ACTA NEUROBIOL. EXP. 1982, 42: 2 W 2

Lecture delivered at the Symposium "Brain and behavior" held in Jabionna near Warsaw

May 1981

ELECTROPHYSIOLOGICAL ANALYSIS OF CONDITIONED TASTE AVERSION IN RATS

Gustav BROZEK

Institute of Physiology, Czechoslovak Academy of Sciences, VideiiskB 1083, 142 20 Prague 4, Czechoslovakia

Key words: conditioned taste aversion, unit activity, gustatory pathway, amygdala, hypothalamus

Abstract. The memory mechanisms of conditioned taste aversion (CTA) were examined using electrophysiological methods. Presentation of the aversive taste to CTA trained animals inhibited unit activity in gustatory cortex, amygdala and ventromedial hypothalamus and caused delayed excitation of neurons in lateral hypothalamus. Lick-triggered rewarding stimulation of medial forebrain bundle substituted the taste CS in CTA experiments. The same stimulation triggered by nose-poking failed to be associated with subsequent poisoning. Importance of specific brain areas for CTA retrieval was assessed by the effect of lick-triggered stimulation of the examined site on gustatory discrimination. The dis- ruption threshold was lowest in amygdala and lateral hypothalamus. Stimulation of other brain structures did not interfere with gustatory discrimination at current intensities subthreshold for disruption of lick- ing. Unilateral electrical stimulation of vestibular nuclei following ingestion of saccharin elicited marked aversion to this taste.

INTRODUCTION

The basic task of research into the mechanisms of learning and memory is to solve the fundamental enigma how the originally ineffec- tive (neutral) input (CS) changes into an effective one when associated

with subsequent unconditioned stimulus (US). Most electrophysiological studies of this problem employed traditional models of operant or clas- sical conditioning (bar pressing-18, conditioned eye blink-27, conditioned nictitating membrane retraction-24). Such models are suitable for labo- ratory investigations but are far from natural instances of learning which is a part of adaptative behavior.

During the past twenty years research into the higher nervous acti- vity was considerably influenced by attempts to study learning in more naturalistic setting (15, 22, etc.). Instead of association of an arbitrary CS with an arbitrary US, memory is considered to be a specialized function, subjected to similar evolutionary influences as other vital phenomena and, therefore, optimally adjusted for coping with highly predictable situations, most relevant for the survival of the species.

This trend, represented by contemporary ethology, offers to brain scientists unique models of learning and memory and discloses many unprecedented features of these phenomena, but requires that they are studied within definite constraints. One of the most important tasks of neuroethology is to develop laboratory versions of the ethological experiments which would make it possible to investigate the underlying brain mechanisms with sophisticated analytical methods. Particularly important in this respect are the neural processes controlling food in- take, represented by conditioned taste aversion (CTA), capability of rats to avoid a novel food or liquid the ingestion of which has lead to poi- soning (3, 11, 16, 21). Recognition of the aversive taste stops consump- t ~ o n of the flavoured fluid. On the other hand, rats do not avoid drink- ing from a noisy spout or from spout of a particular shape (10). Re- search into the mechanism of CTA lead to the formulation of the prin- ciples of "preparedness" or belongingness (21, 22) to explain the pre- ferential association of visceral symptoms of poisoning with preceding gustatory or olfactory experiences. The purpose of the present paper is to review recent progress in the electrophysiological analysis of CTA and to discus6 not only the results obtained, but also some tentative approaches.

UNIT ACTIVITY STUDIES

Most unit activity studies examine differences between activity of caive and trained animals. They are based on the belief (18), that elec- trophysiological analysis of propagation of the CS volley through the brain can reveal the site where the signal leaves the route followed in naive animals and enters th~e other one, eventually leading to activation of the conditioned reflex (CR). The d i f f e ~ ~ n c e s due to learning can be established in the same animal tested before and after acquisition, but are more commonly studied by comparison of groups of naive and train- ed animals.

