Acta physiol. scand, 1977. 99. 270-280 F r o m the Institute of Zoophysiology, University of Oslo, Blindern, Norway
Spatial Distribution of the EOG in the Rat; a Variation with Odour Quality BY
G. THOMMESEN and K. B.
DQVING
Received 1 July 1976
Abstract THOMMESEN, G . and K. B. D B V I N G . Spatial distribution of the EOG in the rat; a variation with odour quality. Acta physiol. scand. 1977. 99. 270-280. The spatial distribution of olfactory receptors in the rat has been studied by simultaneous recordings of the electroolfactograms (EOGs) from areas of the olfactory mucosa. The potentials were recorded from the dorsal surface of the cribriform plate, leaving the rnucosa and the nasal cavities intact. The ratio between the peak amplitude of the EOG potentials served as a measure of the relative sensitivity of one area against the other. The results obtained by stimulating with 31 substances a t 38 positions demonstrate a non-homogeneous distribution of different receptors. Each substance gives a spatial pattern of response efficiency. The response distributions are mapped on the cribriform plate for the different odours.
The existence of different olfactory receptors in vertebrates has been demonstrated in frogs (Gesteland et al. 1963, Altner and Boeckh 1967, Duchamp et al. 1974) and in the tortoise (Mathews 1972). A differential spatial distribution of receptor cells of different specificity to olfactory stimuli is a possible basis for the functional organization of this sensory pathway within the vertebrates. Adrian (1951) found that the different odours evoked maximum responses in different areas of the rabbit olfactory bulb. His findings were confirmed by Mozell and Pfaffman ( I 954). The spatial distribution of responses to odours within the olfactory system have also been demonstrated by other methods. Thus prolonged exposure to an odour causes a characteristic pattern of morphological changes named selective degeneration (Dsving and Pinching 1973, Pinching and Dsving 1974). Odour stimulation is associated with an increased metabolic activity which is unevenly spatially distributed (Sharp et al. 1975). Le Gros Clark (1951) made lesions in the glomerulus layer of the olfactory bulb of rabbits. On the basis of the retrograde degeneration patterns in the olfactory mucosa he concluded that the projection of the olfactory epithelium on the olfactory bulb was somatotopic. This was confirmed by Land (1973) looking at the anterograde degeneration in the glomeruli after sectioning of small bundles of the olfactory nerve. The projections showed however, to be divergent and partially overlapping. 270
271
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
Dorsal Fig. 1. Medial view of the left side endoturbinals of the rat head. The distribution of the olfactory epithelium is indicated by stippling. The upper left projection shows the cribriform plate in a dorsal view with a typical arrangement of the major holes.
4nterior
From these observations and the observations on the spatial distribution of olfactory bulb responses one can assume a non-homogeneous distribution of receptors within the epithelium. By simultaneous recordings of the electro-olfactogram (EOG) at different sites of the sensory epithelium such a non-homogeneous distribution was demonstrated in frog (Mustaparta 1971, Daval, Leveteau and MacLeod 1969). We wanted to compare the pattern of selective degeneration with a possible pattern of non-homogeneous distribution of receptor cells. In the present study we show a spatial distribution of the different types of receptor cells in the rat by analysis of the relative amplitudes of the EOGs recorded from different areas of the cribriform plate.
Materials and methods The strain of experimental animals was the same as used in the studies on the selective degeneration occurring in the olfactory bulb of rat after prolonged odour exposure (Pinching and Deving 1974). White female rats of the Wistar strain were used. They were kept in semi-germfree conditions in isolation chambers to avoid rhinitis. The rats were between 180 and 230 g (18-23 weeks old) when used in the expts. The experimental animal was anesthetized with urethane given intraperitoneally (1.5 g per kg b.wt.). To separate the sniffing action from the respiration a tracheostomy was carried out, and a polyethylene catheter was inserted through the tracheostoma into the choana. The animal was fastened in a headholder. The skull was opened dorsally and the olfactory bulb and the frontal part of the cerebrum was removed to expose the dorsal surface of the cribriform plate as indicated in Fig. 1. Lymph and cerebro-spinal fluid was continuously removed by cellulose wicks. Deep body temperature of the preparation was maintained at 37.3"C. During unilateral recordings, the contralateral (right) nostril was plugged with vaseline. Recording Two recording electrodes made contact with the basal side of the olfactory epithelium at the holes of the cribriform plate. Monopolar recordings were made against a common indifferent electrode placed on the skull. The electrodes were broken glass capillaries, tip diameter 25-100 ,urn, filled with agar-Ringer and inserted in electrode holders containing sintred Ag/AgC1 (WPI, Hamden, Conn., USA). The potentials were pre-amplified by a DC-amplifier, with input impedance of 10 MOhm, and recorded on a pen recorder Beckman RS Dynograph 2 channel with DC-couplers; frequency response: DC to 20 Hz).
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G . THOMMESEN AND K. B. DBVlNG
TABLE 1. List of stimuli.
