Hippocampal lesions slow extinction of a conditioned taste aversion in rats

Physiology & Behavior, Voi. 23, pp. 217-222. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.

Hippocampal Lesions Slow Extinction of a Conditioned Taste Aversion in Rats D A N I E L P. K I M B L E , R U T H B R E M I L L E R A N D L Y N N S C H R O E D E R 1

Department of Psychology, University of Oregon, Eugene, OR 97403 AND W I L L I A M P. S M O T H E R M A N

Oregon State University, Corvallis, OR 97330 R e c e i v e d 28 August 1978 KIMBLE, D. P., R. BREMILLER, L. SCHROEDER AND W. P. SMOTHERMAN. Hippocampal lesions slow extinction o f a conditioned taste aversion in rats. PHYSIOL. BEHAV. 2,3(2) 217-222, 1979.--Rats were subjected to either a bilateral removal of the superior cervical ganglia or to a sham operation of the neck. In subsequent operations, these animals underwent either bilateral dorsal hippoeampal lesions, bilateral neopallial lesions or no further operation. Removal of the superior cervical ganglia prevented the anomalous innervation of the remaining hippocampal tissue. All animals then learned a conditioned taste aversion to a sweetened milk solution (CS) following LiCl-induced gastrointestinal illness (UCS). The rats with hippocampal lesions were significantly slower to extinguish the aversion conditioning across the 14 day recovery period than were controls. There was no effect of any manipulation on the acquisition of the conditioned taste aversion, and there were no behavioral consequences of the presence or absence of the anomalous innervation. The slower extinction of the taste aversion shown by the hippocampal lesioned rats was discussed in terms of a lessened impact of the pre-illness exposure to the CS in these animals.

Hippocampus Superior cervical ganglia aversion CS preexposure

Anomalous noradrenergic innervation

CONDITIONED taste aversion is a particularly interesting behavioral phenomena because of the extended time between CS and UCS presentations over which successful conditioning can occur, and because of the apparent specificity of the association of illness with a previously presented gustatory CS [7,8]. This behavior seems to be widespread throughout the animal kingdom, and of obvious evolutionary significance [6,19]. A number of investigators have published reports concerning the effects of hippocampal lesions on this phenomena. Although some similarity is present in the results of these various investigations, no consistent pattern has so far been obtained. For example, Best and Orr [2] found that electrolytic lesions of the posteroventral hippocampus did not interfere with the development of a conditioned aversion to a novel saccharin solution which was followed by apomorphine-induced gastrointestinal illness, while anterodorsal hippocampus lesions extending into the fimbfia did interfere with such conditioning. Miller et al. [18], on the other hand, found no effect of near total hippocampal lesion on the acquisition of a conditioned taste aversion paired with cyclophosphamide-induced illness, but found that the aversion did extinguish more rapidly in the

Conditioned taste

lesioned rats. Krane et al. [15], in an extensive study, found that large electrolytic lesions of the hippocampus can retard the conditioning of a taste aversion to a solution of physiological saline followed by apomorphine-induced illness, perhaps by altering the neophobic tendency of rats to the novel flavored CS. Since there have been many procedural differences among these studies (lesion extent and placement, behavioral testing procedures, illness-inducing methods, etc.), it is not clear just what effects hippocampal lesions may have on conditioned taste aversion. We have been examining the behavioral effects of hippocampal lesions in rats for several years [10, 11, 12]. Recently, we were interested to learn of Loy and Moore's discovery that following lesions of the anterior portion of the hippocampus in rats, there was an extensive anomalous innervation of the remaining hippocampal tissue [16]. This anomalous innervation has also been observed in rats following similar anterior hippocampal or septal area lesions by Stenevi and Bjtrklund [24]. This novel growth is noradrenergic, and was discovered using flourescence histochemical procedures [4,16]. The fibers are coarse, intensely fluorescent and resemble peripheral noradrenergic fibers. These fibers are believed to sprout from axons whose cell

1This project was supported in part by BRSC Grants RR 07079 and 07080 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health to Oregon State University and the University of Oregon.

