144
Brain Research, 133 (7977) 144-149 © Elsevier/North-Holland Biomedical Press
Olfactory receptor units - - a mammalian preparation
ROBERT C. GESTELAND and CHARLES D. SIGWART* Department of Biological Sciences, Northwestern University, Evanston, IlL 60201 (U.S.A.)
(Accepted May 25th, 1977)
A persistent obstacle in vertebrate olfactory receptor research has been the lack of a mammalian preparation in which responses of single receptor units can be reliably recorded. Most physiological data has come from frogs. In these animals summated receptor potentials (the electro-olfactogram or EOG), summated action potential activity in the olfactory nerve, and responses of single receptor neurons have been studied 3,5-10. However, frogs show little behavioral response to olfactory stimulation. Therefore, the neural basis for odor discrimination has been experimentally inaccessible. We report here that responses of single receptor neurons in the young rat can be easily and reliably recorded with extracellular electrodes for periods of time long enough to allow presentation of a large number of stimuli at a variety of intensities. EOGs are easily recorded as well. Sperm-positive female rats were ordered from Holtzman Co., (Madison, Wisc.). Pups were taken 1-30 days post-partum and rapidly decapitated. The excised head was mounted snout-up in a chamber continuously flushed with a moist 95 ~ oxygen-5 ~o carbon dioxide gas mixture. The dorsal skin and cartilage of the nose and the dorsal epithelium of the nasal cavity were surgically removed as quickly as possible (typically about 2 min). The success of the preparation appears to be critically dependent upon maintenance of an adequate oxygen supply to the receptor neurons from the high partial pressure oxygen atmosphere after circulation of the blood is interrupted. When air is substituted for the 95 ~ oxygen atmosphere, stimulus-evoked activity declines rapidly. (We have found that the stability of an in vitro amphibian nose preparation is also improved when the oxygen partial pressure is increased and, in some cases, when the tissue temperature is reduced to about 15 °C.) For electrical connection to the preparation a chlorided-silver indifferent electrode constituted the floor of the chamber and was separated from direct contact with the tissue with a piece of filter paper moistened with 0.9 ~ saline. Two recording electrodes were inserted into the chamber through holes in the transparent top. One electrode was a glass pipette with a 10-30 # m diameter tip which was filled with a stiff gelatin * Present address: Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267, U.S.A.
145 solution made up in 0.9 % saline. This electrode was brought into contact w'th the surface of the olfactory epithelium and recorded the stimulus-evoked EOG. The second electrode, used for recording single unit action potentials, was either a metal-filled, platinum black-plated 3-6 # m tip diameter pipette or a micropipette filled with 0.9 % saline. Metal microelectrodes were prepared immediately prior to the start of the experiment and selected for low tip impedance as measured by the wideband noise amplitude with the electrode touching the mucus surface 4. Micropipettes were selected for tip resistances between 30 and 100 M ~ . EOG and micropipette electrodes were direct-coupled to a solid state amplifier with an input resistance greater than 1012 and input current less than 10-13 A. Metal electrodes were coupled through a 1500 pF capacitor to a similar amplifier, the input of which was shunted by 40 M f L A continuously flowing moist 95 % oxygen-5 % carbon dioxide carrier stream was directed at the olfactory epithelium. To stimulate, a portion of this stream was deflected into a stimulus-containing vial, forcing out head space vapor which was mixed into the carrier stream a short distance upstream from the exit port. Amyl acetate, L-butanol, butyric acid, dichloroethane, ethyl n-butyrate, methyl n-butyrate, pyridine and triethylamine at concentrations between 1 × 10-4 and 6 × 10-2 of saturation were effective stimuli. No EOG or unit responses were detectable from a blank vial or as a result of turning the carrier stream on and off. The characteristics of the EOG of the young rat are similar to those observed in other animals. Most of the odorous chemical compounds which we tried evoked a measurable response at vapor concentrations commonly used in olfactory experiments. Male and female urine, both clearly odorous, were not effective stimuli. The EOG evoked by stimulation with ethyl n-butyrate had a rise time of approximately 0.5 sec and amplitude proportional to stimulus concentration at low levels of stimulus intensity. At high intensities adaptation occurred, the EOG rising to an initial peak and then declining during the continued stimulus presentation. These responses are shown in Fig. 1A. Over