J. Physiol. (1978), 282, pp. 521-540

521

With 11 text-figure8 Printed in Great Britain

RESPONSES OF OLFACTORY RECEPTOR CELLS TO STEP PULSES OF ODOUR AT DIFFERENT CONCENTRATIONS IN THE SALAMANDER

BY THOMAS V. GETCHELL* AND GORDON M. SHEPHERD From the Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A.

(Received 17 January 1978) SUMMARY

1. The response properties of single olfactory receptor cells in the salamander have been analysed in unitary recordings obtained with platinum-black metal-filled micro-electrodes. 2. Stimulation has been carried out using an apparatus which delivers odour pulses of abrupt onset, steady plateau and abrupt termination. The pulses have been monitored near the site of stimulation on the olfactory epithelium during the experiments. 3. The main type of response was a discharge of impulses that was time-locked to the stimulus pulse. The pattern of the responses consisted of a relatively brief latency of onset, a rapid rise in impulse frequency, a continuation of impulse firing during the plateau of the pulse, and an abrupt termination of the discharge correlated with the termination of the pulse. 4. There was a clear relationship between the receptor responses and odour concentration. In general, impulse firing frequency increased with increasing odour concentration. The firing frequency ranged from approximately 1-3 impulse's/sec at threshold, up to 20 impulses/sec at the highest concentration. 5. Two types of reduced impulse activity were observed. One occurred after the termination of the pulse and lasted 1-3 sec; this was a common occurrence. The other type was seen during a pulse as a reduction of impulse activity compared to the background level; this type was rarely observed. 6. The receptor responses resembled those of mitral cells in the olfactory bulb to odour pulses in their sensitivity to odour concentration. They differed in that mitral cells show primary response categories consisting of brief excitation followed by suppression, and pure suppression, that are rarely seen at the receptor level. These differences may be ascribed to synaptic interactions in the olfactory bulb. 7. It is concluded that the majority of receptor cells have a stereotyped discharge response pattern and a systematic relation to odour concentration. These properties appear to reflect the simple time course of the odour pulses used in these experiments. This represents an initial step toward analysing olfactory coding at the receptor level using stimuli controlled in a manner similar to that used in other sensory systems. * Present address: Morin Laboratory Department of Anatomy, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, U.S.A.

522

522T. V.

GETCHELL AND G. M. SHEPHERD INTRODUCTION

Studies of the responses of vertebrate olfactory receptor cells to odour stimulation have in general shown a diversity of impulse discharge patterns (Gesteland, Lettvin, Pitts & Rojas, 1963; Shibuya & Shibuya, 1963; Takagi & Omura, 1963; Gesteland, Lettvin & & Boeckh, 1967; Shibuya, 1965; Shibuya & Tucker, 1967; 1969; O'Connell & Mozell, 1969; Sutterlin & Sutterlin, 1971; Suzuki & Tucker, 1971; Mathews, 1972; Daval, Leveteau, MacLeod, Holley, Duchamp & Revial, 1972; Shibuya & Tonosaki, 1272; Getchell, 1973, 1974a; Holley, Duchamp, Revial, Juge & MacLeod, 1974; Blank, 1974; Holley, 1974; MacLeod, 1974; Gesteland, 1976). This diversity has been so great that it has hindered a clear understanding of the basic response properties of the receptor cells. A central problem in all these studies has been the fact that the olfactory stimulus was difficult to deliver and monitor. It is possible therefore that some of the response diversity has reflected differences in experimental methods. In other sensory systems, analysis of receptor function has been based on the use of standardized step pulses of stimuli with precisely controlled time courses (e.g. Ottoson & Shepherd, 1971; Levick, 1972). We have therefore used step pulses of odour to analyse olfactory receptor cell responses in the salamander. In addition, we have monitored the pulses near the site of stimulation of the olfactory mucosa. The results show that, with stimuli controlled in this manner, certain basic response properties of the receptor cells can be identified more clearly than has been previously possible. In this paper we are primarily concerned with the properties related to changes in odour concentration; a subsequent paper will deal with those related to changes in stimulus duration. The results also permit one to begin to define the sequence of processing that takes place from the receptors to the second order neurones in the salamander olfactory bulb, which have recently been studied under similar stimulus conditions (Kauer & Shepherd, 1977).