Copyright © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/080217-02501. i0/0

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KIMBLE, BREMILLER AND SCHROEDER

bodies are in the superior cervical ganglion. These fibers normally innervate pial blood vessels. As a result of the hippocampal denervation and/or the surgical trauma, these fibers sprout and invade the hippocampal formation tissue caudal to the lesion, distributing themselves throughout the remaining hippocampal tissue, particularly in the area dentata and in the CA3 zone of the dorsal hippocampus [16,24]. Subsequent electron microscope examination of this anomalous innervation indicates that synapses are in all likelihood being formed, particularly in the hilar region of the dentate gyms (R. Loy, personal communication). This anomalous innervation can be completely eliminated by bilateral removal of the source of the fibers, the superior cervical ganglia of the sympathetic nervous system [13, 16, 24]. As Loy and Moore state: " . . . it is not clear whether the innervation is functional. If it is, such anomalous growth could have significant implications for the sequelae to injury of the central nervous system of both experimental animals and man" [16]. The potential significance of this anomalous innervation led us to prepare rats with and without this growth and to examine their behavior in tasks likely to be relevant to hippocampal function. In this paper we report on conditioned taste aversion in such animals.

METHOD

Animals The animals were 30 male Spragne-Dawley albino rats (CD, random-bred) from the Charles River Co., Wilmington, MA. They were maintained in individual cages on a 12 hr diurnal cycle, with the dark cycle from 7:00 p,m. until 7:00 a.m. The animals were approximately 75-80 days of age at the time of the first surgical operation (superior cervical ganglionectomy). Food and water were available on an ad lib basis. Prior to the present experiment, all animals had been tested repeatedly in an open field for locomotor activity, in an unbaited T-maze for spontaneous alternation tendencies and in a Hebb-Williams apparatus for spatial maze acquisition [13].

Surgery The anomalous innervation of the hippocampus can be prevented by removal of the source of the fibers, the superior cervical ganglia. Fifteen of the animals in the present study underwent bilateral removal of the superior cervical ganglia prior to any .further manipulation. Anesthesia in all cases was induced by sodium pentobarbital (Nembutal) in doses of 50 mg/kg, IP. Atropine sulfate, in doses of about 0.04 mg/animal was administered to reduce mouth and throat secretions. The ganghonectomy procedure was worked out in a series of pilot operations. Basically, the ganglion is located using a binocular operating microscope and gently pulled away from the rest of the sympathetic chain. In the rat it is located just dorsal to the junction of the internal and external carotid. After a little practice, the ganglion can be easily and quickly removed with virtually no bleeding, providing of course, that no rupture of the carotid takes place. Further surgical details on the ganglionectomy are published elsewhere [13]. Those animals not undergoing superior cervical ganglionectomy were given a sham ganglionectomy. They were anesthetized, administered atropine and then subjected to an operation in which the neck incision was made, submaxillary and anterior sublingnal

glands gently pulled to one side, and sufficient blunt dissection carried out to visualize the carotid artery. Approximately one week after undergoing bilateral superior cervical ganglionectomy, twelve animals sustained bilateral dorsal hippocampal lesions. Eight other animals sustained bilateral lesions of the neopallium overlying the dorsal hippocampus. All operations were performed in one stage by aspiration, as described previously [10,12]. Ten additional animals underwent no further operations and served as unoperated controls. Five of these rats had, however, undergone bilateral superior cervical ganglionectomy, All operations, including ganglionectomies and brain lesions were performed by one of us (DPK). One non-brain-lesioned rat was discarded due to poor health.

Behavioral Procedures Details of the taste aversion conditioning procedure used have been specified previously [21,22]. Briefly, the animals were trained to drink from bottles in their home cage by presenting a sucrose solution (20% by weight) daily for five consecutive days. For the next five days the novel solution which would serve as the CS was presented for 21 min each day. This solution was prepared by adding 40 g of sugar to 384 ml of Carnation evaporated milk diluted3.5:1 with water. The solutions were presented in volumetric bottles which enabled the experimenter to record consumption to the nearest gram. Laboratory chow and tap water were available ad lib. The extensive preexposure regimen has proved successful in insuring that rats begin to drink immediately upon presentation of the CS, thus producing a relatively constant CS-UCS interval across animals [21,22]. On the fifth day of CS presentation all animals received an IP injection (7.5 ml/kg) of 0.15 M LiCI approximately 30 min following the beginning of the drinking session. Animals were removed from their home cage and taken to an adjacent room where they were injected. Animals were placed into individual cages in the testing room for 2 hr after which they were returned to their home cages. All animals were given 48 hr to recover following LiCI injections. On the third day after LiCI injection the subjects were again presented with the sweetened milk solution (CS) for 21 min consumption sessions. This continued for 14 days. All extinction sessions, as was true for pre-iUness testing sessions, took place during the first hour after onset of light in the colony room. All consumption measurements were taken by one experimenter (LS). Water continued to be available ad lib throughout all phases of the experiment

