BY R. G. MAIR From the Walter S. Hunter Laboratory, Department of Psychology,

J. Phyeiol. (1982), 326, pp. 361-369 361 With 3 text-figure8 Printed in Great Britain ADAPTATION OF RAT OLFACTORY BULB NEURONES BY R. G. MAIR From the Walter S. Hunter Laboratory, Department of Psychology, Brown University, Providence, RI 02912, U.S.A. (Received 5 January 1981) SUMMARY 1. Single-unit activity was recorded from olfactory bulb neurones driven by odorous stimuli. Neural responses were quantified as averaged peristimulus time histograms. 2. Successive presentations of the same stimulus evoked similar patterns of activity during each presentation. Some neurones exhibited increased and others decreased excitability in the adapted state. The occurrence of such facilitative or suppressive self-adaptation was not determined by odorant concentration or by the number of action potentials evoked by a given stimulus. Neurones driven by more than one odorant exhibited the same type of adaptation (facilitative or suppressive) for all effective stimuli. 3. When the first odorant differed from the second, the number but not the pattern of action potentials evoked by the second odorant differed from the non-adapted state. Some neurones exhibited increased and others decreased excitability in the cross-adaptation state. Neurones exhibiting suppressive self-adaptation did not exhibit facilitative cross-adaptation and those exhibiting facilitative self-adaptation did not exhibit suppressive cross-adaptation. Instances of asymmetrical crossadaptation were noted in which two odorants differed in the extent to which they affected subsequent sensitivity to each other. The degree of symmetry for a given pair of odorants differed for different neurones. It is concluded that neurones in the olfactory bulb differ in both the type (suppressive or facilitative) and extent of adaptation evoked by a given odorant. INTRODUCTION Most vertebrates sample odorous stimuli periodically during successive sniff cycles. The effect of adaptation on the response to sequential stimulus events is unknown. Psychophysical measures of olfactory adaptation follow a complex set of rules. Stimulation with a strong odorant causes a transient decrease in sensitivity that is greater when the same substance is used to induce adaptation and to test subsequent sensitivity (self-adaptation). The extent of adaptation between different odorous substances (cross-adaptation) is asymmetrical for many pairs of odorants matched for stimulating effectiveness, that is odorant A may have a much larger effect on subsequent sensitivity to odorant B than odorant B has on that to odorant A (Cain &

