Gustatory and Multimodal Neuronal Responses in the Amygdala During Licking and Discrimination of Sensory Stimuli in Awake Rats

Hisao Nishijo, Teruko Uwano, Ryoi Tamura, and Taketoshi Ono

Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Nishijo, Hisao, Teruko Uwano, Ryoi Tamura, and Taketoshi Ono. Gustatory and multimodal neuronal responses in the amygdala during licking and discrimination of sensory stimuli in awake rats. J. Neurophysiol. 79: 21-36, 1998. The amygdala (AM) receives information from various sensory modalities via the neocortex and directly from the thalamus and brain stem and plays an important role in ingestive behaviors. In the present study, neuronal activity was recorded in the AM and amygdalostriatal transition area of rats during discrimination of conditioned sensory stimuli and ingestion of sapid solutions. Of the 420 responsive neurons, 227 responded exclusively to one sensory modality, 120 responded to two or more modalities, and the remaining 73 could not be classified. Among the responsive neurons, 108 responded to oral-sensory stimulation (oral-sensory neurons). In detailed analyses of 84 of these oral-sensory neurons, 24 were classified as taste responsive and were located mainly in the central nucleus of the AM. The other 60 oral-sensory neurons were classified as nontaste oral-sensory neurons and were distributed widely throughout the AM. Both the taste and nontaste oral-sensory neurons also responded to other sensory stimuli. Of the 24 taste neurons, 21 were tested at least with four standard taste solutions. On the basis of the magnitudes of their responses to these sapid stimuli, the taste neurons were classified as follows: seven sucrose-best, four NaCl-best, three citric acid-best, and six quinine HCl-best. The remaining cell responded significantly only to lysine HCl and monosodium glutamate. Multivariate analyses of these 21 taste neurons suggested that, in the AM, taste quality was processed based on palatability. Taken with previous lesion studies, the present results suggest that the AM plays a role in the evaluation of taste palatability and in the association of taste stimuli with other sensory stimuli.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

In rodents, the amygdala (AM) is one of the major recipients of gustatory projections to forebrain (Norgren 1976, 1995). The nucleus of the solitary tract (NST) in the medulla receives taste information from peripheral taste nerves and sends gustatory information to the pontine parabrachial nucleus (PBN) (Hamilton and Norgren 1984; Norgren 1976, 1990). Gustatory neurons in the PBN (Nishijo and Norgren 1990, 1991, 1997; Norgren and Pfaffman 1975) send two parallel ascending paths, one to thalamo-cortical axis and the other directly to the AM (Halsell 1992). The cortical taste area (area 13 in the insula) also projects to the central and lateral nuclei of the AM (Turner and Zimmer 1984).

The AM receives not only gustatory information but also sensory information of all other modalities via the thalamus and sensory association cortices (Mascagni et al. 1993; McDonald and Jackson 1987; Turner and Herkenham 1991; Turner and Zimmer 1984; Turner et al. 1980). It has been suggested that the AM might function to correlate this multimodal sensory information (Geschwind 1965; Johnston 1923). Consistent with this suggestion, AM neurons respond to multimodal and unimodal stimuli in awake monkeys and rats (Muramoto et al. 1993; Nishijo et al. 1988a,b; Sanghera et al. 1979; Uwano et al. 1995). Other studies report AM taste neurons in rabbits (Schwartzbaum and Morse 1978), monkeys (Nishijo et al. 1988a,b; Scott et al. 1993), and rats (Azuma et al. 1984; Yasoshima et al. 1995). Sensory responsiveness of the taste neurons to other sensory modalities, however, has not been reported. With this in mind, neuronal activity was recorded in the AM and the amygdalostriatal transition area of unanesthetized rats during discrimination of various conditioned sensory stimuli associated with reward (sweet solution, intracranial self-stimulation) and aversion (tail pinch) in the present study.

AM taste responses in anesthetized rats have been reported previously (Azuma et al. 1984). Taste neuron responses of awake rats, however, might be different from those of anesthetized rats. Taste responses recorded from the PBN and NST of awake rats differ from those of anesthetized rats (Nakamura and Norgren 1991, 1993; Nishijo and Norgren 1990, 1991, 1997) in the following ways: 1) the spontaneous firing rates of the PBN taste neurons in awake rats were higher than those in anesthetized ones; 2) responses to water were more common in PBN and NST taste neurons in awake rats; 3) in awake rats, PBN taste neurons responded less to sour and bitter chemicals than in anesthetized rats, and in both the PBN and NST, taste neurons of awake rats responded more to sweet chemicals; and 4) taste neurons that responded only to one of four standard sapid chemicals were more common in awake rats. We report here responses of taste neurons in the AM of awake rats using same sapid stimuli that were used in the previous studies in the PBN (Nishijo and Norgren 1990, 1991, 1997) and the NST (Nakamura and Norgren 1991, 1993) of awake rats, and AM taste responses are compared with those in the PBN and NST of awake rats.

It has been reported that decerebration and electrical stimulation or inactivation of the forebrain modulated activity of taste neurons in the NST and PBN (Di Lorenzo 1990; Hayama et al. 1985; Mark et al. 1988; Matsuo et al. 1984; Murzi et al. 1986). It has been suggested that the descending projections from the gustatory cortex, hypothalamus, and the AM to brain stem taste areas (Krettek and Price 1978; Van der Kooy et al. 1984) are responsible for such modulation. Because the AM appears to play a pivotal role in the evaluation of the biological significance of sensory stimuli (Davis 1992, 1994; LeDoux 1987, 1992; Ono and Nishijo 1992; Ono et al. 1995; Rolls 1992), the AM might function as an evaluative filter for taste stimuli. This idea is consistent with the results of our previous neurophysiological studies in awake monkeys and rats (Muramoto et al. 1993; Nishijo et al. 1988a,b; Uwano et al. 1995), in which AM neuronal responses to conditioned visual, auditory, somatosensory, and olfactory stimuli were related to biological significance of the conditioned stimuli. The present study extends this analysis to the gustatory system, using both cued and noncued responses, to determine whether the AM also might play a role in evaluating the reward potential of sapid stimuli.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Subjects and experimental design

Twelve male albino Wistar rats, weighing 280-350 g (SLC, Hamamatsu, Japan), were housed individually in clean 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-dark cycle (on at 7:00, off at 19:00). A rat was placed painlessly in the special stereotaxic apparatus equipped with devices for sensory stimulation (Fig. 1A). Licking was signaled by a photoelectric sensor triggered by the tongue. The stereotaxic apparatus was covered by transparent plastic box with an electric fan in the anterior wall and one in the posterior wall to discharge odor-laden air from the enclosure through an exhaust pipe.