Histological Procedures Following the completion of the behavioral measures, the brain-lesioned animals were sacrificed by decapitation and the unperfused brains removed and frozen in a cryostat at -30°C. Irises were removed from a number of animals that had and had not undergone surgical removal of the superior cervical ganglia. Irises were removed from the eyes in a procedure described by R. Y. Moore (personal communication). The eyeball was removed, cut along the anteriorposterior midline, and the lens removed and discarded. The anterior half of the eye was placed downward and the iris grasped with iris forceps and laid flat on a glass slide. The slides were placed in a dessicator containing phosphorus pentoxide and kept there overnight. The slides were then transferred to a closed jar containing paraformaldehyde

HIPPOCAMPUS AND TASTE AVERSION

219

\

FIG. 1. Frontal sections through posterior-ventral hippocampal formation showing coarse anomalous noradrenergic innervation in rat with anterior hippocampal lesion (on left) and absence of such anomalous growth in rat with similar lesion but prior removal of superior cervical ganglia. which had been previously equilibrated in an atmosphere of 70% relative humidity. After incubating for an hour at 80°C, the slides were removed, coverslipped in mineral oil and examined under a Zeiss fluorescent microscope. The brains were sectioned at either 16 or 32 microns. Alternate sections were then subjected to a modified glyoxylic acid technique to examine for catecholamine content [4]. These sections were also examined under the fluorescent microscope. The alternate sections were stained with thionin according to BreMiller's procedure [3]. RESULTS Histological Results Following the operation to remove the superior cervical ganglia, a marked ptosis of the rat's eyes was observed, due to cutting the superior cervical trunk and the resultant retraction of the eyeball [14]. There were no observed behavioral consequences of this ptosis. Examination of the irises from either normal or shamoperated animals revealed an extensive network of adrenergic fibers under the fluorescent microscope. Irises from animals with superior cervical ganglia removed showed no fluorescence. The brain sections from the hippocampal-lesioned animals which were stained and examined for catecholamine fluorescence showed the extensive pattern of anomalous sympathetic innervation in the remaining hippocampal tissue first described by Loy and Moore [16]. Figure 1 shows relevant brain sections through hippocampal tissue from rats with and without their superior cervical ganglia removed. This anomalous innervation was found throughout the hippocampus and dentate gyms. This growth is seen as a plexus of intensely fluorescent fibers, much thicker and coarser than are found in normal brains or in our rats which had undergone removal of the superior cervical ganglia. There is a normal fluorescence observed in the hippocampal formation which is due to fibers from the brainstem, particularly the locus coeruleus. This can also be seen in Fig. 1. Only this normal fluorescence was observed in those brains from rats

with superior cervical ganglia removed whereas the anomalous pattern was observed in all of the hippocampal lesioned animals not subjected to prior removal of the superior cervical ganglia. No anomalous fluorescence was seen in rats with lesions confined to the neopallium. The thionin stained sections revealed a pattern of hippocampal damage similar to that reported from our laboratory several times previously [10, II, 12]. There was substantial ablation of the anterior and dorsolateral hippocampus and dentate gyrus, with considerable sparing of the more ventral and posterior aspects. Some entorhinal tissue was destroyed in some animals, and the subiculum was partially invaded in all hippocampal-lesioned rats. In the neopallial lesioned rats there was extensive removal of both white and grey matter overlying the hippocampus, but the hippocampal formation itself was not invaded. Taste Aversion Results In order to analyze the pre-illness data, separate three factor repeated measures ANOVAs were computed for sucrose and milk consumption data. The factors were three central surgical manipulations (hippocampal lesion, neopallial lesion, unoperated) x two peripheral surgical manipulations (superior cervical ganglionectomy, sham operation) x five days of testing. Animals increased their consumption of both sucrose F(4,92)=20.11, p<0.001, and milk F(4,92)=32.57, p<0.001 with repeated presentations. The overall ANOVA showed that neither the central surgical manipulations, F=0.21 (2.23), p>0.25, nor the peripheral surgical manipulations, F=l.12 (1,23), p>0.25, approached statistical significance. Table 1 shows the milk consumption data for the five pre-illness days for the six treatment groups. The postillness data were analyzed in a three factor ANOVA: three central surgical manipulations) x two (peripheral surgical manipulations) x 14 (days of testing). Days of testing was treated as a repeated measures factor. The analysis yielded the significant interaction of central surgical manipulations by days of testing, F= 1.72 (26,299), p=0.053. Inspection of Fig. 2 indicates that this difference developed after the first four days of extinction, as the un-