Rats lick at the drinking spout with astonishingly regular frequency of 6 Hz (25, 26). During the tongue-spout contact lasting about 60-80 ms, a small amount of the fluid (5 yl) adheres to the tongue tip and is transfered into the mouth during the subsequent 80-100 ins interval. A rat with well established CTA needs a single lick at the aversive fluid to suppress further drinking. As pointed out by Halpern and Maro- v~itz (14), such single lick discrimination implies that the processing of the afferent gustatory signal, its matching with the information stored in memory and elaboration of the appropriate out,put command must take place before the next lick, i.e. within 110-120 ms. Rats were tested in the gustatory discrimination apparatus (5) exposing the animal si- ~nultaneously to two drinking spouts, one delivering water, the other containing the aversive fluid. The s~pouts were 20 mm apart and acces- sible through small openings allowing photoelectric recording of the tongue-spout contacts. A CTA-trained rat finds rapidly the water con- taining spout and starts drinking thereof. After licking has stabilized, water is suddenly replaced by the aversive fluid. This can be done either by exchanging the position of the spouts, mounted on ,a parallel axis which can be rapidly rotated by 180" with an electromotor, or by provid- i ~ g each spout with independent water and aversive fluid inflows, con- trolled by solenoids. An important prerequisite of stationary retrieval conditions is that prolonged testing does not cause CTA-extinction. High motivation of the animal to avoid the aversive flavour is maintained when the aversive fluid contains poison at concentrations, eliciting mo- derate gastrointestinal disorder after th,e ingestion of a few ml. Isotonic solution of lithium chloride (0.15 M) can serve as a combined CS (salty taste) and US (symptoms of poisoning).

About half of the units recorded from the centers of gustatory path- way, amygdala and hypothalamic nuclei of freely moving rats showed lick-related activity modulation (9). Reactions were elicited both by water and salt licks and th~e histogram shapes were usually similar. The correlates of retrieval were reflected in the incidence of the lick wlated activity in different brain regions. The percentage of units, showing significant lick related PSHs in the gustatory thalamus and cortex was significantly higher in CTA-train~ed rats than in naive ones. This pro- bably reflects increased attention paid to the gustatory stimuli (both water and salt) in the discrimination situation. Incidence of lick related PSHs was higher also in ventromedial hypothalamus and amygdala but not in lateral hypothalamus of CTA trained rats. The latter result is in agreement with an earlier finding (2) that CTA training increases the incidence of unit reactions to perfusion of the mouth cavity with the aversive fluid in ventromedial hypothalamus but reduces it in the lateral llypothalamus. Such reciprocal changes may reflect the inhibition of the

feeding center and activation of the satiety centers in the restrained CTA trained animals.

Responses to the first salt lick were more frequently lower than responses to the preceding water lick in CTA trained rats and this difference was most striking in the ventromedial hypothalamus and .amygdala. Unfortunately it was impossible to relate this effect more closely with the retrieval process, since the later parts of the PSHs

10 Fig. 1. Time relationship between be- 40 havioral manifestation of CTA retrieval

and unit activity in various brain re- gions of CTA trained rats. Top of

VM"/y\ figure: Cumulative distribution of LiCl licks (decreasing curve) and water licks (increasing curve) after spout switch- ing. Co, Population responses of gu- statory cortex; Am, amygdala: VMH, ventromedial hypothalamus; and LH, lateral hypothalamus to switching of the spouts. Ordinate: Percentage of neurons exhibiting statistically signifi-

----d cant excitatory (upward) or inhibitory 70 /::̂-..-- (downward) vals after spout reactions switching at various (abscissa). inter-

o 25b 3'1 768 WIU-. (Modified from Bure5ovB et al. Ref. 9).

were already influenced by the elaboration of the output command. In order to isolate the output command from the processing of the input signal the same unit activity recordings were analysed using spout switching as the triggering signal. PSHs plotted in this way yielded more consistant results (Fig. 1). The prevailing effect in the gustatory cortex, amygdala and ventromedial hypothalamus was inhibition, starting 100-150 ms after spout switching and culminating 100 ms later. Two steps seen in the incidence of inhibitory reactions particularly in amyg- dala and ventromedial hypothalamus probably correspond to trials in which licking stopped after one or two licks, respectively. The late ex- citatory reaction in lateral hypothalamus coincided with the time, when the animal had stopped licking lithium chloride, but has not yet started licking water. I t may reflect the increasing drive to continue drinking. No significant changes were found in lower gustatory centers, which is not surprising if we take into account that these structures are not directly involved in the elaboration of the output command.

EVOKED RESPONSE STUDIES

The main disadvantage of the unit activity approach is the necessity of population comparisons between naive and CTA-trained animals. It would be preferable to record gustatory responses of the same neuron before and after CTA acquisition, but this is impractical with the pre- sently available techniques. I t is feasible, however, to use averaging to follow evoked responses in various centers of the gustatory pathway in the course of CTA conditioning and to determine in this way the locus of the plastic change. Unfortunately, the poorly synchronized gustatory volleys do not elicit so well discernible evoked potentials (EPs) as acous- tic clicks or light flashes. Yamamoto et al. (28) recorded averaged EPs in the gustatory cortex of rats to brief superfusion of the tongue sur- face with flavoured fluids, but the responses were low and only detec- table against background superfusion with water. During licking, the oral cavity is permanently flooded with the flavoured solution and con- tribution of individual licks is obscured. This is the rationale of various attempts to enhance the EP approach by lick-triggered or lick-related non-gustatory stimulation.