a limited intensity range, the EOG peak amplitude varied approximately linearly with the log of stimulus concentration (Fig. 2). Steven's plots (log of EOG peak amplitude vs log of vapor concentration) tended to have similar slopes for homologous substances and different slopes for substances with very different chemical propertieslL We expected to find that the EOG amplitude varied with animal age. Except for the youngest animals (1-3 days post-partum, when the tissue was very small and thus seriously shunted by saline solution in the chamber) there was no statistically significant correlation with age. The viability of the excised receptor organ preparation was tested by delivering a test stimulus (ethyl n-butyrate, 10 #M, 2 sec duration) every 3 min. In 35 preparations, the amplitude declined by 50 % in an average of 90 min. The range was 40-180 min. There was no statistically significant correlation between animal age and preparation longevity. EOG amplitude decline was not linear. In our better preparations the EOG evoked by the standardizing stimulus was invariant for an hour or more, after which a relatively rapid decline in amplitude occurred. In a typical case, the amplitude was constant for 70min, dropped by 10% at l l 0 m i n and by 50% by 155 min. Single unit responses were easily recorded during the initial period of maximum EOG amplitude.
A I
/0.6
/ 0
\3
30
0.5 s
B 0.5 s :t
0
I
0.6
~p
i I
"~
iviimmili
I" i
,_~, :30 .eIJJ , , 0
i i ill
mlii
i i
lli,w.l,li" iiiiii, i Ir ~iivi, ,qi viil
i (
3
ii
-Iv- .....
i
Iiii
it
IlVllVVlimvrwlllv IVivivil"rw "'w~ I
ill-
1~ r
Fig. 1. A: EOGs evoked by various concentrations of ethyl n-butyrate vapors. Concentrations are in ymoles/liter of air. The horizontal bar at the top indicates the stimulus duration. Positive voltage is upward. B: responses of a single receptor cell to stimulation with ethyl n-butyrate. The signal was processed with a threshold circuit to remove baseline noise. Spike amplitude variations result from noise and from inherent changes which occur during periods of rapid firing. Traces marked 0 show spontaneous activity (which was indistinguishable from activity evoked by a blank stimulus vial).
A
8-
"~ 6 o.
E o
_~ 4
O LLI
2 I
I
I
I
2
4
I
I
I
I0
20
40
[Ethyl butyrote] (y.M) Fig. 2. Variation in EOG peak amplitude with logarithm of ethyl n-butyrate vapor concentration. Vertical bars indicate range of values measured for 10 animals,
147
~ o D ° r s a --. l Posterior
1.0 ~')--
0.7 I. 0
Anterior
/
0.05 Fig. 3. Schematic representation of sagittal section through the posterior ethmoturbinal on one side of the head. Numbers indicate relative EOG amplitudes recorded at the dotted locations resulting from 10 t~molar ethyl n-butyrate stimuli lasting for 2 sec. During the declining amplitude E O G period units were encountered less often and evoked responses were variable. The amplitude of the E O G is a function of the population density of receptor neurons in the vicinity of the recording electrode. Fig. 3 shows a schematic drawing of a sagittal section through the olfactory cavity and the posterior ethmoturbinal. The numbers indicate the relative amplitudes of E O G s at different recording electrode positions evoked by 2-sec l0 # M ethyl n-butyrate stimulation. E O G s were not observed at the highest recording system gain when the electrode was centered in areas composed solely of respiratory epithelium. The probability of encountering single units was maximum in regions where the E O G amplitude was maximum. Extracellular action potentials of single units were surprisingly easy to record compared with other vertebrate preparations. As with the EOG, the probability of finding a unit and the description of its response properties appear to be independent of the age of the animal. It was easier to find units with metal microelectrodes than with micropipettes. However, metal electrodes have tips comparable in diameter to that of the cell soma and often record activity from several neighboring cells simultaneously. With careful positioning of the electrode, the amplitude of the spikes from one cell can be maximized with respect to the others and the effects of stimuli on that one cell determined. With saline-filled micropipettes, cells are generally recorded in isolation. On some occasions the recorded signal was unusually large compared to the noise level. We then saw that the three main phases of a triphasic action potential were commonly preceded by one or two small phases with durations comparable to
A .I o:.ll Fig. 4. Wave forms of extracellular action potentials recorded with a 40 M~ micropipette filled with 0.9 ~ NaCl. The electrode tip was located close to the receptor neuron soma. Calibration marks represent 200/~V, positive upward on the vertical axis and l0 msec on the horizontal axis. Phases of the potential are indicated by a', a, l, 2 and 3.