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FIG. 1. Schema of paradigm. A: rats were prepared for chronic recording by forming receptacles of dental cement to accept modified earbars. Electrodes were implanted in the lateral hypothalamic area (LHA) for intracranial self-stimulation (ICSS). Rat was trained to lick when the spout was placed automatically close to its mouth. Licking was signaled by a photoelectric sensor triggered by the tongue. B: conditioned stimulus (tone, light, air puff, or odorized air) was presented for 2.0 s before placing the spout close to the rat's mouth. Tone for licking to produce glucose or sucrose was 1,200 Hz; the tone for licking to produce ICSS was 4, 300 Hz. C: a 2,800-Hz tone signaled aversive stimulation (tail pinch) if the spout was not licked within 2 s after cue presentation in experiment I (exp-I).

In the first experiment (exp-I), four rats were trained to lick a spout that protruded close to its mouth to obtain reward [glucose solution or intracranial self-stimulation (ICSS); Fig. 1B, reward task] or to avoid tail pinch (Fig. 1C, avoidance task). A 2.0-s conditioned tone preceded protrusion of the spout. Tones of 1,200 Hz (tone 1) and 4,300 Hz (tone 2) signaled availability of 0.3 M glucose and ICSS (0.5-s train of 100 Hz, 0.3-ms capacitor-coupled negative square wave pulses), respectively. A 2,800-Hz tone (tone 3) signaled 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 (Ono et al. 1992).

After the rats had learned the tasks (see Training), AM neurons were recorded during performance of the tasks. First, data were acquired during the tone discrimination task, then responsiveness to other nonassociative sensory stimuli was tested as follows (noncontingent test): auditory stimulation such as clicks, computer-synthesized artificial sounds, hand clapping, or human vocalization (unconditioned sounds); 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 vocalization, and hand clapping were introduced because previous results indicated that those sounds were sometimes effective in evoking neuronal responses (Jacobs and McGinty 1972; Nishijo et al. 1988a,b). 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 was estimated to range between 70 and 90 dB. Intraoral infusions of water and sapid stimuli (see further) were not tested in exp-I.

In the second experiment (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 (2,800-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) also were 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 tested further with nine odorants associated with ICSS: five volatile organic compounds consisting of acetophenone, isoamyl acetate (3-methylbutyl acetate), cyclohexanone, p-cymene (p-isopropyl toluene), and 1,8-cineole (Eucalyptol) (Wako Pure Chemical Industry, Saitama, Japan); two food-related essences, cheese and black pepper; and two cosmetic-related essences rose and perfume (Chanel No. 5) (Takasago, Kanagawa, Japan). The first five odorants were similar to those used in a previous study of rats (Buonviso et al. 1992). Each odorant, at saturated vapor pressure, was presented through a polyethylene tube as a conditioned stimulus for 2 or 4 s. Air in the enclosure was exchanged continuously by two fans, and trials were separated by intervals of >= 30 s.

Noncontingent 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 (23°C). Sapid stimuli consisted of four standard solutions (in M): 0.1 NaCl, 0.3 sucrose, 0.01 citric acid, 0.0003 quinine HCl (QHCl), and two other taste stimuli: [0.1 monosodium glutamate (MSG) and 0.2 lysine HCl]. The concentrations of four standard sapid solutions were identical to those used in previous studies of awake rats (Nishijo and Norgren 1990, 1991, 1997). For testing taste, each trial 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 in the previous studies and, in the PBN, evoked taste responses quite similar to those produced by natural licking (Nishijo and Norgren 1990, 1991).

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 head restraint system was similar to that used by Nishijo and Norgren (1990, 1991, 1997),as modified from Nakamura and Ono (1986). After being anesthetized (pentobarbital sodium, 40 mg/kg ip), the rat was mounted in a stereotaxic apparatus with its skull level between the bregma and lambda suture points. The cranium was exposed, 2-3 mm of the temporal end of the temporal muscle was removed bilaterally, and 8-10 small, sterile, stainless screws then were 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 concentric 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 (1986). The concentric bipolar electrode was a varnished, 0.3-mm-diam stainless steel tube with an inner 0.1-mm-diam enameled wire. Electrodes were insulated except for 0.1 mm of the outer pole and 0.3 mm of the inner pole. 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), that were attached rigidly to the earbars of the stereotaxic instrument and served the same purpose as earbars in the recording session. 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-µm diam) were inserted into the genioglossus muscle of the rats used in exp-II to monitor tongue movements. Two intraoral cannulae were implanted by a techniquedescribed by Phillips and Norgren (1970). The polyethylene cannulae (SP-65, Natsume) were inserted just rostral and lateral to the first maxillary molar on each side. The electromyographic (EMG) wires and intraoral cannulae were brought out subcutaneously to the skull and anchored to the cranioplastic cap. After surgery, an antibiotic [gentamicine sulfate, (Gentacin Injection) Schering-Plough, Osaka, Japan] was administered topically and systemically (5 mg im).

After recovery from surgery (10-14 days) and training (2 wk; see next section), rats were reanesthetized (pentobarbital, 40 mg/kg ip) and mounted in the fake earbars. A hole (3-5 mm diam) for chronic recording was drilled through the cranioplastic and the underlying skull (A, -2.0 to -4.0 from bregma; L, 3.0-6.0). The exposed dura was excised and the exposed brain was covered with hydrocortisone ointment (Rinderon-VG; Shionogi, Tokyo) or one or two drops of chloramphenicol solution (0.1 g/ml; Chloromycetin Succinate, Sankyo, Tokyo, Japan). The hole was covered with a sterile Teflon sheet and sealed with an epoxy glue. A second small hole (1.5-mm diam) then was drilled just medial to the hole for recording. A stainless steel wire (130-µm diam), insulated except at the cross section of the tip, was implanted near the medial end of the central nucleus of the AM through the hole to serve as an indifferent electrode. This hole then was filled with cranioplastic acrylic. After the animal recovered (5-7 days), training was reinstituted.

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 a22-h water-deprivation regimen. While in the enclosure (1-2 h daily), they had access to a spout from which they initially learned to take 0.3 M sucrose and, within 1-2 days, other fluids. Subsequently, their heads were fixed rigidly and painlessly by inserting the fake earbars into the impressions in the cranioplastic cap. While restrained, the rats were trained to lick a spout, which automatically was protruded close to their mouths for 2 s, to obtain glucose or sucrose solutions and an ICSS reward. The threshold level for ICSS to maintain licking behavior in the reward task was determined based on the behavioral observation, and any rat for which the threshold exceeded 300 µA was excluded. Rats also were trained to consume distilled water and sucrose solution applied via the intraoral cannulae. The rats then were trained to discriminate between conditioned auditory stimuli to obtain the rewards. Next, the rats were trained to lick a spout, which automatically was 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 then were 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.

