256
Brain Research, 517 (1990) 256-262 Elsevier
BRES 15498
Olfactory discrimination over a wide concentration range. Comparison of receptor cell and bulb neuron abilities Patricia Duchamp-Viret, Andr6 Duchamp and Gilles Sicard Laboratoire de Physiologie neurosensorielle, Universit# Claude-Bernard, CNRS, Villeurbanne (France) (Accepted 27 October 1989) Key words: Olfaction; Receptor cell; Bulb neuron; Convergence; Qualitative discrimination; Intensity coding
Until now, olfactory discrimination had never been investigated using stimuli delivered over a wide concentration range. However, the fact that intensity variations might influence qualitative discrimination has been suggested in numerous physiological and psychophysical studies. The aim of the present work was to investigate qualitative coding mechanisms when stimulus intensity varies. For this purpose, receptor cell and olfactory bulb neuron unit activities were recorded in response to 2-s pulse delivery of 4 odorants available at 20 discrete concentration values over a range from 1 x 10 -6 tO 5.62 × 10-2 of saturation. Two types of mathematical analyses, Pearson's r correlation coefficient calculation and principal component factor analysis, were applied to odor-evoked discharge frequencies. In both receptor cells and bulb neurons, qualitative discrimination abilities were found to increase with stimulus concentration. Furthermore, the results suggest that the olfactory bulb can send a discriminant and specific message at lower concentrations than the olfactory mucosa. The amplifying role of convergence of primary afferences onto olfactory glomeruli could account for this ability of the bulb neurons.
INTRODUCTION N u m e r o u s studies based on electrophysiological recordings of olfactory cells have a t t e m p t e d to describe the principles governing the neural coding of the o d o r a n t quality. These investigations were concerned with the discrimination p r o p e r t i e s of either r e c e p t o r cells 1"1318,27.28 or olfactory bulb n e u r o n s 4-6'21-24'26"27'29'3°. O n the whole, these approaches have contributed to the understanding of olfactory coding mechanisms but as the results were collected from different animal species with different sets of odors, the data r e m a i n e d heterogeneous. To obviate these drawbacks, extensive investigations of qualitative discrimination were p e r f o r m e d in the frog. In these studies, n u m e r o u s responses of r e c e p t o r c e l l s 8'32-36 and of bulb neurons 7'9'1° to large sets of stimuli were recorded. A f t e r analysis of the distributions of receptor cell responses, significant similarities have been deduced in the stimulating p r o p e r t i e s of some odorants. Multidimensional representations of relationships between odorants led the authors to use the concept of ' o d o r a n t g r o u p ' to describe the proximities of some stimuli 8. C o m p a r i n g r e c e p t o r and bulb levels, similar relationships between odorants have emerged. Nevertheless, the
olfactory bulb has been found to improve discrimination of some odorants which were not very well distinguished at the p e r i p h e r y 9. These insights into the basis of discrimination abilities of the two cell populations were obtained by delivering stimuli at single and relatively high concentration levels, an e x p e r i m e n t a l condition which was not the most suitable for bringing out the discrimination performances of the olfactory system. In other experiments, the influence of stimulus intensity on qualitative discrimination has been investigated at the p e r i p h e r a l level 34 and at the bulbar level 1°. In these experiments, 5 c o m p o u n d s were delivered at two concentrations. The message sent by r e c e p t o r cells was clearly affected by changes of stimulus intensity. Compared with the mucosa, the olfactory bulb s e e m e d to stabilize the activation p a t t e r n (i.e. the specific combination of active cells coding for an o d o r a n t quality) when the stimulus intensity was changed. T h e organization of the primary fiber projections onto glomeruli as well as the bulbar information processing through interneurons might explain these observations. H o w e v e r , as the two concentrations tested in these last studies were very close to each other, we felt it was necessary to further investigate the discrimination mechanisms over a wider range of concentrations.
Correspondence: P. Duchamp-Viret, Laboratoire de Physiologic Neurosensorielle, Universit6 Claude-Bernard, Lyon I, F69622 Villeurbanne Cedex, France. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
257 Animal preparation
TABLE I
List of odorant stimuli Name
Code
Origin
Molarityof saturated vapor, M/l, at22 °C
DL-Camphor Isoamyl acetate Limonene Anisole
CAM ISO LIM ANI
Merck Merck Merck Merck
1.12 2.44 1.10 1.98
x x x x
10-5 10~* 10~* 10-4
MATERIALS AND METHODS
Odorants The 4 odorants tested in the present work are listed in Table I. They have been selected from 68 stimuli used in previous studies of qualitative discrimination7'8'32. Three 'odorant groups' were represented: the 'camphor group' by camphor, the 'terpene group' by limonene and the 'aromatic group' by anisole. The fourth type of stimulus employed was isoamyl acetate. In our previous studies, all these compounds were found to be highly effective and clearly distinghuished by olfactory neurons.
Stimulation The stimuli were delivered by a dynamic flow multistage olfaetometer. This device was specially designed to ensure a precise control of the stimulation parameters. A detailed technical description of this device and of its performances has been published37. Twenty different concentration values can be obtained from discrete dilutions of the saturated vapors available at atmospheric pressure from Tedlar bags. The laboratory was kept at a temperature of 22 °C to prevent any variations in the molarity of the odor source. The 20 concentrations available were distributed in the range from 10 -6 to 5.62 X 10-2 of saturation (Table II). Whatever the dilution selected, the stimulus was delivered at a constant flow rate to the olfactory mucosa and consisted of a 2 s square pulse of odor.
Experiments were performed on 92 frogs (Rana ridibunda). Xylocaine (lidocaine 2%) was applied to the dorsal surface of the head. The local anesthesia was tested by pricking the head skin. When the animals failed to react, they were immobilized by a subcutaneous injection of 0.1 ml of D-tubocurarine (0.2%). Xyiocaine was reapplied to the wound areas intermittently throughout the recording session. The frogs were kept at 13 °C throughout the experiments by cold water circulation within the restraining block. Skin respiration was preserved by wrapping the frogs in wet gauze. For the receptor cell recordings, the nasal cavity roof was removed to give access to the ventral mucosa. For the bulb neuron recordings, the ipsilateral bulb was uncovered by resection of the cranial upper wall and the meninges were carefully dissected. Dissection of the nasal cavity was performed specially to allow stimulus delivery to the entire mucosa: a lateral incision was made from the nostril toward the base of the orbit. This incision was widened by dissecting out a rectangular piece of cartilage, which, in this lateral region, is not lined by sensory epithelium.