Order of stimulation
Symbols
Name
Order of stimulation
Symbols
Name
4 5 24
c5 C6 c7 C8 c9 CIO c1I c12 C13 ACE BEN CAM CHL DEC EAA HAL
C yclo pent ano ne Cyclohexanone Cycloheptanone Cyclooctanone Cyclononanone Cyclodecanone Cycloundecanone Cyclododecanone Cyclotridecanone Acetophenone Benzylthiol DL-Camphor Cyclohexanol 1-Decanol Ethyl acetoacetate Heptanal
17 16
HOL ION iBA I PA LI M MEN MBZ MBL MHN MUL MVA NAF NBZ OCT MCC
1-Heptanol p-Ionone Isobornylacetate isopentylacetate ( +)-Limonene L-Menthol Methylbenzoate 2-Methyl-3-butyn-2-01 6- Methyl-5-hepten-2-one 2-Methylundecanal 4-Methy1 valeric acid Naphthalene Nitrobenzene Octanal 1,1,2,2,-tetramethy1-3-,3dichloro-cyclopropane
6
25 1
7
26
3 15 21-3 1 20 7 18
12 27-31
11
27-3 1 8 21 13 22 27-3 1 23 10 9 19
27-3 1 14
Sriniulation The catheter in the choana was connected to a suction pump via a flowmeter and a magnetic valve. The valve was controlled by a programming device giving 1 s suction, 1 s pause, 5 s suction, 4 s pause and f s suction. Suction air flow was held at 7-14 ml/s. The preparation was continuously supplied with purified and humidified air through a glass funnel at a rate of 100 ml/s, producing a clean air-mask around the snout. In the studies of selective degeneration (Pinching and Dsving 1974) were used 44 different odours. For the present investigation 31 of them were found to be potent enough to give basis for discussion. These odours and their order of presentation are listed in Table I. The stimuli were introduced on polyethylene spatulae between the funnel and the snout just before the first (1 s) suction and withdrawn during the first pause. The second (5 s) suction thus served as washing and the last (3 s) suction as a control. In every position the total series of stimuli were used. Grading of stimuli was performed by varying the position of the spatula in the clean air flow. Experimental procedure For positioning the recording electrodes on the preparation the horizontal bony crest dividing the cribriform plate in two approximately equal parts, served as a landmark. One electrode was placed on the left cribriform plate in a position approximately 0.5 mm ventral to this crest and 1 mm from the septa1 extension as seen in Fig. 2 and 3. This electrode will later be referred t o as the “stationary electrode”. The other recording electrode was successively placed at 38 different positions uniformly spread over the left cribriform plate, and will be referred to as the “mobile electrode”. Every preparation was photographed and the electrode positions were plotted on the photograph. Th e different electrode positions are shown in Fig. 6 a . Data treatment The treatment of the results is based on the method used by Mustaparta (1971). The ratio, N between the the EOG amplitude of the mobile electrode and that of the stationary one served as a measure for the stimulating efficiency of each substance at the position of the mobile electrode. This procedure was used to minimize the effects of variable concentrations. The results were transferred to a non-parametric scale by ranking the ratios according to decreasing value at every recording position. The response at the position of the stationary electrode was quantified by a mean rank value for each stimulant. This value is equal to 32 minus the mean of all the rank numbers obtained for that stimulant and regarded as equivalent to the proper rank numbers. For each odorous substance the ranks were noted on position maps similar to those shown in Fig. 5 and 6.
273
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
L imonene
Fig. 2. Difference in response to limonene and methylbenzoate in two positions of the mobile electrode us. the stationary electrode. Electrode positions are shown to the left. Stimulation time is 1 s. Vertical bar is 0.5 mV.
Stirnulotion
I
Washing Control
mnnmmrm n
I -c;I
met hy 1 benzoate
I - 0
Controls
Various forms of control expts. were performed. The stability of the preparation and the experimental conditions were controlled by repeating the first stimulus at the end of each stimulation series. Expts. were made to control the ratio N at various odour intensities. In 17 series several stimulations were made with the same odour a t different intensities. These expts. would indicate possible systematic variations of the ratio N between the amplitudes at the stationary and mobile electrode positions with the concentration of the odour. To control the variability from animal to animal eight pairs of presumed equivalent recording positions were studied.
Results The extension of the olfactory mucosa on the endoturbinal surfaces was studied in several rats and an average distribution is shown in Fig. 1. The difference between the sensory and the respiratory surfaces is clearly indicated by the ciliary movements in the latter epithelium in a freshly killed animal. By recording from the dorsal surface of the cribriform plate as described by Ottoson (1954, 1959) the EOGs are recorded without interfering with the air flow in the nasal cavities. EOGs were positive when recorded from the basal side of the olfactory epithelium (Ottoson 1954, 1959) and behaved like a summated generator potential. If the electrode penetrated the mucosa, the EOG became negative. The time course of the EOG agrees with that described by Ottoson (1954, 1956, 1959). After a latency of approximately 0.2 s the EOG rose to full size in 0.5 to 0.7 s. By prolonged stimulations an adaptation was seen whereby the EOG reduced to about 2/3 of its peak amplitude. Only the peak amplitude has been subject to analysis in the present article. EOG amplitudes to odorous stimulation varied from 0.2 to 2.5 mV. To standardize recording conditions, stimulation procedures were modified so that responses usually did not exceed 1 mV, thereby minimizing stimulation of receptor types with different specificity. Examples of the recordings are shown in Fig. 2. 18 - 775873
274
G. THOMMESEN AND K. B. DQVING
r
Amplitude at p o s. S ( m V ) >
E
].Or LiM
Fig. 3. The peak amplitudes of the electro-olfactogram recorded with different stimulus intensities. The recordings at the different positions A, B and C are simultaneous with those at the stationary electrode position, S. Pos. A and pos. B are from the same animal. The abbreviations in the diagrams refer to the different chemicals listed in Table I. The straight lines are drawn through the origin. Note the difference in stimulation efficiency of limonene between pos. A and pos. B compared with that of the cycloketones.
At four positions along the ventral margin of the cribriform plate, hatched on the position map Fig. 6 a, most responses were too faint to give substantial bases for data treatment. Purified air elicited responses of 0.1-0.2 mV depending upon the flow of air suction, presumably due to imperfect purification. This response disappeared only when the suction air flow was insufficient for odour stimulation. Controls of the reliability of rhe dara
Various expts. were performed to study the reliability of the results. As mentioned in the discussion the ratio N between two recording positions should be independent of stimulus intensity. To test this hypothesis repetitive stimulations were made with varying stimulus concentrations. Fig. 3 shows the amplitudes of the EOG at the stationary electrode position versus those from 3 other areas. Plots have been made for four odours, uiz., two cycloketones, limonene and methyl benzoate. The ratio N is shown as the slope of the line through the origin. The variation is small with different concentrations but the slopes or the ratio
275
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
Stimulus :
.-
,
Open Symbols-Cyclodecanone Cyclohexanone
L
/
1.5 -
cy
2
.o 0
m Fig. 4. The ratios N obtained by stimulation with the same substance at the beginning (N,) and at the end (N2) of each stimulation series. Graph symbols refer to the diferent preparations as indicated, Fig. 6 a . Broken lines show the 95% confidence interval.