Altner

Pitts,

METHODS

Animals. Experiments were carried out on the tiger salamander, Amby8toma tigrinum. The animals were the adult land-phase variety, approximately 14 cm long; they were freshly colsoon after they emerged from hibernation during the months of December to early March. lected A total of thirty.five animals was used. They were initially anaesthetized in an ice bath and either pithed and/or curarized (Kauer, 1974; Kauer & Shepherd, 1977; Getchell, 1977). The use of D-tubocurarine (0-05-0.1 ml., 3 mg/ml.) reduced spontaneous muscle contractions in the cranial region. The ventral sheet of olfactory epithelium in the nasal cavity was exposed by removal of the dorsal wall of the cavity. The cytoarchitecture of the olfactory epithelium of the salamander (Getchell, 1977) and its fine structure in the water-phase salamander land.phase have been described (Graziadei & Monti Graziadei, 1976). Recording8. Single unit extracellular recordings were made with platinum-black metal-filled micropipettes (Gesteland, Howland, Lettvin, & Pitts, 1959). They were fabricated as previously described (Getchell, 1973). They had plated tip diameters of approximately 5 ,sm and resistances of approximately 0-25 The was oriented perpendicular to the mucosal surface in the middle region of the receptor sheet. It was lowered slowly through the epithelium until single unit activity was encountered. A large diameter pipette, approximately 50 filled with saline-gelatin, was positioned on the mucosal surface in the vicinity of the unit recording sites in order to record the summated receptor potential, the electro-olfactogram (Ottoson, 1956; Getchell, 1974b). Examples of unitary (top trace) and summated (middle trace) activity

MQ.

microelectrode

csm,

OLFACTORY RECEPTORS AND ODOUR PULSES

523

are shown in Fig. 1. The responses and stimulus monitor (bottom trace) were recorded on magnetic tape for playback and data analysis. The state of the preparation was carefully monitored by visual inspection of the mucosal surface, the blood flow within the capillary vasculature of the olfactory epithelium, and the reproducibility of the electro-olfactogram and the unit responses. The time interval between the abrupt onset of the CO2 monitor and the initiation of the electro-olfactogram was less than 100 msec when the delivery nozzle was properly oriented toward the recording site (cf. Figs. 1 and 4).

__INM

Fig. 1. Schematic diagram of experimental set-up, showing relations of stimulating nozzles and recording electrodes to exposed surface of the ventral olfactory epithelium of a salamander. Sample tracings are shown of spike potentials and the summated receptor potential (electro-olfactogram), together with the CO2 monitor of a stimulus pulse.

Olfactometer and stimulus delivery. The experimental arrangement for stimulating the olfactory mucosa is shown schematically in Fig. 1. Purified 'zero' air was led into an olfactometer where it was mixed with stimulus vapours at the desired concentration. This stream was led into a special delivery device consisting of concentric nozzles which permitted the odour stream to be directed onto the mucosa and abruptly turned on or terminated by an electrically driven solenoid switch (Kauer & Shepherd, 1975, 1977). An active vacuum ensured that there was no background stimulation during the interstimulus interval and that active removal of molecules after the termination of the pulse occurred. The odour pulse was monitored by mixing it with 5 % CO2 95 % 02 and recording the output of the stimulus delivery nozzle with a Beckman LB-2 CO2 analyzer which sampled the region of the tip of the nozzle over the olfactory mucosa. The monitored stimulus pulse is shown in the lower trace of Fig. 1. The interstimulus interval ranged from 1 to 3 min. The stimulating and monitoring methods are described in detail by Kauer & Shepherd (1975). A possible effect of the CO2 in the carrier stream on olfactory responses has been carefully investigated. Unit studies of second order neurones have shown no evidence of significant effects on responses of individual calls to carrier stream+ C00 pulse alone (Kauer & Shepherd, 1975),

524

T. V. GETCHELL AND G. M. SHEPHERD the response categories in the population of recorded cells (Kauer &

or on our own

524

Shepherd,

experiments,

1977).

evidence was found for only one receptor unit which (see Fig. 4) and breath. The other units in our receptor showed no discernible effect of the carrier-CO2 pulse alone. The odours used as stimuli in the present study are listed in Table first ten compounds are part of a family of structurally related compounds which are of methoxy benzene. The present use of these compounds is part of a longer term the diversity structure-activity relations. This group is of special interest because ofstructure. As sensations evoked by substitution of different side chains on the basic (eugenol) spicy through the odour from medicinal (guaiacol) comment, qualities ranged sweet (vanillin). In addition, two compounds, n-butanol and iso-amyl acetate, were these, together with eugenol, were included in the study to allow comparison of(1977). evoked by these stimuli in the olfactory bulb in the study of Kauer & Shepherd vapour pressure of the compounds, expressed in mm Hg at 23 'C, and the calculated of the saturated vapour, are also given in the Table. The olfactometer was calibrated to the values obtained by Kauer (1974) using a gas chromatograph.

control

to the carrier stream-CO2 pulse

In

responded

population

Stimulul selection.

1.

