Neuronal responsiveness to various sensory stimuli, and associative learning in the rat amygdala

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Neuroscience Vol. 68, No. 2, pp. 339-361, 1995 Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

N E U R O N A L RESPONSIVENESS TO VARIOUS SENSORY STIMULI, A N D ASSOCIATIVE L E A R N I N G IN THE RAT AMYGDALA T. U W A N O , H. N I S H I J O , T. O N O * a n d R. T A M U R A Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-01, Japan Abstract--Neuronal activities were recorded from the amygdala and amygdalostriatal transition area of behaving rats during discrimination of conditioned auditory, visual, olfactory, and somatosensory stimuli associated with positive and/or negative reinforcements. Neurons were also tested with taste solution and various sensory stimuli that were not associated with reinforcement. Of the 1195 neurons tested, 475 responded to one or more sensory stimuli. Of these, 256 neurons responded exclusively to a unimodal sensory stimulus, 128 to multimodal sensory stimuli, and the remaining 91 could not be classified. Distribution of unimodal neurons was correlated with anatomical projections to the amygdala from sensory thalamus or sensory cortices. Multimodal neurons were located mainly in the basolateral and central nuclei of the amgydala. Response latencies of neurons in the basolateral nucleus were longer than those in other nuclei and neurons in the central nucleus had both short and long latencies. Neurons responsive to a given stimulus were more frequently encountered in the amygdalas of the trained rats than in those of the rats not trained to associate that stimulus with a reinforcement. Multimodal neurons that responded to conditioned and/or unconditioned stimuli used in the associative learned tasks were concentrated in the basolateral and central nuclei. The results indicate that some amygdalar neurons receive exclusive single sensory information, and the others receive information from two or more sensory inputs. Considering the long latencies and multimodal responsiveness, the basolateral and central nuclei of the amygdala might be foci where various kinds of sensory information converge. It is also suggested that the basolateral and central nuclei of the amygdala have critical roles in associative learning to relate sensory information to reinforcement or affective significance.

It has been suggested t h a t the a m y g d a l a is critical in various kinds of m o t i v a t e d a n d e m o t i o n a l behaviour, a n d related a u t o n o m i c responses. 2°'38'41'81 A n a t o m i cally, the a m y g d a l a provides a link between sensory system a n d b r a i n s t e m executive systems for e m o t i o n a l behaviour, a n d a u t o n o m i c a n d endocrine responses. 1'29~43'68~77In these situations, sensory inform a t i o n m a y achieve m o t i v a t i o n a l a n d e m o t i o n a l significance u p o n its projection to the amygdala. 54'81 A m y g d a l a r lesion as well as deafferentation of the a m y g d a l a has decreased e m o t i o n a l or b e h a v i o u r a l responses to the deafferented sensory stimuli (see review by Davis TM a n d LeDoux43'44). Visual disconnection o f the m o n k e y a m y g d a l a 2°-35 resulted in deficits in e m o t i o n a l reaction to visually a r o u s i n g stimuli, a l t h o u g h reactions to o t h e r sensory stimuli were normal. Similarly, rats with a u d i t o r y deafferentation of the a m y g d a l a did n o t react to the auditory stimuli associated with shock. 71 All this evidence indicates t h a t sensory access to the a m y g d a l a is i m p o r t a n t to e m o t i o n a l processing. Recent a n a t o m i c a l studies o f rodents suggest that modality-specific a n d / o r m u l t i m o d a l i n f o r m a t i o n *To whom correspondence should be addressed. Abbreviation: ICSS, intracranial self-stimulation 339

from t e m p o r a l sensory association cortices a n d the t h a l a m u s project topographically to the amygdala. 51'52"68-76'78 It has been suggested, at least, t h a t thalamo-amygdalar projection was modalityspecific. TM A study using the a n t e r o g r a d e tracer, Phaseols vulgaris leucoagglutinin, suggested t h a t the corticoamygdalar projection is m u l t i m o d a l , 5~ a l t h o u g h a n earlier study suggested t h a t this projection m i g h t be modality-specific, v5'78 Thus, it seems reasonable to conclude t h a t the a m y g d a l a is able to receive various kinds of u n i m o d a l a n d m u l t i m o d a l sensory i n f o r m a t i o n . Consistently, the neurophysiological studies indicated t h a t some a m y g d a l a r neurons responded to various sensory stimuli w h e t h e r or n o t they are associated with reinforcement, a n d others responded only to stimuli t h a t indicate reinforcem e n t . 12"5°'55'59'6°'63"65 Results of these studies are consistent with the a m y g d a l a ' s function as a focus of i n f o r m a t i o n convergence to integrate e m o t i o n a l behaviour. M o s t neurophysiological studies o f the rat a m y g d a l a used only a u d i t o r y stimuli with or w i t h o u t reinforcement, ~°'55'7° but m a n y a n a t o m i c a l studies suggest the convergence o f sensory i n f o r m a t i o n in the rat amygdala. 5~'68'76 Previously, we reported t o p o g r a p h i c localization of b o t h u n i m o d a l a n d

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multimodal neurons in the m o n k e y amygdala. 59'6° However, anatomical connections between sensory systems and the amygdala in monkeys may differ from those in rats. 42'51'62 Few experiments have been designed to systematically test responses to various sensory stimuli in the same amygdalar neuron in rats. The first objective o f the present study was the recording o f neuronal activity in the rat amygdala and amygdalostriatal transition area during discrimination o f visual, auditory, somatosensory, and olfactory conditioned stimuli associated with reward (sweet solution or intracranial self-stimulation) or aversion (tail pinch), and during the ingestion o f various taste solutions. The second objective was investigation o f the role o f the amygdala in conditioned associative learning. The amygdalar role in e m o t i o n is not confined to innate emotional responses, 9 but encompasses emotional responses acquired by conditioning. Recent lesion studies in rats indicated that lesions in the lateral, basolateral, or central nuclei disturbed learning o f c o n d i t i o n e d responses to sensory stimuli associated with shock o r r e w a r d . 14'21'22'26'27'33'34'45 Unit recording results indicate that sensory tuning o f auditory neurons in the rat auditory cortex shifted toward the frequency o f the conditioned acoustic stimuli after conditioning to shock, w It has been suggested that this shift is mediated t h r o u g h amygdalar projections to the cortices, by which inform a t i o n a b o u t significance o f the sensory stimuli is transmitted. 24'8° These studies suggest that amygdalar neurons could encode specific sensory stimuli that have been associated by learning with a reinforcement. Synaptic plasticity in the amygdala has been implicated by evidence o f long-term p o t e n t i a t i o n J 6'~7 However, there have been few neurophysiological studies o f m o d u l a t i o n o f neuronal responses by conditioned associative learning in the rat amygdala. F o r this purpose, neuronal responses to sensory stimuli were c o m p a r e d between animals with and without conditioning to the sensory stimuli. We report here that some amygdalar neurons r e s p o n d e d exclusively to unimodal, or to multimodal stimuli, and were distributed topographically. Learning task-related multimodal neurons were mainly observed in the basolateral and central nuclei o f the amygdala. The experiments described here have been reported in part in abstract form. 79

EXPERIMENTAL PROCEDURES

Subjects and experimental design Fifteen male albino Wistar rats weighing 280-350 g (SLC, Hamamatsu, Japan) were used. Rats were individually housed in clear cages with free access to water and laboratory chow. The housing area was temperature controlled at 23°C and maintained on a 24 h light,lark cycle (on at 7.00, off at 19.00). A rat was painlessly placed in the special stereotaxic apparatus with a device for various sensory stimulation (Fig. 1A). Licking was signalled by a photoelectric sensor triggered by the tongue. The stereotaxic apparatus was covered by transparent plastic with an electric fan in the anterior wall and one in the posterior wall to