-
1.0-
0.5
1.0 Ratio N,
1.5
2.0
N varies from one electrode position to the other. The diagram demonstrates the general tendency of the ratio to be constant over a large range of concentrations for the same stimulus and electrode position. In 32 runs covering several recording positions, 3-8 consecutive stimulations were made with the same substance. The standard deviation of the ratio N of the EOG amplitude at the mobile electrode to that at the stationary one, with the same substance and position, showed a mean value of 0.07. This standard deviation is less than that obtained for the total series of stimuli in any recording position, which ranged from 0.08 to 0.42, the lowest values in medial positions. The stability of the preparation and the reproducibility of the results were also controlled by repeating the first stimulation at the end of each series. This was done in all cases except for the 4 ventral positions mentioned above. Fig. 4 shows the ratio N for the first ( N l ) and the last ( N e )stimulation with the same substance in the 34 stimulation series. In all but one case the control stimulus was cyclodecanone. At three positions only, the difference between the ratios fall outside a 95 % confidence interval found by using the above value of SD(N). At none of the positions included in this presentation the differences fall outside the 98 % confidence interval centered at zero. The test confirms that the preparations did not change significantly in sensitivity throughout the expts. The results consist of measurements made on five animals, 4 to 12 recording positions in each (Fig. 6 a). The positions cover overlapping areas of the cribriform plate. Due to individual variations none of the positions could be regarded as completely congruent in different animals. Some recording positions were however, assumed to be equivaient from one preparation to another, indicated by overlapping symbols in Fig. 6 a. We have made eight comparisons, calculating the Spearman rank correlation coefficients. In seven of these comparisons the ranking of odours were positively correlated with signif-
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G. THOMMESEN AND K. B. DBVING
icance levels less than 0.5 per cent. In only one comparison concerning positions centrally in the dorsal half of the cribriform plate there was no significant correlation. These controls indicate that the results included in this presentation were reproducible from one preparation to another. Stirnuluting with different substance5
At all the different positions studied the ratio of the EOG amplitude at the mobile us. the stationary electrode position varied with odour quality. Recordings of the EOG from different positions on the cribriform plate are shown in Fig. 2. Recording pairs with positions marked with the same symbols were recorded simultaneously. As seen limonene gave the smallest amplitude at the stationary electrode position in both cases. Methyl-benzoate gave the highest response at the “mobile” position in the upper pair but for the EOGs at the lower pair of positions, the highest response was at the stationary electrode. Relative shifts in stimulation efficiency are also demonstrated in Fig. 4 for limonene and two of the cycloketone\. Conipurisons o f EOGs from left and right sides
3 series of simultaneous recordings have been made with the 2 recording electrodes symmetrically placed on the left and the right side cribriform plate. In this case, the ratio N was found to be similar for all odours. The variations were not greater than expected from random variations in stimulus concentrations on the two sides, inaccuracy in response measurements or the uncertainty in finding exactly equivalent positions on nearly symmetrical structures. There is therefore no basis for assuming qualitative differences in sensitivity distribution between the olfactory epithelium of the right and the left side. Mapping of response distribution
The N values of each position of the mobile electrode were ranked, giving the number 1 to the highest ratio and rank number 31 to the lowest. The rank numbers were plotted on the map of the cribriform plate, one map for each stimulus used. 4 examples of such maps are given in the upper row of diagrams in Fig. 5. In an attempt to visualize the sensitivity distribution in the olfactory epithelium as it appears by recording from the cribriform plate, the maps were simplified by hatching. Areas with rank numbers 1-9 have been crosshatched as a sign of high stimulus efficiency. Areas with rank numbers 10-19 have been covered with simple hatching and areas with rank numbers 20-31 are unhatched as signs of medium and low response respectively. In Fig. 5 this arbitrary and simplified visualization of the data is shown for acetophenone, cycloundecanone, camphor and heptanal. The four maps represent 4 different types of distribution pattern which are fairly typical. Of the remaining 27 substances, 24 gave distribution maps which had visual similarity to one of the four maps shown. Similarity to the acetonphenone map was found for the lower cycloketones of 5-8 carbon atoms, cyclohexanol, menthol, methylbenzoate, 4-methyl valeric acid, naphthalene and nitrobenzene. Responses to decanol, cyclodecanone, cyclododecanone and cyclotridecanone were distributed similarly to the map for cycloundecanone. Only the
277
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
Acetopheno ne
Cycloundecanone
D,L-Camphor
'1619
l5
Heptanal
14
2
17.2
1
208
10
15 2
15
22
18
6
2
7
Fig. 5. Examples of response distributions to four different odours. Upper row: the rank numbers obtained in every position. Low rank numbers show high response ratio. Lower row: Simplified response distribution maps. Areas with rank numbers lower than 20 are hatched, areas with rank numbers lower than 10 are crosshatched.
response distribution of methylheptenone shows any similarity to that of camphor. The maps obtained for benzylthiole, cyclononanone, ethyl acetoacetate, isobornylacetate, isopentylacetate, limonene, methylundecanal, octanal and tetra-methyl-dichloro-cyclo-propane bear the closest resemblance to the heptanal map. The responses to p-ionone, methylbutynol and heptanol were so uniformly distributed that no pattern will be proposed. The majority of 26 substances can also be seen to divide in two groups with response distribution maps approximately complementary to each other. One group of 16 odours represented by acetophenone and cycloundecanone, gives the strongest response dorsally or dorsomedially. The group of 10 here represented by heptanal give the strongest responses ventrally or laterally. Another way of representing the results is shown in Fig. 6. In a map of the cribriform plate the recording positions are marked with different symbols for each preparation (Fig.
278
G. THOMMESEN AND K. B. D0VING
E l e c t r o d e positions
Rank n o . 1
Rank n o 3
Rank n o 2
CSIACE
I BA
a
b.
C.
d.
Fig. 6 . Schematic diagrams of the left cribriform plate (see Fig. 1). a shows the different electrode positions. The different symbols refer to different preparations. Black symbols show the positions of the stationary electrode. Hatched symbols mark positions where only a few substances elicited a measurable response. h, c and d show what substances obtained rank number 1, 2 and 3 respectively (see text).
6 a). In the following maps the odours are plotted which had the first, second and third rank in each position. The maps show the same grouping of substances as mentioned above and shown in Fig. 5 .