The

derivatives study of

a

of

odour

general

tested;

responses

molarity

according

TABLE 1. List of odours used in this study, with vapour pressures at calculated molarities saturated vapour. The asterisks indicate compounds also used by Kauer & Shepherd study of the salamander olfactory bulb Vapour pressure Molarity (m) of the Odour saturated vapour (mmHg, 3 0C in

1. Vanillin 2. Piperonal 3. Guaiacol 4.

iso-eugenol

5. Eugenol* 6. Safrole 7. Anisole 8. Anethole 9. Estragole 10. Anisaldehyde

23 0 003 0-013 0-13 0-015 0-025 0 07 3'3 0-08 0-14 180-04

0

1-6 x 10-7 70 x 10-7 70 x 10-6 1 10-7 1-3 x 10-6 3 6x 10-6 1.8 10-4 4-2 x 7-8 x 3 2 07 x

88x x

10-6 10-6 10-6 7x 10-4 11. n-butanol* 2-7 x 10-4 12.iso-amyl acetate* 50 The sequence in the experiments was as follows: when a unit was isolated and recording conditions were stable, its responses to each of the odours in Table1 were tested using a survey As described previously (Getchell, 1974b; Getchell & Getchell, 1977), this instruolfactometer. ment provides for delivery of a sequence of odours in a relatively quantitative manner and without significant cross-contamination between the different odours used. After effective odours had been identified, one was selected for systematic study using the pulse olfactometer described above.

RESULTS

recordings and spontaneous activity. Recording depths ranged from 30 to 200jum below the mucosal surface. Spike amplitudes ranged up to 1 mV in amplitude, and the spikes displayed either one of three basic conformations, diphasic frog (+, -) diphasic (-, +), or triphasic (+, -, +), as previously described for the common conformation was triolfactory receptors (Getchell, 1973). The mostfrom receptor cell axons deep in theon phasic spike (Fig. 2A) presumably recorded of electrode impingement effects the Criteria for evaluating epithelium. olfactory described (Getchell, previously as conformation were and activity spontaneous spike spikes, presumably amplitude Even smaller the best included recordings 1973). Unit

OLFACTOR Y RECEPTORS AND ODOUR PULSES 525 recorded from receptors in the vicinity of the recording site. Although single unit recordings were preferred, the multiunit recordings did provide a criterion to indicate a generally healthy preparation. Rates of spontaneous activity varied widely, from less than 1 to 120 spikes/min, with an average firing frequency of approximately 20 spikes/min. These are similar to the values obtained for frog olfactory receptors with these methods (Getchell, 1974a). Patterns of activity also varied greatly, from slow asynchronous firing (Fig. 2B) to rhythmic bursts of 2 or 3 spikes (Fig. 2C), or fairly regular discharges (Fig. 2D). The rate and pattern of spontaneous activity remained fairly constant for a given unit over the periods of testing with odours, which lasted up to 3 hr. However, in some units the background activity increased or decreased gradually during the interstimulus intervals. Long intervals, i.e. up to 3 min, were used in such cases to minimize these changes. Also, high concentrations of odours, that is, greater than 10-1 dilution of the saturated vapour, were avoided for this reason. In several cases an apparently silent unit was revealed by a gradual increase in background activity during the course of odour testing (see Fig. 5). A

C

10 msec

B

1 sec

D

1 sec 1sec Fig. 2. Examples of spontaneous unit spikes recorded in olfactory epithelium: A, fast sweep shows a typical triphasic spike conformation; B-D, slow sweeps show typical variations in rates and patterns of spontaneous activity.

Stability of responses to odours. Stable recordings have been obtained from ninetynine olfactory receptors. A total of forty-five receptors responded to at least one of the twelve odours delivered through the survey olfactometer (see Methods). When a receptor responded to more than one odour, the odour which evoked the clearest change in activity was selected for study first using the controlled and monitored pulses. Response stability was of particular concern. Variations in responses to odours could be due to several factors: receptor fatigue, alterations in the level of receptor excitability due to changes in the microenvironment of the extracellular mucus, or to changes in the stimulus pulse which could alter the response pattern. In order to reduce these variables, the interstimulus interval was at least one minute, and supramaximal stimulus strengths, as judged by spike decrement in the response, were avoided. In addition, systematic observations of the consistency of the mucus on the epithelial surface, the vascular flow in the nasal region and reproducibility of the electro-olfactogram were made. With these precautions, the responses to the step pulses were remarkably stable with repeated odour applications.

T. V. GETCHELL AND G. M. SHEPHERD 526 Typical results are shown in Fig. 3 for responses recorded from a receptor to a near-threshold 1 sec pulse of estragole. The threshold for excitation was estimated from the intensity-response function to be approximately 3*9 x 1o-8 M. It can be seen that the latency of onset, burst duration, and spike frequencies are similar in all the responses. These results demonstrate not only the stability of the response pattern but also the reproducibility of the odour step pulses delivered by the olfactometer, as can be seen in the stimulus monitors in the lower traces. MM"UMN A

-

D

-_

E

|

B I

0 5 sec

C_ Fig. 3. Unitary responses recorded sequentially to 1 see pulse of estragole. Sweeps A-E show reproducibility of the pulse monitor and the response pattern. Interstimulus intervals: 1 min.