Electrical taste. Weijnen and Mendelson (25) reviewed literature on the so called current licking, i.e. tendency of rats to lick a dry metal rod, delivering low current (1 to 5 yA) to the tongue. Weijnen (25) re- ported that current licking can be associatled with subsequent LiCl poi- sonlng and that the ensuing conditioned aversion is manifested by avoidance of electrical tongue stimulation. In collaboration with Dr. Weijnen we have attempted to record the EPs elicited by electrical

3 - Acta Neurobiol. Exp. 1\82

pulses applied to the tongue, in various centers of the gustatory path- way and to asses the potential value of this technique for CTA studies.

Rats with metal electrodes implanted in appropriate brain regions were placed in a plexiglas box with metal floor. Small opening allowed the animals to reach with the tongue the stainless steel spout. Contact betwleen tongue and spout was detected by a lick sensor (25) and used for triggering a constant current stimulator delivering properly delayed stimuli between the spout and floor. Stimulator output, spout and floor were well insulated from the ground. Miniature symmetric FET follow- ers were attached to animal's head to reduce artefacts. In this prelimi- nary experiments every lick triggered electrical stimulation of the ton- gue. Evoked responses from various areas were averaged (n = 400). Figure 2 shows the typical response recorded with amygdala electrodes

Fig. 2. Averaged (n = 400) electrical activity from amygdala synchronised with licking. Dashed line - licking without stimulation, solid and dotted lines anodal and cathodal stimulation of tongue, respectively. Bipolar recording, stimulation by

pulse (10 ms, 100 pA) delayed 1 ms after tongue-spout contact.

(bipolar recording, distance between tips approximately 0.5 mm). The dashed curve corresponds to lioking alone, the full and dotted lines to licking with a superimposed anodal and cathodal stimulus, respectively. Note the stimulus artefact at beginning of record. The electrically evoked responses can be obtained by subtraction of the dashed and full curves.

A disadvantage of stimulation triggered by every lick is the relati- vely high frequency (6-7 Hz) of stimuli and the low amplitude of evoked responses which are not well discernible in the background activity. More promising is triggering of stimulation by every 4th or 8th lick yielding responses better suited for CTA analysis.

Intracranial stimulation (ICS) as a taste substitute. Another possibi- lity of enhancing the EP analysis of CTA is to form aversion not to a particular taste, but to licking accompanied by electrical stimulation of a specific brain area. Greenshaw and BureS (13) have recently de- monstrated the essential feasibility of this approach. In their experi- ments, rats were trained to obtain their daily ration of water by licking a spout equipped with a photoelectric lickometer. After the basic water intake had been established, they received after every eighth lick elec- trical stimulation of various hypothalamic or subthalamic loci (50 Hz sine wave current, 400 ms, 50 to 100 PA root mean square). The stimu- lation intensity was adjusted so as not to interfere with licking. Imme- diately after the drinking +ICS session the animals were poisoned with LiCl. Water drinking sessions alternated with water drinking +ICS sessions associated with poisoning. Figure 3 shows absence of ICS- induced interference with licking and of ICS-accompanied water in the first retrieval test.

REWARDING NONREWARDING ICS 165 T

'S-LiCI ICS W W.ICS..LiCI W*lCS

Fig. 3. Conditioned aversion to lick-triggered electrical intracranial stimulation (ICS). The columns indicate the cumulative number of licks during 20 min in percentages of control values. The vertical bars are SEM values. ICS is triggered

by every 8th lick. For explanation see text.

Clearer results were obtained with rewarding ICS of lateral hypo- thalamus and medial forebrain bundle. In this case, water drinking could be omitted while the rat continued licking a dry spout to obtain trains of electrical stimuli as the only reward. Association of the lick triggered self-stimulation with poisoning resulted in a marked drop of the self-stimulation rate already in the next session (Fig. 3) and was only slowly extinguished. Same ICS triggered by another operant (nose-

poking into a hole in the wall) was not diminished by acquisition of CTA to licktriggered stimulation.