Discussion The general properties of the electro-olfactogram (EOG) in frog have been analysed in detail by Ottoson (1956). The objections raised by various authors concerning the features of the EOG and its relation to the receptor mechanisms have been discussed by Ottoson (1971). I n the present study we have considered the EOG to represent the electrotonic projection of the sum of generator potentials produced by those receptor cells which respond to the stimulus. The laws for the spread of electrical potentials from a dipole layer in a volume conductor imply that the ratio between the potentials recorded from two different points is a function only of the shape and location of the dipole layer, but not of the voltage. In the present work it is assumed that the recording of a sum of generator potentials from the olfactory mucosa follows the same rule, as long as only one homogeneous population of olfactory receptors are activated. From this assumption follows that during stimulation with different odours, the ratio between the EOG potentials recorded from 2 different points in the olfactory epithelium will vary if and only if the epithelium contains receptor populations with different spectra of sensitivity and different spatial distribution. The ratio will be independent of the concentration of the stimulant within certain limits; i.e., as long as receptor types with different thresholds are not activated, so that only one receptor population is involved. Even under ideal circumstances the ratio between the two recorded potentials will vary, however, due
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
279
to random variation in the electrical noise of the recording devices and the uncertainty in the reading of the potentials. The EOG may be recorded anywhere in the proximity of the olfactory epithelium. The most convenient recording site in the mammal is the nearly two-dimensional cribriform plate. By the described preparation procedure the EOG potentials are recorded without interference with the nasal flow pattern since the nasal cavities are intact. The recordings are also free of electrode potential artefacts as the electrode tips are not exposed to the stimulating substances. The recorded potentials are electrotonic projections of potentials from a highly folded olfactory epithelium serving as a volume conductor. The potentials are not generated by the olfactory axons projecting through the holes of the cribriform plate, and the exact location of the excited receptor cells within the epithelium can not be deduced from the distribution of responses. In spite of these reservations it is reasonable to presume that the different parts of the olfactory epithelium project mainly to nearby parts of the cribriform plate. For instance, a strong response in the dorsal area of the cribriform plate is most likely caused by activation of receptors mainly located in the roof of the nasal cavity or on the upper turbinates. Adrian (1951) suggested that the spatial differences in the bulbar responses to different odours were caused by non-homogeneous distribution of olfactory receptors of different specificity. The interpretation assumed that the neuronal projection from the olfactory mucosa to the 01factory bulb was more or less somatotopic as in fact was demonstrated the same year by Le Gros Clark (1951). Spatial differences in the distribution of different olfactory receptors were confirmed by Mustaparta in 1971 as evident in the frog.
The reliability of the distribution maps In the present study several types of control experiments were performed; the variation of the ratio N at the same position for different odour intensities, the stability of one preparation throughout the expt. and the reproducibility from one animal to another. The results of these control experiments demonstrate the consistency of the reported data. They also obviate further repetitions of the experiments at homologous positions in different animals. Contamination of the odorous substances is another possible cause of error. Aldehydes in general are susceptibleto oxidation producing the corresponding acids, the effect of which may have distorted the response distribution maps of the three aldehydes. The cyclononanone sample is known to contain impurities, presumably di-ketones and aldehydes. It is therefore interesting to note that cyclononanone, as the only one of the cycloketones, places itself in the group of substances which also includes the three aldehydes.
Conclusions The present results show that receptor neurones of different spectra of sensitivity are differentially distributed in the mammalian olfactory mucosa as well. In spite of the complex folding of this epithelium, the spatial distribution of receptor responses is clear. None of
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the response distribution maps were identical. On the basis of their shape, the maps may be grouped, as belonging to a small number of patterns, some of which make up pairs of almost complementary response distributions. A further analysis of the data on the similarity in response distributions will appear in a subsequent article. Difference in the distribution of the EOG does not indicate that there are discrete areas of uniform specificity. If a finite number of receptor types exists, the receptors most likely cover overlapping fields in the olfactory mucosa. The non-homogeneous distribution of olfactory receptors of different specificity implies that if two substances show different response distributions, they most likely are perceived by the animal as being different. The reverse statement is not necessarily true. Receptors sensitive to different odours may be intermingled so as to produce the same EOG distribut ion. This study has been supported by the Norwegian Research Council for Science and the Humanities.
References ADRIAN,E. D., Olfactory discrimination. Ann. psychol. 1951. 50. 107-113. ALTNER, H. and 3 . BOECKH,fiber das Reaktionsspektrum von Rezeptoren aus der Riechschleimhaut von Wasserfroschen (Rana esculenta). Z. rergl. Physiol. 1967. 55. 299-306. and P. MACLEOD, Electroolfactogramme local et discrimination olfactive chez DAVAL,G., J. LEVETEAU la Grenouille. 1. Physiof. (Paris) 1969. 61. Suppl. 2. Selective degeneration of neurones in the oifactory bulb following DQVING,K. B. and A. J. PINCHING, prolonged odour exposure. Brain Res. 1973. 52. 115-129. DUCHAMP, A., M. F. RIVIAL,A. HOLLEYand P. MACLEOD,Odour discrimination by frog olfactory receptors. Chem. Scnses and flouor 1974. 1. 213-233. R. C., 7. Y. LETTVIN, W. H. PITTSand H. ROJAS,Odour specificities of the frog’s olfactory GESTELAND, receptors. Olfaction and Tasre. Pergamon Press, Oxford, 1963. 1. 19-34. LAND,L. J., Localized projection of olfactory nerves to rabbit olfactory bulb. Bruin Res. 1973. 63. 153-1 66. , E., The projection of the olfactory epithelium on the olfactory bulb in the rabbit. LE GROSC L A R KW. J. Neurol. Neurosurg. Psychiat. 1951. 14. 1-10. MATHEWS, D. F., Response Patterns of Single Neurons in the Tortoise Olfactory Epithelium and Olfactory Bulb. J . gen. Physiol. 1972. 60. 166-180. C., The afferent neural processes in odor perception. Ann. N . Y. Acad. MOZELL,M. M. and PFAFFMANN, Science 1954. 58. 96-108. MUSTAPARTA, H., Spatial Distribution of Receptor-Responses to Stimulation with Different Odours. A c f a phvsiol. scund. 1971. 82. 154-166. D., Sustained potentials evoked by olfactory stimulation. Acra physiol. scand. 1954. 32. 384-386. OTTOSON, D., Analysis of the electrical activity of the olfactory epithelium. Acru physiol. scand. 1956. 35. OTTOSON, Suppl. 122. 1-83. OTTOSON,D., Studies on slow potentials in the rabbit’s olfactory bulb and nasal mucosa. A c f a ph.wioI. scand. 1959. 47. 136-148. OTTOSON, D., The electro-olfactogram. A review of studies on the receptor potential of the olfactory organ. In Handbook of Sensory Physiology. Springer Verlag. 1971. 4. 95-1 3 1. A. J. and K. B. DQVING, Selective degeneration in the rat olfactory bulb following exposure to PINCHING, different odours. Brain Res. 1974. 82. 195-204. SHARP,F. R., J. S . KAUERand G. SHEPHERD, Local sites of activityrelated glucose metabolism in rat olfactory bulb during olfactory stimulation. Bruin Res. 1975. 98. 596-600.