R.. 362 0.MAIR Engen, 1969; Koster, 1971). Self-adaptation results in a greater decrease in sensitivity than cross-adaptation; however, the extent of cross-adaptation can be asymmetrical and thus not simply related to the propensity of odorants to stimulate common receptor sites or neural structures. Adaptation with low intensity (Berglund, 1977) or threshold level stimuli in humans (Engen & Bosack, 1969; Corbit & Engen, 1971) or threshold level stimuli in rats (Laing & Mackay-Sim, 1975) can increase subsequent sensitivity to other odorants (cross-facilitation). Thus, psychophysical measures of olfactory adaptation must reflect more than simple receptor fatigue. Olfactory receptors exhibit decreased excitability in the adapted state. When an odorous stimulus is presented twice within a limited period of time, less neural activity is evoked by the second presentation (Ottoson, 1956; Boxtel & Koster, 1978; Baylin & Moulton, i )79). Baylin & Moulton (1979) recorded single-unit activity from salamander olfactory receptors under cross-adaptation conditions. They report that cross-adaptation diminishes the number of evoked action potentials and that this effect is always asymmetrical. Response properties of olfactory receptors can account for the occurrence of asymmetrical cross-adaptation but not of cross-facilitation. At least two types of synaptic mechanisms within the olfactory bulb could cause facilitation. First, olfactory bulb neurones make extensive synaptic interactions with interneurones (Shepard, 1972) which could induce a state of increased mitral cell excitability. Secondly, the massive convergence of input from primary to second-order neurones could result in summation of excitation evoked from different receptors during successive stimulus events. The present experiment concern the responses of olfactory bulb neurones during two sequential stimulus events. Self-adaptation was recorded for odorants presented at varying inter-stimulus intervals. Stimulus-specific components of adaptation were studied by measuring the extent of cross-adaptation between different odorants. METHODS Recordings were made from pathogen-free male albino rats (Charles River CD strain) weighing 300-500 gms. The experimental preparation and recording procedures were the same as those described by Mair (1982). Neural activity was converted into averaged peristimulus time (p.s.t.) histograms based on responses evoked in eight trials with the same stimuli. Action potentials were counted in 100 msec time bins by the method described by Mair (1982). In a typical trial, neural activity was recorded prior to stimulus onset (spontaneous activity), during a 2 sec adapting stimulus event, during an inter-stimulus interval, during a 2 sec test stimulus event, and afterwards until the end of the sweep. The odorants used in this experiment were methyl ethyl ketone, ethyl acetate, n-butanol and benzaldehyde. Stimuliwere delivered byapolytetrafluoroethyleneandglassair-dilutionolfactometer (refer to Fig. 1 in Mair, 1982). In the adaptation mode the olfactometer delivered two sequential stimuli with a variable inter-stimulus interval. Timer 1 opened the solenoid to the adaptation manifold, selecting the adapting odorant, allowing it to mix into delivery line 2 and come to steady state. Timer 1 also started the signal averager sweep control. After a 7 sec delay, timer 2 activated solenoids A,B,C and D, diverting the odorous mixture in delivery line 2 to the animal and causing air to be drawn through the nose by connecting a vacuum via a cannula in the animal's choana. At the offset of the adapting stimulus, timer 3 opened an odorant-selection solenoid to allow the second (test) stimulus to mix into delivery line 2. Timer 4 was turned on after a variable period of time and activated solenoids A,B,C, and D to deliver the test stimulus to the rat. Once a suitable unit was isolated it was tested with a low and a high concentration of each of the test odorants. Odorants which drove the cells were first used for intensity-coding experiments

OLFACTORY BULB ADAPTATION (Mair, 1982). Adaptation was then studied by presenting two stimuli within p.s.t. histogram trial. A 2-3 min interval was interposed between each of the eight trials averaged. A consistent inter-trial interval was chosen for each neurone based on the time required to return to spontaneous activity. When self-adaptation effects were measured (adapting and test stimuli being the same) p.s.t. histograms were generated for several inter-stimulus intervals. Longer and shorter inter-stimulus intervals were tested in counterbalanced order. If a given unit was driven by two or more of the odorants, cross-adaptation effects were measured. This was done by generating two p.s.t. histograms. In one, odorant A was presented before odorant B and in the other odorant B preceded odorant A. The inter-stimulus intervals for all cross-adaptation trials were about 7 sec long. This interval was short enough to produce measurable adaptation and long enough to avoid crosscontamination in the olfactometer. RESULTS Odorant-evoked responses were categorized according to the scheme described by Mair (1982). Self-adaptation effects were recorded for seventeen type I responding cells, six type II responding cells, five type III responding cells and six cells exhibiting after-responses. Cross-adaptation effects were recorded for thirteen pairings of odorants from a total of ten neurones. The presentation of an adapting stimulus did not change the type of response evoked by a test stimulus. Type I responding neurones continued to produce type I responses. The number of evoked action potentials was affected by adaptation. For self-adaptation, units were observed showing less (suppression), equal, or more (facilitation) activity when driven by a stimulus in the adapted state. The occurrence of suppression or facilitation was not consistently related to either the number of evoked action potentials or to stimulus concentration. Neurones driven by more than one odorant exhibited the same type of adaptation behaviour (facilitation or suppressive) for all effective stimuli. When inter-stimulus intervals were lengthened from 1 to 15 sec, some units exhibited clearly diminished adaptation effects and others did not. For self-adaptation, test and adapting stimuli were identical. Adaptation effects were thus studied by directly comparing the responses evoked by these two stimuli. The validity of this measure rests on the assumption that neurones are normally responsive when they are driven by the adapting stimulus. The p.s.t. histograms in this experiment were based on eight successive trials. In three instances, the responses evoked by the adapting stimulus declined markedly during later averaging trials. In each of these instances the interval between adapting and test stimuli was 2 sec or less. In all other cases, the responses evoked by adapting stimuli were unaffected by the averaging procedures. Figure 1 presents adaptation data from two neurones making type I responses to ethyl acetate. For both units, the responses evoked by adapting stimuli were consistent across trials, demonstrating the reliability of the response measures employed. The unit on the left made a multi-peaked response to 0023 ethyl acetate and provides an example of a suppressive adaptation effect. At inter-stimulus intervals of 1P0, 2-0 and 6-2 seconds the initial peak evoked by the test stimulus was unclear and the second peak was smaller than in the response to the adapting stimulus. Although the initial peak was not clear, the latency of the second peak from stimulus onset was similar to the latency of the response to the adapting stimulus. When the inter-stimulus interval was increased to 14-2 sec (the lower histogram) the responses evoked by the test adapting stimuli were equivalent. 363