discharge odour-laden air from the enclosure through an exhaust pipe. There were three paradigms, described as experiment-I (Exp-I), experiment-If (Exp-lI) and experiment-Ill (Exp-III), and using the tasks shown in Fig. 1B and C. In Exp-I, a rat was trained to lick a spout that protruded close to its mouth to obtain reward [glucose solution or intracranial self-stimulation (ICSS)] (Fig. 1B), or to avoid tail pinch (Fig. IC). A 2s conditioned tone preceded protrusion of the spout. Tones of 1200 Hz (Tone 1) and 4300 Hz (Tone 2) signalled availability of 0.3 M glucose and ICSS (0.5 s train of 100 Hz, 0.3 ms capacitorcoupled negative square-wave pulses), respectively. A 2800 Hz tone (Tone 3) signalled a weak tail pinch if the spout was not licked within 2 s after its presentation. The tail pinch was a mild 2 s compression between two acrylic plates activated by an electromagnet,e4 After the rats had learned the tasks described above (see Training), amygdalar neurons were recorded during performance of the tasks. First, data were acquired during the tone-discrimination task, then clinical tests for other nonassociative sensory stimuli were performed as follows: auditory stimulation such as clicks, computer-synthesized artificial sounds, hand clapping, or human voice; somatosensory stimulation on various parts such as the head, body, and paws with a cotton tipped probe or an air puff; visual stimulation with a flash light. For auditory stimulation, complex sounds such as mewing, human voice, and hand clapping were introduced since previous results indicated that those sounds were sometimes effective in evoking neuronal r e s p o n s e s . 36,59,6° The intensity of the conditioned tones, clicks, and computer-synthesized tones was controlled at 80 dB; the intensity of complex sounds made by the experimenter were estimated to range between 70 and 90 dB. Four rats were used in Exp-I. In Exp-II each single neuron was tested with conditioned stimuli, including auditory, visual, somatosensory, and olfactory stimuli. Auditory discrimination was the same as in Exp-I except that 0.3 M sucrose solution was used instead of glucose solution, and Tone 3 (2800 Hz) was used as a neutral sound (associated with neither reward nor aversion). Either a 2 s stimulation of light (visual), or an air puff (somatosensory) was also associated, respectively, with a sucrose solution or ICSS reward. Eight rats were used in Exp-II. Neurons recorded from three of the rats used in Exp-II were further tested with nine odourants associated with ICSS; five volatile organic compounds consisting of acetophenone, isomyl acetate (3-methylbutyl acetate), cyclohexanone, p-cymene (p-isopropyl toluene), and 1,8-cineole (Eucalyptol) (Wako Pure Chemical Industry Ltd, Saitama, Japan), and two food-related, cheese and black pepper essences, and two cosmetic-related, rose and perfume (Chanel No. 5 type) essences (Takasago Int. Corp., Kanagawa, Japan). The first five odorants were similar to those used in a previous study of rats.t3 Each odorant, at saturated vapour pressure, was presented through a polyethylene tube as a conditioned stimulus for 2 or 4 s. Air in the enclosure was continuously exchanged by two fans, and trials were separated by intervals of at least 30 s. Clinical tests of auditory and somatosensory stimulation in Exp-II were the same as those in Exp-I. For taste stimulation, each sapid solution was infused through intraoral cannulae at room temperature. Taste stimuli consisted of four standard solutions; NaC1, 0.1 M; sucrose, 0.3 M; citric acid, 0.01 M; quinine HCI, 0.0003 M, and two other taste stimuli; monosodium glutamate, 0.1 M; lysine HC1, 0.2 M. The concentrations of four standard taste solutions were similar to those used in previous studies of awake ratsJ 758 Each trial for testing a taste solution consisted of delivering 0.05 ml of distilled water, a similar amount of a sapid stimulus, and then at least one water rinse of the same volume. The minimum interval between water and stimulus application was 15 s, and between one taste stimulus and the next, 45 s. These infusion procedures were similar to those

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Fig. 1. Schema of paradigm. (A) The stereotaxic apparatus was covered by transparent plastic plates with electric fans in the anterior and posterior walls to move odourized air through and from the enclosure. Rats were prepared for chronic recording by forming receptacles of dental cement to accept fake earbars. Electrodes were implanted in the posterior lateral hypothalamus for ICSS. The rat was trained to lick when the spout was automatically placed close to its mouth. Licking was signalled by a photoelectric sensor triggered by the tongue. (B) Conditioned sensory stimulus (tone, light, air puff, or odorant) was presented for 2 s (in the case of odourant, 2 or 4 s) prior to placing the spout close to the rat's mouth. A 1200 Hz (Tone 1) signalled that licking would produce glucose or sucrose; 4300 Hz (Tone 2) signalled ICSS; and 2800 Hz (Tone 3) was not associated with any reinforcements in Exp-II. (C) In Exp-I, 2800 Hz (Tone 3) signalled aversive stimulation (tail pinch), if the spout was not licked within 2 s after cue presentation. in the previous studies, and evoked taste responses quite similar to those evoked by natural licking. 57.58 In Exp-lll, a rat licked a spout to obtain sucrose solution or ICSS reward. The sensory stimuli were the same as in Exp-II (tones, light, air puff, odorants), however, they were presented randomly and were independent of spout protrusion. Therefore, no fixed temporal relationship existed between the protrusion of the spout and the sensory stimulation. The clinical tests of auditory and somatosensory stimulation in Exp-IIl were the same as those in Exp-I. Three rats were used in Exp-III.

Surgery Surgery was performed under aseptic conditions in two stages. First, a cranioplastic cap and two intraoral catheters were attached to the skull. After a recovery and a training period, a permanent indifferent electrode was implanted. The system for head restraint by Nishijo and Norgren, 57'58 modified from a method described by N a k a m u r a and Ono, 56 was used. After being anaesthetized (sodium pentobarbital, 40 mg/kg, i.p.), the rat was m o u n t e d in a stereotaxic apparatus with its skull level between the bregma and lambda suture points. The cranium was exposed, 2-3 m m of the temporal end of the temporal muscle was removed bilaterally, and eight to 10 small, sterile, stainless screws were then

threaded into holes in the skull to serve as anchors for cranioplastic acrylic. Stainless steel wires were soldered onto two screws to serve as a ground. Two bipolar electrodes for intracranial stimulation were implanted in the lateral hypothalamic medial forebrain bundle (A, - 4 . 3 from bregma; L, + 1.2; V, 8.5) according to the atlas of Paxinos and Watson. ~ After covering the cut end of the temporal muscle with the skin, the cranioplastic acrylic was built up on the skull, and molded around the conical ends of two sets of double stainless steel rods (fake earbars), which served the same purpose of earbars in the recording session, and were rigidly attached to the earbars of the stereotaxic instruments. A short length of 27-gauge stainless steel tubing was embedded in the cranioplastic acrylic near bregma to serve as a reference pin during chronic recording. Two stainless wires (50 p m diameter) were inserted into the genioglossus muscle to monitor tongue movements. Two intraoral cannulae were implanted by a technique described by Phillips and Norgren. 67 The polyethylene cannulae (SP65, Natsume) were inserted just rostral and lateral to the first maxillary molar on each side. The electromyograph wires and intraoral cannulae were brought out subcutaneously to the skull, and anchored to the cranioplastic cap. After surgery, an antibiotic (gentamicine sulfate, Gentacin c" Injection, Schering-Plough, Osaka, Japan) was administered topically and systemically (5 mg, i.m.).

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After recovery from sugery (10-14 days) and training (two weeks; see below), rats were reanaesthetized (sodium pentobarbital, 40mg/kg, i.p.) and mounted by the fake earbars. A hole (3-5 mm diameter) for chronic recording was drilled through the cranioplastic and the underlying skull (A, - 2 . 0 to - 4 . 0 from bregma: L, 3.0 to 6.0). The exposed dura was excised, and the hole was covered with hydrocortisone ointment (Rinderon VG '" ointment, Shionogi Co., Ltd, Tokyo, Japan), or one or two drops of chloramphenicol (Chloromycetin 'R Succinate, Sankyo Co., Ltd., Tokyo, Japan) solution (0.1 g/ml) were dropped in the hole. The hole was covered with a sterile teflon sheet, and sealed with an epoxy glue. A second small hole (1.5 mm diam.) was then drilled just medial to the hole for recording. A stainless steel wire (130 p m diam.), insulated except at the cross section of the tip, was implanted near the medial end of the central nucleus of the amygdala through the hole to serve as an indifferent electrode. This hole was then filled with cranioplastic acrylic. After the animal recovered (five to seven days), it was placed back on the water-deprivation regimen.

Training Before surgery, the rats were acclimated by handling and accustomed to being placed into a small, plastic restraining cage for brief periods. After recovery from the first stage of surgery, the rats were reacclimated to the plastic enclosure and placed on a 22 h water-deprivation regimen. While in the enclosure (1-2 h daily), they had access to a spout from which they learned to take fluids, initially 0.3 M sucrose, within one to two days. Subsequently, their heads were rigidly and painlessly fixed by inserting the fake earbars into the impressions in the cranioplastic cap. While restrained, the rats were trained to lick a spout, which was automatically protruded close to its mouth for 2 s, to obtain glucose or sucrose solutions and an ICSS reward. The threshold level for ICSS was determined, and any rat for which the threshold exceeded 300/tA was excluded. Rats were also trained to consume distilled water and sucrose solution applied via the intraoral cannulae. The rats were then trained to discriminate between conditioned auditory stimuli to obtain the rewards. Next, the rats were trained to lick a spout that was automatically protruded for 2 s without a conditioned auditory stimulus, to avoid a tail pinch. If the rat did not lick within 2 s after spout protrusion, a tail pinch was delivered. No rewards were delivered during these trials, so any response was made for avoidance only. The rats were then trained to discriminate between conditioned auditory stimuli to avoid a tail pinch. Training with either positive or negative reinforcement was carried out separately in one block of 10 or 20 trials. Finally, rats were trained to lick a spout after other conditioned stimuli in Exp-II. The total number of trials per day was 400-500 in 4 h from 20.00 to 24.00. Throughout the training and recording period, a rat was permitted to ingest 20-30 ml of water while in the restrainer. If it failed to take a total volume of 30 ml water while restrained, it was given the remainder when it was returned in its home cage. All experimental procedures were carried out in accordance with the National Institutes of Health "Guide for the care and use of laboratory animals".