Of particular concern was the relation between the response pattern and the orientation of the simulus delivery nozzle to the epithelial surface. Previous results (Getchell, 1974b; Getchell & Getchell, 1977) have shown that the wave form of the electro-olfactogram can vary as a function of the orientation of the stimulus. Fig. 4 shows the effect of changing nozzle position on a unitary response recorded from the mid-ventral olfactory epithelium approximately 1 mm from the posterior margin of the nasal cavity. The nozzle tip was directed posteriorly along a line which passed through the recording site, and was subsequently moved anteriorly for successive stimulus applications. At position A, with the nozzle tip positioned 2-5 mm above and 1 mm anterior from the recording site, the response consisted of a brief latency excitatory burst of spikes. The initial phasic response was separated from the tonic phase by a pause approximately 200 msec in duration. This basic pattern was present with the nozzle moved to position B, 1.5 mm away. At position C the early phasic component was reduced, and at positions D and E the response was only minimal or absent. Note that there was a progressive shift in the onset latency, but the response always terminated coincident with the termination of the pulses. Upon

OLFACTORY RECEPTORS AND ODOUR PULSES 527 subsequent repositioning of the nozzle to position A the basic response pattern was again recorded (trace A'). These results show that the nozzle system functioned as intended to give a defined stimulus pulse in time and to a certain extent in space. The details of the temporal Olfactory

0 05 sec

,mm -I

A'

A

Snout C

D

Fig. 4. Schematic illustration of the effect of changing position of the stimulating nozzle on the pattern of unitary responses. See text.

pattern of impulses clearly depend on the orientation of the nozzle to the recording site. The unit illustrated in Fig. 4 was unusual; it was the only unit that responded to the carrier stream-CO2 pulse alone, and it was the only one which showed distinctive phasic and tonic components, separated by a pause in firing (cf. Ottoson &

T. V. GETCHELL AND a. M. SHEPHERD Shepherd, 1971). The other responsive units were specific for odour stimuli listed in Table 1, and their responses were less clearly delineated into phasic and tonic components. The stimulus delivery nozzle was routinely oriented to give the optimal response, as in Fig. 4A, in a given unit with an odour at a given concentration. The nozzle position was not changed during study of the unit's response properties.

528

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1

2 3 Time (sec)

M

t

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Isec

-

Fig. 5. Unit responses to odour pulses of anisole of increasing concentration. A, recorded traces showing background (a, b) and response (c-f ). B, instantaneous frequency plots of responses in A. Stimulus duration shown by bar at bottom of graph. Occurrence of initial spike discharge indicated by (0) and plotted as reciprocal of the latency following the onset of the pulse.

Excitatory responses to odours of increasing concentration. Of the units which responded to odours, the most common response pattern was that of a simple discharge or burst of spikes. A typical example is shown in Fig. 5. Background activity

OLFACTORY RECEPTORS AND ODOUR PULSES 529 is shown in traces a and b. A definite response to anisole occurred at a concentration of 3-6 x 10-7 Iq (d). At higher concentrations (e, f) the firing frequencies increased, but the onset latency and burst duration remained relatively constant. These properties are plotted in the graph of Fig. 5B. Note the strong dependence of firing frequency on concentration, with a peak ranging from approximately 3-14 impulses/ see. Also, there was a slight shortening of latency of onset and termination of the burst at higher concentrations. The bursts were followed by a period during which spike firing was absent. This period of impulse inactivity was remarkably constant over the concentration range, lasting approximately 2-5 sec. Note that the spike amplitudes during background activity and odour stimulation are similar. This, the period of inactivity is not associated with decrementing spikes during the response (Shibuya & Tucker, 1967; Mathews, 1972; Holley et al. 1974; Gesteland, 1976). This was a consistent finding in our results. An excitatory response pattern evoked in a different unit, the large amplitude spike, by a different odour is shown in Fig. 6. The stimulus was piperonal, and a definite excitatory response was elicited at a concentration of 7 x 10-9 M (d). The threshold of this unit to piperanol was almost 2 log steps lower than that of the previous unit to anisole. Despite this difference in unit, odour, and threshold, the response patterns and the corresponding frequency plots can be seen to be remarkably similar. One difference is that the onset latency became considerably shorter as the concentration was increased from 7 x 10-9(d) to 1-4 x 10-8 M (e). However, there was only a slight change with the increase from e to f. Note the relatively constant time of termination of the burst, independent of concentration, and also the ensuing period of impulse inactivity lasting 2-2-5 sec. In some units the timing of the termination of the response was dependent on the concentration of the stimulating substance. This is illustrated in Fig. 7. It can be seen that two units were simultaneously recorded in this case, as indicated by large and small amplitude spikes. The large unit gave a definite response to nbutanol at a concentration of 1P5 x 10-5 M (d). The onset latency was relatively long, 1500 msec, and the response terminated 700 msec after the termination of the pulse. At a higher concentration (trace e), the onset latency shortened to 800 msec, similar to the results shown in Fig. 6. However, the cessation of this discharge also occurred earlier and appeared essentially coterminous with the end of the stimulus pulse. This is similar to the result previously shown in Fig. 4. Instantaneous frequency plots are shown in Fig. 7B. In addition to the points already mentioned, it may be seen that, at the higher concentrations, the initial phase of the response consisted of a high frequency discharge (0). This was followed by a phase during which the impulse frequency overlapped with that found at the lower odour concentrations (0), and this phase then terminated early, as already mentioned. Note also the slight decrement in the amplitude of the initial spikes in this response (Fig. 7 e). The properties of decrement and early termination may be related to the relatively high stimulus intensity. Further evidence on this point, obtained by varying the duration of pulses, will be given in the following paper. Plots of the initial latencies are shown in Fig. 7 C for both the large ( 0) and small () amplitude units. Note the lower threshold of the small unit, and the systematic