364 R. G. MAIR 00 0-023 EtAc 00019 EtAc CLI 0 - m 0 _ 0 0-0of0,~ 0 of Fig. 1. Averaged p.s.t. histograms recorded from neurones making type I responses driven by the same stimulus presented twice in succession (self-adaptation). The rate of action potential firing is plotted on the ordinate and time on the abscissa. Each histogram respresents a period of 25'6 sec. All stimulus events were 2 sec in duration and are represented by the heavy black lines under the abscissa. The unit on the left was driven by paired pulses of 0 023 ethyl acetate (EtAc) at inter-stimulus intervals of 1 0, 2-0, 6-2 and 14-2 sec (for histograms from top to bottom, respectively). This unit is an example of suppressive adaptation. The unit on the right was driven by paired pulses of 0 019 ethyl acetate at inter-stimulus intervals of 1-0, 2-0, 5-6 and 13X3 sec and is an example of facilitative adaptation. The histograms on the right-hand side of Fig. 1 depict the activity of a unit excited by ethyl acetate and showing a facilitative adaptation effect. The adapting stimulus evoked a multi-peaked on-response followed by a period of diminished activity. Test and adapting stimuli evoked similar patterns of activity. When the inter-stimulus interval was 1 sec, the test stimulus evoked an initial burst of action potentials that was smaller and a second burst that was equivalent to that evoked by the adapting

OLFACTORY BULB ADAPTATION 365 stimulus. Relative to the level of activity prior to stimulus onset, the response to the test stimulus was much larger than that to the adapting stimulus. When the inter-stimulus interval was increased to 2 sec, the absolute amplitudes of both peaks evoked by the test stimulus were larger than those evoked by the adapting stimulus. When the test stimulus followed the adapting stimulus by 5-6 or 13-1 see the second but not the first peaks evoked by the test stimuli were larger. 0039 But 0-036 EtAc._ (n _ 0 0 - -- Fig. 2. P.s.t. histograms from two type II responding neurones driven by the same stimulus presented twice in succession (self-adaptation). The unit on the left was driven by paired pulses of 0039 n-butanol at inter-stimulus intervals of 1 0, 2-0, 6-0 and 14-4 sec (for histograms from top to bottom, respectively) and provides an example of facilitative adaptation. The unit on the right was driven by paired pulses of 0-036 ethyl acetate and is an example of suppressive adaptation. All stimulus events were 2 sec in duration. Fig. 2 displays results from two units giving type II responses. The unit on the left responded to 0 039 n-butanol and provides an example of facilitative adaptation. The responses evoked by the adapting stimuli are consistent except when the interval between stimuli is 1 sec (top histogram). This unit exhibited diminished responsiveness following stimulation with the two stimuli closely approximated that was more pronounced during later averaging trials. The decrease in responsiveness was apparent in the lower spontaneous activity and decreased post-inhibitory excitation associated with the interval of 1 sec. The timing of post-inhibitory excitation was consistent for all adapting stimuli. Test stimuli evoked more action potentials during the post-inhibitory excitation than did the adapting stimuli at all intervals. The unit shown on the right-hand side of Fig. 2 responded to 0-036 ethyl acetate