Electrophysiological recording

An individual rat usually was 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 MOmega at 1,000 Hz) was inserted stereotaxically stepwise with a pulse motor-driven manipulator (SM-20, Narishige) into various parts of the AM and amygdalostriatal transition area. Extracellular neuronal and EMG activities were passed through a dual channel differential amplifier with a preamplifier (MEG-2100, Nihon Kohden), monitored on an oscilloscope, and recorded on magnetic tape (DFR-3715, Sony Magnescale). Onset of somatosensory stimulation in the noncontingent test and intraoral infusion of sapid solution were noted on magnetic tape by voice commentary. Neuronal activity was counted by a two-level voltage discriminator. The analog 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 (PC-98 Bp, NEC) 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.

Data analysis of responses during conditioned tasks and other stimuli

Both neuronal and behavioral 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 4 s), and a rewarding or aversive stimulation period (2 s). Neuronal activities were compared by one way analysis of variance (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) also were 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 (Nishijo et al. 1988a,b; Richardson and Thompson 1984). Significant responses during ingestion of sweet solutions in the reward task (i.e., reinforcement period) in analysis I were defined as at least oral sensory (taste, nontaste, or unknown; see further for details). Any significant responses to olfactory stimuli in analysis I were defined as olfactory when the neuronal response to somatosensory stimulus (air puff) was not significant in analysis I. Responses to olfactory stimuli, when the neuronal response to somatosensory stimulus (air puff) was also significant in analysis I, were defined as olfactory only if there was significant difference in analysis III. The significance level was P < 0.05. Responses of the AM neurons with or without oral-sensory responses in the conditioned tasks were further analyzed in terms of the noncontingent tests (see further text for details).

Data analysis of responses to unconditioned somatosensory and auditory stimuli in the noncontingent test

For neuronal responses to tactile stimulation with a cotton tipped probe or an air puff in the noncontingent test, all data analyses were based on neuronal activity in 1.0-s samples after onset of stimulation. Responses to tactile stimulation were considered to be significant if the neuronal activity increased or decreased >= 2.0 SD from the mean of the spontaneous activity. Neuronal responses to auditory stimulation in the noncontingent test were analyzed similarly.

Data analysis of responses to intraoral infusion of water and sapid stimuli in the noncontingent test

Only neuronal responses to intraoral infusion of water and sapid stimuli in the noncontingent test of the exp-II were used. The statistical methods used for this section were essentially identical to those in previously published papers (Nakamura and Norgren 1991, 1993; Nishijo and Norgren 1990, 1991, 1997). For taste responses to intraoral infusions, all data analyses were based on the neuronal activity in 5.0-s samples after the onset of the infusion. When more than one sample was available for a particular taste stimulus, the mean was used because the correlations between the response profiles to sapid chemicals for multiples trials were high. Spontaneous activity and responses to prestimulus water were calculated from multiple samples. Water and stimulus responses were calculated during the 5.0 s, 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 5.0-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 >= 2.0 SD from the mean of the prestimulus water response. Similarly, a water response was considered to be significant if the neuronal activity increased or decreased >= 2.0 SD from the mean of the spontaneous activity of the neuron. In this noncontingent test, neurons that responded significantly to an infusion of either water or taste stimuli were defined as at least oral sensory.

To compare these data with earlier samples, two forms of analysis used only responses to the four standard stimuli that were common to all five studies (Nakamura and Norgren 1991, 1993; Nishijo and Norgren 1990, 1991, 1997). First, each neuron was categorized on the basis of its best stimulus (i.e., the chemical that produced the greatest number of spikes in 5.0 s), its second-best stimulus, and any effective stimuli (i.e., the chemical that produced a significant response using our criterion). Second, for each neuron, the breadth of responsiveness was calculated from the formula for entropy based on the excitatory component of the activity generated by each of the four standard chemicals (i.e., NaCl, sucrose, citric acid, and quinine HCl) (Smith and Travers 1979). The entropy measure (H) of each neuron was given by the following formula
<IT>H</IT> = −1.661 <LIM><OP>∑</OP><LL><SUB><IT>i=</IT>1</SUB></LL><UL>4</UL></LIM>pi(log pi)
where pi is the relative response to each of the four standard taste stimuli. Because this formula cannot accept negative numbers, the absolute value (Pritchard et al. 1989; Travers and Smith 1979) or dominant component, which corresponded to excitatory component (Van Buskirk and Smith 1981) when responses were all excitatory, of each corrected response was used. For calculation of entropy values using the dominant components, excitatory components were used for the neurons in which excitatory responses were dominant, and absolute values of inhibitory components, for the neurons in which inhibitory responses were dominant.

The neural data also were treated with several multivariate analyses that used the responses to all six sapid stimuli. The cluster analysis used Pearson's product-moment correlation coefficients and the average linkage method (SYSTAT statistical package). The multidimensional scaling (MDS) employed the metric ratio and Euclidean model (SYSTAT statistical package, Guttman scaling method). In the MDS, the data were normalized so that the maximum response of each neuron to the stimuli was the same (Erickson et al. 1993). The dissimilarity (Euclidean distance) between each possible pair of chemicals or neurons was calculated and analyzed using the MDS program. The statistical criteria, categories, and numerical analyses were identical to those used in the two previous studies except that Euclidean distance was used instead of correlation coefficient in the MDS (Nishijo and Norgren 1990, 1991) and all identical to those used in the most recent studies (Nishijo and Norgren 1997).

Classification of oral-sensory neurons

Oral-sensory neurons were defined from responses during either the reward task (exp-I and -II) or the noncontingent test with intraoral infusions (exp-II). Oral-sensory neurons were categorized further into taste, nontaste, or unknown oral-sensory neurons based on their responses to intraoral infusion of water and sapid solutions. The AM oral-sensory neurons were defined as taste responsive (taste oral sensory) only if they significantly responded more to infusion of a sapid solution than to water in exp-II. The remaining AM oral-sensory neurons in exp-II were defined as nontaste oral sensory because infusions of sapid stimuli did not evoke responses significantly different from those of water. Therefore all oral-sensory neurons in exp-II were categorized into either taste or nontaste oral-sensory neurons. In exp-I, all except one oral-sensory neurons were categorized into unknown oral sensory because the AM neurons were not tested with infusions of water and sapid stimuli.