Electrophysiological recording and analysis An individual rat was usually tested every other day. After being placed in the enclosure, the ointment was removed, and a glass-insulated tungsten microelectrode (Z = 1.0 1.5 M ~ at 1000 Hz) was stereotaxically inserted stepwise with a pulse motor-driven manipulator (SM-20, Narishige) into various parts of the amygdala and amygdalostriatal transition area. Extracellular neuronal and electromyograph activities were passed through a dual channel differential amplifier with a preamplifier (M EG-2100, Nihon Kohden), monitored on an oscilloscope, and recorded on magnetic tape (DFR-3715, Sony Magnescale). Onset of

somatosensory stimulation in the clinical test, and intraoral infusion of taste solution were noted on magnetic tape by voice commentary. Neuronal activity was counted by a two-level voltage discriminator. The analogue signal, the trigger levels, and the output of the discriminator were monitored continuously on an oscilloscope during analysis. The discriminator output pulses were accumulated, and displayed as peristimulus histograms by an on-line minicomputer (ATAC-450, Nihon Kohden). Another computer (MC 6300, Concurrent Computer Co.) stored the events and times of the trigger signals, output pulses from the discriminator, and lick signals for display of rasters and histograms off line. Both neuronal and behavioural data in each trial were counted from the peristimulus histograms in successive 100 ms bins for three periods: a pretrial control period (3 s), conditioned sensory stimulation period (2 or 4s), and a rewarding or aversive stimulation period (2 s). Neuronal activities were compared by one way ANOVA among discharge rates in the control period, conditioned sensory stimulation periods with different modalities, and a reinforcement (rewarding or aversive) period. Neuronal responses were determined from the discharge rates in the control periods and those in each conditioned sensory stimulation or reinforcement period, by the post hoc test (Tukey test) (analysis I). Comparisons between possible pairs of auditory responses (analysis II), and those between possible pairs of olfactory responses (analysis III) were also made by Tukey test. Neuronal response to each conditioned sensory stimulus was defined by analysis I. Some responses that contained both increases and decreases in firing rate were evaluated by visual inspection of the peristimulus time histograms, s9'6°'69 Significant responses during ingestion of sweet solution (i.e. reinforcement period) in analysis I were defined as oral sensory. If these neurons significantly responded more to infusion of taste solution than to that of water, they were defined as taste responsive (see below in detail). Responses to olfactory stimuli, when the neuronal response to somatosensory stimulus (air puff) was also significant in analysis I, were defined as olfactory if there was significant difference in analysis I11. The significance level was P < 0.05. For taste responses to intraoral infusion, all data analyses were based on neuronal activities in 5 s samples after infusion. When more than one sample was available for a particular taste stimulus, the mean was used. Spontaneous activity and responses to prestimulus water were calculated from multiple samples. Water and stimulus responses were calculated during the 5 s period beginning with the onset of a prestimulus water or a stimulus infusion. Depending on the analysis, two different response measures were employed: a raw score (the mean neuronal activity in a 5 s period) or a corrected score. For water, the corrected score was the raw score minus the spontaneous rate; and for a taste stimulus, it was the raw score minus the raw water response. A response to a taste stimulus was considered to be significant if the neuronal activity increased or decreased at least 2.0 S.D. from the mean of the prestimulus water response. Similarly, a water response was considered to be significant if the neural activity increased or decreased at least 2.0 S.D. from the mean of the spontaneous activity of the neuron. For neuronal responses to tactile stimulation by a cotton tipped probe or an air puff in the clinical test, all data analyses were based on neuronal activity in 1 s samples after onset of stimulation. Responses to tactile stimulation were considered to be significant if the neuronal activity increased or decreased at least 2.0 S.D. from the mean of the spontaneous activity. Neuronal responses to auditory stimulation in the clinical test were similarly analysed. The response latency of each neuron that responded during a conditioned sensory stimulation period was defined

Amygdalar neuron responses to conditioned sensory stimuli as the time between the computer-generated, synchronizing trigger signal that represented the onset of sensory stimulation and the time when the firing rate exceeded + 2.0 S.D. from the mean spontaneous firing rate determined from a histogram with 1-5 ms bins. The response latency to auditory stimuli in the clinical test was similarly measured since timing of auditory stimulation could be precisely determined from recorded sound data.

Histology After the last recording session, a rat was reanaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and several small electrolytic lesions (20/~A for 20 s) were made stereotaxically around the recorded sites with a glass-insulated tungsten microelectrode. Rats were then given a further overdose of anaesthetic and perfused transcardially with heparinized 0.9% saline following by 10% buffered formalin. The brain was removed, and cut into 30/~m frontal sections with a freezing microtome. Sections were stained with Cresyl Violet. All marking and stimulation sites were then carefully verified microscopically. Positions of neurons were stereotaxically located on the real tissue sections, and then compared to the atlas of Paxinos and Watson. 66

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auditory stimulation by a pure tone and a click, visual stimulation by a light, oral sensory stimulation by a taste solution, and s o m a t o s e n s o r y stimulation by an air puff and a cotton tipped probe. The 196 neurons recorded from three o f eight rats used in Exp-II, and all 156 neurons recorded in Exp-III were further tested with nine odourants. O f the 1195 neurons, 475 (39.7%) [Exp-I, 145 (37.4%); Exp-II, 275 (42.4%); Exp-III, 55 (35.3%)] r e s p o n d e d to one or m o r e sensory stimuli. Responses in two experiments were c o m b i n e d and analysed in terms o f responses to each sensory stimulation. O f the 475 responsive neurons, 256 responded exclusively to one sensory modality (85, auditory; 6, visual; 72, oral sensory; 80, somatosensory; 13, olfactory), 128 r e s p o n d e d to various c o m b i n a t i o n s o f each sensory modality, and the remaining 91 could not be classified. The n u m b e r s o f various type neurons responding to uni- and multim o d a l sensory stimuli are summarized in Table 1.

Responses to auditory stimuli

RESULTS

Over a period o f one to three m o n t h s for each rat, recordings were m a d e f r o m 1195 neurons (Exp-I, 388; Exp-II, 651; Exp-III, 156) in the amygdala and amygdalostriatal transition areas. Each n e u r o n was tested with various sensory modality stimuli, i.e.

O f 180 (37.9% o f responsive neurons) auditionresponsive neurons, 85 (72, excitatory; 13, inhibitory) r e s p o n d e d exclusively to auditory stimuli (unimodal), and 95 (69, excitatory; 26, inhibitory) responded to various sensory stimuli (multimodal). O f these

Table 1. Classification of sensory-reponsive neurons in and around the rat amygdala* A. Unimodal

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N~: Numbers of neurons responding to given stimuli. *In Exp-l, Exp-II and Exp-III 1195 neurons were tested with auditory, visual, somatosensory, and oral sensory stimuli. Of these, 196 in Exp-II and all 156 in Exp-III were further tested with nine odorants (see Experimental Procedures). One neuron in Exp-I and two in Exp-II were tested with a specific odorant. A, auditory; V, visual; SS, somatosensory; OS, oral sensory; OLF, olfactory.

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audition-responsive neurons, 102 responded to conditioned auditory stimuli, 48 to click and/or other complex sounds, and 30 to both. Responses to click or other auditory stimuli not associated with reinforcement usually disappeared or decreased when stimulus was presented repeatedly in several trials. Of the 49 neurons that responded to conditioned auditory stimuli in Exp-I, 15 responded differentially to tones that were associated with the positive- and negative-reinforcements. The neuronal responses depicted in Fig. 2 were recorded from an auditory specific neuron that responded differentially to conditioned auditory stimuli in Exp-II. The neuron responded strongly to Tone 1 associated with sucrose (A), and less to Tone 2 that predicted ICSS (B) or Tone 3 with no reward (C). The neuronal activity decreased very much to its spontaneous level during ingestion of sucrose solution after a period of conditioning tone (A). This insensitivity to taste stimuli was confirmed later by intraoral infusion of taste solution. The activity of the neuron also did not change during other conditioned stimuli such as light (D) and air puff (E). When tested by repeated stimulation with clicks, the neuron responded phasically in at least five trials. Tactile stimulation with a cotton tipped probe as well as infusion of taste solutions did not affect neuronal activity (not shown). Responses to somatosensory stimuli