148 those of the major components. These are clearly seen in the photographic record shown in Fig. 4. The electrode tip was located about 80 # m below the surface of the mucus, the depth at which the neuron soma lie. The photograph was taken during a period of stimulus-evoked activity. At maximal firing rates the large positive phase labeled 3 diminishes in amplitude rapidly after a few seconds of evoked activity. There were no concurrent changes in the major phases 1 and 2 or the early small phases a' and a. We suppose that the multiphasic nature of the action potential is related to changes in propagation velocity associated with the abrupt change in cross-sectional area of the cell in the soma region 11. Different stimulus substances can have widely different effects on any particular cell. Some excite the cell, some suppress spontaneous activity, and some have no effect over the 2 or 3 log-unit range of concentrations which we used. Excitatory effects vary with stimulus concentration in a regular (but unusual) way. This is illustrated in Fig. lB. At low intensities the firing rate increases soon after stimulus onset and continues for a period equal to or greater than the stimulus duration. As the concentration is increased the firing rate and the number of evoked spikes increases and the latency decreases. At still higher concentrations there is a short onset burst followed by an extended period of suppressed activity. This is sometimes followed by a second burst of activity and sometimes by a return to the spontaneous firing pattern. At high stimulus intensities the onset burst may consist of a single spike. We used several measures of evoked activity, the number of evoked spikes, the maximum instantaneous spike frequency (i.e., the reciprocal of the shortest interspike interval), and the time integral of the response (the product of the number of evoked spikes and the duration of the response). All show a maximum at some particular concentration of an excitatory stimulus and decreasing magnitude at both lower and higher concentrations. Responses of a single cell are repeatable for at least 30 min as long as intense stimulation is avoided. With repeated intense stimulation, the number of evoked spikes diminishes with each successive stimulus presentation. After 5-10 stimulus events, the cell becomes unresponsive to all stimuli but will still occasionally fire spontaneously. Rat pups of the ages used in these experiments exhibited exploratory curiosity when odors (including all of the stimuli listed) were presented. They were attracted to odors of the mother and of the nest. It has been shown that the olfactory epithelium and the olfactory bulb from this rat strain can be grown in organ culture from cranial anlagen tissue1, 2. Electrophysiological responses to odors can be recorded from the cultured nose 2. The young rat appears to be uniquely useful for physiological, developmental and behavioral studies of the olfactory sense. This work was supported in part by National Science Foundation Grant BNS7502339. 1 Farbman, A. I., Differentiation of olfactory receptors in combination with olfactory bulb in organ culture, Neurosci. Abstr., 2 (1976) 147. 2 Farbman, A. 1. and Gesteland, R. C., Developmental and electrophysiological studies of olfactory mucosa in organ culture. In D. Denton and J. Coghlan (Eds.), Olfaction and Taste, Vol. V, Academic Press, New York, 1975, pp. 107-110.