Electrophysiological recordings Unit activity of receptor cells and bulb neurons was recorded using glass microelectrodes filled with an alloy of Wood's metal and indium and plated electrolytically with platinum black at the tip 12. Their impedances ranged from 1 to 4 MI~ at 1000 Hz. Neural activity was amplified by a low noise amplifier (bandwidth 300-3000 Hz; input impedance 500 MO), displayed on an oscilloscope and stored on magnetic tapes for subsequent processing. Receptor cells were recorded in various regions of the ventral epithelium. Bulb neurons were recorded, at a depth of 300-1200/~m, in the anterior part of the bulb, where the mitral cell layer is dorsoventrally oriented in the frog. Previous experiments using a collision test 11 have identified the neurons recorded in this region as output neurons. Identical experimental procedures were applied to both cell populations. The concentration-response relations were studied only for the stimuli which elicited excitatory responses since this type of response had previously been found to be more specific of the stimulus quality than were inhibitory responses9. For the data analysis, we took into account only the cells tested with the four stimuli.
TABLE II
List of the available concentrations and their respective codes Concentration as afraction of saturated vapor at 22 °C 1x 1.7 x 3.16 x 5.62 x 1x 1.7 x 3.16 x 5.62 x
10-6 10-6 10-6 10-6 10-5 10-s 10-5 10-5
1 x 10 -4
1.7 x 3.16 x 5.62 x 1x 1.7 x 3.16 x 5.62 x
104 10-4 10-4 10-3 10 -3
10-3 10 -3
1 x 10 -2
1.7 x 10-2 3.16 x 10-2 5.62 x 10-2
Code 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
Response determination After spike triggering, the interspike intervals were measured off-line from magnetic tape and the values were stored and processed on a microcomputer. For each stimulation sequence, corresponding to one cell stimulated with one stimulus at one concentration, a data file was created. Each file consisted of the successive interspike values, 30 s before and 30 s after the 2 s odor delivery. To determine the neural response, the Mann-Whitney U-test was applied to each stimulation sequence, in order to identify those poststimulus interspike intervals which show a distribution that stochastically differs from the prestimulus distribution. A fully detailed description of this statistical processing has been given in a previous paper 11. The responses were quantified as the average of the instantaneous frequency of each interspike interval included in the response. The matrices of the response frequencies were then mathematically processed to analyse the interactions between stimulus intensity and qualitative discrimination. A first matrix was made up of the responses of 28 receptor cell responses. The 34 variables corresponded to the 4 stimuli delivered at the concentrations coded as 3, 8, 9, 10, 12, 14, 16, 18 and 20 in Table II. For camphor, concentrations 3 and 8 were not represented due to the small number of responses collected. The total number of divisions in the matrix was 952 (28 cells x 34 stimulations). A second matrix was made of the responses of 30 bulb neurons. For the 4 stimuli, the concentrations were 3, 8, 9, 10, 12, 14, 16, 18
258
CAM 18
20
1
16 14 12
3
C20 C 18
20~8"
9
120
le
-118
C 16
10
ANI
32 %
10 9
--
~
12
14
ISO
16 9
C 14 -
18
-
A18
8 I 16 -
-
A16
-
A14
- - - -
A12
C12 El
10
12
14
18
114
L20 -
LIM
L 18
Fig. 1. Correlations between the responses evoked in the receptor cell population by the different odorant concentrations. Odorant names are abbreviated according to the code in Table I; concentrations are coded according to Table II. Two classes of significant r values are represented; 0.48 < r < 0.8, thin lines; r > 0.8, thick lines. All these values are significant at the 0.1% threshold.
and 20. The total number of divisions was 1080 (30 cells × 36 stimulations). RESULTS
Comparison of stimulus concentrations in pairs for the two cell populations Distributions of the responses evoked in the cell populations either by two concentrations of an odorant or by two concentrations of two different odorants were compared by calculating the Pearson's 'r' correlation coefficient for all the
CAM ._
14
12
10 ! 2O
18 16
9 10
14
ANI 12,
1= ISO
14 le
0
11
8 8 "
20 8 10
12
14
C 10 Cg
L le L 14 L 12. 10, 9 L8.5,3
112
I 10 A 10
19
18, 5 . 3 A9.8.5,3
Fig. 3. Representation of the different concentrations of the stimuli with respect to the first factor of the principal component analysis applied to the responses of the receptor cell population (associated variance: 32%). C, camphor; I, isoamyle acetate; L, limonene; A, anisole. Concentrations are coded according to Table II.
pairs of columns (i.e. stimulations) in the matrices. For receptor cells, 116 out of the 561 pairs of concentrations (20.6%) were found to be significantly correlated at the 0.001 threshold (Fig. 1). In this complex network of correlations, the different concentrations of camphor were found to be intercorrelated and distinguished from the other compounds. For isoamyle acetate, limonene or anisole, significant correlations were observed between closely related concentration values but not systematically between high and low concentrations. In addition, significant interstimulus correlations were observed, specially among the lowest concentrations. For bulb neurons, 109 out of the 630 pairs of concentrations (17.3%) were found to be significantly correlated at the 0.001 threshold (Fig. 2). Numerous significant r values were found for concentration pairs of a given odorant. By comparison with receptor cells, the different concentrations of an odorant are more strongly intercorrelated. Moreover there are fewer significant links between odorants. Only, the lowest concentration of each compound appears to be isolated from the other concentrations and systematically correlated with that of the other compounds.
m
LIM Fig. 2. Correlations between the responses evoked in the bulb neuron population by the different concentrations of the odorants. Same legend as Fig. 1.
Representation of olfactory spaces The matrices of average instantaneous frequencies were submitted to a principal component analysis 3"19. This method provides a synthetic view of the relation-
259
'T" lon~/.' 12
CAM
2
I
i r 1 2211
T/L/3e-i,,s
,.3
,
271l
Fig. 4. Principal component analysis of receptor cell responses: three-dimensional space defined by factors 2, 3 and 4 (associated variances in %). Odorant names are abbreviated as in Table I; concentrations are coded as in Table If.
lowest concentrations being near the center of the space and the highest concentrations at the periphery. Factor 2 opposes camphor and anisole on the one hand and isoamyle acetate on the other hand. The discrimination between anisole and isoamyle acetate is obtained by taking into account the third factor. Factor 4 discriminates low concentrations of limonene from the other three compounds. For the bulb neurons, the olfactory space corresponding to factors 1, 2 and 3, accounting for 37%, 31% and 12% respectively of the total variance, is represented in Fig. 5. Camphor and isoamyle acetate are discriminated by the first factor. These two compounds are opposed to
ships between the different variables (i.e. the different concentrations of the 4 odorants tested). In the olfactory space representations, distances between representative points of stimuli reflect the degree of similarity of their action on cell populations. For receptor cells, the four odorants are distributed along the first axis (32% of the variance) according to the stimulus intensity (Fig. 3). The olfactory space defined by factors 2, 3 and 4, accounting for 27%, 22% and 8%, respectively, of the total variance, is represented in Fig. 4. In this space, the four sets of points corresponding to the 4 odorants are arranged along 4 axes. The position of points along the axes depends on concentration: the
3 18
/
12~
20
20
r"
,f
t.l',j,, . Io'o " i l l 4 ~Pi78., ; / ° ,
/
,,;
,so/ /~
/
16(?
o
"_._ "-
,ltil,":o
J
,/
12141~ 7 1'
,18 /
/
1 37~ Fig. 5. Principal component analysis of bulb neuron responses: three-dimensional space defined by factors 1, 2 and 3 (assodated variances in %). Odorant names are abbreviated as in Table I; concentrations are coded as in Table [I.