A m o n g 180 (37.9%) somesthesia-responsive neurons, 80 (73, excited; 7, inhibited) responded exclusively to somatosensory stimuli only (unimodal), and 100 (84, excited; 16, inhibited) responded to various sensory stimuli (multimodal). O f these 180 neurons, 88 responded to the air puff, 77 responded to tactile stimulation with a cotton tipped probe, and 15 responded to both. Most of the receptive fields of the 92 somesthesia-responsive neurons that responded to the cotton tipped probe included a part of head and/or neck. O f these, receptive fields of 85 neurons were bilateral, and those of seven were unilateral (2, ipsilateral; 5, contralateral). An example of a unimodal somesthesia-responsive neuron in Exp-II that responded exclusively to an air puff preceding ICSS is shown in Fig. 3. The neuronal activity increased in response to presentation of a conditioned air puff stimulus (E). The activity did not change when any of three other different conditioned tones, such as Tone 1 preducting sucrose solution (A), Tone 2 predicting ICSS (B), or Tone 3 without reward (C); or light preceding ICSS (D) were presented. Tactile stimulation with a cotton tipped probe as well as infusion of taste solutions did not evoke significant neuronal response (not shown). Figure 4 is an example of neuronal responses exclusively to tactile stimulation with a cotton tipped probe in Exp-II. The neuronal activity increased phasically in response to tactile stimulation on the animal's nose (A11), but not to stimuli of other parts

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of the body ( A I - 1 0 , 12). Fig. 4B summarizes the magnitude of responses to the tactile stimulation described above. This neuron did not respond in the conditioned tasks in Exp-II, nor to infusion of taste solutions (not shown).

Responses to visual stimuli O f 63 (13.3%) vision-responsive neurons, only 6 (3, excitatory; 3, inhibitory) were unimodal and 57 (40, excitatory; 17, inhibitory) were multimodal. Thus,

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Neurons that responded to glucose or sucrose solution during the ingestion period in the reward task and/or during infusion of sapid stimuli were designated oral sensory. O f 118 (24.8%) oral sensory neurons, 72 (67, excitatory; 5, inhibitory) responded exclusively to oral sensory stimuli (unimodal), and 46 (35, excitatory, 11, inhibitory) responded to other sensory stimuli (multimodal). Of these 118 oral sensory neurons, 83 neurons could be further classified into two subcategories (non-taste oral sensory, and taste) based on the data from intraoral infusion. In detailed analysis of these 83 neurons, 23 were classified as taste neurons inasmuch as these neurons responded more strongly to taste stimuli than to water. O f the 23 taste neurons, 17 (all excited) responded only to oral sensory stimuli including taste stimuli, 6 (5, excitatory; 1, inhibitory) responded also to other sensory stimuli. Fig. 5 shows examples of taste neuron responses during ingestion of sucrose in Exp-II. This neuron responded during licking of sucrose solution, but not to the conditioned tone associated with sucrose solution (A). This neuron did not respond in other tasks (not shown). The neuron also responded while licking sucrose solution from a spatula, but not while licking a spatula without sucrose solution (B). This indicated that the neuron responded to taste, but not to tactile

stimulation of the tongue. In clinical testing, the neuron did not respond to tactile stimulation of various parts of the body (B). Neuronal activity also increased when sucrose solution was delivered through an intraoral cannulae, but did not change when water or NaC1 solution was applied. Insensitivity of the neuron to water was confirmed by licking water instead of sucrose solution in the conditioned task (C). Responses to oO~actory stimuli

There were 13 (all excited) unimodal, and nine (all excited) multimodal olfaction-responsive neurons. Examples of neuronal responses that were evident during the presentation of odorants are shown in Fig. 6. This neuron did not respond during stimulation by Tone 1 associated with sucrose (Aa), Tone 2 with ICSS (Ab), Tone 3 with no reward (Ac), nor light with ICSS (Ad). However, it did respond to the presentation of various odorants associated with ICSS (Ba-i). This neuron was judged to be olfactory since it did not respond to a non-odourized air-puff preceding ICSS (Bj). Responses of this neuron to various conditioned stimuli are summarized in Fig. 6C. There were significant differences among responses to various odorants. Post hoc comparison between these odorants indicated responses to pcymene were significantly larger than those to odorants other than acetophenone, and responses to acetophenone were significantly larger than those to odorants other than p-cymene, cheese, or

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Responses to muhimodal stimuli As indicated above, 128 n e u r o n s responded to two or more sensory modalities in various c o m b i n a t i o n (Table 1). O f these, 78 r e s p o n d e d to b o t h c o n d i t i o n e d a n d u n c o n d i t i o n e d stimuli in one trial, or to two or more c o n d i t i o n e d stimuli. O f these 78 neurons, 70 did not r e s p o n d to the stimuli used in the clinical trials. Figure 7 shows examples o f responses by one n e u r o n to three sensory modalities. T h e n e u r o n re-

The latencies in the various sensory modalities t h a t could be analysed are s h o w n in Fig. 8. There were significant differences in latencies between u n i m o d a l a n d m u l t i m o d a l neurons. The latency of the unim o d a l audition-responsive n e u r o n s ranged from 8 to 85 (40.4 + 4.08, m e a n + S.E.M., n = 29) ms, a n d from 8 to 370 (80.9 + 14.3, n = 34) ms for the multim o d a l audition-responsive neurons. The latencies of u n i m o d a l audition-responsive n e u r o n s were significantly shorter t h a n those of the m u l t i m o d a l n e u r o n s (Student's t-test, P < 0 . 0 5 ) (A). Latencies of the u n i m o d a l vision-responsive n e u r o n s ranged from 55 to 138 ( 1 0 2 . 7 + 2 4 . 7 , n = 3 ) m s a n d those of the m u l t i m o d a l vision-responsive n e u r o n s ranged from 12 to 270 ( 7 9 . 0 + 17.4, n = 1 4 ) m s (B). Latencies of u n i m o d a l somesthesia-responsive n e u r o n s ranged from 20 to 210 (114.8 + 15.6, n = 1 2 ) m s a n d those o f m u l t i m o d a l somesthesia-responsive n e u r o n s ranged from 30 to 560 (i17.5 + 32.9, n --- 16)ms (C). T h e r e was no significant difference between the latencies of the u n i m o d a l a n d the m u l t i m o d a l in b o t h vision- a n d somesthesia-responsive n e u r o n s (Student's t-test, P > 0.05). There were also significant differences a m o n g latencies o f responses to different sensory modalities (one way A N O V A , d.f. = 2, F = 4.781, P < 0.05). Response latencies o f audition-responsive n e u r o n s were significantly shorter t h a n those of somesthesiaresponsive n e u r o n s (Tukey test, P < 0 . 0 1 ) . Differences between latencies in o t h e r modalities were n o t significant (Tukey test, P > 0.05). W h e n d a t a were confined to u n i m o d a l sensory-responsive n e u r o n s (one way A N O V A , d.f. = 2 , F = 19.33, P < 0 . 0 1 ) , latencies of u n i m o d a l a u d i t i o n - n e u r o n s were shorter t h a n those o f u n i m o d a l somesthesia-responsive (Tukey test, P < 0.01) a n d u n i m o d a l vision-responsive n e u r o n s (Tukey test, P < 0.05), a n d in multim o d a l n e u r o n s there were no statistically significant differences between latencies in each m o d a l i t y (one way A N O V A , d.f. = 2, F = 0.932, P > 0.05). There were significant differences in latencies of auditory responses a m o n g three a m y g d a l a r subnuclei: the lateral nucleus (L), the basolateral nucleus (B1), a n d the central nucleus (Ce), a n d the amygdalostriatal transition area (ASt) including the ventral part of the c a u d a t e - p u t a m e n (one way A N O V A , d.f. = 3, F = 9.286, P < 0.01) (Fig. 9). A post hoc test indicated t h a t latencies to auditory stimuli were significantly longer in the basolateral nucleus t h a n in the amygdalostriatal transition area (Tukey test, P<0.01), or the lateral nucleus (Tukey test,

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349

neurons were similar to those of unimodal and multimodal audition-responsive neurons, except that somesthesia-responsive neurons were located in both dorsal and ventral parts of the amygdala, while audition-responsive neurons were located mainly in the dorsal part. As shown in Fig. 13, there were 14 vision-responsive neurons in the amygdalostriatal transition area; 1 was unimodal (open circle) and 13 were multimodal (filled circles). Only five unimodal vision-responsive and 44 multimodal vision-responsive neurons were located in the amygdala. Multimodal vision-responsive neurons were often observed in the central and basolateral nuclei. Thirteen unimodal olfactionresponsive neurons (Fig. 13, open inverted triangles) were recorded from the ventral part of the amygdala. O f l0 multimodal olfaction-responsive neurons (filled inverted triangles), two were recorded from the amygdalostriatal transition area, and eight from the amygdala. Distributions of oral sensory neurons including both non-taste plus taste neurons (circles + triangles), and taste neurons only (triangles only) are shown in Fig. 14. Oral sensory neurons were located mainly in the amygdala. O f 102 oral sensory neurons recorded from the amygdala, 64 that were unimodal were located in the central, basolateral, and cortical nuclei. The remaining 38 multimodal neurons were distributed mainly in the basolateral and central nuclei. In the amygdalostriatal transition area, eight neurons responded to oral sensory stimuli only, and eight to other sensory stimuli as well as to oral sensory stimuli. A m o n g data limited to taste neurons (triangles), there were 15 unimodal taste neurons in the amygdala, most of which were in the central nucleus, and six multimodal taste neurons distributed widely in the amygdala including the central and basolateral nuclei. In the amygdalostriatal transition area, there were only two unimodal taste neurons and no multimodal neurons. O f 128 multimodal neurons, 78 responded to various combinations of the conditioned and unconditioned stimuli used in the conditioned tasks (see "Responses to multimodal stimuli"). M o r e than 90% of these task-related multimodal neurons were located in the basolateral, central, and lateral nuclei of