Histology

After the last recording session, a rat was reanesthetized with pentobarbital sodium (50 mg/kg ip), and several small electrolytic lesions (20 µA for 20 s) were made stereotaxically around the recorded sites with a glass-insulated tungsten microelectrode. Rats then were given a further overdose of anesthetic and perfused transcardially with heparinized 0.9% saline followed by 10% buffered formalin. The brain was removed and cut into 30-µm frontal sections with a freezing microtome. Sections were stained with cresyl violet. All marking and stimulation sites then were verified carefully microscopically. Positions of neurons were located stereotaxically on the real tissue sections, and then compared with the atlas of Paxinos and Watson (1986).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

General responsiveness

During periods of 1-3 mo, depending on the rat, recordings were made from 1,039 neurons (exp-I, 388; exp-II, 651) in the AM and amygdalostriatal transition area. Analysis of EMGs in exp-II indicated that no motor-related neuronal responses were found in the AM. Although some neurons responded during licking and ingestion of glucose or sucrose solution in the conditioned tasks and during intraoral infusion of water and/or sapid solutions, their activity did not rise when EMG activity increased in the intertrial interval (see also Fig. 4). The absence of motor-related neuronal responses in the AM in the present study is consistent with the results in our previous neurophysiological studies in rats (Uwano et al. 1995) and monkeys (Nishijo et al. 1988a,b).


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FIG. 4. Raw records of a taste neuron during intraoral infusions. A-E: raw records of neuronal spikes (a) and raw records of electromyograms (EMGs) from genioglossus muscle (b). Each arrow, infusion onset.

Each neuron was tested with stimulation of different modalities, i.e., auditory stimulation with a pure tone and a click, visual stimulation with a light, oral-sensory stimulation with a taste solutions, and somatosensory stimulation with an air puff and a cotton tipped probe. In three of the eight rats, 196 neurons in exp-II were further tested with nine odorants. Of the 1,039 neurons, 420 (40.4%) [exp-I, 145 (37.4%); exp-II, 275 (42.2%)] responded to one or more sensory stimuli. Of the 420 responsive neurons, 227 responded exclusively to one sensory modality [75, auditory (Aud); 5, visual (Vis); 62, oral sensory (OS); 75, somatosensory (SS); 10, olfactory], 120 responded to various combinations of the stimuli, and the remaining 73 could not be classified. Figure 2 shows an example of an AM neuron that responded to conditioned and unconditioned stimuli during the conditioned task in exp-I. The neuron responded to both tone 1 associated with glucose solution and glucose solution itself (Fig. 2A) but did not respond to other conditioned tones and the unconditioned stimulus (ICSS, tail pinch; Fig. 2, B and C). The neuron also failed to respond to other stimuli used in the noncontingent tests---unconditioned sounds, light flash, air puff, and stroking with a cotton-tipped probe (not shown).


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FIG. 2. An example of the amygdala (AM) neurons that responded to the conditioned tone and glucose solution during the conditioned tasks. A-C: raster displays and summed histograms of the neuronal responses to tone 1 (1,200 Hz) predicting glucose solution (A), tone 2 (4,300 Hz) predicting ICSS (B), and tone 3 (2,800 Hz) predicting tail pinch (C). Each dot below a raster line indicates one lick, each of top histograms shows summed neuronal responses, and the bottom histograms show summed licks. Open and hatched rectangles at top indicate duration of conditioned stimulus and time of reinforcement, respectively. Tri, number of trials; Glu, glucose solution. Time scale: seconds; conditioned stimulus onset at time 0; minus is pretrial control. Each histogram bin, 100 ms.

Of these 420 cells, 108 responded to oral-sensory stimulations (62, OS; 16, OS + Aud; 19, OS + SS; 4, OS +Aud + SS; 1, OS + SS + Vis; 6, OS + Aud + SS + Vis). Of these 108 oral-sensory neurons, 84 could be further classified as taste and nontaste oral-sensory based on the data from intraoral infusions. Twenty-four cells were classified as taste sensitive, because they responded more strongly to gustatory stimuli than to water, and 60 neurons as nontaste oral-sensory neurons (Table 1). Of the 84 oral-sensory neurons tested with intraoral infusions, 11 (8, nontaste oral sensory; 3, taste oral sensory) responded to conditioned tones, and 5 (4, nontaste oral sensory; 1, taste oral sensory) responded to unconditioned sounds.

 
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TABLE 1. Classification of 108 oral-sensory neurons in terms of sensory responsiveness to other modalities

Classification and response profiles of nontaste oral-sensory neurons

Of the 60 oral-sensory neurons identified as nontaste, 29 (25, excited; 4, inhibited; 48.3%, 29/60) responded exclusively during licking of sucrose solution in the reward task and/or during licking after intraoral infusion of a sapid solution and 31 (22, excited; 9, inhibited; 51.7%, 31/60) responded also to other sensory stimuli.

Figure 3 shows an example of a multimodal nontaste, oral-sensory neuron. In the reward task, the neuron did not respond during licking of a spout to obtain 0.3 M sucrose solution (Fig. 3A, Suc licking). Compared with the spontaneous rate, the neuron did respond significantly to infusions of water and all four taste solutions (Fig. 3A). The responses to four taste solutions did not differ significantly from those to water and, thus, this cell was classified as nontaste, oral-sensory neurons. The neuron failed to respond at all during active licking required for the conditioning trials, but it did respond to a tactile stimulation applied to an area around the mouth and nose (Fig. 3B). The neuron did not respond to unconditioned auditory stimuli such as clicks (not shown). Based on this pattern of activity, this neuron was categorized as a nontaste oral-sensory neuron with somatosensory responsiveness (OS + SS).


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FIG. 3. An example of a nontaste oral-sensory AM neuron. A: response profile of an AM neuron for 50-µl intraoral infusions of sapid solutions [0.1 M NaCl, 0.3 M sucrose (Suc), 0.03 M citric acid (CA), 0.0003 M quinine hydrochloride (QHCl), 0.1 M monosodium glutamate (MSG), and 0.2 M lysine hydrochloride (Lys)] and for licking of sucrose solution from a spout in the reward task (Suc licking). Neuronal spikes were counted for 5.0 s after the infusion of a liquid. Hatched columns, significant response (deviated >2.0 SD from the spontaneous firing rate); open columns, nonsignificant taste response (<2.0 SD corresponding water responses). Error bar indicates SE of mean firing rate. B: neuronal activity during tactile stimulation. Each column indicates mean firing rate for 1 s after tactile stimulation. Hatched columns, deviated >= SD from the spontaneous firing rate; dotted area, receptive field. This neuron responded to a tactile stimulus around a mouth and a nose.

Classification and response profiles of taste neurons

Of the 24 taste neurons, 17 (all excited; 70.8%) responded only to oral-sensory stimuli including taste, 7 (6, excitatory; 1, inhibitory; 29.2%) also responded to other sensory stimuli. The spontaneous firing rates of 24 taste neurons ranged from 0.23 to 17.24 spikes/s (4.83 ± 1.14; mean ± SE). Raw records of a unimodal taste neuron responding during intraoral infusions appears in Fig. 4. The neuron responded briskly to 0.0003 M QHCl (Fig. 4E) but not to water (Fig. 4A), 0.3 M sucrose (Fig. 4B), 0.1 M NaCl (Fig. 4C), nor to 0.01 M citric acid (Fig. 4D). Tongue muscle EMG activity did not obviously correlate with the ongoing neuronal discharge. The response profile of this neuron during intraoral infusions is depicted in Fig. 5A. The neuron responded selectively to QHCl. Neuronal activity of the same neuron during the reward task is illustrated in Fig. 5B. The neuron did not respond during conditioned trials using auditory (Fig. 5Ba), somatosensory (Fig. 5Bb), and visual (Fig. 5Bc) stimuli nor during licking a spout to obtain 0.3 M sucrose. Furthermore, this neuron did not respond to other sensory stimuli, such as clicks or tactile stimuli by a cotton tipped probe (not shown). Thus the neuron responded only to QHCl infused through an intraoral cannula.