149 3 Gesteland, R. C., Neural coding in olfactory receptor cells. In L. Beidler (Ed.), Handbook of Sensory Physiology, 1Iol. IV, Chemical Senses 1, Olfaction, Springer-Verlag, Heidelberg, 1971, pp. 132-150. 4 Gesteland, R. C., Techniques for investigating single unit activity in the vertebrate olfactory epithelium. In D. Moulton, A. Turk and J. Johnston (Eds.), Methods in Olfactory Research, Academic Press, New York, 1975, pp. 269-321. 5 Gesteland, R. C., Physiology of olfactory reception. In R. Llimis and W. Precht (Eds.), Frog Neurobiology, Springer-Verlag, Heidelberg, 1976, pp. 234-250. 6 Gesteland, R. C., Lettvin, J. Y. and Pitts, W. H., Chemical transmission in the nose of the frog, J. Physiol. (Lond.), 181 (1965) 525-559. 7 Holley, A., Duchamp, A., Revial, M. and Juge, A., Qualitative and quantitative discrimination in frog olfactory receptors: analysis from electrophysiological data, Ann. N.Y. Acad. Sci., 237 (1974) 102-114. 8 Mozell, M. M., The spatiotemporal analysis of odorants at the level of the olfactory receptor sheet, J. gen. Physiol., 56 (1966) 25-41. 9 Ottoson, D., Analysis of the electrical activity of the olfactory epithelium, Acta physiol, scand., 35, Suppl. 122 (1956) 1-83. 10 Ottoson, D., The electro-olfactogram. In L. Beidler (Ed.), Handbook of Sensory Physiology, Vol. IV, Chemical Senses 1, Olfaction, Springer-Verlag, Heidelberg, 1971, pp. 95-131. 11 Ram6n, F., Moore, J., Joyner, R. and Westerfield, M., Squid giant axons, a model for the neuron soma?, Biophys. J., 16 (1976) 953-963. 12 Sigwart, C. D., Electrophysiology of the olfactory epithelium in the rat, Ph.D. Thesis, Northwestern University, Evanston, I11., 1976.
Brain Research, 133 (7977) 144-149 © Elsevier/North-Holland Biomedical Press
Olfactory receptor units - - a mammalian preparation
ROBERT C. GESTELAND and CHARLES D. SIGWART* Department of Biological Sciences, Northwestern University, Evanston, IlL 60201 (U.S.A.)
(Accepted May 25th, 1977)
A persistent obstacle in vertebrate olfactory receptor research has been the lack of a mammalian preparation in which responses of single receptor units can be reliably recorded. Most physiological data has come from frogs. In these animals summated receptor potentials (the electro-olfactogram or EOG), summated action potential activity in the olfactory nerve, and responses of single receptor neurons have been studied 3,5-10. However, frogs show little behavioral response to olfactory stimulation. Therefore, the neural basis for odor discrimination has been experimentally inaccessible. We report here that responses of single receptor neurons in the young rat can be easily and reliably recorded with extracellular electrodes for periods of time long enough to allow presentation of a large number of stimuli at a variety of intensities. EOGs are easily recorded as well. Sperm-positive female rats were ordered from Holtzman Co., (Madison, Wisc.). Pups were taken 1-30 days post-partum and rapidly decapitated. The excised head was mounted snout-up in a chamber continuously flushed with a moist 95 ~ oxygen-5 ~o carbon dioxide gas mixture. The dorsal skin and cartilage of the nose and the dorsal epithelium of the nasal cavity were surgically removed as quickly as possible (typically about 2 min). The success of the preparation appears to be critically dependent upon maintenance of an adequate oxygen supply to the receptor neurons from the high partial pressure oxygen atmosphere after circulation of the blood is interrupted. When air is substituted for the 95 ~ oxygen atmosphere, stimulus-evoked activity declines rapidly. (We have found that the stability of an in vitro amphibian nose preparation is also improved when the oxygen partial pressure is increased and, in some cases, when the tissue temperature is reduced to about 15 °C.) For electrical connection to the preparation a chlorided-silver indifferent electrode constituted the floor of the chamber and was separated from direct contact with the tissue with a piece of filter paper moistened with 0.9 ~ saline. Two recording electrodes were inserted into the chamber through holes in the transparent top. One electrode was a glass pipette with a 10-30 # m diameter tip which was filled with a stiff gelatin * Present address: Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267, U.S.A.