530 T. V. GETCHELL AND G. M. SHEPHERD decrease in latency as a function of increasing concentration. At the highest concentration, the latencies for the two units were similar. A

(a) Bkg. 16

14

B

k

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1

a

2

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3

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Time (sec)

(f)

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M

1

sec

Fig. 6. Responses of a different unit to odour pulses of piperonal of increasing concentration. Recordings in A, instantaneous frequency plots in B. Note similar response pattern to that in Fig. 5.

Variations on burst pattern. The simple excitatory responses just described were observed in thirty-five of the forty-five cells responding to stimuli. They consisted essentially of a discharge of impulses that increased in frequency with increasing concentration. The characteristic sequence of interspike intervals during the excitatory discharge is similar to that of frog olfactory receptors (Getchell, 1974a). The duration of the burst for a given pulse duration was relatively constant. There were two main variations on this pattern: an increase, or a decrease, in the burst duration as a function of increasing odour concentration. An example of

531 OLFACTORY RECEPTORS AND ODOUR PULSES a decreasing burst duration is shown in Fig. 8. In this experiment a brief 200 msec odour pulse was used. The threshold response (c) consisted of a slow discharge of 5 impulses barely discernible from the background activity. At higher concentrations (d and e), the receptor discharged at a higher rate and the burst duration shortened. The evolution of these patterns is shown graphically in B. Note the increase in frequency, from about 2 impulses/sec to about 10 impulses/sec, and the associated decrease in burst duration from about 2 see to about 0-8 see as the odour concen-

tration was increased.

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I 0-5sec Fig. 7. Simultaneous recording of activity of two unit spikes. A, responses to n-butanol pulses of increasing concentration. B, instantaneous frequency plots of large spikes (0) and small spikes (@). C, latency changes as a function of odour concentration for large (0) and small (0) amplitude spike responses.

In contrast, an example of an increase in burst duration is shown in Fig. 9. There is a questionable response in c and a clear response in d consisting of a burst of about 2 sec in duration. In e the concentration was raised still further and the response can be seen to consist of a sequence of impulse activity: an initial period during which the receptor discharged at a high frequency with decrementing spike amplitudes, a

T. V. GETCHELL AND G. M. SHEPHERD 532 period during which the spikes were of very low amplitude, followed by a somewhat lower frequency discharge and recovery of the spike amplitudes. The over-all duration of the burst discharge is more than 4 sec. A

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Fig. 8. Unit responses to brief pulses (0-2 sec) of anisole of increasing concentration. A, recorded traces of background activity (a) and responses (b-d). B, instantaneous frequency plots of responses shown in A. Pulse duration indicated by bar.