Fig. 6. Neuron that responded during stimulation with odorant (Exp-II). (A) Histograms of neuronal responses to Tone l with sucrose (a), Tone 2 with ICSS (b), Tone 3 without reward (c), and light with ICSS (d). (B) Responses of same neuron to odorized air (odorant) with ICSS (a~) and non-odourized air puff with ICSS (j). Each histogram shows neuronal responses accumulated for five trials. Other descriptions as for Fig. 2. (C) Response profiles to various odorants, and other conditioned sensory stimuli during 2 s presentation. Odorants used for stimulation were: CYC, cyclohexanone; ISO, isoamyl acetate; ACE, acetophenone; CIN, 1,8-cineole; CYM, p-cymene; CH, cheese flavour; BP, black pepper flavour; RO, rose essence; PF, perfume essence typed Chanel No. 5. This neuron did not respond to conditioned auditory or visual stimuli, nor to rewards. Neuronal responses to odorants were significantly larger than those to corresponding non-odourized air (Tukey test, P < 0.01 for each odorant). Post hoc comparison of odorants indicated responses to CYM were significantly larger than those all odorants expect ACE, and responses to ACE were significantly larger than those to all odorants other than CYM, CH, and BP. Values are means + S.E.M.

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responses to sensory stimuli. One difference between Exp-II and Exp-I or Exp-III was that the light and air puff were associated with the reinforcements in Exp-II, whereas these two sensory stimuli were not associated with reinforcement in Exp-I and -III. Only 6.2% (9/145) in Exp-I and 3.6% (2/55) in Exp-III of the sensory-responsive neurons responded to light, whereas 18.9% (52/275) of the sensoryresponsive neurons responded to light in Exp-II. The percentage of vision-responsive neurons in Exp-II was statistically different from those in Exp-I and Exp-III (Fisher's exact probability test, P <0.01). Data samples from the amygdala and from the amygdalostriatal transition area were analysed separately (Table 2). In the amygdala, 3.7% (3/81) of sensory-responsive neurons in Exp-I and 4.8% (2/42) of those neurons in Exp-III responded to visual stimuli, while in Exp-II 20.2% (44/218) responded to visual stimuli. The differences between the percentage of vision-responsive neurons in Exp-II and Exp-I, and that between Exp-II and Exp-III were statistically significant (Fisher's exact probability test, P < 0.01). In the amygdalostriatal transition area, the proportions of vision-responsive neurons among the sensory-responsive neurons were not significantly different among Exp-I, Exp-II and Exp-III (Fisher's exact probability test, P > 0.05).

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sive neurons was significantly larger in Exp-II (71/218, 32.6%) than those in Exp-I (16/81, 19.8%) and in Exp-III (4/42, 9.5%) (Fisher's exact probability test, P < 0.05), and the ratio of vision-responsive neurons in Exp-I, Exp-lI, and Exp-III became similar if multimodal neurons were dropped from the data (Fisher's exact probability test, P > 0.05). DISCUSSION

Auditory responses Auditory information comes directly to the amygdala from subcortical areas, such as the medial

Table 2. Comparison of sensory-responsive neurons in each of three modalities among Exp-I, Exp-II and Exp-III Auditory ASt

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division of the medial geniculate body, the suprageniculate nucleus, and the posterior intralaminar nucleus, 46'47'75'76 o r via the auditory association cortices such as ventral parts of area TE 1, areas TE3 and TE2, and area 35a in the perirhinal periallocortex in rats. 46,SLTaThese direct and indirect sensory afferents have the same target areas, i.e. the caudate-putamen, the amygdalostriatal transition area, the lateral nucleus, and to a lesser extent, the basolateral nucleus.46'51 Distribution of unimodal auditionresponsive neurons in the present study agrees very closely with those anatomical projections. Although no other sensory stimuli were tested, neurons responding to auditory stimuli have also been reported in the amygdalostriatal transition area, the caudateputamen, and the lateral, basolateral and central nuclei of the amygdala of awake rats. 12'55Among the auditory responses, excitation predominated in the present study, which is consistent with previous studies in awake animals) 2'55'59,6°This may be due to the mediation of neurotransmission from sensory areas by the excitatory transmitter, glutamate, in the lateral nucleus.23.48 The present study does not reveal the routes (i.e. thalamoamygdalar and thalamo-cortico-amygdalar) by which these unimodal neurons receive the sensory information. It has been estimated that the shortest auditory latencies by direct thalamic routes were 12-15 ms, and latencies over neocortical routes were 18-25 ms in the lateral nucleus and the amygdalostriatal transition area. ~°'~2 In the present study, both latencies were observed, and longer latencies (exceeding 70 ms) were also seen in the lateral, basolateral, and central nuclei, consistent with previous studies ~°'H'55that indicate other multisynaptic pathways. S o m a t o s e n s o r y responses

Anatomically, the caudat~putamen, the amygdalostriatal transition area, and the lateral nucleus of the amygdala receive somatosensory information from the same nuclei of the thalamus, as auditory information, such as the suprageniculate nucleus, medial division of the medial geniculate body, and the posterior intralaminar nucleusfl7 In addition, the central, medial and basomedial nuclei receive somatosensory information from the medial posterior complex of the thalamus. 47'76 The parabrachial nucleus in the pons, which receives spinal and trigeminal somatosensory projectionsf9 also sends afferents to the central nucleus.6"25 Neocortical somesthesiarelated areas such as areas TE2 and TE3, and area 13p in the perirhinal periallocortex project to the caudate-putamen, the amygdalostriatal transition area and lateral nucleus, and to a lesser extent, the central and the basolateral nuclei.46"51'7s Distribution of unimodal somasthesia-responsive neurons in the present study is correlated with these anatomical projections. Although responses to other sensory stimuli were not demonstrated, neurons in the central, basolateral, basomedial, and lateral nuclei, or in

the amygdalostriatal transition area responded to somatosensory stimuli in anaesthetized, v'8'7° and awake rats? 5 Romanski et al. TM reported that the mean latency of somatosensory responses in the lateral nucleus was 17 ms, while a value of 61 ms was found in the present study. However, the shortest latencies in the present study were comparable to those of Romanski et al. v° The difference in the mean latency of somatosensory responses between the two studies might be ascribed to differences in somatosensory stimulations that were used. In the present study, an air-puff was used while Romanski et al. 7° utilized an electric foot shock. Visual responses

Subcortical visual information from the lateral posterior nucleus of the thalamus goes to the lateral nucleus of the amygdala and the amygdalostriatal transition area. 47Neocortical visual information from area TE2, and areas 35 in the perirhinal periallocortex goes to the arnygdalostriatal transition area, and the lateral, basolateral, and central nuclei.46'5t'78 Although unimodal vision-responsive neurons have been found in these nuclei, their numbers are very few; only five were found in the present study. The finding of few vision-responsive neurons in the present study is consistent with previous studies of cats. In cats, auditory stimuli were more effective than visual stimuli in evoking neuronal responses. 63'72 In contrast to rats and cats, vision-responsive neurons were predominant in monkeys. 59'6° This speciesrelated difference may be due to the primate's greater dependence on visual stimulation)° Relatively large numbers of multimodal visual conditioned neurons were found in the basolateral and central nuclei in Exp-II. It is reported that potentiation of the acoustic startle responses by visual as well as auditory conditioned stimuli were mediated through the basolateral nucleus.4°'53 O r a l s e n s o r y responses

We observed oral sensory neurons, mainly in the central nucleus of the amygdala, and to a lesser extent, in the amygdalostriatal transition area, the lateral, medial, basomedial, and basolateral nuclei. From the pontine parabrachial nucleus, which sends two parallel ascending paths, thalamo-cortically and directly to the amygdala, 3~ taste and/or lingual somatosensory information comes to the central 6'6~'62 and lateral nuclei.25 The parvicellular part of the ventral posteromedial nucleus of the thalamus (the thalamic taste area) projects to the central and lateral nuclei,76 and area 13 in the perirhinal periallocortex (the cortical taste area) projects to the central and lateral nuclei.TM The distribution of unimodal oral sensory neurons were well correlated with these anatomical projections. In the present study, 83 of 108 oral sensory neurons could be further classified into two subcategories. Of these 83 oral sensory neurons,