145 solution made up in 0.9 % saline. This electrode was brought into contact w'th the surface of the olfactory epithelium and recorded the stimulus-evoked EOG. The second electrode, used for recording single unit action potentials, was either a metal-filled, platinum black-plated 3-6 # m tip diameter pipette or a micropipette filled with 0.9 % saline. Metal microelectrodes were prepared immediately prior to the start of the experiment and selected for low tip impedance as measured by the wideband noise amplitude with the electrode touching the mucus surface 4. Micropipettes were selected for tip resistances between 30 and 100 M ~ . EOG and micropipette electrodes were direct-coupled to a solid state amplifier with an input resistance greater than 1012 and input current less than 10-13 A. Metal electrodes were coupled through a 1500 pF capacitor to a similar amplifier, the input of which was shunted by 40 M f L A continuously flowing moist 95 % oxygen-5 % carbon dioxide carrier stream was directed at the olfactory epithelium. To stimulate, a portion of this stream was deflected into a stimulus-containing vial, forcing out head space vapor which was mixed into the carrier stream a short distance upstream from the exit port. Amyl acetate, L-butanol, butyric acid, dichloroethane, ethyl n-butyrate, methyl n-butyrate, pyridine and triethylamine at concentrations between 1 × 10-4 and 6 × 10-2 of saturation were effective stimuli. No EOG or unit responses were detectable from a blank vial or as a result of turning the carrier stream on and off. The characteristics of the EOG of the young rat are similar to those observed in other animals. Most of the odorous chemical compounds which we tried evoked a measurable response at vapor concentrations commonly used in olfactory experiments. Male and female urine, both clearly odorous, were not effective stimuli. The EOG evoked by stimulation with ethyl n-butyrate had a rise time of approximately 0.5 sec and amplitude proportional to stimulus concentration at low levels of stimulus intensity. At high intensities adaptation occurred, the EOG rising to an initial peak and then declining during the continued stimulus presentation. These responses are shown in Fig. 1A. Over a limited intensity range, the EOG peak amplitude varied approximately linearly with the log of stimulus concentration (Fig. 2). Steven's plots (log of EOG peak amplitude vs log of vapor concentration) tended to have similar slopes for homologous substances and different slopes for substances with very different chemical propertieslL We expected to find that the EOG amplitude varied with animal age. Except for the youngest animals (1-3 days post-partum, when the tissue was very small and thus seriously shunted by saline solution in the chamber) there was no statistically significant correlation with age. The viability of the excised receptor organ preparation was tested by delivering a test stimulus (ethyl n-butyrate, 10 #M, 2 sec duration) every 3 min. In 35 preparations, the amplitude declined by 50 % in an average of 90 min. The range was 40-180 min. There was no statistically significant correlation between animal age and preparation longevity. EOG amplitude decline was not linear. In our better preparations the EOG evoked by the standardizing stimulus was invariant for an hour or more, after which a relatively rapid decline in amplitude occurred. In a typical case, the amplitude was constant for 70min, dropped by 10% at l l 0 m i n and by 50% by 155 min. Single unit responses were easily recorded during the initial period of maximum EOG amplitude.