These types of responses were uncommon. A marked decrease in burst duration observed in three units; definite prolongation was seen in only two units. The example of prolongation accompanied by spike decrement shown in Fig. 9 was obtained with guaiacol at a concentration of 7 x 10-7 M. This concentration corresponds to a flow dilution of 10-1 of the saturated vapour. This was the highest concentration used in these experiments. There is reason to believe that the responses obtained at this level represent some degree of over-stimulation beyond the physiological range. Response latencies. Thus far, changes in frequency, response patterns and burst duration as a function of changes in concentration have been described. Another property which depended on stimulus concentration was the latency of onset of the response. In general, this decreased with increasing stimulus concentration. This was

533 OLFACTORY RECEPTORS AND ODOUR PULSES aspect of the response is summarized graphically for a number of units in Fig. 10. The time of occurrence of the first spike is plotted for different concentrations and the lines connect the points for a given experiment. It can be seen that in some units (Fig. 1OA) the change in latency was relatively restricted. For example, in the responses elicited by guaiacol (1) the latency decreased from about 1300 to about 950 msec over a 3 log unit change in concentration. In contrast, in the responses elicited by anethole ( x ) the latency decreased from 2700 msec to 700 msec over less than 1 log unit. The intensity-latency response function for a given unit also varied at different points on the concentration curve, as is shown in the responses op"N"

(a) Bkg.

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M

(d) .M

"Wqwwmpp-lmwgmvf Fri" (b) 7xlO-10 M

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-I 05 sec

(c) 7x10-9

M

Fig. 9. Unit responses to brief pulses (03 see) of guaiacol of increasing concentration. Note prolonged discharge, and decrement of spike amplitude in e.

elicited by n-butanol ( 0, *). It was of interest that two units which responded to guaiacol (c, c) both had relatively steep curves over a wide concentration range. The remainder of this unit population had rather shallow curves. Two unitary responses simultaneously recorded at the same site ( *) showed distinctive relationships to increasing concentration. Finally, two units recorded from different animals which responded to two similar odours (c, estragole; x, anethole) had rather similar, relatively flat, curves over the same narrow concentration range. The responses of different units to the same stimulus are shown graphically in Fig. lOB and C. The same properties already discussed are also evident in these responses. The relation between latency and concentration is steep in some units and shallow in others, and variable over the concentration range tested in still others. The family of curves in Fig. lOB appears remarkably homogeneous. Two of the units responding to safrole (0, 0) were recorded simultaneously at the same site. These curves are quite similar over the concentration range. 0,

534 T. V. GETCHELL AND G. M. SHEPHERD Some units had relatively brief latencies. The latency of the unit (0) shown in Fig. lOB ranged from 04 to 0.1 sec. A unit which responded with a very brief latency is illustrated in Fig. 11. Simultaneous recordings from several units were obtained, as is shown by the different amplitude spikes. However, the smallest spikes formed a relatively homogeneous population and it can be seen that the discharge of these spikes began shortly after the onset of the pulse of the odorant safrole. The latency changed with concentration from 80 to 20 msec over a concentration range from 1 8 x 10-8 to 3 6 x 10-8M. Notethatthe discharge lasted throughout the odour pulse and several seconds afterwards. In contrast, the unit giving the largest amplitude spike responded at a relatively long latency which decreased with increasing concentration from 2-4 to 1-4 sec. The extremely limited concentration ranges over which these latency changes took place is remarkable. The dramatic changes in latency and firing pattern occurred over a total range of less than one log unit. (A)

10-4

10-5 -

10 6

(B) Safrole

10-6

10-7

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~~~~~~~~10-8_

-

0I 5

1-0 1-5 20 Latency (sec)

2-5

30

05

1.0 1.5 2-0 Latency (sec)

2-5

Fig. 10. Plots of variations in initial spike latency with changing odour concentration. A, response latencies of different units to n-butanol (0, *), guaiacol (O, (I), anethole (x ), and estragole ([l). B, response latencies of different units (*, 0, O], A) to the same stimulus (safrole), C, response latencies of different units (L, V, 0, A) to same Stimulus (anisole).

Reduced activity associated with odour pultes. It has already been noted that impulse activity was often reduced or abolished in the period immediately following a stimulus pulse. This of course was more clearly seen at higher levels of background activity. We were also interested in determining whether impulse activity was reduced or abolished during an odour pulse. This was an infrequent finding; only five of the unit population gave any indication of this type of response. In one case there appeared to be a change from excitation at a low concentration to the absence of

OLFACTORY RECEPTORS AND ODOUR PULSES 535 impulses at moderate odour concentrations. In two other cases, there was a reduction in impulse activity during the pulses at all levels of concentration. The normal levels of background spontaneous activity were typically less than 60 spikes/min. Units with lower rates of activity, i.e. less than 20 spikes/min, were usually chosen in order to study more clearly initial response latencies and impulse discharge patterns. Because of this low background activity we did not expect to observe clear reductions in activity associated with the stimulus pulse. In addition, in the present paper our focus is on relatively short duration odour pulses, i.e. less

(a) Bkg

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(b) 1-8x10 -M

(e) 3 2x10B M

(c) 2-5x1O-8

(t) 3-6x10-8

M

M

M

0 5 sec Fig. 11. Simultaneous recordings, illustrating very short latency of onset of small spike activity, and long onset latency of large spike activity. Stimulus: Safrole.

than 3 sec. Against background activity of approximately 1 spike every 3 see only a statistical analysis on great numbers of repeated runs could establish a reduction or absence of activity during such short pulses. Given this fact, the most effective manipulation was to increase the pulse duration. In the following paper (Getchell & Shepherd, 1978) we will show how this has been used to obtain evidence for this response type which was a rare occurrence in this series of experiments.