Amygdalar neuron responses to conditioned sensory stimuli 23 were classified as taste neurons, and 60 as nontaste oral sensory neurons. Azuma et aL 2 reported that many amygdalar neurons in anaesthetized rats responded to lingual somatosensory stimuli other than taste such as cold, warm or mechanical stimulation of the tongue. In the present study, all solutions were applied at room temperature, so non-taste oral sensory neurons might respond to such mechanical and thermal sensory stimuli. Olfactory responses

Olfactory information goes to the anterior amygdalar area and the medial and cortical nuclei directly from the olfactory bulb, and into the basomedial and basolateral, and central nuclei from the piriform cortex. TM Distribution of unimodal olfaction-responsive neurons was correlated with olfactory projections. Previously, it was reported that many amygdalar neurons recorded from virtually all subnuclei of the amygdala responded to olfactory stimuli in anaesthetized rats. ~5 The profiles of responses of the neurons to various kinds of odourants in that study were very broad. This is consistent with those in the present study, and receptive fields of audition-responsive neurons in the amygdala were similarly broad/° In awake monkeys, some vision-responsive neurons responded to all objects that had affective significance. 59'6° Thus, broad sensory tuning might be a characteristic of amygdalar neurons. M u l t i m o d a l responses

In the present study, multimodal responsive neurons were observed mainly in the basolateral and central nuclei of the amygdala, and the amygdalostriatal transition area. These multimodal neurons were divided into two types; one type responded to plural sensory modalities that were not included in the conditioned learning, and the other type responded to combinations of conditioned and unconditioned stimuli that were used in the conditioned associative paradigms. The neurons, that responded to plural sensory modalities in the present study and were not involved in conditioned learning were located in the amygdalostriatal transition area, whereas neurons that responded to stimuli related to conditioned learning were mainly located in the basolateral and central nuclei of the amygdala. Previously, Romanski et al. 7° reported some multimodal neurons in the lateral nucleus of the amygdala in anaesthetized rats, which responded to both auditory and somatosensory stimuli that were not used as sets of conditioned and unconditioned stimuli. Consistently, convergence between somatosensory and other sensory stimuli were observed in the lateral nucleus in the present study. However, more multimodal neurons were recorded in the amygdalostriatal transition area than in the lateral nucleus of the amygdala in the previous and the present studies. The multimodal neurons recorded in the amygdalostriatal transition area in the present study were comparable to the multimodal

357

neurons in the amygdalostriatal transition area reported by Romanski et al. TM On the other hand, neurons that responded to stimuli related to conditioned learning were reported in the basolateral and central amygdalar nuclei in monkeys 59'6°and in rats) 5 Ben-Ari and colleagues4~5'43also reported multimodal neurons in the lateral nucleus of immobilized cats. However, most multimodal neurons reported by them had inhibitory responses, which were rarely found in the present and other 59'6°'7° studies of the lateral nucleus. In the present study, the conditioned sensory stimuli were all very familiar to the animals since they were also presented in the training period. Furthermore, electric foot shock, which was reported, by Ben-Ari and colleagues, 4'5'43 to be the most effective stimulus for evoking neuronal responses, was not used in the present study. The differences in response direction (excitation or inhibition) and sensory convergence between the previous 4'5'43 and the present studies might be ascribed to the differences in the experimental designs. Recent anatomical studies support multimodal responsiveness in the basolateral and central nuclei. The basolateral nucleus receives afferents from the lateral nucleus 73 which in turn, receives various sensory afferents from the thalamus and the neocortices. 51"52'76'78 The central nucleus receives afferents from basolateral, lateral, and basomedial nuclei. 6s This anatomical evidence of convergent inputs supports multimodal responsiveness in the central and basolateral nuclei. In accordance with this anatomical evidence, some neurons in the basolateral and central nuclei in the present study had longer latencies than those in the amygdalostriatal transition area of the lateral nucleus. Recently, Bordi et al) 2 also reported long latencies in these nuclei although no sensory responsiveness to other than auditory stimuli was known. Among the multimodal responses, excitatory responses were predominant. Most projection neurons in the lateral nucleus were glutamatergic, 23 and most synapses of the projections on the basolateral neurons were asymmetrical. 73 These studies suggest that neurotransmission between the lateral and basolateral nuclei is excitatory, which is consistent with the present results, at least in the basolateral nucleus. Spontaneous firing rate

The mean spontaneous firing rates in the present study (lateral nucleus of the amygdala, 7.6 spikes/s; basolateral, 8.9 spikes/s central, 10.5 spikes/s) were relatively higher than those reported previously, 12'55 although the exact spontaneous firing rate for each nucleus was not determined in the previous studies. In our preliminary study, there was a tendency for the spontaneous firing rate of an amygdalar neuron to become higher when the neuron was tested with the learned sensory stimuli to which it responded (Nishijo et al., unpublished observation). Furthermore, the spontaneous firing rates of some neurons have been

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reported to change over long periods of time after stimulus presentation? These facts suggest that the experimental design could affect the spontaneous rates of amygdalar neurons. Other experimental conditions such as subject preparation (freely moving or restrained animals) and recording electrodes (tungsten, glass, nichrome, etc.), may also contribute to differences in spontaneous firing rates. Further studies are required to determine which factors contribute to the increase in spontaneous firing rates.

Amygdalar plasticity It has been reported that receptive fields of audition-responsive neurons in the neocortex could be changed plastically by conditioned associative learning. 8° This conclusion can now be extended to the amygdala, and to other sensory modalities. Animals recorded in the present study had already learned auditory conditioned stimuli. In the present study, many amygdalar neurons responded to conditioned tones at frequencies below 5 kHz in Exp-I and Exp-II. However, in an earlier study, most amygdalar neurons in naive awake rats responded to tones above 10 kHz. H It is suggested that the sensory tuning of audition-responsive neurons in the lateral amygdala was changed by conditioning/2'68a Furthermore, the proportions of amygdalar neurons that responded to auditory stimuli were significantly larger in Exp-I and Exp-II, in which the auditory stimuli were associated with reinforcements, than that in Exp-III in which the auditory stimuli were not associated with any reinforcements. In addition, the proportions of amygdalar neurons that responded to visual and somatosensory stimuli were significantly larger in Exp-II, in which those sensory stimuli were associated with reinforcements, than in Exp-I and Exp-III, in which they were not associated with reinforcements. The results suggest that plastic changes can also occur in the amygdala during conditioning, and these plastic changes in the amygdala might be essential to the associative learning discussed below. First, there have been many reports of amygdalar lesions that caused deficits in learning of conditioned associative paradigms (see review by Davis TM and LeDoux43'44). Lesions in the amygdala have been especially critical, and those in the ventral part of the caudate-putamen, including the amygdalostriatal transition area have been less so, although both areas receive similar sensory projections.45 Results in the present study are consistent with those in previous behavioural studies. In the present study, evidence of plasticity was observed in the amygdala, but none were evident in the ventral part of the caudateputamen. Second, it was reported that neuronal responses in the amygdala were plactically modulated during associative learning by rabbits. 39 In monkeys, responses of some amygdalar neurons to unfamiliar objects habituated within a few trials when such unfamiliar objects were not associated with reinforce-

ments, but the neurons became responsive to the stimuli when the stimuli were associated with reinforcement/9'6° In rats, amygdalar neurons that responded to an unconditioned stimulus became responsive to the corresponding conditioned stimulus after associative conditioning/5 These previous lesion and neurophysiological studies support the present results. It has been suggested that, in classical conditioning or in the learning process of recognizing an affective stimulus, inputs from conditioned and unconditioned stimuli, or those from different sensory modalities are correlated. 28'32'37 In the present study, the ratio of multimodal amygdalar neurons to all responsive neurons was significantly larger in Exp-II than in Exp-I. This suggests that learning produces neurophysiological changes in the convergence of multimodal stimuli in the amygdala. Taken together, the present results suggest that the amygdala is a critical focus of convergence of various sensory stimuli, and modification of synapses on amygdalar neurons from such sensory inputs might underlie mechanisms in conditioned associative learning.

CONCLUSION

Neuronal activity in the amygdala in response to various sensory stimuli was recorded to clarify whether two or more sensory modalities might reach a single neuron. The results demonstrate, first, that some amygdalar neurons receive specific sensory modalities, and some neurons receive multiple sensory modalities. Multimodal neurons that responded to conditioned and/or unconditioned stimuli in association tasks were found in the basolateral and central nuclei of the amygdala. Second, more neurons responded to a given stimulus in rats trained to associate that stimulus with a reinforcement than in rats that were not trained. This increase in sensoryresponsive neurons was correlated with the increase in multimodal neurons. Third, some neurons in the basolateral and central nuclei of the amygdala had longer onset latencies than those in other nuclei. The results suggest that basolateral and central nuclei are foci where various sensory modalities converge, and these nuclei of the amygdala might perform critical functions in associative learning to relate sensory information to reinforcment or affective significance.