260 anisole by taking into account factor 2. The third factor allows anisole to be discriminated from limonene. Factor 4 (not represented) indicates a clear distinction between the highest concentrations of limonene and its lowest concentrations which are grouped in the center of the space with the lowest concentrations of the other odorants. As in the space related to the responses of receptor cells, the 4 sets of points representing the 4 odorants are distributed along 4 directions. From the center to the periphery, the points are ranked according to increasing concentrations. DISCUSSION This work provides the first comparative description of discrimination abilities of receptor cells and bulb neurons as a function of stimulus intensity.
Comparison of stimuli in pairs In the olfactory mucosa, the correlations observed between low and medium concentrations of different compounds and the lack of cohesion inside a given set of odorant concentrations bring out the difficulties of receptor cell population to discriminate the stimuli in the range of concentration from 10-6 to 1.7 x 10-4 of saturation (with the exception of camphor). The dynamics of cell recruitment as a function of stimulus intensity has been recently analyzed H. For receptor cells, it appeared that, depending on the nature of the stimulus, 5-20% of the cell population were involved in the activation pattern at concentrations lower than 10 -4 of saturated vapor. In this concentration range, because of the small fraction of the cell population involved in the activation pattern, it could be assumed that receptor cells send a message with a low signal/noise ratio. Thus, before the olfactory bulb processing, we cannot statistically extract the specific pattern encoding for stimulus quality. In the bulb, there is considerable cohesion among the different concentrations of a given stimulus while there are few correlations between odorants. Thus, whatever the intensity, each compound seems to be better recognized as a given quality and more sharply discriminated from other compounds. However, the lowest concentrations of the 4 odorants tested do not appear to be clearly discriminated from each other. By contrast with the receptor cell level, at 10 -6 of saturation, the recruitment of bulb neurons was found to involve 10-40% of the cell population, depending on the nature of the stimulus ~1. From these data, it can be assumed that, once bulbar processing has been carried out, the message is more salient, specially when stimuli are delivered at low concentrations.
Representation of olfactory spaces Factor analyses provide other kinds of information and lead to further discussion of the results. Considering the responses of the two cell populations, the 4 stimuli are clearly individualized in the olfactory spaces. In addition, we mentioned that the concentrations for a given compound are ranked from the center to the periphery of the space according to increasing concentrations. The better an odorant is distinguished, the farther its point is from the center of the space and so the probability of discrimination seems to increase with stimulus concentration. This result is of importance in the knowledge of olfactory coding. It can be discussed in terms of the setting up of the activation pattern. The continuity of cell recruitment implies that the activation pattern is progressively built up as a function of stimulus intensity: more and more cells, among those which are responsive to a given stimulus, are involved. For increasing concentrations, it can be assumed that the activation pattern becomes more and more specific for a given stimulus, thus probably representing a more and more detailed description of the odorants. Furthermore, according to the olfactory spaces described here, this pattern keeps its specificity at the highest intensity tested (5.62 x 10-2 of saturation). This improvement of discrimination probabilities with increasing intensity is closely associated with the fact that cell recruitment is a continuous process: we have never observed cells responding at low concentrations which failed to respond at high concentrations. Furthermore, from the present data, it is clear that the sensory message does not undergo any damage at high intensity. Thus, our findings are not in agreement with the hypothesis put forward by some authors 21'25 who assumed that a distinct combination of active cells encoded the olfactory stimulus at each intensity level. At the receptor level it is worth noting that the first factor of the analysis ranks the representative points of stimuli according to the strength of the responses, whatever the nature of the stimulus. In the space defined by subsequent factors 2, 3 and 4, four subsets of points aligned according to 4 directions are identified. At the olfactory bulb level, the analysis shows that the first factor is directly involved in qualitative discrimination. This difference between the olfactory spaces is related to discrimination abilities observed at the bulbar level. The convergence of primary afferences onto glomeruli seems to result in shifts of the discrimination thresholds of odors towards lower concentrations. It has been demonstrated that the convergence was functionally involved in the amplification of the primary signal and therefore, in an increase of the detection power 11. Because of this amplification, the dynamics of cell recruitment was found to begin and to end at lower concentration levels in the
261 olfactory bulb than in the mucosa. Thus, at a given concentration, the fraction of the cell p o p u l a t i o n involved in a stimulus activation p a t t e r n is larger in the bulb than in the mucosa. T h e i m p r o v e m e n t o b s e r v e d in the bulb strongly underlines the d e p e n d e n c e which exists b e t w e e n stimulus intensity and discrimination abilities: in the bulb, as a c o n s e q u e n c e of p r i m a r y signal amplification, these abilities s e e m less d e p e n d e n t on intensity than in the mucosa. Quality versus intensity In previous studies of o d o r discrimination 7-1°'32'33'36, the qualitative group concept has been introduced. It was p o i n t e d out that possibilities of discrimination r e m a i n e d inside a qualitative group. A s an example, two enantiomers, like D-carvone and L-carvone, e n c o d e d by very similar activation patterns, were found to be distinguished by bulb neurons 7 and r e c e p t o r cells 35. In the same way, in the p r e s e n t study, the different concentrations of a given o d o r evoking similar patterns could nevertheless be discriminated from one another. This REFERENCES 1 Baylin, E, Temporal patterns and selectivity in the unitary responses of olfactory receptors in the tiger salamander to odor stimulation, J. Gen. Physiol., 74 (1975) 17-36. 