366 R. G. MAIR A B N._V QL n/ A: 0-018 MEK B: 0-031 EtAc A: 0-036 MEK B: 0-036 EtAc B A C A: 0*017 MEK B: 0-017 But 1001 D A: 0-035 MEK B: 0-035 EtAc OL A B 0A B A Fig. 3. A-D, p.s.t. histograms recorded for four cross-adaptation pairings from four different neurones. All stimulus events were 2 sec in duration. Odorants included methyl ethyl ketone (MEK), ethyl acetate (EtAc), and n-butanol (But). Stimulus substance and concentration (fractional vapour saturation) is indicated for each of the pairings. The inter-stimulus intervals were: A, 6-7; B, 7-0; C, 6-0; and D, 6-3 sec. The histograms in A are an example of asymmetrical facilitation, those in B show asymmetrical suppression, those in C symmetrical suppression, and those in D symmetrical facilitation. with a type II on-response and a phasic after-response. This unit provides an example of suppressive adaptation. Similar responses were evoked by the adapting stimulus at all inter-stimulus intervals, and the test stimulus always evoked a smaller post-inhibitory excitatory burst than did the adapting stimulus. The phasic aftercomponent showed less variation between adapting and test responses.

OLFACTORY BULB ADAPTATION Cross-adaptation was studied by presenting a series of trials with odorant A before B and a series with B before A. Cross-adaptation effects were measured by comparing the response to an odorant when it was presented first in the sequence (as the adapting stimulus) to the response when it was presented second (as the test stimulus). Both suppressive and facilitative cross-adaptation were noted. In general, units showing suppressive self-adaptation effects showed either suppressive cross-adaptation or no cross-adaptation depending on the test and adapting stimuli chosen. Units showing facilitation self-adaptation did not show suppressive cross-adaptation. Cases of both symmetrical and asymmetrical cross-adaptation were seen. For a given pair of odorants, symmetry in cross-adaptation effectiveness differed for different neurones. Four examples of cross-adaptation are illustrated in Fig. 3. The unit depicted in Fig. 3A made type I responses to methyl ethyl ketone (A) and ethyl acetate (B) and provides an example of asymmetrical facilitation. The peak responding rate to 0-031 ethyl acetate was not affected by previous exposure to 0O018 methyl ethyl ketone although there was a greater decrease in activity after each of the peaks in the adapted condition. In contrast, stimulation with 0O018 methyl ethyl ketone evoked more action potentials when it was preceded by a pulse of 0031 ethyl acetate. The unit in Fig. 3B made a type II response to methyl ethyl ketone and to ethyl acetate, and is an example of asymmetrical suppression. Post-inhibitory excitation evoked by methyl ethyl ketone was eliminated when it was preceded by 0036 ethyl acetate. The response to 0-036 ethyl acetate was unaffected by previous exposure to 0036 methyl ethyl ketone. Note the phasic after-response when this unit was driven by 0O036 ethyl acetate. The unit pictured in Fig. 3C made a type I response to 0017 methyl ethyl ketone and 0017 n-butanol and is an example of symmetrical suppression. Pre-exposure to 0017 n-butanol decreased the number of action potentials subsequently evoked by 0017 methyl ethyl ketone. Likewise, pre-exposure to 0017 methyl ethyl ketone decreased the subsequent response to 0017 n-butanol. It is of interest that the phasic after-responses associated with these stimuli were relatively unaffected. The unit shown in Fig. 3D made a type I response to methyl ethyl ketone and ethyl acetate and provides an example of symmetrical facilitation. Stimulation with 0035 methyl ethyl ketone increased the number of action potentials subsequently evoked by 0035 ethyl acetate. Stimulation with 0035 ethyl acetate also facilitated the subsequent response to 0035 methyl ethyl ketone. 367 DISCUSSION When two identical stimuli are presented successively, the response of an olfactory bulb neurone evoked during the second presentation may be larger (facilitation) or smaller (suppression) than the response evoked during the first presentation. The occurrence of facilitation or suppression is not related to odorant concentration or to the amount of activity that a particular stimulus evokes. Likewise, the type of adaptation is not stimulus-specific. Neurones driven by more than one odorant exhibit the same type of adaptation under both self- and cross-adaptation conditions. The type of adaptation observed depends on the properties of the particular neurone recorded. Olfactory receptor cells exhibit suppressive adaptation under both