220

K I M B L E , B R E M I L L E R A N D SCHROEDER TABLE

1

AMOUNT OF CS CONSUMED (ML): PRECONDITIONING DAYS 1-5; DAY I OF POST-ILLNESS

Group Groups

Days 4

1

2

3

10.2 6.8

12.5 7.4

18.5 13.2

NeopaUial Sham Neopallial-SCG

6.7 6.7

10.7 11.0

Hippocampal-Sham Hippocampal-SCG

7.5 7.5

8.0 8.2

Unop-Sham Unop-SCG

5

l(Post-illness)

23.0 18.4

24.0 17.0

6.0 4.0

13.5 14.5

17.5 17.0

22.0 20.5

5.7 2.2

15.8 9.3

20.7 14.2

18.0 19.3

3.8 4.0

operated and neopallial lesioned rats recovered their preillness consumption levels at a faster rate from Day 5 onward, compared with the hippocampal-lesioned rats. Initial acquisition of the taste aversion was evaluated by comparing the amount of milk consumed on the first postillness day with the amount consumed on the last preiUness day (the day of conditioning). All of the nine unoperated controls, seven of the eight neopallial-lesioned rats and 10/12 of the hippocampal-lesioned rats drank less (typically much less) on the first post-illness day. The two hippocampallesioned rats which did not drink less drank exactly the same amount on the two days, and one neopallial-lesioned rat drank 1 ml more on the first postiUness day than it had on the day of conditioning. These data were tested for statistical significance using a Binomial test [20]. The probabilities associated with these results were p <0.002 for the unoperated controls, p<0.035 for the neopallial lesioned rats, and p <0.019 for the hippocampal lesioned rats. As a further test of the acquisition data, individual corn-

parisons of the three central surgical groups on the first postillness day yielded no statistically significant differences in the absolute change in amount of CS (milk) consumed from the last preillness day to the first postillness day. These data were evaluated using a Mann-Whitney U test [20]. The U values associated with these tests were U=52.5 for the unoperated hippocampal-lesion comparison; U =37.5 for the unoperated neopallial-lesion comparison: and U = 4 7 for the neopallial-lesion hippocampal-lesion comparison. As can be seen from Fig. 2, rats with hippocampal damage (with or without superior cervical ganglionectomy) were significantly slower to recover preoperative consumption measures (i.e., they showed a slower extinction of the conditioned taste aversion). In fact, rats with hippocampal damage did not recover these levels during the duration of the 14 day extinction period. Both the unoperated controls and the rats with neopallial lesions had recovered their preoperative consumption levels by Day 11. There was no suggestion in the data that the presence or

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FIG. 2. Consumption of CS during 14 day extinction period. Open circles=normal controls: closed circles=neopallial lesioned controls: triangles=hippocampal lesioned rats.

HIPPOCAMPUS AND TASTE AVERSION absence of the anomalous sympathetic innervation of the remaining hippocampal tissue could be linked to any behavioral changes.