DISCUSSION

Response patterns. One of the principal results of this study is that the impulse responses to step odour pulses have a relatively stereotyped temporal pattern. The main pattern is a discharge of impulses that is time-locked to the stimulus pulse. The salient properties of this discharge include the following: a relatively brief

T. V. GETCHELL AND G. M. SHEPHERD latency of onset; a rapid rise in impulse frequency; a persistence of impulse firing during the plateau of the pulse; and an abrupt termination of the discharge which is correlated with the termination of the pulse. In this study we have usually restricted ourselves to relatively brief pulses of up to 3 sec. During this time the impulse discharge characteristically builds up to a peak and then rapidly falls off to a lower level. The peak frequencies ranged from 10 to 20 impulses/sec and declined to 1-4 impulses/sec. One can therefore identify this as a phasic response property of the olfactory receptor. The subsequent adaptive properties of the receptors during longer duration pulses will be described in the following paper. A second main result of this study is that there is a clear and relatively stereotyped relationship between the receptor response and odour concentration. In general, impulse firing frequency increases with increasing odour concentration. The firing frequency ranges from approximately 1-3 impulses/sec at threshold, up to 20 impulses/sec at the highest concentration. Units differed in the concentration range to which they were sensitive. Some units showed response changes, from threshold to near maximum, over a concentration range as small as 0 5 log units. Other units were sensitive over a range of as much as 4 log units. Thus, it appears that the majority of individual receptors transmit information about stimulus concentration. This information appears to be encoded in the initial period of the impulse response to an applied odour, and different receptors vary in their sensitivity and dynamic range. An interesting finding was that with increasing concentration the onset latency of the response typically decreased. This property, however, varied considerably for the whole population of units. In some units the latency change was limited to about 300 msec, while in others a change of 1-2 sec could occur. The concentration range over which these changes took place also varied, being as narrow as 0*5 log units or as wide as 4 log units. This could be seen in the very steep or very flat curves, in which latency was plotted against concentration (see Fig. 10). In a few receptors, the latency of onset was very brief, from 20 to 125 msec. Thresholds also varied for different units for different stimuli, from as low as 3.5 x 10-10 M for guaiacol to as high as 3-7 x 10-5 M for n-butanol. These ranges for threshold values are similar to those previously reported for frog olfactory receptors (Getchell, 1974a). The thresholds of different units to the same stimulus, or of different stimuli to the same unit, could vary by as much as 4 log units. The variation in threshold may reflect in part the different numbers of molecular receptors for a given odour on the membrane of different receptor cells (Getchell, 1974; Getchell & Getchell, 1974). It should be noted that this is the concentration calculated from the flow dilution within the calibrated olfactometer, and we have no direct information about the actual concentration within the mucosa or at the mucus-receptor membrane interface. The physicochemical factors which may directly influence concentration gradients of olfactory molecules in the mucus have been discussed elsewhere (Getchell & Getchell, 1977; Hornung & Mozell, 1977). The present values are of added significance in being obtained under precisely controlled stimulus conditions. A characteristic finding was the abrupt termination of the discharge. In the main 536