2 Berglund, B., Berglund, U. and LindvaU, T., Psychological processing of odor mixtures, Psychol. Rev., 83 (1976) 432-441. 3 Bieber, S.L. and Smith, D.V., Multivariate analysis of sensory data: a comparison of methods, Chem. Senses, 18 (1986) 19-47. 4 Doving, K.B., Studies on responses of bulbar neurons of frog to different odor stimuli, Rev. Laryngol., 86 (1965) 845-854. 5 Doving, K.B., The influence of olfactory stimuli upon the activity of secondary neurons in the burbot (Lota Iota L.), Acta Physiol. Scand., 66 (1966) 290-299. 6 Doving, K.B., An electrophysiologicalstudy of odor similarities of homologous substances, J. Physiol. (Lond.), 186 (1966) 97-109. 7 Duchamp, A., Electrophysioiogical responses of olfactory bulb neurons to odor stimuli in the frog. A comparison with receptor cells, Chem. Senses, 7 (1982) 191-210. 8 Duchamp, A., Revial, M.E, Holley, A. and Mac Leod, P., Odor discrimination by frog olfactory receptors, Chem. Senses, 1 (1974) 213-233. 9 Duchamp, A. and Sicard, G., Odour discrimination by olfactory bulb neurons: statistical analysis of electrophysiological responses and comparison with odour discrimination by receptor cells, Chem. Senses, 9 (1984) 1-14. 10 Duchamp, A. and Sicard, G., Influence of stimulus intensity on odour discrimination by olfactory bulb neurons as compared with receptor cells, Chem. Senses, 8 (1984) 355-366. 11 Duchamp-Viret, P., Duchamp, A. and Vigouroux, M., Amplifying role of convergence in olfactory system. A comparison of receptor cell and second order neuron sensitivities, J. Neurophysiol., 61 (1989) 1085-1094. 12 Gesteland, R.C., Comments on microelectrodes, Proc. LR.E., 47 (1959) 1856-1862. 13 Gesteland, R.C., Neural coding in olfactory receptor cells. In L.M. Beidler (Ed.), Handbook of Sensory Physiology, Vol. IV, Chemical Senses, 1, Springer, Berlin, 1971, pp. 132-150. 14 Gesteland, R.C., Physiology of olfactory reception. In R. Llinas and W. Precht (Eds.), Frog Neurobiology, Springer, Berlin,
possibility does not imply that different concentrations of an o d o r are systematically p e r c e i v e d as different qualities, but in some cases, shifts of perceived quality can be expected. A few studies have r e l a t e d the intensity to the quality of an odorant. Previously, the influence of quality on perceived intensity 2 and conversely, the influence of intensity on o d o r quality 2° have been studied using psychophysical experiments. T h e authors concluded that variations in stimulus intensity m a y be perceived as differences in stimulus quality, a conclusion in a g r e e m e n t with the empiric experience of p e r f u m e r s 31. In terms of neural coding, the possibility of discriminating two activation p a t t e r n s e v o k e d by different concentrations of a given stimulus might supply information to the olfactory system a b o u t both intensity and quaiity. This means that perceived quality could be involved in the evaluation of stimulus intensity. Acknowledgements. We should like to thank Professor A. Holley for his critical review of the manuscript. 1976, pp. 234-250. 15 Gesteland, R.C., Lettvin, J.Y., Pitts, V.H. and Rojas, H. Odor specificities of the frog's olfactory receptors. In Y. Zotterman (Ed.), Olfaction and Taste, Vol. I, Pergamon, Oxford, 1963, pp. 19-34. 16 Getchell, T.V., Analysis of unitary spikes recorded extracellularly from frog olfactory receptor cells and axons, J. Physiol. (Lond.), 234 (1973) 533-551. 17 Getchell, T.V. and Shepherd, G.M., Responses of olfactory receptor cells to step pulses of odour at different concentrations in the salamander, J. Physiol. (Lond.), 282 (1978) 521-540. 18 Getchell, T.V. and Shepherd, G.M., Adaptative properties of olfactory receptors analyzed with odour pulses of varying duration, J. Physiol. (Lond.), 282 (1978) 541-560. 19 Gower, J.C., Some distance properties of latent root and vector methods used in multivariate analysis, Biometrika, 53 (1966) 325-338. 20 Gross-Isseroff, R. and Lancet, D., Concentration-dependent changes of perceived odor quality, Chem. Senses, 13 (1988) 191-204. 21 Kauer, J.S., Response patterns of amphibian olfactory bulb neurons to odour stimulation, J. Physiol. (Lond.), 243 (1974) 695-715. 22 Kauer, J.S. and Moulton, D.G., Responses of olfactory bulb neurons to odour stimulation of small nasal areas in the salamander, J. Physiol. (Lond.), 243 (1974) 717-737. 23 Kauer, J.S. and Shepherd, G.M., Olfactory stimulation with controlled and monitored step pulses of odour, Brain Research, 85 (1975) 108-113. 24 Kauer, J.S. and Shepherd, G.M., Analysis of the onset phase of olfactory bulb unit responses to odour pulses in the salamander, J. Physiol. (Lond.), 272 (1977) 495-516. 25 Lidow, M.S., Gesteland, R.C., Shipley, M.T. and Kleen, J.S., Comparative study of immature and mature olfactory receptor cells in adult frogs, Dev. Brain Res., 31 (1987) 243-258. 26 Mair, R.G., Response properties of rat olfactory bulb neurons, J. Physiol. (Lond.), 326 (1982) 341-359. 27 Mathews, D.F., Response pattern of single-neurons in the tortoise olfactory epithelium and olfactory bulb, J. Gen. Physiol., 60 (1972) 166-180. 28 Mathews, D.E, Response pattern of single units in the olfactory
262 bulb of the rat to odor, Brain Research, 47 (1972) 389-400. 29 Meredith, M., The analysis of response similarity in single neurons of the goldfish olfactory bulb using amino-acids as odor stimuli, Chem. Senses, 6 (1981) 277-293. 30 Meredith, M. and Moulton, D.G., Patterned response to odor in single neurons on goldfish olfactory bulb: influence of odor quality and other stimulus parameters, J. Gen. Physiol., 71 (1978) 615-643. 31 Moncrieff, R.W., The Chemical Senses, 2nd edn., Hill, 1967, p. 538, 32 Reviai, M.E, Duchamp, A. and Holley, A., Odour discrimination by frog olfactory receptors: a second study, Chem. Senses, 3 (1978) 7-21. 33 Reviai, M.E, Duchamp, A., Holley, A. and Mac Leod, P., Frog olfaction: odour groups acceptor distribution and receptor
categories, Chem. Senses, 3 (1978) 23-33. 34 Revial, M.E, Sicard, G., Duchamp, A. and Hoiley, A., New studies on odour discrimination in the frog's olfactory receptor cells. I. Experimental results, Chem. Senses, 7 (1982) 175-190. 35 Revial, M.E, Sicard, G., Duchamp, A. and Hoiley, A., New studies on odour discrimination in the frog's olfactory receptor cells. II. Mathematical analysis of electrophysiological responses, Chem. Senses, 8 (1983) 179-194. 36 Sicard, G. and Holley, A., Receptor cell responses to odorants: similarities and differences among odorants, Brain Research, 292 (1984) 283-296. 37 Vigouroux, M., Viret, P. and Duchamp, A., A wide concentration range olfactometer for delivery of short reproducible odor pulses, J. Neurosci. Methods, 24 (1988) 57-63.
