change in quality with concentration in humans (pentanal and 2-hexanone), the proportion of active glomeruli in the rat olfactory bulb increased signiWcantly as concentration increased. With odorants for which
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there is relatively little qualitative change in humans with increasing concentration (pentanoic acid, methyl pentanoate, and pentanol), the pattern of glomerular activity in rats remained largely unchanged as concentration increased. Although these imaging studies provide evidence of recruitment of receptors with increasing concentration and important information as to the neural mechanisms underlying coding of both odor quality and intensity they are not adequate for quantiWcation of the concentration range between detection and recognition thresholds. The restricted concentration range of one log unit that we found between detection threshold and plateau performance in discriminating between aliphatic aldehydes is similar to that reported for responses of isolated olfactory receptor cells to increasing odorant concentrations (Firestein et al. 1990) though in situ studies have revealed wider ranges (Grosmaitre et al. 2006). In future studies we will assess whether the concentration range between detection and plateau performance of discrimination is substance classspeciWc and/or a function of the size of the repertoire of olfactory receptors, which may directly aVect the recruitment of additional receptors with increasing concentration. Acknowledgments GMS is supported by NIH grant (5 R01 DC00086-38) and the Human Brain Project. The experiments reported here comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication no. 86-23, revised 1985) and were performed according to a protocol approved by the Yale University Institutional Animal Care and Use Committee.
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