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FIG. 5. Neuron that responded exclusively to taste oral-sensory stimulation (gustation) depicted in Fig. 4. A: response profile of an AM neuron for 50-µl intraoral infusions of sapid solutions [0.1 M NaCl, 0.3 M sucrose (Suc), 0.03 M citric acid (CA), 0.0003 M quinine hydrochloride (QHCl), 0.1 M monosodium glutamate (MSG), and 0.2 M lysine hydrochloride (Lys)] and for licking of sucrose solution from a spout in the conditioned task (Suc licking). , deviated >2.0 SD from responses to water. Neuron significantly responded only to QHCl. B: neuronal activity during the reward task. This neuron did not respond to conditioned stimuli (a, tone 1; b, air puff; c, light) or to licking for sucrose solution. Other descriptions as for Fig. 2.

Figure 6 provides an example of another taste neuron type that responded broadly to sapid chemicals. This neuron did not respond to any conditioned sensory stimuli in the reward task but responded during licking of sucrose solution (not shown). The neuron also responded to infusions of water and sapid solutions including sucrose (Fig. 6Aa), NaCl (Fig. 6Ab), citric acid (Fig. 6Ac), and QHCl (Fig. 6Ad). Response profile of the neuron is summarized in Fig. 6B. Compared with its spontaneous activity, the neuron responded significantly to water; compared with water, it responded to all the sapid stimuli save NaCl. Thus the neuron responded significantly to sucrose, citric acid, QHCl, MSG, and lysine HCl when compared with water infusion.


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FIG. 6. Activity of taste-related neuron with broad response spectrum. A, a-d: peristimulus histograms of intraoral infusion of each of 4 standard sapid stimuli. black-down-triangle , onset of each infusion. Each histogram bin, 100 ms. B: response profiles for the 4 standard sapid stimuli, MSG, and Lys. , deviated >2.0 SD from the spontaneous firing rate; , deviated >2.0 SD from responses to water. Other descriptions as for Fig. 2.

Of the 24 taste oral-sensory neurons, 21 were tested at least with four standard taste solutions. Based on the magnitudes of their responses to these four standard sapid stimuli, the taste neurons were classified as follows: 4 NaCl-best, 7 sucrose-best, 3 citric acid-best, and 6 QHCl-best. The remaining neuron responded significantly only to lysine HCl and MSG. In Fig. 7, response profiles are arranged in descending order, beginning with the NaCl-best neurons on the left followed by the sucrose-best cells, citric acid-best cells, QHCl-best cells, and the lone lysine HCl-best cell. The sapid chemicals elicited excitatory responses in most neurons. Two sucrose-best cells and the lysine HCl-best neuron were inhibitory. Of these 21 taste neurons, 5 responded to only one sapid stimulus each and 4 responded significantly to water. The mean response profiles of the four best-stimulus categories and all taste neurons are shown in Fig. 8. Based on the overall mean response profile, the order of effectiveness of the taste solutions was QHCl > citric acid > sucrose > NaCl.


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FIG. 7. Response profile of 21 AM taste neurons to 4 standard sapid stimuli, and other stimuli. Taste neurons grouped into best-stimulus categories and arranged within those categories in descending order of response magnitude to the best stimulus. Taste responses were adjusted for water responses and responses to water for spontaneous firing rates. Bottom: spontaneous discharge rates with unit number. black-square, significant responses: taste responses, 2.0 SD more than corresponding water responses; water responses, 2.0 SD more above spontaneous rate. Bars above the zero line indicate excitatory responses; bars below, inhibitory ones; * not tested with a given stimulus. Suc, sucrose; CA, citric acid; QHCl, quinine HCl; MSG, monosodium glutamate; Lys, lysine HCl; SP, spontaneous firing rate.


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FIG. 8. Mean response profiles of taste neurons by best-stimulus category. A: NaCl-best neurons; B: sucrose-best neurons; C: citric acid-best neurons; D: QHCl-best neurons; E: mean response profile of all taste neurons. Other descriptions as for Fig. 7.

Each neuron was further categorized on the basis of its second-best stimulus and any effective stimuli (i.e., the chemical that produced a significant response using our criterion) (Table 2).

 
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TABLE 2. Breadth of responsiveness (entropy) for each categories of taste neurons

Breadth of responsiveness

The relationship of response category to breadth of response can be expressed numerically in terms of entropy (Smith and Travers 1979). The entropy measure was calculated from both the absolute values and the dominant components of the taste responses to four standard sapid stimuli (Table 2 and Fig. 9). Because excitatory responses were dominant in most taste neurons whereas inhibitory responses were dominant in three taste neurons (i.e., units 9, 11, and 21 in Fig. 7), we calculated entropy values using dominant, but not excitatory, components of taste responses of all taste neurons. To calculate entropy measure using dominant components of taste neurons, excitatory components of taste responses were used for the neurons in which excitatory responses were dominant, whereas absolute values of inhibitory components of taste responses were used in the neurons in which inhibitory responses were dominant. These two values calculated with absolute and dominant components of taste responses are given for comparison with other reports. Entropy values using the dominant components correspond to those using excitatory components of the taste responses in the PBN and NST because only excitatory components of the responses reached significance in the PBN (Nishijo and Norgren 1990, 1991) and NST (Nakamura and Norgren 1991, 1993).


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FIG. 9. Breath of responsiveness (entropy) of 21 AM taste neurons. A: distribution of entropy measures calculated from absolute value of (corrected) taste responses. B: entropy values based on the dominant component of taste responses only.

The entropy measures of most neurons ranged from 0.6 to 1.0. The mean entropies for dominant and absolute components of all neuronal responses were 0.742 ± 0.038 and 0.812 ± 0.031, respectively. The mean entropies for dominant and absolute components for specific neurons were 0.740 ± 0.039 and 0.803 ± 0.031, respectively. There were no significant differences in entropy measures among four best-stimulus categories in both dominant [1-way ANOVA: F(3,16) = 0.054, P > 0.05] and absolute [1-way ANOVA: F(3,16) = 0.316, P > 0.05] values.