Brain Research, 517 (1990) 256-262 Elsevier
BRES 15498
Olfactory discrimination over a wide concentration range. Comparison of receptor cell and bulb neuron abilities Patricia Duchamp-Viret, Andr6 Duchamp and Gilles Sicard Laboratoire de Physiologie neurosensorielle, Universit# Claude-Bernard, CNRS, Villeurbanne (France) (Accepted 27 October 1989) Key words: Olfaction; Receptor cell; Bulb neuron; Convergence; Qualitative discrimination; Intensity coding
Until now, olfactory discrimination had never been investigated using stimuli delivered over a wide concentration range. However, the fact that intensity variations might influence qualitative discrimination has been suggested in numerous physiological and psychophysical studies. The aim of the present work was to investigate qualitative coding mechanisms when stimulus intensity varies. For this purpose, receptor cell and olfactory bulb neuron unit activities were recorded in response to 2-s pulse delivery of 4 odorants available at 20 discrete concentration values over a range from 1 x 10 -6 tO 5.62 × 10-2 of saturation. Two types of mathematical analyses, Pearson's r correlation coefficient calculation and principal component factor analysis, were applied to odor-evoked discharge frequencies. In both receptor cells and bulb neurons, qualitative discrimination abilities were found to increase with stimulus concentration. Furthermore, the results suggest that the olfactory bulb can send a discriminant and specific message at lower concentrations than the olfactory mucosa. The amplifying role of convergence of primary afferences onto olfactory glomeruli could account for this ability of the bulb neurons.
INTRODUCTION N u m e r o u s studies based on electrophysiological recordings of olfactory cells have a t t e m p t e d to describe the principles governing the neural coding of the o d o r a n t quality. These investigations were concerned with the discrimination p r o p e r t i e s of either r e c e p t o r cells 1"1318,27.28 or olfactory bulb n e u r o n s 4-6'21-24'26"27'29'3°. O n the whole, these approaches have contributed to the understanding of olfactory coding mechanisms but as the results were collected from different animal species with different sets of odors, the data r e m a i n e d heterogeneous. To obviate these drawbacks, extensive investigations of qualitative discrimination were p e r f o r m e d in the frog. In these studies, n u m e r o u s responses of r e c e p t o r c e l l s 8'32-36 and of bulb neurons 7'9'1° to large sets of stimuli were recorded. A f t e r analysis of the distributions of receptor cell responses, significant similarities have been deduced in the stimulating p r o p e r t i e s of some odorants. Multidimensional representations of relationships between odorants led the authors to use the concept of ' o d o r a n t g r o u p ' to describe the proximities of some stimuli 8. C o m p a r i n g r e c e p t o r and bulb levels, similar relationships between odorants have emerged. Nevertheless, the
olfactory bulb has been found to improve discrimination of some odorants which were not very well distinguished at the p e r i p h e r y 9. These insights into the basis of discrimination abilities of the two cell populations were obtained by delivering stimuli at single and relatively high concentration levels, an e x p e r i m e n t a l condition which was not the most suitable for bringing out the discrimination performances of the olfactory system. In other experiments, the influence of stimulus intensity on qualitative discrimination has been investigated at the p e r i p h e r a l level 34 and at the bulbar level 1°. In these experiments, 5 c o m p o u n d s were delivered at two concentrations. The message sent by r e c e p t o r cells was clearly affected by changes of stimulus intensity. Compared with the mucosa, the olfactory bulb s e e m e d to stabilize the activation p a t t e r n (i.e. the specific combination of active cells coding for an o d o r a n t quality) when the stimulus intensity was changed. T h e organization of the primary fiber projections onto glomeruli as well as the bulbar information processing through interneurons might explain these observations. H o w e v e r , as the two concentrations tested in these last studies were very close to each other, we felt it was necessary to further investigate the discrimination mechanisms over a wider range of concentrations.
Correspondence: P. Duchamp-Viret, Laboratoire de Physiologic Neurosensorielle, Universit6 Claude-Bernard, Lyon I, F69622 Villeurbanne Cedex, France. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
257 Animal preparation
TABLE I
List of odorant stimuli Name
Code
Origin
Molarityof saturated vapor, M/l, at22 °C
DL-Camphor Isoamyl acetate Limonene Anisole
CAM ISO LIM ANI
Merck Merck Merck Merck
1.12 2.44 1.10 1.98
x x x x
10-5 10~* 10~* 10-4
MATERIALS AND METHODS
Odorants The 4 odorants tested in the present work are listed in Table I. They have been selected from 68 stimuli used in previous studies of qualitative discrimination7'8'32. Three 'odorant groups' were represented: the 'camphor group' by camphor, the 'terpene group' by limonene and the 'aromatic group' by anisole. The fourth type of stimulus employed was isoamyl acetate. In our previous studies, all these compounds were found to be highly effective and clearly distinghuished by olfactory neurons.
Stimulation The stimuli were delivered by a dynamic flow multistage olfaetometer. This device was specially designed to ensure a precise control of the stimulation parameters. A detailed technical description of this device and of its performances has been published37. Twenty different concentration values can be obtained from discrete dilutions of the saturated vapors available at atmospheric pressure from Tedlar bags. The laboratory was kept at a temperature of 22 °C to prevent any variations in the molarity of the odor source. The 20 concentrations available were distributed in the range from 10 -6 to 5.62 X 10-2 of saturation (Table II). Whatever the dilution selected, the stimulus was delivered at a constant flow rate to the olfactory mucosa and consisted of a 2 s square pulse of odor.
Experiments were performed on 92 frogs (Rana ridibunda). Xylocaine (lidocaine 2%) was applied to the dorsal surface of the head. The local anesthesia was tested by pricking the head skin. When the animals failed to react, they were immobilized by a subcutaneous injection of 0.1 ml of D-tubocurarine (0.2%). Xyiocaine was reapplied to the wound areas intermittently throughout the recording session. The frogs were kept at 13 °C throughout the experiments by cold water circulation within the restraining block. Skin respiration was preserved by wrapping the frogs in wet gauze. For the receptor cell recordings, the nasal cavity roof was removed to give access to the ventral mucosa. For the bulb neuron recordings, the ipsilateral bulb was uncovered by resection of the cranial upper wall and the meninges were carefully dissected. Dissection of the nasal cavity was performed specially to allow stimulus delivery to the entire mucosa: a lateral incision was made from the nostril toward the base of the orbit. This incision was widened by dissecting out a rectangular piece of cartilage, which, in this lateral region, is not lined by sensory epithelium.