The above experiments suggest that (i) rewarding ICS is associated with poisoning more efficiently than neutral ICS and (ii) aversion is formed not to ICS alone but to the licking/ICS complex. The first state- ment is understandable in the light of the hedonic properties of taste stimuli, which usually have a strong emotional content and are inter- preted as pleasant or repulsive. The emotional state induced by the rewarding ICS is obviously well "prepared" for being interpreted as a property of licking, i.e. as a kind of "taste". The operant-specific character of the aversion indicates that the plastic change is not due to decreased excitability at the stimulation site, but rather to changes occurring at some point of convergence between the CS-elicited and lick-related signals. This notion is an important hint for the future orien- tation of the electrophysiological analysis of the above phenomena.

Excitability testing by lick-triggered CS. In the previous paragraph electrical stimulation served as CS or as a component of a complex CS, but CTA mechanisms can also be explored by neutral ICS applied dur- ing various phases of CTA retrieval. Aleksanyan and Broiek (1) estab- lished a strong CTA to isotonic LiCl and tested the resulting rejection of a salty fluid in a gustatory discrimination apparatus. The rats car- rying stimulating and recording electrodes in the amygdala, ventro- medial and lateral hypothalamus and gustatory cortex received regular lick-triggered single pulse stimulation (0.1 ms) of one of these struc- tures, while EPs were recorded from the other sites. Intensity of single electrical pulses trigge~ed by the onset of every fifth lick were adjusted to elicit submaximal evoked response in the target structure. After a variable number of licks (30 to 40), the solid state programming equip- ment switched the fluids in the spouts in such a way that the next stimulus was applied at the onset of the second (usually the last) LiCl lick (Fig. 4, upper part). Trials in which the animals stopped drinking after a single LiCl lick were not evaluated. Responses to the last stimu- lus triggered by the water lick (S,), to the stimulus triggered by the LiCl lick (S,) and to subsequent stimulus (S3) were separately averaged with a computer (n = 32) and compared with the responses elicited by lick independent stimuli. The latter were applied at regular 1 s intervals and elicited usually higher averaged EPs than to lick triggered stimuli. The effect is obviously due to increased activity in the target structure during gustatory discrimination. The stimulus, triggered by the second LiCl lick at a time when the retrieval process was already well ad- vanced, elicited in most cases similar averaged evoked responses as the preceding stimulus, triggered by water lick. Only in three structures were the differences statistically significant. Average EPs to LiC1-lick

triggered stimulation of the gustatory pathway (nucleus of solitary tract, parabrachial gustatory area, gustatory thalamus) and of the gu- statory cortex were higher in the amygdala and ventromedial hypo- thalamus, respectively. Stimulation of the lateral hypothalamus elicited

Fig. 4. Averaged evoked responses (AERs) in amygdala to lick-independent or lick- triggered electrical stimulation of lateral hypothalamus.

Above: scheme of the lick-stimulus synchronisation. Upward and downward deflec- tions-water and LiCl licks, respectively. The changes of level indicate spout switching. Below: upper and lower rows: AERs for lick-independent stimuli before and after drinking. Middle row: AERs to stimuli triggered by licks SI, S,

and S3 according to the above scheme.

lower evoked response in the amygdala (see Fig. 4, lower part, middle row). The next stimulus (triggered by water lick) usually elicited res- ponses tending to return to preswitch amplitude. The interpretation of results is complicated, because the effect can be due to excitability changes in the area of stimulation and/or in the target structum. The latter possibility applies to augmented responses in the amygdala and ventromedial hypothalamus: responses to testing stimulus are better discernible against the inhibition prevailing in these brain regions after spout switching. On the other hand, the decrease of amygdalar respon- ses to stimulation of lateral hypothalamus cannot be explained by the above mechanism, but rather by occlusion in the area of stimulation and/or in the connections between lateral hypothalamus and the area of recording.

RETRIEVAL DISRUPTION BY INTERFERING ICS

Whereas excitability testing requires that ICS does not influence CTA retrieval, importance of the stimulated site for correct taste dis- crimination performance can be assessed by increasing the stimulus in-

tensity and determining the disruption threshold. Broiek et al. (6) used lick-triggered stimulation of lateral hypothalamus or amygdala for esti- mation of impairment of CTA retrieval in the gustatory discrimination apparatus mentioned earlier (5). Lick-triggered monopolar stimuli we- re applied to selected brain structures and the intensity of stimulation was gradually increased until discrimination was disrupted or until the animal stopped drinking. Without stimulation the rat recognized LiCl after 1 to 3 licks, so that LiCl formed only 3 to 10°/o of total volume of fluids consumed. This percentage increased during stimulation above 20% (Fig. 5). Sometimes the animals ignored the spout switching alto-

S

"Lu ilm ru "' . .