Acknowledgements--Wethank Dr A. Simpson (Showa University) for help in preparing the manuscript, and Takasago Int. Corp. (Kanagawa) for kindly supplying us the odorants. This work was supported partly by the Japanese Ministry of Education, Scienceand Culture Grants-in Aid for Scientific Research (05267103, 06454706, 06680786, 07244207), Human Frontier Science Program for 3rd fiscal year, and by Uehara Memorial Foundation.

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REFERENCES 1. Amaral D. G., Price J. L., Pitkanen A. and Carmichael S. T. (1992) Anatomical organization of the primate amygdaloid complex. In The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction (ed. Aggleton J. P.), pp. 1-66. A John Wiley and Sons, New York. 2. Azuma S., Yamamoto T. and Kawamura Y. (1984) Studies on gustatory responses of amygdaloid neurons in rats. Expl Brain Res. 56, 12-22. 3. Ben-Ari Y. and Le Gal La Salle G. (1972) Plasticity at unitary level. II. Modifications during sensory-sensoryassociation procedures. Electroenceph. clin. Neurophysiol. 32, 667-679. 4. Ben-Ari Y. and Le Gal La Salle G. (1974) Lateral amygdala unit activity: II. Habituating and non-habituating neurons. Electroenceph. clin. Neurophysiol. 37, 463-372. 5. Ben-Ari Y., Le Gal La Salle G. and Champagnat J. C. (1974) Lateral amygdala unit activity: I. Relationship between spontaneous and evoked activity. Electroenceph. clin. Neurophysiol. 37, 449-461. 6. Bernard J.-F., Alden M. and Besson J.-M. (1993) The organization of the efferent projections from the pontine parabrachial area to the amygdaloid complex: A phaseolus vulgaris leucoagglutinin (PHA-L) study in the rat. J. comp. Neurol. 329, 201-229. 7. Bernard J. F., Huang G. F. and Besson J. M. (1990) Effect of noxious somesthetic stimulation on the activity of neurons of the nucleus centralis of the amygdala. Brain Res. 523, 347-350. 8. Bernard J. F., Huang G. F. and Besson J. M. (1992) Nucleus centralis of the amygdala and the globus pallidus ventralis: Electrophysiological evidence for an involvement in pain process. J. Neurophysiol. 68, 551 569. 9. Blanchard D. C. and Blanchard R. J. (1972) Innate and conditioned reactions to threat in rats with amygdaloid lesions. J. comp. physiol. Psychol. 81, 281-290. 10. Bordi F. and LeDoux J. (1992) Sensory tuning beyond the sensory system: an initial analysis of auditory responses properties of neurons in the lateral amygdaloid nucleus and overlying areas of the striatum. J. Neurosci. 12, 2493-2503. 11. Bordi F. and LeDoux J. E. (1993) Sensory-specific conditioned plasticity in lateral amygdala neurons. Soc. Neurosci. Abst. 19, 1227. 12. Bordi F., LeDoux J., Clugnet M. C. and Pavlides C. (1993) Single-unit activity in the lateral nucleus of the amygdala and overlying areas of the striatum in freely behaving rats: rates, discharge patterns, and responses to acoustic stimuli. Behav. Neurosci. 107, 757 769. 13, Buonviso N., Chaput M. A. and Berthommier F. (1992) Temporal pattern analyses in pairs of neighboring mitral cells. J. Neurophysiol. 68, 417-424. 14. Cador M., Robbins T. W. and Everitt B. J. (1989) Involvement of the amygdala in stimulus-reward associations: Interaction with the ventral striatum. Neuroscience. 30, 77-86. 15. Cain D. P. and Bindra D. (1972) Responses of amygdala single units to odors in the rat. Expl Neurol. 35, 98-110. 16. Chapman P. F., Kairiss E. W., Keenan, C. L. and Brown T. H. (1990) Long-term synaptic potentiation in the amygdala. Synapse 6, 271-278. 17. Clugnet M. C. and LeDoux J. E. (1990) Synaptic plasticity in fear conditioned circuits: Induction of LTP in the lateral nucleus of the amygdala by stimulation of the medial geniculate body. J. Neurosci. 10, 2828-2824. 18. Davis M. (1992) The role of the amygdala in conditioned fear. In The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction (ed. Aggleton J. P.), pp. 255-305. A John Wiley and Sons, New York. 19. Diamond D. M. and Weinberger N. M. (1989) Role of context in the expression of learning-induced plasticity of single neurons in auditory cortex. Behav. Neurosci. 103, 471-494. 20. Downer J. D. C. (1961) Changes in visual gnostic function and emotional behavior following unilateral temporal lobe damage in the "split-brain" monkey. Nature 191, 513~51. 21. Everitt B. J., Cador M. and Robbins T. W. (1989) Interactions between the amygdala and ventral striatum in stimulus-reward associations: Studies using second-order schedule of sexual reinforcement. Neuroscience 30, 63-75. 22. Everitt B. J., Morris K. A., O'Brien A. and Robbins T. W. (1991) The basolateral amygdala-ventral striatal system and conditioned place preference: further evidence of limbic-striatal interactions underlying reward-related processes. Neuroscience 42, 1-18. 23. Farb C., Aoki C., Milner T., Kaneko T. and LeDoux J. E. (1992) Glutamate immunoreactive terminals in the lateral amygdaloid nucleus: a possible substrate for emotional memory. Brain Res. 593, 145-158. 24. Friston K. J., Tononi G., Reeke G. N. Jr, Sporns O. and Edelman G. M. (1994) Value-dependent selection in the brain: stimulation in a synthetic neural model. Neuroscience 59, 229-243. 25. Fulwiler C. E. and Saper C. B. (1984) Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev. 7, 229-259. 26. Gallagher M., Graham P. W. and Holland P. C. (1990) The amygdala central nucleus and appetitive pavlovian conditioning: Lesions impair one class of conditioned behavior. J. Neurosci. 10, 190~1911. 27. Gentile C. G., Jarrel T. W., Teich A., McCabe P. M. and Schneiderman N. (1986) The role of amygdaloid central nucleus in the retention of differential pavlovian conditioning of bradycardia in rabbits. Behav. Brain Res. 20, 263-273. 28. Geschwind N. (1965) Disconnexion syndromes in animals and man. Brain 88, 237-294. 29. Gloor P. (1960) Amygdala. In Handbook of Physiology, Neurophysiology (ed. Field J.), pp. 1395 1420. American Physiological Society, Washington DC. 30. Grossman S. P. 0962) A Textbook o f Physiological Psychology. Wiley, New York. 31. Halsell C. B. (1992) Organization of parabrachial nucleus efferents to the thalamus and amygdala in the golden hamster. J. comp. Neurol. 317, 57 78. 32. Hawkins R. D., Clark G. A. and Kandel E. R. (1987) Cell biological studies of learning in simple vertebrate and invertebrate systems. In Handbook of Physiology. l: The Nervous System (ed. Mountcastle V. B.), Vol. 5, pp. 25-83. American Physiological Society, Bethesda. 33. Hitchcock J. M. and Davis M. (1986) Lesions of the amygdala, but not of the cerebellum or red nucleus, block conditioned fear as measured with the potentiated startle paradigm. Behav. Neurosci. 100, 11-22. 34. Hitchcock J. M. and Davis M. (1991) Efferent pathway of the amygdala involved in conditioned fear as measures with the fear-potentiated startle paradigm. Behav. Neurosci. 105, 82(~842.