DISCUSSION

The extensive hippocampal lesions produced in the present experiment did not interfere with the acquisition of a conditioned taste aversion following LiCI induced illness. However, the hippocampal-lesioned rats were significantly slower to extinguish this conditioning, as compared with either the neopallial lesioned rats or the unoperated controls. Thus, while our results agree with those of several other investigators that even extensive hippocampal lesions do not seriously interfere with the formation of the association between the gustatory CS and gastrointestinal illness, our results on extinction are different from those reported earlier. In the study most similar in terms of extinction data, Miller et al. [18] found that rats with hippocampal lesions comparable to those in the present study extinguished more rapidly than did either cortical controls or normal controls. Procedural differences exist between the Miller et al. study and our own. In their paradigm animals had almost continual access to both the CS solution (0.05% sodium saccharin) and tap water. In our testing situation access to the CS solution was available for only 21 rain each day. The longer time for "sampling" of the CS in their study might be suggested as an explanation for the difference in our studies. However, examination of our data showed that only one hippocampal lesioned rat failed to drink from the bottle containing the CS on the first extinction day (all of the neopallial-lesioned and unoperated rats in our experiment drank at least 1 ml of the CS on the first postillness testing session). Thus, it is somewhat unlikely that reluctance to taste the CS could account adequately for the differences between the Miller et al. study and our own. While different substances were used to induce the gastrointestinal illness (they used cyclophosphamide, we used LiC1), it is not obvious how this could produce any behavioral differences. One procedural difference may be particularly important: the degree of preillness exposure to the CS solution. In the Miller et al. study the animals were exposed to the CS for only about 15 rain before illness was induced. "The solution was available for a minimum of 10 rain and for at least 5 rain after the onset of ingestion" ([18], p. 124). In our study, the rats had five consecutive daily sessions of 21 rain to drink the CS solution. Perhaps the degree of preillness exposure in our study accelerated the extinction process in our normal and neopallial-lesioned rats, but less so in our hippocampal-lesioned rats. It is difficult to compare our extinction data with that of Miller et al. Our normals and neopallial lesioned rats were consuming preillness amounts of the CS by Day 11 of testing, while those of Miller et al. took about 6 weeks on average to extinguish (by their criterion of two consecutive days over which the mean saccharin consumption equalled or exceeded 50% of the total fluid intake). If the degree of preexposure does influence the faster extinction of the conditioned taste aversion in our normal and neopallial lesioned rats, then presumably our hippocampal lesioned rats were less influenced by this preillness exposure. Recently, after the completion of the present experi-

221 ments, McFarland et al. [17] have published results which also suggest that preconditioning CS exposure had less of an effect on rats with hippocampal lesions in taste aversion conditioning than it did for controls. They found that the preconditioning CS exposure (preexposure) did retard acquisition in their control animals, but not in their rats with hippocampal lesions. While their experimental design did not allow for an examination of extinction of the conditioning, there is no reason to expect that, given the normal acquisition of the aversion in the preexposed hippocampal-lesioned animals, they would not also have been slower to extinguish this response (as in the present study) when compared to the preexposed controls which showed less conditioning. In comparing the McFarland et al. data with our own, one obvious discrepancy is that they found that the CS preexposure retarded conditioning among their controls, an effect we did not see. In the present results, five CS exposures were given. Unpublished results in one of our laboratories (WPS) indicate that using the present conditioning procedures, conditioning can be retarded among controls, but that a minimum of nine preexposure sessions are required to observe this effect. Extinction, on the other hand, may be more sensitive to such preexposure manipulations. This sensitivity has already been demonstrated in the case of ACTH injections during extinction. ACTH injections, like hippocampal lesions, slow the extinction but have no effect on the acquisition of conditioned taste aversion [9]. The abnormal response to CS preexposure may be an important aspect of the behavior of rats with hippocampal lesions in other conditioning situations. Several years ago Ackil e t a l . [1] found that 30 preexposures of the stimulus later used as the CS in a two-way shuttle-avoidance task (800 Hz, 80 dB tone) slowed acquisition and speeded extinction in the unoperated and neopallial-lesioned animals, but not in rats with hippocampal lesions. They interpreted their findings in terms of the role of the hippocampus in attentional and/or inhibitory mechanisms [5,11]. Similarly, Solomon and Moore [23] found that hippocampal lesions eliminate the latent inhibition effect of CS preexposure in rabbits in a nictitating membrane conditioning situation. Thus, in several different conditioning paradigms, hippocampal lesions seem to reduce the impact of CS preexposure. In the present experiments, one may speculate that the long preexposure experience with the CS served to hasten the extinction of the conditioned taste aversion in our animals without hippocampal damage, but was less effective in altering the extinction rate in the hippocampal lesioned rats. Tests of this hypothesis involving parametric manipulations of CS preexposure should shed light on this hypothesis. With respect to the anomalous innervation, we found no evidence that there is any behavioral significance of this abnormal growth. No behavioral differences appeared, either in preillness consumption measures, acquisition of the conditioned taste aversion, or extinction of the conditioning between those rats which showed this growth compared with those in which it was prevented by superior cervical ganglionectomy. The ganglionectomy itself was without any apparent behavioral effect. Thus, while we cannot reject the possibility that this impressive anomalous innervation pattern has some functional effects on the subsequent behavior of the animals, we find no support for this possibility in our present results.

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AND SCHROEDER

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