Electrophysiological recordings Unit activity of receptor cells and bulb neurons was recorded using glass microelectrodes filled with an alloy of Wood's metal and indium and plated electrolytically with platinum black at the tip 12. Their impedances ranged from 1 to 4 MI~ at 1000 Hz. Neural activity was amplified by a low noise amplifier (bandwidth 300-3000 Hz; input impedance 500 MO), displayed on an oscilloscope and stored on magnetic tapes for subsequent processing. Receptor cells were recorded in various regions of the ventral epithelium. Bulb neurons were recorded, at a depth of 300-1200/~m, in the anterior part of the bulb, where the mitral cell layer is dorsoventrally oriented in the frog. Previous experiments using a collision test 11 have identified the neurons recorded in this region as output neurons. Identical experimental procedures were applied to both cell populations. The concentration-response relations were studied only for the stimuli which elicited excitatory responses since this type of response had previously been found to be more specific of the stimulus quality than were inhibitory responses9. For the data analysis, we took into account only the cells tested with the four stimuli.
TABLE II
List of the available concentrations and their respective codes Concentration as afraction of saturated vapor at 22 °C 1x 1.7 x 3.16 x 5.62 x 1x 1.7 x 3.16 x 5.62 x
10-6 10-6 10-6 10-6 10-5 10-s 10-5 10-5
1 x 10 -4
1.7 x 3.16 x 5.62 x 1x 1.7 x 3.16 x 5.62 x
104 10-4 10-4 10-3 10 -3
10-3 10 -3
1 x 10 -2
1.7 x 10-2 3.16 x 10-2 5.62 x 10-2
Code 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
Response determination After spike triggering, the interspike intervals were measured off-line from magnetic tape and the values were stored and processed on a microcomputer. For each stimulation sequence, corresponding to one cell stimulated with one stimulus at one concentration, a data file was created. Each file consisted of the successive interspike values, 30 s before and 30 s after the 2 s odor delivery. To determine the neural response, the Mann-Whitney U-test was applied to each stimulation sequence, in order to identify those poststimulus interspike intervals which show a distribution that stochastically differs from the prestimulus distribution. A fully detailed description of this statistical processing has been given in a previous paper 11. The responses were quantified as the average of the instantaneous frequency of each interspike interval included in the response. The matrices of the response frequencies were then mathematically processed to analyse the interactions between stimulus intensity and qualitative discrimination. A first matrix was made up of the responses of 28 receptor cell responses. The 34 variables corresponded to the 4 stimuli delivered at the concentrations coded as 3, 8, 9, 10, 12, 14, 16, 18 and 20 in Table II. For camphor, concentrations 3 and 8 were not represented due to the small number of responses collected. The total number of divisions in the matrix was 952 (28 cells x 34 stimulations). A second matrix was made of the responses of 30 bulb neurons. For the 4 stimuli, the concentrations were 3, 8, 9, 10, 12, 14, 16, 18
258
CAM 18
20
1
16 14 12
3
C20 C 18
20~8"
9
120
le
-118
C 16
10
ANI
32 %
10 9
--
~
12
14
ISO
16 9
C 14 -
18
-
A18
8 I 16 -
-
A16
-
A14
- - - -
A12
C12 El
10
12
14
18
114
L20 -
LIM
L 18
Fig. 1. Correlations between the responses evoked in the receptor cell population by the different odorant concentrations. Odorant names are abbreviated according to the code in Table I; concentrations are coded according to Table II. Two classes of significant r values are represented; 0.48 < r < 0.8, thin lines; r > 0.8, thick lines. All these values are significant at the 0.1% threshold.
and 20. The total number of divisions was 1080 (30 cells × 36 stimulations). RESULTS
Comparison of stimulus concentrations in pairs for the two cell populations Distributions of the responses evoked in the cell populations either by two concentrations of an odorant or by two concentrations of two different odorants were compared by calculating the Pearson's 'r' correlation coefficient for all the
CAM ._
14
12
10 ! 2O
18 16
9 10
14
ANI 12,
1= ISO
14 le
0
11
8 8 "
20 8 10
12
14
C 10 Cg
L le L 14 L 12. 10, 9 L8.5,3
112
I 10 A 10
19
18, 5 . 3 A9.8.5,3
Fig. 3. Representation of the different concentrations of the stimuli with respect to the first factor of the principal component analysis applied to the responses of the receptor cell population (associated variance: 32%). C, camphor; I, isoamyle acetate; L, limonene; A, anisole. Concentrations are coded according to Table II.
pairs of columns (i.e. stimulations) in the matrices. For receptor cells, 116 out of the 561 pairs of concentrations (20.6%) were found to be significantly correlated at the 0.001 threshold (Fig. 1). In this complex network of correlations, the different concentrations of camphor were found to be intercorrelated and distinguished from the other compounds. For isoamyle acetate, limonene or anisole, significant correlations were observed between closely related concentration values but not systematically between high and low concentrations. In addition, significant interstimulus correlations were observed, specially among the lowest concentrations. For bulb neurons, 109 out of the 630 pairs of concentrations (17.3%) were found to be significantly correlated at the 0.001 threshold (Fig. 2). Numerous significant r values were found for concentration pairs of a given odorant. By comparison with receptor cells, the different concentrations of an odorant are more strongly intercorrelated. Moreover there are fewer significant links between odorants. Only, the lowest concentration of each compound appears to be isolated from the other concentrations and systematically correlated with that of the other compounds.
m
LIM Fig. 2. Correlations between the responses evoked in the bulb neuron population by the different concentrations of the odorants. Same legend as Fig. 1.
Representation of olfactory spaces The matrices of average instantaneous frequencies were submitted to a principal component analysis 3"19. This method provides a synthetic view of the relation-
259
'T" lon~/.' 12
CAM
2
I
i r 1 2211
T/L/3e-i,,s
,.3
,
271l
Fig. 4. Principal component analysis of receptor cell responses: three-dimensional space defined by factors 2, 3 and 4 (associated variances in %). Odorant names are abbreviated as in Table I; concentrations are coded as in Table If.
lowest concentrations being near the center of the space and the highest concentrations at the periphery. Factor 2 opposes camphor and anisole on the one hand and isoamyle acetate on the other hand. The discrimination between anisole and isoamyle acetate is obtained by taking into account the third factor. Factor 4 discriminates low concentrations of limonene from the other three compounds. For the bulb neurons, the olfactory space corresponding to factors 1, 2 and 3, accounting for 37%, 31% and 12% respectively of the total variance, is represented in Fig. 5. Camphor and isoamyle acetate are discriminated by the first factor. These two compounds are opposed to
ships between the different variables (i.e. the different concentrations of the 4 odorants tested). In the olfactory space representations, distances between representative points of stimuli reflect the degree of similarity of their action on cell populations. For receptor cells, the four odorants are distributed along the first axis (32% of the variance) according to the stimulus intensity (Fig. 3). The olfactory space defined by factors 2, 3 and 4, accounting for 27%, 22% and 8%, respectively, of the total variance, is represented in Fig. 4. In this space, the four sets of points corresponding to the 4 odorants are arranged along 4 axes. The position of points along the axes depends on concentration: the
3 18
/
12~
20
20
r"
,f
t.l',j,, . Io'o " i l l 4 ~Pi78., ; / ° ,
/
,,;
,so/ /~
/
16(?
o
"_._ "-
,ltil,":o
J
,/
12141~ 7 1'
,18 /
/
1 37~ Fig. 5. Principal component analysis of bulb neuron responses: three-dimensional space defined by factors 1, 2 and 3 (assodated variances in %). Odorant names are abbreviated as in Table I; concentrations are coded as in Table [I.