368 R.G. MAIR self- and cross-adaptation conditions (Ottoson, 1956; Baylin & Moulton, 1979) and thus receptor responses cannot account for the occurrence of facilitation in neurones of the olfactory bulb. Olfactory bulb neurones exhibit similar adaptation responses (facilitation or suppression) when test and adapting stimuli are the same (selfadaptation) and when they are different (cross-adaptation). Thus the type of adaptation is the same when neurones are driven by different receptive mechanisms during successive stimulus events. Rats normally sample olfactory stimuli by repeatedly sniffing in a stereotyped fashion. Adaptation to successive stimulus events must occur during this odoursampling behaviour. To achieve constancy in perceived odour between sniffs, some aspect of the evoked neural response must be unchanged across presentations. Macrides & Chorover (1972) reported that rodent olfactory bulb neurones develop stimulus-specific patterns of activity during periods of rapid, repetitive stimulation analogous to bouts of exploratory sniffing. The results of the present experiments demonstrate that the pattern of evoked activity is consistent, whereas the amount of evoked activity can vary between successive stimulus events. Mair (1982) showed that the pattern of activity evoked from rat olfactory bulb neurones changes as a monotonic function of stimulus magnitude. In contrast, the number of evoked action potentials is more consistently related to stimulus quality. The constancy in patterns of evoked activity suggests that olfactory sensitivity is not dramatically altered by a brief stimulus event. The variation in amount of evoked activity indicates that either perceived odour quality changes during successive stimulations or that the code for odour quality is more dynamic than an examination of an isolated stimulus event might suggest. The response properties of olfactory bulb neurones can account for the occurrence of asymmetrical cross-adaptation and cross-facilitation in psycophysical studies. Olfactory receptor cells exhibit asymmetrical decrements in evoked activity under cross-adaptation conditions (Baylin & Moulton, 1979). Thus, receptor response properties can account for such effects among second-order olfactory neurones or in psychophysical studies. The relationship between facilitation of olfactory bulb responses and psychophysical cross-facilitation is less certain. First olfactory bulb facilitation occurs under conditions ofboth self- and cross-adaptation. Psychophysical studies have not demonstrated self-facilitation, although few experiments have looked at adaptation following stimulation with weak odorants. Secondly, the amount of activity evoked from a given neurone is more consistently correlated with changes in odour quality than in stimulus concentration. The bulk of evidence for cross-facilitation comes from animal and human psychophysical experiments using detection tasks (Engen & Bosack, 1969; Corbit & Engen, 1971 ; Laing & Mackay-Sim, 1975). Performance on these tasks depends on the ability to detect the difference between a target odorant and a blank stimulus. Measurements of sensitivity with these methods can be facilitated by either increasing the perceived intensity of the target odorant or by improving the discrimination of the target odour quality. Thus, cross-facilitation in detection experiments could result from one stimulus priming neurones in the olfactory bulb to fire more action potentials in response to another stimulus whether that increase is related to the coding of intensity or quality.

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