The Journal of Neurophysiology Vol. 80 No. 2 August 1998, pp. 998-1001
Copyright ©1998 by the American Physiological Society

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Synaptic Correlates of Odor Habituation in the Rat Anterior Piriform Cortex

Donald A. Wilson

Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Wilson, Donald A. Synaptic correlates of odor habituation in the rat anterior piriform cortex. J. Neurophysiol. 80: 998-1001, 1998. Responses of anterior piriform cortex layer II/III neurons to both odors and electrical stimulation of the lateral olfactory tract (LOT) were measured with intracellular recordings in urethan-anesthetized, freely breathing rats. Odor-evoked, respiration-entrained postsynaptic potentials (PSPs) rapidly habituated during a 50-s odor stimulus, then spontaneously recovered within 2 min of odor termination. Associated with the decrease in odor-evoked PSP amplitude was a decrease in the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the LOT. The decrement in LOT-evoked EPSPs recovered with a time course similar to the odor response recovery. These results demonstrate that odor habituation is associated with a decrease in afferent synaptic efficacy in the anterior piriform cortex.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Habituation or adaptation to repetitive sensory input is a simple form of memory found in most sensory systems. Where it has been examined, behavioral and cortical neuron responses habituate more rapidly than first- or second-order neurons in mammalian sensory systems (e.g., olfaction, Hummel et al. 1996; vision, Sclar et al. 1985; audition, Weinberger et al. 1975). Thus, although some decrement in responsiveness of primary or secondary neurons may occur to repetitive stimulation, responses of cortical neurons decrease more than can be accounted for by reduced afferent activity.

This enhanced habituation in higher-order neurons has recently been demonstrated in the rat olfactory system, where decrements of odor-evoked spiking and postsynaptic potentials (PSPs) in anterior piriform cortex (aPCX) layer II/III neurons occur despite relatively maintained input from the main olfactory bulb (Wilson 1998). This habituation in aPCX is odor specific and is expressed as changes in both intensity and temporal pattern of aPCX activity (Wilson 1998). The decrement in aPCX odor-evoked activity relative to afferent input suggests that repetitive odor stimulation induces changes intrinsic to the aPCX.

Based on the well-described synaptic physiology of the piriform cortex (Haberly 1998), and synaptic correlates of habituation in other systems, there are several possible mechanisms for the reduction in aPCX odor-evoked PSPs during habituation including excitatory synaptic depression (e.g., Castellucci et al. 1970; Finlayson and Cynader 1995; Zucker 1972), inhibitory synaptic potentiation (Krasne and Teshiba 1995), and changes in neuromodulatory activity (Hasselmo and Barkai 1995; Paolini and McKenzie 1993). The present study examined whether odor habituation is associated with depression of excitatory afferent input. The results demonstrate that adaptation to prolonged odor stimulation by aPCX neurons is associated with a decrease in afferent synaptic efficacy.

	METHODS

Abstract Introduction Methods Results Discussion References

Male and female Long-Evans hooded rats (150-450 g) obtained from Charles River Labs were used as subjects. Animals were housed in polypropylene cages lined with wood chips. Food and water were available ad libitum. Lights were maintained on a 12:12 light:dark cycle with testing occurring during the light portion of the cycle.

Animals were anesthetized with urethan (1.5 g/kg) and placed in a stereotaxic apparatus. The lateral olfactory tract (LOT) was stimulated at 0.2 Hz with constant current square wave pulses (50-500 µA) with a tungsten monopolar electrode. Intracellular potentials were recorded in the aPCX, ~0-1 mm anterior to Bregma (incisor bar at 3 mm) as previously described (Wilson 1998). Briefly, intracellular recordings were made with glass microelectrodes filled with 2 M potassium acetate (tip resistance 60-150 M), lowered into the aPCX from the dorsal skull surface. Resting membrane potentials were at least 60 mV. Identification of aPCX Layer II/III neurons was done with LOT electrical stimulation, which evokes a short-latency monosynaptic excitatory postsynaptic potential (EPSP) (Haberly 1998). Intracellular recordings were digitized at 5 kHz and analyzed with Spike2 software.

Animals were freely breathing and stimulated with a flow dilution olfactometer. A continuous stream (500 ml/min) of air, passed through an activated charcoal filter and humidified, was blown across the nares of the animal. Odor vapor was added to the airstream with a computer-controlled 4-channel Picospritzer, which forced air through odorant-saturated filter paper at 50 ml/min, creating odor concentrations of 10¹ of saturated vapor. Odorants used included isoamyl acetate, eugenol, anisole, terpineol (all from Sigma), and peppermint (McCormick). Respiratory activity was monitored with a piezoelectric device monitoring chest wall movements. No behavioral or respiratory responses were observed to odor stimulation. Odor stimulation consisted of 2-s test stimuli and 50-s habituating stimuli. Stimulus onset was triggered on the respiratory cycle (at the exhalation/inhalation transition). Due to behavioral evidence of long-term effects of repeated, short-term habituation training (e.g., Grajski and Freeman 1989), animals were habituated to a particular odor only once. Control cells received no habituating odor stimulus, or were stimulated with an odor that produced no detectable response.