260 anisole by taking into account factor 2. The third factor allows anisole to be discriminated from limonene. Factor 4 (not represented) indicates a clear distinction between the highest concentrations of limonene and its lowest concentrations which are grouped in the center of the space with the lowest concentrations of the other odorants. As in the space related to the responses of receptor cells, the 4 sets of points representing the 4 odorants are distributed along 4 directions. From the center to the periphery, the points are ranked according to increasing concentrations. DISCUSSION This work provides the first comparative description of discrimination abilities of receptor cells and bulb neurons as a function of stimulus intensity.
Comparison of stimuli in pairs In the olfactory mucosa, the correlations observed between low and medium concentrations of different compounds and the lack of cohesion inside a given set of odorant concentrations bring out the difficulties of receptor cell population to discriminate the stimuli in the range of concentration from 10-6 to 1.7 x 10-4 of saturation (with the exception of camphor). The dynamics of cell recruitment as a function of stimulus intensity has been recently analyzed H. For receptor cells, it appeared that, depending on the nature of the stimulus, 5-20% of the cell population were involved in the activation pattern at concentrations lower than 10 -4 of saturated vapor. In this concentration range, because of the small fraction of the cell population involved in the activation pattern, it could be assumed that receptor cells send a message with a low signal/noise ratio. Thus, before the olfactory bulb processing, we cannot statistically extract the specific pattern encoding for stimulus quality. In the bulb, there is considerable cohesion among the different concentrations of a given stimulus while there are few correlations between odorants. Thus, whatever the intensity, each compound seems to be better recognized as a given quality and more sharply discriminated from other compounds. However, the lowest concentrations of the 4 odorants tested do not appear to be clearly discriminated from each other. By contrast with the receptor cell level, at 10 -6 of saturation, the recruitment of bulb neurons was found to involve 10-40% of the cell population, depending on the nature of the stimulus ~1. From these data, it can be assumed that, once bulbar processing has been carried out, the message is more salient, specially when stimuli are delivered at low concentrations.
Representation of olfactory spaces Factor analyses provide other kinds of information and lead to further discussion of the results. Considering the responses of the two cell populations, the 4 stimuli are clearly individualized in the olfactory spaces. In addition, we mentioned that the concentrations for a given compound are ranked from the center to the periphery of the space according to increasing concentrations. The better an odorant is distinguished, the farther its point is from the center of the space and so the probability of discrimination seems to increase with stimulus concentration. This result is of importance in the knowledge of olfactory coding. It can be discussed in terms of the setting up of the activation pattern. The continuity of cell recruitment implies that the activation pattern is progressively built up as a function of stimulus intensity: more and more cells, among those which are responsive to a given stimulus, are involved. For increasing concentrations, it can be assumed that the activation pattern becomes more and more specific for a given stimulus, thus probably representing a more and more detailed description of the odorants. Furthermore, according to the olfactory spaces described here, this pattern keeps its specificity at the highest intensity tested (5.62 x 10-2 of saturation). This improvement of discrimination probabilities with increasing intensity is closely associated with the fact that cell recruitment is a continuous process: we have never observed cells responding at low concentrations which failed to respond at high concentrations. Furthermore, from the present data, it is clear that the sensory message does not undergo any damage at high intensity. Thus, our findings are not in agreement with the hypothesis put forward by some authors 21'25 who assumed that a distinct combination of active cells encoded the olfactory stimulus at each intensity level. At the receptor level it is worth noting that the first factor of the analysis ranks the representative points of stimuli according to the strength of the responses, whatever the nature of the stimulus. In the space defined by subsequent factors 2, 3 and 4, four subsets of points aligned according to 4 directions are identified. At the olfactory bulb level, the analysis shows that the first factor is directly involved in qualitative discrimination. This difference between the olfactory spaces is related to discrimination abilities observed at the bulbar level. The convergence of primary afferences onto glomeruli seems to result in shifts of the discrimination thresholds of odors towards lower concentrations. It has been demonstrated that the convergence was functionally involved in the amplification of the primary signal and therefore, in an increase of the detection power 11. Because of this amplification, the dynamics of cell recruitment was found to begin and to end at lower concentration levels in the
261 olfactory bulb than in the mucosa. Thus, at a given concentration, the fraction of the cell p o p u l a t i o n involved in a stimulus activation p a t t e r n is larger in the bulb than in the mucosa. T h e i m p r o v e m e n t o b s e r v e d in the bulb strongly underlines the d e p e n d e n c e which exists b e t w e e n stimulus intensity and discrimination abilities: in the bulb, as a c o n s e q u e n c e of p r i m a r y signal amplification, these abilities s e e m less d e p e n d e n t on intensity than in the mucosa. Quality versus intensity In previous studies of o d o r discrimination 7-1°'32'33'36, the qualitative group concept has been introduced. It was p o i n t e d out that possibilities of discrimination r e m a i n e d inside a qualitative group. A s an example, two enantiomers, like D-carvone and L-carvone, e n c o d e d by very similar activation patterns, were found to be distinguished by bulb neurons 7 and r e c e p t o r cells 35. In the same way, in the p r e s e n t study, the different concentrations of a given o d o r evoking similar patterns could nevertheless be discriminated from one another. This REFERENCES 1 Baylin, E, Temporal patterns and selectivity in the unitary responses of olfactory receptors in the tiger salamander to odor stimulation, J. Gen. Physiol., 74 (1975) 17-36. 