I t C I 0 . . . . . W I 1

LlClL . . . . . Fig. 5. Coded records of the gustatory discrimination behavior of CTA trained rats in control condition (C) and during lick-triggered electrical stimulation of amygdala (S). Upward and downward short rectangular deflections correspond to single licks of water and LiC1, respectively. The changes of level indicate spout switching. Dots above and below the licks indicate intracranial stimulation (1 ms,

20 PA).

gether and continued to lick LiCl until water appeared at the same spout again. With the cessation of stimulation, taste discrimination im- proved again, but prolonged periods of stimulation were followed by slowly subsiding aftereffects. Amygdala was more sensitive to disrup- tion which was elicited from all 21 stimulation sites at low threshold (about 100 PA). About half of the hypothalamic sites did not yield to disruption even with stimulus intensities interfering with licking (around 500 PA). Also stimulation of other brain structures (cerebral cortex, centers of the gustatory pathway) did not deteriorate gustatory discrimination at current intensities subthreshold for the disruption of licking.

ELECTRICAL US

CTA is usually elicited by systemic application of poisons, which may evoke visceral symptoms, signalled through interoceptor affenents, affect the brain through the chemoreceptive zone of area postrema, or pene-

trate across the blood brain barrier and influence the brain centers directly. Electrical correlates of the above mechanism are diffuse and not very specific. Visceral projections to the nucleus of the solitary tract and to the amygdala (17) lead to the assumption that the latter struc- ture may be one of the US targets. Reddy and BureS (19) reported, that LiCl injection (0.15 M, 2O/o body weight) to rats anesthetized with pen- tobarbital causes activation of more than 50°/o amygdalar units, but has little effect on cortical neurons. The nature of the stimulus makes it difficult to decide, however, whether the amygdalar reaction is a direct consequence of activation of visceral afferents or a part of a complex homeostatic reaction.

Substitution of poisoning by electrical stimulation of peripheral ner- ves or nerve centers has not yet been seriously examined. Semenov et al. (23) took advantage of the possibility to induce CTA by labyrinthine stimulation (rotation of the animal in the centrifuge (4, 12) and attemp- ted to use electrical stimulation of vestibular nuclei as the US. A stain- less steel monopolar electrode was implanted in the vestibular complex on one side and silver screw indifferent electrode in the nasal bones. The electrode leads were brought to a miniature socket fixed to the skull with anchoring screws and acrylate. After stabilization of water intake, the animal was offered saccharin and subjected 10 min later to 10 min stimulation of vestibular nuclei (100 Hz, 1 ms) the intensity of which was adjusted to elicit tonal asymmetry, contralateral deviation of the head and lying on the contralateral side. The above signs were a function of current intensity. After discontinuation of stimulation nor- mal posture was immediately resumed. Saccharin preference testing two days later revealed marked aversion to saccharin (Fig. 6) which only slowly subsided over four extinction sessions, alternating with presen- tation of water only. The experiment shows the feasibility of eliciting

extinction sessions. C = control animals; o E = experimental animals. R1 "2 R3 "4

Fig. 6. Decrease of saccharin preference 6 0 -

caused by association of sweet taste with following 10 min unilateral electrical ,,o. stimulation (100 Hz, 1 ms) of vestibular nuclei. Ordinate: relative consumption of saccharin in a two-choice preference ap- 'O-

paratus. Abscissa: R ~ I , four consecutive

yC

--_ --_ -__--- ---------- E -----

CTA by electrical stimulation and employing electrophysiological me- thods to trace the locus of the plastic change also from the US input.

CONCLUSIONS

Although the biological significance and unusual physiological pro- perties of CTA attract increasing attention of brain scientists (7, 8, 20), electrophysiological analysis of the phenomenon is still in the elementary stages. Whereas the first studies concentrated on the electrical correlates of retrieval under conditions of steady state performance in highly overtrained animals, recent experiments show the feasibility of a more dynamic approach which allows to follow the CS evoked activity also during CTA acquisition. The methods employed contribute not only to the elucidation of the CTA mechanism, but to a wider spectrum of re- lated problems, including interpretation of excitability measures, rela- tionship between classical and operant conditioning, and changes of motivational significance of brain stimulation. It is hoped that recording, stimulation and interference studies will specify, together with surgical and functional ablation experiments, the relevant centers and circuits of CTA and prepare conditions for its neurochemical and morphological analysis.

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