In a subset of cells, a single 2-s test stimulus (same odor as the habituating stimulus) was delivered posthabituation (at a different latency in each cell) to determine the time course of spontaneous recovery from odor habituation. Single test stimuli within cells were used to avoid altering the time course of recovery.

LOT-evoked EPSPs were measured by determining the slope of the linear rising phase of EPSP onset. The use of EPSP initial slope measures provided a relatively pure measure of afferent synaptic efficacy, uncontaminated by short-latency inhibitory postsynaptic potentials (IPSPs) (Kapur et al. 1997) and disynaptic EPSPs (Haberly 1998). Slopes were averaged within cells in groups of four consecutive EPSPs for statistical comparisons between groups and time points posthabituation. EPSP slope was expressed as a percent of prehabituation values. LOT stimuli were delivered for at least 1 min before habituation odor onset and continued for up to 10 min postodor offset.

Odor-evoked PSPs were measured as previously described (Wilson 1998) from respiration-triggered averages over 10-s periods at odor onset (0-10 s of stimulation) and odor offset (40-50 s of stimulation; see Fig. 1). PSP amplitude was measured from baseline to peak, away from evoked action potentials. Odor-evoked PSP amplitude was expressed as a percent of initial amplitude (0-10 s). Similar respiration-triggered averages were used to determine the amplitude of PSPs to test odor stimuli.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Representative anterior piriform cortex (aPCX) neuron odor-evoked and lateral olfactory tract (LOT)-evoked responses from a single layer II/III neuron. A: initial response to isoamyl acetate (horizontal bar) was characterized by large Vm oscillations in phase with the respiratory cycle (vertical tick marks). These oscillations, initially suprathreshold for action potentials, rapidly decreased in amplitude. B: respiratory cycle waveform and averaged, cycle-triggered odor-evoked postsynaptic potentials (PSPs) before and during the odor stimulus. Odor-onset (initial 10 s) evoked a large depolarization that was greatly attenuated by the last 10 s of the 50-s stimulus. Restimulation with a 2-s isoamyl acetate stimulus after a 2-min recovery period again evoked a large depolarization. Dashed horizontal line is 79 mV in all traces. Spike heights variable due to averaging. C: averaged LOT-evoked response before and after odor habituation. Vertical tick marks denote location from which slope measurements were made. Note reduction in postodor excitatory PSP (EPSP) slope. D: LOT-evoked response before habituation and at 2 min postodor offset (recovery). E: LOT-evoked response in the same cell at a longer time view.

	RESULTS

Abstract Introduction Methods Results Discussion References

Data from a total of 24 different cells from 15 animals were analyzed, including habituation (n = 17) and control experiments (n = 16), with some cells tested with more than one odor or in more than one condition. No obvious differences were noted in habituation between odors, thus results from all odors were combined. Of the control experiments, 13 were with no odor and 3 were with an odor to which the cell showed no detectable response. There was no significant difference in resting V_m between habituation (74.7 ± 1.7 mV, mean ± SE) and control (74.4 ± 2.1 mV) experiments.

Odor stimulation produced PSP responses that generally occurred phasically over the respiratory cycle. With the depth of anesthesia used here, respiration cycles were generally around 500 ms in duration. As shown in Fig. 1, A and B, these odor-evoked PSPs often exhibited large depolarizations supratheshold for spike initiation, along with other depolarizing and/or hyperpolarizing events within single respiratory cycles. Prolonged odor stimulation (50 s) produced rapid habituation of these PSPs and associated spiking (Fig. 1, A and B), in some cells resulting in a total elimination of a detectable response. For the cell shown in Fig. 1, restimulation with isoamyl acetate 2 min after the end of the habituating stimulus showed a nearly complete recovery of odor-evoked PSP amplitude.

As shown in Fig. 2A, 50-s odor stimulation resulted in a reduction in odor-evoked PSP amplitude over the last 10 s of stimulation to a mean 35.5 ± 6.1% of initial response amplitude [mean initial odor-evoked PSP amplitude at 0-10 s = 7.7 ± 1.6 mV; PSP amplitude at 40-50 s = 2.2 ± 0.4 mV, paired t-test, t(16) = 3.32, P < 0.01]. Test odor stimuli (2 s duration) delivered at various latencies posthabituating stimulus offset showed a recovery of odor-evoked PSP amplitude to initial values within 100-120 s (Fig. 2A).

View larger version (31K): [in this window] [in a new window]   FIG. 2. A: time course of spontaneous recovery from odor habituation. Odor evoked PSP amplitude as a percent of initial amplitude at various time points postodor offset. Time point 0 is the mean odor-evoked PSP amplitude during the last 10 s of odor stimulation for all cells (±SE). Odor-evoked PSP amplitude returned to near prestimulation levels within 120 s postodor offset. Each point represents a different cell. B: mean LOT-evoked EPSP slope as a percent of preodor slope. LOT-evoked EPSP slope was significantly reduced after odor habituation compared with EPSP slope in control cells until 80 s postodor offset (*P < 0.05).