2 Berglund, B., Berglund, U. and LindvaU, T., Psychological processing of odor mixtures, Psychol. Rev., 83 (1976) 432-441. 3 Bieber, S.L. and Smith, D.V., Multivariate analysis of sensory data: a comparison of methods, Chem. Senses, 18 (1986) 19-47. 4 Doving, K.B., Studies on responses of bulbar neurons of frog to different odor stimuli, Rev. Laryngol., 86 (1965) 845-854. 5 Doving, K.B., The influence of olfactory stimuli upon the activity of secondary neurons in the burbot (Lota Iota L.), Acta Physiol. Scand., 66 (1966) 290-299. 6 Doving, K.B., An electrophysiologicalstudy of odor similarities of homologous substances, J. Physiol. (Lond.), 186 (1966) 97-109. 7 Duchamp, A., Electrophysioiogical responses of olfactory bulb neurons to odor stimuli in the frog. A comparison with receptor cells, Chem. Senses, 7 (1982) 191-210. 8 Duchamp, A., Revial, M.E, Holley, A. and Mac Leod, P., Odor discrimination by frog olfactory receptors, Chem. Senses, 1 (1974) 213-233. 9 Duchamp, A. and Sicard, G., Odour discrimination by olfactory bulb neurons: statistical analysis of electrophysiological responses and comparison with odour discrimination by receptor cells, Chem. Senses, 9 (1984) 1-14. 10 Duchamp, A. and Sicard, G., Influence of stimulus intensity on odour discrimination by olfactory bulb neurons as compared with receptor cells, Chem. Senses, 8 (1984) 355-366. 11 Duchamp-Viret, P., Duchamp, A. and Vigouroux, M., Amplifying role of convergence in olfactory system. A comparison of receptor cell and second order neuron sensitivities, J. Neurophysiol., 61 (1989) 1085-1094. 12 Gesteland, R.C., Comments on microelectrodes, Proc. LR.E., 47 (1959) 1856-1862. 13 Gesteland, R.C., Neural coding in olfactory receptor cells. In L.M. Beidler (Ed.), Handbook of Sensory Physiology, Vol. IV, Chemical Senses, 1, Springer, Berlin, 1971, pp. 132-150. 14 Gesteland, R.C., Physiology of olfactory reception. In R. Llinas and W. Precht (Eds.), Frog Neurobiology, Springer, Berlin,
possibility does not imply that different concentrations of an o d o r are systematically p e r c e i v e d as different qualities, but in some cases, shifts of perceived quality can be expected. A few studies have r e l a t e d the intensity to the quality of an odorant. Previously, the influence of quality on perceived intensity 2 and conversely, the influence of intensity on o d o r quality 2° have been studied using psychophysical experiments. T h e authors concluded that variations in stimulus intensity m a y be perceived as differences in stimulus quality, a conclusion in a g r e e m e n t with the empiric experience of p e r f u m e r s 31. In terms of neural coding, the possibility of discriminating two activation p a t t e r n s e v o k e d by different concentrations of a given stimulus might supply information to the olfactory system a b o u t both intensity and quaiity. This means that perceived quality could be involved in the evaluation of stimulus intensity. Acknowledgements. We should like to thank Professor A. Holley for his critical review of the manuscript. 1976, pp. 234-250. 15 Gesteland, R.C., Lettvin, J.Y., Pitts, V.H. and Rojas, H. Odor specificities of the frog's olfactory receptors. In Y. Zotterman (Ed.), Olfaction and Taste, Vol. I, Pergamon, Oxford, 1963, pp. 19-34. 16 Getchell, T.V., Analysis of unitary spikes recorded extracellularly from frog olfactory receptor cells and axons, J. Physiol. (Lond.), 234 (1973) 533-551. 17 Getchell, T.V. and Shepherd, G.M., Responses of olfactory receptor cells to step pulses of odour at different concentrations in the salamander, J. Physiol. (Lond.), 282 (1978) 521-540. 18 Getchell, T.V. and Shepherd, G.M., Adaptative properties of olfactory receptors analyzed with odour pulses of varying duration, J. Physiol. (Lond.), 282 (1978) 541-560. 19 Gower, J.C., Some distance properties of latent root and vector methods used in multivariate analysis, Biometrika, 53 (1966) 325-338. 20 Gross-Isseroff, R. and Lancet, D., Concentration-dependent changes of perceived odor quality, Chem. Senses, 13 (1988) 191-204. 21 Kauer, J.S., Response patterns of amphibian olfactory bulb neurons to odour stimulation, J. Physiol. (Lond.), 243 (1974) 695-715. 22 Kauer, J.S. and Moulton, D.G., Responses of olfactory bulb neurons to odour stimulation of small nasal areas in the salamander, J. Physiol. (Lond.), 243 (1974) 717-737. 23 Kauer, J.S. and Shepherd, G.M., Olfactory stimulation with controlled and monitored step pulses of odour, Brain Research, 85 (1975) 108-113. 24 Kauer, J.S. and Shepherd, G.M., Analysis of the onset phase of olfactory bulb unit responses to odour pulses in the salamander, J. Physiol. (Lond.), 272 (1977) 495-516. 25 Lidow, M.S., Gesteland, R.C., Shipley, M.T. and Kleen, J.S., Comparative study of immature and mature olfactory receptor cells in adult frogs, Dev. Brain Res., 31 (1987) 243-258. 26 Mair, R.G., Response properties of rat olfactory bulb neurons, J. Physiol. (Lond.), 326 (1982) 341-359. 27 Mathews, D.F., Response pattern of single-neurons in the tortoise olfactory epithelium and olfactory bulb, J. Gen. Physiol., 60 (1972) 166-180. 28 Mathews, D.E, Response pattern of single units in the olfactory
262 bulb of the rat to odor, Brain Research, 47 (1972) 389-400. 29 Meredith, M., The analysis of response similarity in single neurons of the goldfish olfactory bulb using amino-acids as odor stimuli, Chem. Senses, 6 (1981) 277-293. 30 Meredith, M. and Moulton, D.G., Patterned response to odor in single neurons on goldfish olfactory bulb: influence of odor quality and other stimulus parameters, J. Gen. Physiol., 71 (1978) 615-643. 31 Moncrieff, R.W., The Chemical Senses, 2nd edn., Hill, 1967, p. 538, 32 Reviai, M.E, Duchamp, A. and Holley, A., Odour discrimination by frog olfactory receptors: a second study, Chem. Senses, 3 (1978) 7-21. 33 Reviai, M.E, Duchamp, A., Holley, A. and Mac Leod, P., Frog olfaction: odour groups acceptor distribution and receptor
categories, Chem. Senses, 3 (1978) 23-33. 34 Revial, M.E, Sicard, G., Duchamp, A. and Hoiley, A., New studies on odour discrimination in the frog's olfactory receptor cells. I. Experimental results, Chem. Senses, 7 (1982) 175-190. 35 Revial, M.E, Sicard, G., Duchamp, A. and Hoiley, A., New studies on odour discrimination in the frog's olfactory receptor cells. II. Mathematical analysis of electrophysiological responses, Chem. Senses, 8 (1983) 179-194. 36 Sicard, G. and Holley, A., Receptor cell responses to odorants: similarities and differences among odorants, Brain Research, 292 (1984) 283-296. 37 Vigouroux, M., Viret, P. and Duchamp, A., A wide concentration range olfactometer for delivery of short reproducible odor pulses, J. Neurosci. Methods, 24 (1988) 57-63.