Interneuron relationships of the taste neurons

The results of a cluster analysis derived from Pearson's product-moment correlations with the use of an average linkage method are illustrated in Fig. 10. The unit numbers and their response categories are listed on the right. In the dendrogram, the level at which two neurons, or two clusters of neurons, join together indicated their shared correlation coefficient. Thus the similarity indicated by 1 on the abscissa means an identical response profile. Smaller values of cluster similarity indicate more dissimilar relationships between neurons or groups of neurons. The relationship between number of clusters and cluster similarity during amalgamation process is illustrated in Fig. 10, inset. The point indicated by an arrow, where a sudden decrease in similarity occurs when amalgamation process proceeds (Bieber and Smith 1986), provided the basis for the six clusters in the present study. Of these six clusters, four are comparable to four best-stimulus categories: NaCl (N), sucrose (S), citric acid (C), and QHCl (Q) (Fig. 10). The remaining two clusters contains the one lysine HCl-best (L) and a QHCl-best (unit 18) neuron. This lone QHCl-best neuron was merged into a QHCl-best category.


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FIG. 10. Dendrogram of 21 AM taste neurons resulting from a hierarchical cluster analysis by the use of Pearson's product-moment correlation coefficients and the average linkage method. Right: each neuron's number and response categories. Abscissa: cluster similarity between neurons or cluster. Inset: relationships between number of clusters and cluster similarity in the amalgamation process. Elbow in the curve (right-arrow) gives estimated number of clusters (Bieber and Smith 1986).

The interneuron relationships of 21 taste neurons also were analyzed by multidimensional scaling (Fig. 11). Euclidean distances between each possible pair of neurons were used for multidimensional scaling. The Guttman's coefficients of alienation, which represents goodness of fit, were 0.384, 0.113, 0.066, and 0.044, respectively, for dimensions 1-4. Based on the Guttman's coefficients of alienation, two-dimensional space was chosen (Bieber and Smith 1986; Smith et al. 1983a,b). Neurons in each of the standard best-stimulus categories lie close to one another near zero on both dimensions. Despite this common ground, the best-stimulus categories distribute on different aspects of the two dimensions. Thus the sucrose-best cells separate on the minus half of dimension 1, the NaCl-best neurons, on the minus half of dimension 2, and the QHCl-best neurons, on the plus half of dimension 2. 


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FIG. 11. Distribution of 21 AM neurons in a 2-D space resulting from multidimensional scaling using Euclidean distance. Each number beside each symbol corresponds to each unit number indicated in Fig. 7. black-square, black-diamond , and bullet , specific neurons.

Interstimulus relationships

Table 3 shows Pearson's correlation coefficients between sapid chemicals. Sucrose was correlated with NaCl (gamma  = 0.440) most among the four basic chemicals and least with QHCl (gamma  = 0.138). NaCl was most correlated with citric acid (gamma  = 0.672) and less with sucrose (gamma  = 0.440) and QHCl (gamma  = 0.573). Citric acid was most correlated with QHCl (gamma  = 0.905) and less with NaCl (gamma  = 0.672). QHCl was most correlated with citric acid (gamma  = 0.905) and least with sucrose (gamma  = 0.138). This pattern of interstimulus correlation coefficients suggest that taste quality is organized based on palatability; taste stimuli could be arranged in one dimension in that sucrose (most palatable), NaCl, citric acid, and QHCl (least palatable) are plotted sequentially in an one-dimensional line. If this one-dimensional arrangement of taste chemicals is true, pairs of neighboring chemicals were correlated highly as indicated in Table 3. The remaining two chemicals (i.e., MSG and lysine HCl) were correlated highly with NaCl and citric acid (gamma  > 0.800), which are less palatable than sucrose and less aversive than QHCl.

 
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TABLE 3. Across-neuron correlation coefficients between stimulus pairs

The interstimulus relationships also were examined by multidimensional scaling using Euclidean distances between each possible pair of sapid chemicals (Fig. 12). The Guttman's coefficients of alienation (goodness of fit) for the first four dimensions were 0.0016, 0.0038, 0.0004, and 0.0000, respectively. Although the four standard taste solutions spread apart in three-dimensional space, the distance between sucrose and QHCl was longest. Lysine HCl was located at a position close to citric acid, MSG, and NaCl. These relationships were largely consistent with those indicated by correlation analysis noted above. In fact, if one dimension was used, these chemicals were arranged as described in the correlational analysis (not shown).


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FIG. 12. Distribution of sapid chemicals in a 3-D space resulting from multidimensional scaling using Euclidean distance of 21 AM taste neurons. Suc, sucrose; CA, citric acid; QHCl, quinine HCl; MSG, monosodium glutamate; Lys, lysine HCl.

Localization

Distributions of oral-sensory neurons are shown in Fig. 13. Oral-sensory neurons were confined primarily to the AM. There was some tendency for these cells to concentrate in the central nucleus of the AM and the amygdalostriatal transition zone immediately dorsal to it. This was most apparent for the taste neurons that responded only to sapid stimuli (unimodal taste oral-sensory neurons: open circle  in Fig. 13B). Eleven of the 17 such cells (65%) were located in or adjacent to the anterodorsal quadrant of the central nucleus. The nontaste oral-sensory neurons were located widely in the AM (Fig. 13A). On the other hand, multimodal nontaste and taste neurons (bullet , black-triangle, black-square, black-down-triangle , and black-diamond ) were located mainly in the basolateral and central nuclei of the AM and a few neurons in other nuclei of the AM and the amygdalostriatal transition area (Fig. 13, A and B). It should be noted that taste neurons in four best-stimulus categories intermingled, and there were no topographic distributions of those neurons in the AM in terms of best-stimulus category. Other oral-sensory neurons, the taste responsiveness of which were not determined, also were located in the central nucleus of the AM (Fig. 13C).


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FIG. 13. Distributions of neurons that responded to nontaste (A), taste (B), and unknown (C) oral-sensory neurons. Frontal sections, based on the atlas of Paxinos and Watson (1986), are arranged rostrocaudally top left to bottom right. Each value below each section indicates distance (mm) from the bregma. ASt, amygdalostriatal transition area; L, lateral nucleus; Bl, basolateral nucleus; Bm, basomedial nucleus; Ce, central nucleus; Me, medial nucleus; Co, cortical nucleus; AHA, amygdalo-hippocampal transition area; shaded regions, lateral ventricle.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrated unimodal and multimodal sensory responsiveness of AM oral-sensory neurons in awake behaving rats. These responses are compared with those in the brain stem and cortical taste areas of anesthetized and awake rats using various neurophysiological and multivariate analyses. The comparison characterized the responsiveness of AM oral-sensory neurons as follows: low response rate to sapid stimuli, relatively strong responses to QHCl, broad tuning to basic sapid stimuli, taste coding based on palatability, and multimodal responses to sensory stimuli other than gustatory stimuli. These characteristics are discussed in relation to a role of the AM in evaluation of food-related stimuli.