Spatial Distribution of the EOG in the Rat; a Variation with Odour Quality BY
G. THOMMESEN and K. B.
DQVING
Received 1 July 1976
Abstract THOMMESEN, G . and K. B. D B V I N G . Spatial distribution of the EOG in the rat; a variation with odour quality. Acta physiol. scand. 1977. 99. 270-280. The spatial distribution of olfactory receptors in the rat has been studied by simultaneous recordings of the electroolfactograms (EOGs) from areas of the olfactory mucosa. The potentials were recorded from the dorsal surface of the cribriform plate, leaving the rnucosa and the nasal cavities intact. The ratio between the peak amplitude of the EOG potentials served as a measure of the relative sensitivity of one area against the other. The results obtained by stimulating with 31 substances a t 38 positions demonstrate a non-homogeneous distribution of different receptors. Each substance gives a spatial pattern of response efficiency. The response distributions are mapped on the cribriform plate for the different odours.
The existence of different olfactory receptors in vertebrates has been demonstrated in frogs (Gesteland et al. 1963, Altner and Boeckh 1967, Duchamp et al. 1974) and in the tortoise (Mathews 1972). A differential spatial distribution of receptor cells of different specificity to olfactory stimuli is a possible basis for the functional organization of this sensory pathway within the vertebrates. Adrian (1951) found that the different odours evoked maximum responses in different areas of the rabbit olfactory bulb. His findings were confirmed by Mozell and Pfaffman ( I 954). The spatial distribution of responses to odours within the olfactory system have also been demonstrated by other methods. Thus prolonged exposure to an odour causes a characteristic pattern of morphological changes named selective degeneration (Dsving and Pinching 1973, Pinching and Dsving 1974). Odour stimulation is associated with an increased metabolic activity which is unevenly spatially distributed (Sharp et al. 1975). Le Gros Clark (1951) made lesions in the glomerulus layer of the olfactory bulb of rabbits. On the basis of the retrograde degeneration patterns in the olfactory mucosa he concluded that the projection of the olfactory epithelium on the olfactory bulb was somatotopic. This was confirmed by Land (1973) looking at the anterograde degeneration in the glomeruli after sectioning of small bundles of the olfactory nerve. The projections showed however, to be divergent and partially overlapping. 270
271
OLFACTORY RECEPTOR DISTRIBUTION IN RAT
Dorsal Fig. 1. Medial view of the left side endoturbinals of the rat head. The distribution of the olfactory epithelium is indicated by stippling. The upper left projection shows the cribriform plate in a dorsal view with a typical arrangement of the major holes.
4nterior
From these observations and the observations on the spatial distribution of olfactory bulb responses one can assume a non-homogeneous distribution of receptors within the epithelium. By simultaneous recordings of the electro-olfactogram (EOG) at different sites of the sensory epithelium such a non-homogeneous distribution was demonstrated in frog (Mustaparta 1971, Daval, Leveteau and MacLeod 1969). We wanted to compare the pattern of selective degeneration with a possible pattern of non-homogeneous distribution of receptor cells. In the present study we show a spatial distribution of the different types of receptor cells in the rat by analysis of the relative amplitudes of the EOGs recorded from different areas of the cribriform plate.
Materials and methods The strain of experimental animals was the same as used in the studies on the selective degeneration occurring in the olfactory bulb of rat after prolonged odour exposure (Pinching and Deving 1974). White female rats of the Wistar strain were used. They were kept in semi-germfree conditions in isolation chambers to avoid rhinitis. The rats were between 180 and 230 g (18-23 weeks old) when used in the expts. The experimental animal was anesthetized with urethane given intraperitoneally (1.5 g per kg b.wt.). To separate the sniffing action from the respiration a tracheostomy was carried out, and a polyethylene catheter was inserted through the tracheostoma into the choana. The animal was fastened in a headholder. The skull was opened dorsally and the olfactory bulb and the frontal part of the cerebrum was removed to expose the dorsal surface of the cribriform plate as indicated in Fig. 1. Lymph and cerebro-spinal fluid was continuously removed by cellulose wicks. Deep body temperature of the preparation was maintained at 37.3"C. During unilateral recordings, the contralateral (right) nostril was plugged with vaseline. Recording Two recording electrodes made contact with the basal side of the olfactory epithelium at the holes of the cribriform plate. Monopolar recordings were made against a common indifferent electrode placed on the skull. The electrodes were broken glass capillaries, tip diameter 25-100 ,urn, filled with agar-Ringer and inserted in electrode holders containing sintred Ag/AgC1 (WPI, Hamden, Conn., USA). The potentials were pre-amplified by a DC-amplifier, with input impedance of 10 MOhm, and recorded on a pen recorder Beckman RS Dynograph 2 channel with DC-couplers; frequency response: DC to 20 Hz).
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G . THOMMESEN AND K. B. DBVlNG
TABLE 1. List of stimuli.