360

T. Uwano et al.

35. Horel J. A. and Keating E. G. (1969) Partial Klfiver-Bucy syndrome produced by cortical disconnection. Brain Res. 16, 281 284. 36. Jacobs B. L. and McGinty D. J. (1972) Participation of the amygdala in complex stimulus recognition and behavioral inhibition: Evidence from unit studies. Brain Res. 36, 431436. 37. Johnston J. B. (1923) Further contributions to the study of the evolution of the forebrain. J. comp. Neurol. 35, 33748 I. 38. Kaada B. R. (1972) Stimulation and regional ablation of the amygdaloid complex with reference to functional representations. In The Neurobiology of the Amygdala. (ed. Elefthriou B. E.), pp. 205 281. Plenum Press, New York. 39. Kapp B. S., Pascoe J. P. and Bixler M. A. (1984) The amygdala: A neuroanatomical systems approach to its contributions to aversive conditioning. In The neuropsyehology of Memory (eds Butters N. and Squire L. R.), pp. 473488. Guilford Press, New York. 40. Kim M., Campeau S., Falls W. A. and Davis M. (1993) Infusion of the non-NMDA receptor antagonist CNQX into the amygdala blocks the expression of fear-potentiated startle. Behav. neural Biol. 59, 5-8. 41. Klfiver H. and Bucy P. C. (1939) Preliminary analysis of functions of the temporal lobes in monkeys. Archs. Neurol, Psyehiat. 42, 979 1000. 42. Kudo M., Glendenning K. K., Frost S. B. and Masterton R. B. (1986) Origin of mammalian thalamocortical projections. I. Telencephalic projection of the medial geniculate body in the opossum (Didelphis virginiana). J. comp. Neurol. 245, 176-197. 43. LeDoux J. E. (1987) Emotion. In Handbook of Physiology. l: The Nervous System (ed. Mountcastle V. B.), Vol. 5, pp. 419-459. American Physiological Society, Bethesda. 44. LeDoux J. E. 0992) Brain mechanisms of emotion and emotional learning. Curt. Opin. Neurobiol. 2, [91-197. 45. LeDoux J. E., Cicchetti P., Xagoraris A. and Romanski L. M. (1990) The lateral amygdaloid nucleus: sensory interface of the amygdala in fear conditioning. J. Neurosci. 10, 1062-1069. 46. LeDoux J. E., Farb C. R. and Romanski L. M. (1991) Overlapping projections to the amygdala and striatum from auditory processing areas of the thalamus and cortex. Neurosci. Lett. 134, 139 144. 47. LeDoux J. E., Farb C. and Ruggiero D. A. (1990) Topographic organization of neurons in the acoustic thalamus that project to the amygdala. J. Neurosci. 10, 1043-1054. 48. Le Gal La Salle G. and Ben-Ari Y. (1981) Unit activity in the amygdaloid complex: a review. In The Amygdaloid Complex (ed. Ben-Ari Y.), pp. 227-237. Elsevier/North-Holland Biomedical Press, New York. 49. Ma W. and Peschanski M. (1988) Spinal and trigeminal projections to the parabrachial nucleus in the rat: Electron-microscopic evidence of a spino-ponto-amygdalian somatosensory pathway. Somatosensory Res. 5, 247 257. 50. Maren S., Poremba A. and Gabriel M. (1991) Basolateral amygdaloid multi-unit neuronal correlates of discriminative avoidance learning in rabbits. Brain Res. 549, 311 316. 51. Mascagni F., McDonald A. J. and Coleman J. R. (1993) Corticoamygdaloid and corticocortical projections of the rat temporal cortex: A phaseolus vulgalis leucoagglutinin study. Neuroscience 57, 697715. 52. McDonald A. J. and Jackson T. R. (1987) Amygdaloid connections with posterior insular and temporal cortical areas in the rat. J. comp. Neurol. 262, 59 77. 53. Miserendino M. J. D., Sananes C. B., Melia K. R. and Davis M. (1990) Blocking of acquisition but not expression of conditioned fear-potentiated startle by NMDA antagonists in the amygdala, Nature 345, 716 718. 54. Mishkin M. and Aggleton J. (1981) Multiple functional contributions of the amygdala in the monkey. In The Amygdaloid Complex (ed. Ben-Ari Y.), pp. 409420. Elsevier/North-Holland Biomedical, Amsterdam. 55. Muramoto K., Ono T., Nishijo H., and Fukuda M. (1993) Rat amygdaloid neuron responses during auditory discrimination. Neuroscience 52, 621~536. 56. Nakamura K. and Ono T. (1986) Lateral hypothalamus neuron involvement in integration of natural and artificial rewards and cue signals. J. Neurophysiol. 55, 163-181. 57. Nishijo H. and Norgren R. (1990) Responses from parabrachial gustatory neurons in behaving rats. J. Neurophysiol. 63, 707 724. 58. Nishijo H. and Norgren R. (1991) Parabrachial gustatory neural activity during licking by rats. J. Neurophysiol. 66, 974~985. 59. Nishijo H., Ono T. and Nishino H. (1988) Single neuron responses in amygdala of alert monkey during complex sensory stimulation with affective significance. J. Neurosei. 8, 3570 3583. 60. Nishijo H., Ono T. and Nishino H. (1988) Topographic distribution of modality-specific amygdalar neurons in alert monkey. J. Neurosci. 8, 355(~3569. 61. Norgren R. (1976) Taste pathway to hypothalamus and amygdala. J. comp. Neurol. 166, 17 30. 62. Norgren R. (1990) Gustatory System. In The Human Nervous System (ed. Paxinos G.), pp. 845-861. Academic Press, San Diego. 63. O'Keefe J. and Bouma H. (1969) Complex sensory properties of certain amygdala units in the freely moving cat. Expl Nuerol. 23, 384-398. 64. Ono T., Nakamura K., Fukuda M. and Kobayashi T. (1992) Catecholamine and acetylcholine sensitivity of rat lateral hypothalamic neurons related to learning. J. Neurophysiol. 67, 265 279. 65. Pascoe J. P. and Kapp B. S. (1985) Electrophysiological characteristics of amygdaloid central nucleus neurons during Pavlovian fear conditioning in the rabbit. Behav. Brain Res. 16, 117 133. 66. Paxinos G. and Watson C. The Rat Brain in Stereotaxic Coordinations. 2nd ed., Academic Press, San Diego. 67. Phillips M. I. and Norgren R. E. (1970) A rapid method for permanent implantation of an intraoral fistula in rats. Behav. Res. Meth. Instrum. 2, 124. 68. Price J. L., Russchen F. T. and Amaral D, G. (1987) The limbic region. If: The amygdaloid complex. In Handbook of Chemical Neuroanatomy (eds Bj6rklund A. and H6kfelt T.), Vol. 5, pp. 279--388. Elsevier, Amsterdam. 68a. Quirk G. J., Tao P., Repa J. C. and LeDoux J. E. (1994) Simultaneously recorded neurons in the lateral amygdala of freely moving rats during fear conditioning. Soc. Neurosci. Abst. 20, 1007. 69. Richardson R. T. and Thompson R. F. (1984) Amygdaloid unit activity during classical conditioning of the nictating membrane response in rabbit. Physiol. Behav. 32, 527-539. 70. Romanski L. M., Clugnet M.-C., Bordi F. and LeDoux J. E. (1993) Somatosensory and auditory convergence in the lateral nucleus of the amygdala. Behav. Neurosci. 107, 444450.

Amygdalar neuron responses to conditioned sensory stimuli

361

71. Romanski L. M. and LeDoux J. E. (1992) Equipotentiality of thalamo-amygdala and thalamo-cortico-amygdala circuits in auditory fear conditioning. J. Neurosci. 12, 4501-4509. 72. Sawa M. and Delgado J. M. R. (1963) Amygdala unitary activity in the unrestrained cat. Electroenceph. clin. Neurophysiol. 15, 637-350. 73. Stefanacci L., Farb C. R., Pitkanen A., Go G., LeDoux J. E. and Amaral D. G. (1992) Projections from the lateral nucleus to the basal nucleus of the amygdala: a light and electron microscopic PHA-L study in the rat. J. comp. Neurol. 323, 5864501. 74. Switzer R. C., de Olmos J. and Heimer L. (1985) Olfactory system. In The Rat Nervous System (ed. Paxinos G.), pp. 1-36. Academic Press, Sydney. 75. Turner B. H. (1981) The cortical sequence and terminal distribution of sensory related afferents to the amygdaloid complex of the rat and monkey. In The AmygdaloidComplex (ed. Ben-Ari Y.), pp. 51~62. Elsevier/North-Holland, Amsterdam. 76. Turner B. H. and Herkenham M. (1991) Thalamoamygdaloid projections in the rat: a test of the amygdala's role in sensory processing. J. eomp. Neurol. 313, 295 325. 77. Turner B. H., Mishkin M. and Knapp M. (1980) Organization of the amygdalopetal projections from modality-specific cortical association areas in the monkey. J. comp. Neurol. 191, 515-543. 78. Turner B. H. and Zimmer J. (1984) The architecture and some of the interconnections of the rat's amygdala and lateral periallocortex. J. comp. Neurol. 227, 540-557. 79. Uwano T., Nishijo H., Ono T., Nakamura K. and Torii K. (1992) Amygdalar neuronal responses during cross-modal association task in awake rats. Soc. Neurosci. Abstr. 18, 518. 80. Weinberger N. M., Ashe J. H., Metherate R., McKenna T. M., Diamond D. M., Bakin J. S., Lennartz R. C. and Cassady J. M. (1990) Neural adaptive information processing: a preliminary model of receptive-field plasticity in auditory cortex during pavlovian conditioning. In Learning and computational neuroscience: Foundations of Adaptive Networks. (eds Gabriel M. and Moore J.), pp. 91-138. MIT Press, Cambridge. 81. Weiskrantz L. (1956) Behavioral changes associated with ablation of the amygdaloid complex in monkeys. J. comp. physiol. Psychol. 49, 381-391. (Accepted 2 March 1995)