A I
/0.6
/ 0
\3
30
0.5 s
B 0.5 s :t
0
I
0.6
~p
i I
"~
iviimmili
I" i
,_~, :30 .eIJJ , , 0
i i ill
mlii
i i
lli,w.l,li" iiiiii, i Ir ~iivi, ,qi viil
i (
3
ii
-Iv- .....
i
Iiii
it
IlVllVVlimvrwlllv IVivivil"rw "'w~ I
ill-
1~ r
Fig. 1. A: EOGs evoked by various concentrations of ethyl n-butyrate vapors. Concentrations are in ymoles/liter of air. The horizontal bar at the top indicates the stimulus duration. Positive voltage is upward. B: responses of a single receptor cell to stimulation with ethyl n-butyrate. The signal was processed with a threshold circuit to remove baseline noise. Spike amplitude variations result from noise and from inherent changes which occur during periods of rapid firing. Traces marked 0 show spontaneous activity (which was indistinguishable from activity evoked by a blank stimulus vial).
A
8-
"~ 6 o.
E o
_~ 4
O LLI
2 I
I
I
I
2
4
I
I
I
I0
20
40
[Ethyl butyrote] (y.M) Fig. 2. Variation in EOG peak amplitude with logarithm of ethyl n-butyrate vapor concentration. Vertical bars indicate range of values measured for 10 animals,
147
~ o D ° r s a --. l Posterior
1.0 ~')--
0.7 I. 0
Anterior
/
0.05 Fig. 3. Schematic representation of sagittal section through the posterior ethmoturbinal on one side of the head. Numbers indicate relative EOG amplitudes recorded at the dotted locations resulting from 10 t~molar ethyl n-butyrate stimuli lasting for 2 sec. During the declining amplitude E O G period units were encountered less often and evoked responses were variable. The amplitude of the E O G is a function of the population density of receptor neurons in the vicinity of the recording electrode. Fig. 3 shows a schematic drawing of a sagittal section through the olfactory cavity and the posterior ethmoturbinal. The numbers indicate the relative amplitudes of E O G s at different recording electrode positions evoked by 2-sec l0 # M ethyl n-butyrate stimulation. E O G s were not observed at the highest recording system gain when the electrode was centered in areas composed solely of respiratory epithelium. The probability of encountering single units was maximum in regions where the E O G amplitude was maximum. Extracellular action potentials of single units were surprisingly easy to record compared with other vertebrate preparations. As with the EOG, the probability of finding a unit and the description of its response properties appear to be independent of the age of the animal. It was easier to find units with metal microelectrodes than with micropipettes. However, metal electrodes have tips comparable in diameter to that of the cell soma and often record activity from several neighboring cells simultaneously. With careful positioning of the electrode, the amplitude of the spikes from one cell can be maximized with respect to the others and the effects of stimuli on that one cell determined. With saline-filled micropipettes, cells are generally recorded in isolation. On some occasions the recorded signal was unusually large compared to the noise level. We then saw that the three main phases of a triphasic action potential were commonly preceded by one or two small phases with durations comparable to
A .I o:.ll Fig. 4. Wave forms of extracellular action potentials recorded with a 40 M~ micropipette filled with 0.9 ~ NaCl. The electrode tip was located close to the receptor neuron soma. Calibration marks represent 200/~V, positive upward on the vertical axis and l0 msec on the horizontal axis. Phases of the potential are indicated by a', a, l, 2 and 3.