Order of stimulation
Symbols
Name
Order of stimulation
Symbols
Name
4 5 24
c5 C6 c7 C8 c9 CIO c1I c12 C13 ACE BEN CAM CHL DEC EAA HAL
C yclo pent ano ne Cyclohexanone Cycloheptanone Cyclooctanone Cyclononanone Cyclodecanone Cycloundecanone Cyclododecanone Cyclotridecanone Acetophenone Benzylthiol DL-Camphor Cyclohexanol 1-Decanol Ethyl acetoacetate Heptanal
17 16
HOL ION iBA I PA LI M MEN MBZ MBL MHN MUL MVA NAF NBZ OCT MCC
1-Heptanol p-Ionone Isobornylacetate isopentylacetate ( +)-Limonene L-Menthol Methylbenzoate 2-Methyl-3-butyn-2-01 6- Methyl-5-hepten-2-one 2-Methylundecanal 4-Methy1 valeric acid Naphthalene Nitrobenzene Octanal 1,1,2,2,-tetramethy1-3-,3dichloro-cyclopropane
6
25 1
7
26
3 15 21-3 1 20 7 18
12 27-31
11
27-3 1 8 21 13 22 27-3 1 23 10 9 19
27-3 1 14
Sriniulation The catheter in the choana was connected to a suction pump via a flowmeter and a magnetic valve. The valve was controlled by a programming device giving 1 s suction, 1 s pause, 5 s suction, 4 s pause and f s suction. Suction air flow was held at 7-14 ml/s. The preparation was continuously supplied with purified and humidified air through a glass funnel at a rate of 100 ml/s, producing a clean air-mask around the snout. In the studies of selective degeneration (Pinching and Dsving 1974) were used 44 different odours. For the present investigation 31 of them were found to be potent enough to give basis for discussion. These odours and their order of presentation are listed in Table I. The stimuli were introduced on polyethylene spatulae between the funnel and the snout just before the first (1 s) suction and withdrawn during the first pause. The second (5 s) suction thus served as washing and the last (3 s) suction as a control. In every position the total series of stimuli were used. Grading of stimuli was performed by varying the position of the spatula in the clean air flow. Experimental procedure For positioning the recording electrodes on the preparation the horizontal bony crest dividing the cribriform plate in two approximately equal parts, served as a landmark. One electrode was placed on the left cribriform plate in a position approximately 0.5 mm ventral to this crest and 1 mm from the septa1 extension as seen in Fig. 2 and 3. This electrode will later be referred t o as the “stationary electrode”. The other recording electrode was successively placed at 38 different positions uniformly spread over the left cribriform plate, and will be referred to as the “mobile electrode”. Every preparation was photographed and the electrode positions were plotted on the photograph. Th e different electrode positions are shown in Fig. 6 a . Data treatment The treatment of the results is based on the method used by Mustaparta (1971). The ratio, N between the the EOG amplitude of the mobile electrode and that of the stationary one served as a measure for the stimulating efficiency of each substance at the position of the mobile electrode. This procedure was used to minimize the effects of variable concentrations. The results were transferred to a non-parametric scale by ranking the ratios according to decreasing value at every recording position. The response at the position of the stationary electrode was quantified by a mean rank value for each stimulant. This value is equal to 32 minus the mean of all the rank numbers obtained for that stimulant and regarded as equivalent to the proper rank numbers. For each odorous substance the ranks were noted on position maps similar to those shown in Fig. 5 and 6.
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L imonene
Fig. 2. Difference in response to limonene and methylbenzoate in two positions of the mobile electrode us. the stationary electrode. Electrode positions are shown to the left. Stimulation time is 1 s. Vertical bar is 0.5 mV.
Stirnulotion
I
Washing Control
mnnmmrm n
I -c;I
met hy 1 benzoate
I - 0
Controls
Various forms of control expts. were performed. The stability of the preparation and the experimental conditions were controlled by repeating the first stimulus at the end of each stimulation series. Expts. were made to control the ratio N at various odour intensities. In 17 series several stimulations were made with the same odour a t different intensities. These expts. would indicate possible systematic variations of the ratio N between the amplitudes at the stationary and mobile electrode positions with the concentration of the odour. To control the variability from animal to animal eight pairs of presumed equivalent recording positions were studied.
Results The extension of the olfactory mucosa on the endoturbinal surfaces was studied in several rats and an average distribution is shown in Fig. 1. The difference between the sensory and the respiratory surfaces is clearly indicated by the ciliary movements in the latter epithelium in a freshly killed animal. By recording from the dorsal surface of the cribriform plate as described by Ottoson (1954, 1959) the EOGs are recorded without interfering with the air flow in the nasal cavities. EOGs were positive when recorded from the basal side of the olfactory epithelium (Ottoson 1954, 1959) and behaved like a summated generator potential. If the electrode penetrated the mucosa, the EOG became negative. The time course of the EOG agrees with that described by Ottoson (1954, 1956, 1959). After a latency of approximately 0.2 s the EOG rose to full size in 0.5 to 0.7 s. By prolonged stimulations an adaptation was seen whereby the EOG reduced to about 2/3 of its peak amplitude. Only the peak amplitude has been subject to analysis in the present article. EOG amplitudes to odorous stimulation varied from 0.2 to 2.5 mV. To standardize recording conditions, stimulation procedures were modified so that responses usually did not exceed 1 mV, thereby minimizing stimulation of receptor types with different specificity. Examples of the recordings are shown in Fig. 2. 18 - 775873
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r
Amplitude at p o s. S ( m V ) >
E
].Or LiM
Fig. 3. The peak amplitudes of the electro-olfactogram recorded with different stimulus intensities. The recordings at the different positions A, B and C are simultaneous with those at the stationary electrode position, S. Pos. A and pos. B are from the same animal. The abbreviations in the diagrams refer to the different chemicals listed in Table I. The straight lines are drawn through the origin. Note the difference in stimulation efficiency of limonene between pos. A and pos. B compared with that of the cycloketones.
At four positions along the ventral margin of the cribriform plate, hatched on the position map Fig. 6 a, most responses were too faint to give substantial bases for data treatment. Purified air elicited responses of 0.1-0.2 mV depending upon the flow of air suction, presumably due to imperfect purification. This response disappeared only when the suction air flow was insufficient for odour stimulation. Controls of the reliability of rhe dara
Various expts. were performed to study the reliability of the results. As mentioned in the discussion the ratio N between two recording positions should be independent of stimulus intensity. To test this hypothesis repetitive stimulations were made with varying stimulus concentrations. Fig. 3 shows the amplitudes of the EOG at the stationary electrode position versus those from 3 other areas. Plots have been made for four odours, uiz., two cycloketones, limonene and methyl benzoate. The ratio N is shown as the slope of the line through the origin. The variation is small with different concentrations but the slopes or the ratio
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Stimulus :
.-
,
Open Symbols-Cyclodecanone Cyclohexanone
L
/
1.5 -
cy
2
.o 0
m Fig. 4. The ratios N obtained by stimulation with the same substance at the beginning (N,) and at the end (N2) of each stimulation series. Graph symbols refer to the diferent preparations as indicated, Fig. 6 a . Broken lines show the 95% confidence interval.