General characteristics of taste neurons

The mean spontaneous firing rate of the AM taste neurons was 4.83 spikes/s. The spontaneous firing rates of AM taste neurons in anesthetized animals have not been reported (e.g., Azuma et al. 1984). This firing rate was lower than those of taste neurons in the PBN of awake rats (10.8-13.4 spikes/s) (Nishijo and Norgren 1990, 1991, 1997), in the AM of awake monkeys (8.2 spikes/s) (Scott et al. 1993), and in the thalamus of awake monkeys (9.4 spikes/s) (Pritchard et al. 1989) and was comparable with those of taste neurons in the NST (4.1 spikes/s) (Nakamura and Norgren 1991) and the cortical taste area (4.4-5.6 spikes/s) (Yamamoto et al. 1989) of awake rats.

In the present study, water elicited significant responses in 19.0% (4/21) of the taste neurons. "Water" responses were common when taste neurons were recorded from awake rats (Nakamura and Norgren 1991, 1993; Nishijo and Norgren 1990, 1991, 1997; Nishijo et al. 1991). Water responses may arise from tactile stimulation produced during tongue movements and from thermal stimulation produced by room temperature solutions (Nishijo and Norgren 1990). In anesthetized rats, AM taste neurons consistently responded to tactile and thermal stimuli of the tongue (Azuma et al. 1984).

Taste responsiveness of AM taste neurons

In the present study, the response magnitude of AM taste neurons to sapid stimuli was relatively low; except for QHCl, responses to best stimuli were <10 spikes/s. Quinine-best neurons responded to QHCl at 15.7 ± 6.1 spikes/s. This low response rate in the AM contrasts with the high rates of brain stem taste neurons of awake rats (Nakamura and Norgren 1991, 1993; Nishijo and Norgren 1990, 1991, 1997; Nishijo et al. 1991), decerebrated rats (Di Lorenzo 1990), and anesthetized rats and hamsters (Ganchrow and Erickson 1970; Smith et al. 1983a,b; Woolston and Erickson 1979). It is similar to those of cortical taste neurons in anesthetized and awake rats (Yamamoto et al. 1985, 1989) and that of AM taste neurons in anesthetized rats (Azuma et al. 1984). These results suggest existence of two neuronal types in the gustatory system of rats. It has been reported that two similar types of neurons emerge in the olfactory cortex of frogs (Duchamp-Viret et al. 1996). One type of the cortical neurons (group 1) respond to a stimulus with high response rate that was comparable with that of mitral cells in the olfactory bulb, and the other type of the cortical neurons (group 2) have low response rate to a stimulus. The group 2 neurons are rare in the lower stage of the olfactory system such as the olfactory bulb (Duchamp-Viret et al. 1996). These group 2 neurons were suggested to be involved in odor quality coding but less involved in intensity coding (Duchamp-Viret et al. 1996). These results suggest that the cortical taste neurons with low response rate in rats might correspond to the group 2 neurons in the olfactory cortex of frogs, and the AM taste neurons might receive information from the cortical taste neurons. However, further studies are necessary to elucidate whether or not these taste neurons in the cortical taste area and AM in rats are involved in quality rather than intensity coding.

In the present sample, QHCl-best neurons constituted 37.7% of the gustatory responsive cells, almost equivalent to the sucrose-best subset. In anesthetized rats, HCl-best (37.1%) and NaCl-best (28.6%) dominated AM taste neurons, and QHCl-best cells played a minor role (11.4%) (Azuma et al. 1984). In the central gustatory system---the NST (Ganchrow and Erickson 1970; Nakamura and Norgren 1991, 1993; Woolston and Erickson 1979), PBN (Nishijo and Norgren 1990, 1991, 1997; Smith et al. 1983a,b), thalamic (Pritchard et al. 1989), and cortical (Yamamoto et al. 1984, 1989) taste areas---sucrose-best and NaCl-best dominated and QHCl-best cells were rare. In the peripheral taste nerves, sucrose-best and NaCl-best neurons dominate in the chorda tympani (Frank et al. 1983), whereas QHCl-best neurons are common in the glossopharyngeal nerve (Frank 1991; Hanamori et al. 1988).

Responses to QHCl in the AM might be ascribed to nonspecific responses to aversive stimuli because QHCl is bitter to humans and evokes characteristic aversive behavioral responses in rats (Grill and Norgren 1978a,b). Other aversive stimuli such as light tail pinch, however, did not evoke responses in those neurons. Consistently, six of nine AM taste neurons in awake rats preferentially respond to QHCl (Yasoshima et al. 1995), and 20% of AM taste neurons of awake monkeys are QHCl-best neurons (Scott et al. 1993). Taken together, these results imply that the AM is distinctive because responsiveness to bitter stimuli (aversive taste) is encoded preferentially there. Interestingly, it has been reported that correlation coefficient between Na saccharin and QHCl decreased, whereas the correlation coefficient between sucrose and Na saccharin did not change in the NST of chronic decerebrated rats (Mark et al. 1988). This specific decrease in response to the bitter component of Na saccharin might be attributed to interruption of centrifugal projections from the AM to the NST (Kretteck and Price 1978; Van der Kooy et al. 1984). Furthermore, preferential coding of aversive stimuli such as bitter taste in gustatory modality in the present study is consistent with the previous human neuropsychological studies in which preferential coding of aversive or negative stimuli in visual (Adolphs et al. 1994, 1995; Grodd et al. 1995; Morris et al. 1996) and olfactory (Zald and Pardo 1997) modalities has been reported in the AM.

Using dominant and absolute components, the mean entropy measures of AM taste neurons were 0.742 and 0.812, respectively. When excitatory components were used, the mean entropy value in the AM (0.742) in the present study was higher than those in the chorda tympani axons (0.588) (Yamamoto et al. 1984, 1989), NST (0.60) (Nakamura and Norgren 1991), PBN (0.58) (Nishijo and Norgren 1990), and cortical gustatory area (0.54) (Yamamoto et al. 1984, 1989). On the other hand, using absolute values, the mean entropy measure of rat AM taste neurons in the present study (0.812) was comparable with that of the AM taste neurons of awake monkeys (0.82) (Scott et al. 1993). These results suggest that the AM might be less important in discrimination of taste quality than other taste areas.