537 OLFACTORY RECEPTORS AND ODOUR PULSES category of responses this was always locked to the termination of the pulse. The timing varied; it could be as short as 100 msec (Fig. 7) or as long as 1P5 sec (Fig. 5) after the pulse. This is an important property because it may reflect in part a balance between the molecular mechanisms of activation and inactivation of the receptor cell. Further evidence on this point will be presented in the following study of longer duration stimulus pulses. It is especially noteworthy that in the main category of excitatory responses the impulse discharge did not terminate prior to the end of the pulse as one proceeded to higher concentrations. This is in contrast to several reports in the literature of bursts of impulses which appear to decrease in duration with increasing concentration (Shibuya & Tucker, 1967; Mathews, 1972; Daval et al. 1972; Holley et al. 1974; Gesteland, 1976). Careful inspection of the published recordings suggests that the apparent decreased response duration could be complicated by other factors, e.g. spike decrement. We found only one unit in which spike decrement was a prominent feature (Fig. 9). It appears that when the stimulus is held at a steady plateau, as in the present study, most receptor cells are able to respond for the duration of the stimulus pulse over a wide concentration range with little variability in the amplitude of individual spikes. The results also confirm previous studies (Getchell, 1973, 1974a) which reported the absence of a change in spike conformation and spike decrement during excitatory responses recorded from frog olfactory receptors. Finally we may note that the period of stimulation is typically followed by a period of inactivity, as judged by the absence of impulses. This characteristically lasted for a period of 1-3 sec. In some cases this period changed to some extent with increasing concentration, whereas in other cases it was relatively unaffected by changing concentration. This period also appears to be independent of the prestimulus rate of spontaneous activity. The foregoing remarks apply to the category of response type which accounted for the great majority (- 80 %) of the units in our sample. Examples of other response types were observed, such as excitatory bursts which either were prolonged or shortened in duration with increasing concentration. These were rare, and we conclude that they either represent uncommon subtypes of the normal excitatory response, or perhaps reflect unsatisfactory recording conditions, or impaired access of the stimulus to the receptors. Five units responded to the odour pulses with a reduction in activity. The same possible explanations apply to these reponses. There is, however, the additional point, that the units were selected to some degree for their relatively low background rates of activity. As noted above, a low level of background activity makes this type of response less obvious, particularly at threshold. We have no further evidence regarding this type of response, other than that in these experiments it was rare. Comparison with other studies. We have already noted that the responses to controlled and monitored odour pulses do not display the variability in patterns of impulse discharge that have been reported in several other studies (see references in Introduction). In view of the differences in stimulus delivery systems and the lack of a stimulus monitor in those studies, it does not seem feasible to compare directly the results. However, we can begin to identify certain possible sources of the variability. One factor is obviously the time course of the stimulus. Another is

T. V. GETCHELL AND G. M. SHEPHERD the intensity - the importance of avoiding high concentrations cannot be overstressed. A third factor is local recording conditions, for example, sites of recording within the epithelium and the types of electrodes used. It is clear that the orientation of the stimulus delivery nozzle can affect the response pattern. Finally may be mentioned the state of the preparation. These by no means exhaust the possible variables, but they represent a start at specifying the conditions under which characteristic receptor responses are obtained. From the present study on the salamander, one may conclude that with temporally defined stimulus pulses the responses recorded from olfactory receptors have a stereotyped discharge pattern, and a systematic relationship to odour concentration. This is significant, because the wide variety of responses previously reported has suggested that molecular discrimination in the olfactory periphery appeared to be so complex as to represent mechanisms of coding significantly different in kind or principle from those observed in other sensory systems. Our results show instead that the relation between stimulus pulses and unitary responses in individual receptors may be simpler than previously believed, in terms of the properties mentioned above. Comparison with olfactory bulb. It is of interest to compare the present results with those obtained in a study of mitral cell responses in the salamander olfactory bulb, using similar techniques for controlling and monitoring step pulses of odour (Kauer & Shepherd, 1975, 1977). Although the studies were conducted separately on different experimental setups, and the selection of odours was different, with the exception of three overlapping compounds, the two studies afford some interesting comparisons. The majority of bulbar unit responses recorded at the mitral cell body layer consisted of two main types of patterns: an initially excitatory response, long lasting at threshold, which became brief at higher concentrations; and a category of purely suppressive responses. Each of these categories comprised about one third of the total number of responses, with the remaining one third being categorized as no response (cf. also Kauer, 1974; Kauer & Moulton, 1974). An outstanding characteristic was the limited response categories, the stereotyped discharge patterns, and the systematic changes in response frequency and duration with increasing concentration. Thus, the use of step pulses has yielded stereotyped response patterns in bulbar neurones as well as in receptor cells. The types of responses at these two sites are similar in certain respects and distinctly different in others. An important similarity is the high sensitivity of the responses to concentration of the stimulating odour. It appears, therefore, that stimulus concentration is detected by individual receptors, and transmitted by the impulse discharge in the receptor axons to the initral cells. After synaptic integration, the mitral cell further transmits information about concentration in the frequency domain to higher centres in the olfactory cortex. An important difference in the responses at the two levels is that impulse discharges of mitral cells at higher concentrations are very brief, and are followed by periods of strong suppression during the pulse stimulus (Kauer, 1974; Kauer & Shepherd, 1977). This pattern was never observed in the main response category at receptor level. In addition, the category of purely suppressive responses that is so prominent in mitral cells, was rarely seen at the receptor level. These differences 538

539 OLFACTORY RECEPTORS AND ODOUR PULSES may be ascribed to synaptic circuits in the bulb, as has been discussed by Kauer & Shepherd (1977, cf. also Getchell & Shepherd, 1975a, b). The authors thank Dr Marilyn L. Getchell and Dr John Kauer for valuable discussions and critical review of the manuscript. The research was supported by NIH grant NS-11667 and NSF grant BNS-76-81404 to T. V. Getchell and NIH grant NS-07609 to G. M. Shepherd. REFERENCES

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