-
1.0-
0.5
1.0 Ratio N,
1.5
2.0
N varies from one electrode position to the other. The diagram demonstrates the general tendency of the ratio to be constant over a large range of concentrations for the same stimulus and electrode position. In 32 runs covering several recording positions, 3-8 consecutive stimulations were made with the same substance. The standard deviation of the ratio N of the EOG amplitude at the mobile electrode to that at the stationary one, with the same substance and position, showed a mean value of 0.07. This standard deviation is less than that obtained for the total series of stimuli in any recording position, which ranged from 0.08 to 0.42, the lowest values in medial positions. The stability of the preparation and the reproducibility of the results were also controlled by repeating the first stimulation at the end of each series. This was done in all cases except for the 4 ventral positions mentioned above. Fig. 4 shows the ratio N for the first ( N l ) and the last ( N e )stimulation with the same substance in the 34 stimulation series. In all but one case the control stimulus was cyclodecanone. At three positions only, the difference between the ratios fall outside a 95 % confidence interval found by using the above value of SD(N). At none of the positions included in this presentation the differences fall outside the 98 % confidence interval centered at zero. The test confirms that the preparations did not change significantly in sensitivity throughout the expts. The results consist of measurements made on five animals, 4 to 12 recording positions in each (Fig. 6 a). The positions cover overlapping areas of the cribriform plate. Due to individual variations none of the positions could be regarded as completely congruent in different animals. Some recording positions were however, assumed to be equivaient from one preparation to another, indicated by overlapping symbols in Fig. 6 a. We have made eight comparisons, calculating the Spearman rank correlation coefficients. In seven of these comparisons the ranking of odours were positively correlated with signif-
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icance levels less than 0.5 per cent. In only one comparison concerning positions centrally in the dorsal half of the cribriform plate there was no significant correlation. These controls indicate that the results included in this presentation were reproducible from one preparation to another. Stirnuluting with different substance5
At all the different positions studied the ratio of the EOG amplitude at the mobile us. the stationary electrode position varied with odour quality. Recordings of the EOG from different positions on the cribriform plate are shown in Fig. 2. Recording pairs with positions marked with the same symbols were recorded simultaneously. As seen limonene gave the smallest amplitude at the stationary electrode position in both cases. Methyl-benzoate gave the highest response at the “mobile” position in the upper pair but for the EOGs at the lower pair of positions, the highest response was at the stationary electrode. Relative shifts in stimulation efficiency are also demonstrated in Fig. 4 for limonene and two of the cycloketone\. Conipurisons o f EOGs from left and right sides
3 series of simultaneous recordings have been made with the 2 recording electrodes symmetrically placed on the left and the right side cribriform plate. In this case, the ratio N was found to be similar for all odours. The variations were not greater than expected from random variations in stimulus concentrations on the two sides, inaccuracy in response measurements or the uncertainty in finding exactly equivalent positions on nearly symmetrical structures. There is therefore no basis for assuming qualitative differences in sensitivity distribution between the olfactory epithelium of the right and the left side. Mapping of response distribution
The N values of each position of the mobile electrode were ranked, giving the number 1 to the highest ratio and rank number 31 to the lowest. The rank numbers were plotted on the map of the cribriform plate, one map for each stimulus used. 4 examples of such maps are given in the upper row of diagrams in Fig. 5. In an attempt to visualize the sensitivity distribution in the olfactory epithelium as it appears by recording from the cribriform plate, the maps were simplified by hatching. Areas with rank numbers 1-9 have been crosshatched as a sign of high stimulus efficiency. Areas with rank numbers 10-19 have been covered with simple hatching and areas with rank numbers 20-31 are unhatched as signs of medium and low response respectively. In Fig. 5 this arbitrary and simplified visualization of the data is shown for acetophenone, cycloundecanone, camphor and heptanal. The four maps represent 4 different types of distribution pattern which are fairly typical. Of the remaining 27 substances, 24 gave distribution maps which had visual similarity to one of the four maps shown. Similarity to the acetonphenone map was found for the lower cycloketones of 5-8 carbon atoms, cyclohexanol, menthol, methylbenzoate, 4-methyl valeric acid, naphthalene and nitrobenzene. Responses to decanol, cyclodecanone, cyclododecanone and cyclotridecanone were distributed similarly to the map for cycloundecanone. Only the
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Acetopheno ne
Cycloundecanone
D,L-Camphor
'1619
l5
Heptanal
14
2
17.2
1
208
10
15 2
15
22
18
6
2
7
Fig. 5. Examples of response distributions to four different odours. Upper row: the rank numbers obtained in every position. Low rank numbers show high response ratio. Lower row: Simplified response distribution maps. Areas with rank numbers lower than 20 are hatched, areas with rank numbers lower than 10 are crosshatched.
response distribution of methylheptenone shows any similarity to that of camphor. The maps obtained for benzylthiole, cyclononanone, ethyl acetoacetate, isobornylacetate, isopentylacetate, limonene, methylundecanal, octanal and tetra-methyl-dichloro-cyclo-propane bear the closest resemblance to the heptanal map. The responses to p-ionone, methylbutynol and heptanol were so uniformly distributed that no pattern will be proposed. The majority of 26 substances can also be seen to divide in two groups with response distribution maps approximately complementary to each other. One group of 16 odours represented by acetophenone and cycloundecanone, gives the strongest response dorsally or dorsomedially. The group of 10 here represented by heptanal give the strongest responses ventrally or laterally. Another way of representing the results is shown in Fig. 6. In a map of the cribriform plate the recording positions are marked with different symbols for each preparation (Fig.
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E l e c t r o d e positions
Rank n o . 1
Rank n o 3
Rank n o 2
CSIACE
I BA
a
b.
C.
d.
Fig. 6 . Schematic diagrams of the left cribriform plate (see Fig. 1). a shows the different electrode positions. The different symbols refer to different preparations. Black symbols show the positions of the stationary electrode. Hatched symbols mark positions where only a few substances elicited a measurable response. h, c and d show what substances obtained rank number 1, 2 and 3 respectively (see text).
6 a). In the following maps the odours are plotted which had the first, second and third rank in each position. The maps show the same grouping of substances as mentioned above and shown in Fig. 5 .