148 those of the major components. These are clearly seen in the photographic record shown in Fig. 4. The electrode tip was located about 80 # m below the surface of the mucus, the depth at which the neuron soma lie. The photograph was taken during a period of stimulus-evoked activity. At maximal firing rates the large positive phase labeled 3 diminishes in amplitude rapidly after a few seconds of evoked activity. There were no concurrent changes in the major phases 1 and 2 or the early small phases a' and a. We suppose that the multiphasic nature of the action potential is related to changes in propagation velocity associated with the abrupt change in cross-sectional area of the cell in the soma region 11. Different stimulus substances can have widely different effects on any particular cell. Some excite the cell, some suppress spontaneous activity, and some have no effect over the 2 or 3 log-unit range of concentrations which we used. Excitatory effects vary with stimulus concentration in a regular (but unusual) way. This is illustrated in Fig. lB. At low intensities the firing rate increases soon after stimulus onset and continues for a period equal to or greater than the stimulus duration. As the concentration is increased the firing rate and the number of evoked spikes increases and the latency decreases. At still higher concentrations there is a short onset burst followed by an extended period of suppressed activity. This is sometimes followed by a second burst of activity and sometimes by a return to the spontaneous firing pattern. At high stimulus intensities the onset burst may consist of a single spike. We used several measures of evoked activity, the number of evoked spikes, the maximum instantaneous spike frequency (i.e., the reciprocal of the shortest interspike interval), and the time integral of the response (the product of the number of evoked spikes and the duration of the response). All show a maximum at some particular concentration of an excitatory stimulus and decreasing magnitude at both lower and higher concentrations. Responses of a single cell are repeatable for at least 30 min as long as intense stimulation is avoided. With repeated intense stimulation, the number of evoked spikes diminishes with each successive stimulus presentation. After 5-10 stimulus events, the cell becomes unresponsive to all stimuli but will still occasionally fire spontaneously. Rat pups of the ages used in these experiments exhibited exploratory curiosity when odors (including all of the stimuli listed) were presented. They were attracted to odors of the mother and of the nest. It has been shown that the olfactory epithelium and the olfactory bulb from this rat strain can be grown in organ culture from cranial anlagen tissue1, 2. Electrophysiological responses to odors can be recorded from the cultured nose 2. The young rat appears to be uniquely useful for physiological, developmental and behavioral studies of the olfactory sense. This work was supported in part by National Science Foundation Grant BNS7502339. 1 Farbman, A. I., Differentiation of olfactory receptors in combination with olfactory bulb in organ culture, Neurosci. Abstr., 2 (1976) 147. 2 Farbman, A. 1. and Gesteland, R. C., Developmental and electrophysiological studies of olfactory mucosa in organ culture. In D. Denton and J. Coghlan (Eds.), Olfaction and Taste, Vol. V, Academic Press, New York, 1975, pp. 107-110.
149 3 Gesteland, R. C., Neural coding in olfactory receptor cells. In L. Beidler (Ed.), Handbook of Sensory Physiology, 1Iol. IV, Chemical Senses 1, Olfaction, Springer-Verlag, Heidelberg, 1971, pp. 132-150. 4 Gesteland, R. C., Techniques for investigating single unit activity in the vertebrate olfactory epithelium. In D. Moulton, A. Turk and J. Johnston (Eds.), Methods in Olfactory Research, Academic Press, New York, 1975, pp. 269-321. 5 Gesteland, R. C., Physiology of olfactory reception. In R. Llimis and W. Precht (Eds.), Frog Neurobiology, Springer-Verlag, Heidelberg, 1976, pp. 234-250. 6 Gesteland, R. C., Lettvin, J. Y. and Pitts, W. H., Chemical transmission in the nose of the frog, J. Physiol. (Lond.), 181 (1965) 525-559. 7 Holley, A., Duchamp, A., Revial, M. and Juge, A., Qualitative and quantitative discrimination in frog olfactory receptors: analysis from electrophysiological data, Ann. N.Y. Acad. Sci., 237 (1974) 102-114. 8 Mozell, M. M., The spatiotemporal analysis of odorants at the level of the olfactory receptor sheet, J. gen. Physiol., 56 (1966) 25-41. 9 Ottoson, D., Analysis of the electrical activity of the olfactory epithelium, Acta physiol, scand., 35, Suppl. 122 (1956) 1-83. 10 Ottoson, D., The electro-olfactogram. In L. Beidler (Ed.), Handbook of Sensory Physiology, Vol. IV, Chemical Senses 1, Olfaction, Springer-Verlag, Heidelberg, 1971, pp. 95-131. 11 Ram6n, F., Moore, J., Joyner, R. and Westerfield, M., Squid giant axons, a model for the neuron soma?, Biophys. J., 16 (1976) 953-963. 12 Sigwart, C. D., Electrophysiology of the olfactory epithelium in the rat, Ph.D. Thesis, Northwestern University, Evanston, I11., 1976.