Taste stimulus relationships

In the present study, the MSG had moderate correlation coefficients with most sapid chemicals including NaCl(gamma  = 0.862) and sucrose (gamma  = 0.433). Moderate correlation coefficients with NaCl and sucrose were consistent with those in the previous study in the PBN of awake rats (Nishijo et al. 1991). Lysine HCl at concentration of 0.2 M was not preferable solution for rats (Pritchard and Scott 1982). Lysine HCl was correlated moderately with most aversive stimuli (QHCl) (gamma  = 0.716), highly correlated with less aversive chemicals such as citric acid (gamma  = 0.852), and moderately preferable solutions such as MSG (gamma  = 0.890) and NaCl (gamma  = 0.813). This pattern of interstimulus correlation coefficients suggests that, in the AM, taste coding is based on palatability (see also RESULTS). This pattern of correlation coefficients between the sapid stimuli is different from that derived from the PBN taste neurons of awake rats (Nishijo and Norgren 1990, 1997). In the AM, correlation coefficients between basic sapid stimuli with similar degree of palatability were larger than those in the PBN, while correlation coefficients between basic sapid stimuli with different degree of palatability were low in both the AM and PBN (Fig. 14). For example, correlation coefficients between NaCl and sucrose (palatable solutions) were -0.129 and -0.126 in the PBN (Nishijo and Norgren 1990, 1997), which were significantly smaller that (0.440) in the present study (two-tailed t-test after Fisher's Z transformation, P < 0.05). Correlation coefficients between citric acid and QHCl (aversive solutions) were 0.769 and 0.386 in the PBN (Nishijo and Norgren 1990, 1997), which were significantly smaller than that (0.905) in the present study (two-tailed t-test after Fisher's Z transformation, P < 0.05). Furthermore, correlation coefficients between sucrose (most palatable) and QHCl (most aversive) was low (0.138) in the AM, which was statistically not different from those (0.03 and 0.019) in the PBN (Nishijo and Norgren 1990, 1997) (two-tailed t-test after Fisher's Z transformation, P > 0.05). These results strongly suggest difference in taste coding between the PBN and AM, i.e., taste quality versus palatability.


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FIG. 14. Comparison of average across-stimulus correlation coefficients between the AM and pontine parabrachial nucleus (PBN) in awake rats. Correlation coefficients in the PBN were cited from the previous studies in awake rats (Nishijo and Norgren 1990, 1997). Other descriptions as for Fig. 12. Note that correlation coefficients between sucrose (palatable solution) and QHCl (aversive solution) were low in both the AM and the PBN, whereas those between sucrose and NaCl (palatable solutions) and those between citric acid and QHCl (aversive solutions) were significantly larger in the AM than the PBN (2-tailed t-test after Z transformation, P < 0.05).

Thus the correlation coefficients (Table 3) and the multidimensional scaling (Fig. 12) derived from these data suggest that taste is encoded in the AM based on the palatability of the sapid chemicals. Behavioral studies, however, contradict the simplest version of this inference because lesions of the central nucleus of the AM have little effect on responsiveness to the four standard sapid stimuli (Galaverna et al. 1993; Kiefer and Grijalva 1980). In fact, basic oromotor responsiveness to gustatory stimuli are nearly normal in chronically decerebrated rats (Grill and Norgren 1978a,b). Lesions of the central nucleus of the AM do alter the relationship between oromotor responses to taste and the actual consumption of the stimuli (Seeley et al. 1993). Larger lesions of the AM attenuate behavioral responses to both preferred and aversive sapid stimuli and alter conditioned taste aversion (Kemble and Schwartzbaum 1969; Yamamoto et al. 1995). Finally, fiber-sparing lesions of the AM in monkeys changed their food preferences (Murray et al. 1996). Thus gustatory sensory activity reaches the AM, and, apparently, this information may be used in the ongoing process of evaluation. The strong reciprocal connections between the central nucleus of the AM and the brain stem taste nuclei imply that, whatever information that is being added in the AM, it is likely to be involved in modifying ascending gustatory neuronal activity.

Localization and sensory responsiveness to other sensory stimuli of the oral-sensory neurons

Nontaste oral-sensory neurons were distributed widely in the AM. The neurons could respond to somatosensory stimuli including tactile and/or thermal stimuli on a tongue during licking. Anatomic studies indicated that almost all subnuclei of the AM, including the central, basolateral, lateral, basomedial, and medial nuclei, and the amygdalostriatal transition area receive unimodal and multimodal somatosensory information from the medial posterior complex of the thalamus (LeDoux et al. 1990, 1991; Turner and Herkenham 1991), the parabrachial nucleus in the pons (Bernard et al. 1993; Fulwiler and Saper 1984), and cortical somesthesia-related areas such as areas TE2 and TE3, and area 13p in the insula (LeDoux et al. 1991; Mascagni et al. 1993; Turner and Zimmer 1984). These broad anatomic inputs of somatosensory afferents might contribute to the present results in which nontaste oral-sensory responses were recorded from various areas of the AM.

On the other hand, unimodal taste neurons were located topographically within the AM, that is, mainly in the central nucleus and sparsely in the lateral nucleus. Consistently, anatomic studies indicated relatively restricted gustatory projection to the central and lateral nuclei of the AM (see INTRODUCTION). The multimodal taste neurons were located in the basolateral and basomedial nuclei of the AM in the present study. Although responses to other sensory modalities were not tested, taste-responsive neurons in those nuclei were reported previously in awake rats and monkeys (Scott et al. 1993; Yasoshima et al. 1995). Multimodal responsiveness of taste- and/or oral sensation-responsive neurons was reported previously in monkeys, although these neurons were not tested with four standard sapid chemicals (Nishijo et al. 1988a,b). The AM was suggested to function to correlate this multimodal sensory information (Geschwind 1965; Johnston 1923). For example, "psychic blindness and oral tendency," major symptoms of the Klüver-Bucy syndrome that appear after lesions of the temporal cortex including the AM, are ascribed to gustatory-visual disconnection (Geschwind 1965). These multimodal neurons might contribute to association of gustatory sense with other sensory information. It has been reported that responses of AM multimodal taste- and/or oral sensation-responsive neurons to nontaste conditioned stimuli were modulated by association of different sapid chemicals with those nontaste conditioned stimuli, and behavioral responses to those conditioned stimuli were well correlated to modulation of neuronal responses (Muramoto et al. 1993; Nishijo et al. 1988a,b). These evidence along with the present results suggests that the AM is important in recognition of food-related stimuli based on association of oral sensation including taste with other sensory stimuli. Thus it appears that the AM influences the meaning of food-related stimuli, much as it does stimuli in other modalities.

    ACKNOWLEDGEMENTS

  We thank Dr. R. Norgren (Pennsylvania State University, Hershey) for valuable comments on this manuscript.

  This work was supported partly by the Japanese Ministry of Education, Science and Culture Grants-in-Aid for Scientific Research (08408036, 08279105, 08279215, 08234209, 07244103, and 08680884) and by Funds for Comprehensive Research on Aging and Health.

    FOOTNOTES

  Address for reprint requests: H. Nishijo, Dept. of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-01, Japan.

  Received 27 February 1997; accepted in final form 12 September 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society