LOT-evoked, monosynaptic EPSPs displayed a similar decrease and recovery following odor habituation. As shown in Fig. 1E, electrical stimulation of the LOT evoked a short-latency EPSP, often suprathreshold for spike generation, followed by an IPSP. Slope measurements from the linear portion of EPSP onset showed a reduction in LOT-evoked EPSPs following odor habituation compared with preodor measures. In the cell shown in Fig. 1, this EPSP reduction was sufficient to block action-potential generation (Fig. 1C), although blockade of LOT-evoked spiking was not a common observation. Within 2 min, the LOT-evoked EPSP returned to near prehabituation levels (Fig. 1D).

As shown in Fig. 2B, 50 s of odor stimulation resulted in a reduction in LOT-evoked EPSP slope immediately postodor to a mean 76.8 ± 4.9% of preodor slope [t-test, t(31) = 4.73, P < 0.01]. EPSP slope in habituated cells was significantly reduced compared with control cells at the 40- and 60-s time points (t-tests, P < 0.05), returning, statistically, to control levels at 80 s postodor offset.

A subset of cells was tested in both control and habituation conditions (n = 8 replicates in 5 cells). A paired t-test showed that, as with the between-cell comparisons above, odor habituation significantly reduced LOT-evoked EPSP slope compared with control stimulation within single cells [EPSP slope 20 s postodor as a percent of preodor slope, habituation = 87.1 ± 3.2%, control = 101.0 ± 3.9%; paired t-test, t(7) = 4.40, P < 0.01].

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present results demonstrate that habituation of odor responses in the aPCX is correlated with a decrease in efficacy of afferent excitatory synapses from the olfactory bulb. Furthermore, the time course of spontaneous recovery for odor responses and for LOT-evoked responses is similar, with both returning to prehabituation levels within 2 min. Interestingly, the time course of recovery was similar to that reported for recovery of adapted PSPs to visual (Giaschi et al. 1993) and electrical (Finlayson and Cynader 1995) stimulation in the rat visual cortex.

The decrease in odor-evoked PSPs by habituation was far greater than that seen for LOT-evoked EPSPs. Although odor-evoked PSPs were reduced to 35% of initial amplitude, and occasionally completely eliminated, LOT-evoked EPSPs were generally reduced to no less than 70% of prehabituation levels. This difference most likely reflects at least three factors. First, habituation of odor PSPs reflects both changes intrinsic to the aPCX and decrements in afferent activity (mitral/tufted cell habituation). The combination of a relatively small decrease in afferent activity and a relatively small decrease in synaptic strength could result in the more dramatic reduction in odor-evoked PSPs observed. For example, the effects of afferent depression could be amplified if voltage-dependent N-methyl-D-aspartate-mediated currents comprise a significant portion of the odor-evoked PSP, or if the afferent depression results in a shift in balance between excitation and feed-forward/feedback inhibition.

Second, electrical stimulation of the LOT is nonspecific compared with the probable effects of odor stimulation. Thus an odor stimulus presumably activates only a small subset of afferent synapses on aPCX pyramidal cells, whereas electrical stimulation of the LOT should activate a much larger, although overlapping, set of synapses. Previous work has demonstrated that habituation of aPCX responses is odor specific (Wilson 1998). Electrical stimulation following habituation to a single odor, therefore, would activate both habituated and nonhabituated synapses, resulting in a less pronounced reduction in total EPSP size. An analysis of the magnitude of odor-evoked PSP habituation and LOT-evoked EPSP reduction within cells showed no statistically significant correlation (data not shown), presumably for the same reason.

The third factor influencing aPCX odor habituation may be modification of other inputs to layer II/III neurons. For example, association fibers from other parts of the olfactory cortex have been shown to be relatively more plastic than LOT afferent synapses and have been implicated in models of memory formation in piriform cortex (Haberly 1985). Thus a decrease in the disynaptic EPSP, in addition to the afferent synaptic depression, could contribute to odor PSP habituation. Modulatory inputs to the piriform, such as acetylcholine (Hasselmo and Barkai 1995), have also been implicated in synaptic plasticity in this structure.

The short-term afferent synaptic depression reported here has not been previously described in the piriform cortex with electrical stimulation paradigms in vivo or in vitro. A variety of mechanisms for this short-term depression can be postulated including postsynaptic changes such as long-term depression, and/or presynaptic changes such as autoreceptor suppression of afferent neurotransmitter release (Hasselmo and Bower 1991).

In summary, habituation of aPCX neurons to repetitive odor stimuli is associated with a decrease in afferent synaptic strength. Additional studies will be required to determine the mechanism of this change in synaptic efficacy associated with odor stimulation, and its role in habituation of odor-evoked PSPs.

	FOOTNOTES

  Received 20 March 1998; accepted in final form 7 May 1998.

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