Discussion The general properties of the electro-olfactogram (EOG) in frog have been analysed in detail by Ottoson (1956). The objections raised by various authors concerning the features of the EOG and its relation to the receptor mechanisms have been discussed by Ottoson (1971). I n the present study we have considered the EOG to represent the electrotonic projection of the sum of generator potentials produced by those receptor cells which respond to the stimulus. The laws for the spread of electrical potentials from a dipole layer in a volume conductor imply that the ratio between the potentials recorded from two different points is a function only of the shape and location of the dipole layer, but not of the voltage. In the present work it is assumed that the recording of a sum of generator potentials from the olfactory mucosa follows the same rule, as long as only one homogeneous population of olfactory receptors are activated. From this assumption follows that during stimulation with different odours, the ratio between the EOG potentials recorded from 2 different points in the olfactory epithelium will vary if and only if the epithelium contains receptor populations with different spectra of sensitivity and different spatial distribution. The ratio will be independent of the concentration of the stimulant within certain limits; i.e., as long as receptor types with different thresholds are not activated, so that only one receptor population is involved. Even under ideal circumstances the ratio between the two recorded potentials will vary, however, due
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279
to random variation in the electrical noise of the recording devices and the uncertainty in the reading of the potentials. The EOG may be recorded anywhere in the proximity of the olfactory epithelium. The most convenient recording site in the mammal is the nearly two-dimensional cribriform plate. By the described preparation procedure the EOG potentials are recorded without interference with the nasal flow pattern since the nasal cavities are intact. The recordings are also free of electrode potential artefacts as the electrode tips are not exposed to the stimulating substances. The recorded potentials are electrotonic projections of potentials from a highly folded olfactory epithelium serving as a volume conductor. The potentials are not generated by the olfactory axons projecting through the holes of the cribriform plate, and the exact location of the excited receptor cells within the epithelium can not be deduced from the distribution of responses. In spite of these reservations it is reasonable to presume that the different parts of the olfactory epithelium project mainly to nearby parts of the cribriform plate. For instance, a strong response in the dorsal area of the cribriform plate is most likely caused by activation of receptors mainly located in the roof of the nasal cavity or on the upper turbinates. Adrian (1951) suggested that the spatial differences in the bulbar responses to different odours were caused by non-homogeneous distribution of olfactory receptors of different specificity. The interpretation assumed that the neuronal projection from the olfactory mucosa to the 01factory bulb was more or less somatotopic as in fact was demonstrated the same year by Le Gros Clark (1951). Spatial differences in the distribution of different olfactory receptors were confirmed by Mustaparta in 1971 as evident in the frog.
The reliability of the distribution maps In the present study several types of control experiments were performed; the variation of the ratio N at the same position for different odour intensities, the stability of one preparation throughout the expt. and the reproducibility from one animal to another. The results of these control experiments demonstrate the consistency of the reported data. They also obviate further repetitions of the experiments at homologous positions in different animals. Contamination of the odorous substances is another possible cause of error. Aldehydes in general are susceptibleto oxidation producing the corresponding acids, the effect of which may have distorted the response distribution maps of the three aldehydes. The cyclononanone sample is known to contain impurities, presumably di-ketones and aldehydes. It is therefore interesting to note that cyclononanone, as the only one of the cycloketones, places itself in the group of substances which also includes the three aldehydes.
Conclusions The present results show that receptor neurones of different spectra of sensitivity are differentially distributed in the mammalian olfactory mucosa as well. In spite of the complex folding of this epithelium, the spatial distribution of receptor responses is clear. None of
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the response distribution maps were identical. On the basis of their shape, the maps may be grouped, as belonging to a small number of patterns, some of which make up pairs of almost complementary response distributions. A further analysis of the data on the similarity in response distributions will appear in a subsequent article. Difference in the distribution of the EOG does not indicate that there are discrete areas of uniform specificity. If a finite number of receptor types exists, the receptors most likely cover overlapping fields in the olfactory mucosa. The non-homogeneous distribution of olfactory receptors of different specificity implies that if two substances show different response distributions, they most likely are perceived by the animal as being different. The reverse statement is not necessarily true. Receptors sensitive to different odours may be intermingled so as to produce the same EOG distribut ion. This study has been supported by the Norwegian Research Council for Science and the Humanities.
References ADRIAN,E. D., Olfactory discrimination. Ann. psychol. 1951. 50. 107-113. ALTNER, H. and 3 . BOECKH,fiber das Reaktionsspektrum von Rezeptoren aus der Riechschleimhaut von Wasserfroschen (Rana esculenta). Z. rergl. Physiol. 1967. 55. 299-306. and P. MACLEOD, Electroolfactogramme local et discrimination olfactive chez DAVAL,G., J. LEVETEAU la Grenouille. 1. Physiof. (Paris) 1969. 61. Suppl. 2. Selective degeneration of neurones in the oifactory bulb following DQVING,K. B. and A. J. PINCHING, prolonged odour exposure. Brain Res. 1973. 52. 115-129. DUCHAMP, A., M. F. RIVIAL,A. HOLLEYand P. MACLEOD,Odour discrimination by frog olfactory receptors. Chem. Scnses and flouor 1974. 1. 213-233. R. C., 7. Y. LETTVIN, W. H. PITTSand H. ROJAS,Odour specificities of the frog’s olfactory GESTELAND, receptors. Olfaction and Tasre. Pergamon Press, Oxford, 1963. 1. 19-34. LAND,L. J., Localized projection of olfactory nerves to rabbit olfactory bulb. Bruin Res. 1973. 63. 153-1 66. , E., The projection of the olfactory epithelium on the olfactory bulb in the rabbit. LE GROSC L A R KW. J. Neurol. Neurosurg. Psychiat. 1951. 14. 1-10. MATHEWS, D. F., Response Patterns of Single Neurons in the Tortoise Olfactory Epithelium and Olfactory Bulb. J . gen. Physiol. 1972. 60. 166-180. C., The afferent neural processes in odor perception. Ann. N . Y. Acad. MOZELL,M. M. and PFAFFMANN, Science 1954. 58. 96-108. MUSTAPARTA, H., Spatial Distribution of Receptor-Responses to Stimulation with Different Odours. A c f a phvsiol. scund. 1971. 82. 154-166. D., Sustained potentials evoked by olfactory stimulation. Acra physiol. scand. 1954. 32. 384-386. OTTOSON, D., Analysis of the electrical activity of the olfactory epithelium. Acru physiol. scand. 1956. 35. OTTOSON, Suppl. 122. 1-83. OTTOSON,D., Studies on slow potentials in the rabbit’s olfactory bulb and nasal mucosa. A c f a ph.wioI. scand. 1959. 47. 136-148. OTTOSON, D., The electro-olfactogram. A review of studies on the receptor potential of the olfactory organ. In Handbook of Sensory Physiology. Springer Verlag. 1971. 4. 95-1 3 1. A. J. and K. B. DQVING, Selective degeneration in the rat olfactory bulb following exposure to PINCHING, different odours. Brain Res. 1974. 82. 195-204. SHARP,F. R., J. S . KAUERand G. SHEPHERD, Local sites of activityrelated glucose metabolism in rat olfactory bulb during olfactory stimulation. Bruin Res. 1975. 98. 596-600.
