Amygdala and dorsal hippocampus lesions block the effects of GABAergic drugs on memory storage

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Brain Research, 55 t ( 1991) 11)4-t 09 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 000689939116652,~

BRES 16652

Amygdala and dorsal hippocampus lesions block the effects of GABAergic drugs on memory storage Martine Ammassari-Teule 1, Flaminia Pavone 1, Claudio Castellano I and James L. McGaugh 2 l Istituto di Psieobiologia e Psicofarmacologia del CNR, Roma (Italy) and eCenterfor the Neurobiology of Learning and Memory and Department of Psychobiology, University of California, lrvine, CA (U.S.A.)

(Accepted 8 January 1991) Key words: Amygdala; Bicuculline; Caudate; Hippocampus; Inhibitory avoidance; Lesion; Memory; Muscimol

These experiments examined the effects of posttraining systemic administration of the GABAergic agonist muscimol and the GABAergic antagonist bicuculline on retention in mice with bilateral lesions of the amygdala, dorsal hippocampus or caudate nucleus. Unoperated male CD1 mice and mice with either sham lesions or electrolytically induced lesions of these 3 brain regions were trained in a one-trial inhibitory avoidance task and, immediately after training, received i.p. injections of either muscimol, (1.0, 2.0 or 3.0 mg/kg), bicuculline, (0.25, 0.5 or 1.0 mg/kg), or control solutions. Retention was tested 24 h after training. Lesions of the 3 brain regions produced comparable impairment of retention. In the unoperated controls and sham controls muscimol and bicuculline produced dose-dependent impairment and enhancement, respectively, of retention. The drug effects on retention were blocked by lesions of the amygdala and hippocampus, but were not blocked by lesions of the caudate nucleus. These findings are consistent with other recent evidence suggesting that the amygdala and hippocampus are involved in mediating posttraining neuromodulatory influences on memory storage. INTRODUCTION Retention of newly acquired information can be influenced by posttraining administration of drugs affecting neuromodulatory and neurotransmitter systems 26. Several lines of evidence suggest that drugs and hormones affect m e m o r y through influences involving the amygdala and hippocampus. It is well documented that m e m o r y can be modulated by systemic as well as intra-amygdala administration of drugs affecting opiate and adrenergic receptor systems 14'15'21'23"25'2s. Furthermore, the memory-modulating effects of several treatments are blocked by lesions of the stria terminalis, a major amygdaloid pathway z°'24'25'27. There is also evidence indicating that memory can be influenced by treatments affecting cholinergic and dopaminergic receptors within the hippocampus 18'19"3°. Such findings suggest that the amygdala and hippocampus may be involved in integrating neuromodulatory influences on memory storage. Treatments affecting G A B A e r g i c systems have also been found to influence memory. When administered systemically (i.p.) shortly after training GABAergic agonists (muscimol and baclofen) and antagonists (bicuculline and picrotoxin) impair and enhance, respectively, subsequent retention 7. Such effects have been obtained

in experiments using a variety of learning tasks using appetitive as well as aversive motivation 1'2'4'9"16'32. Further, in previous experiments we have found that, in the doses used to enhance retention, posttraining i.p. injections of bicuculline and picrotoxin are not aversive 4. Thus, the effects of G A B A e r g i c drugs on retention cannot be attributed to aversive influences of the drugs. It seems clear that the memory-modulating effects of systemic injections of these G A B A e r g i c drugs are due to central influences, as they readily pass the blood-brain barrier. Further, we have found that retention is not influenced by posttraining systemic injections of bicuculline methiodide, a drug which does not readily cross the blood-brain barrier 4. Other recent findings suggest that peripherally administered G A B A e r g i c drugs may influence memory through influences on amygdala and hippocampus: G A B A e r g i c drugs also affect retention when administered directly into the amgydala or hippocampus. The effects of intra-amygdala injections of G A B A e r g i c drugs are comparable to those found with systemic injections: muscimol and bicuculline have impairing and enhancing effects, respectively, on retention 5,s. Further, retention is impaired by posttraining intra-hippocampal administration of the G A B A e r g i c antagonist picrotoxin TM. If, as the findings of these recent studies suggest, the

Correspondence: J.L. McGaugh, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, U.S.A.

105 memory-modulating effects of GABAergic drugs are due to influences involving the amygdala and hippocampus, such effects should be blocked by lesions of these brain regions. The present experiments examined this implication. Further, as previous findings indicate retention is not influenced by injections of GABAergic drugs into the caudate nucleus5 these experiments also examined the effect of lesions of the caudate. It was expected that lesions of the caudate would not block the memorymodulating effects of GABAergic drugs.

MATERIALS AND METHODS

Subjecls The subjects were 240 male CD1 mice (Charles River Laboratories, Como, Italy). They weighed approximately 25 g, were caged in groups of 8 with food and water available ad lib and were maintained on a 12h:12h light/dark cycle (lights on at 07.00 h) at a constant temperature of 21 °C for two weeks prior to the surgical treatments and subsequent behavioral experiments.

Statistics The step-through latencies of the unoperated mice given different drug treatments were analyzed with one-way ANOVAs. Retention test latencies of the lesioned and sham-lesioned groups given control and drug injections were also assessed with one-way ANOVAs. Dose-dependent effects were evaluated by subsequent betweengroup comparisons using a Duncan multiple-range test.

Drug administration Immediately after training the mice were injected with a control solution or a solution of the GABAergic antagonist bicuculline (0.25, 0.5 or 1.0 mg/kg) or the GABAergic agonist muscimol (1.0 2.0 or 3.0 mg/kg). Bicuculline (Sigma Chemical Corp., St. Louis, MO) was first dissolved in a few drops of 0. l N HC1 and then diluted to the final volume with a saline (0.9% NaCI) solution. Muscimol (Sigma Chemical Corp.) was dissolved in a saline solution. Saline (S) and a buffer solution (saline + 0.1 N HC1, pH 6.5) (V = vehicle control for bicuculline solution) were used for control treatments. All drug and control solutions were injected i.p., in a volume of 10 ml/kg.

Surgery and histology Mice in some groups received electrolytic lesions of the amygdala, hippocampus or caudate-putamen. Controls for the surgery received sham operations. Under chloral hydrate (400 mg/kg) anesthesia, a stainless steel electrode (0.2 mm diameter), that was insulated except at the tip, was inserted bilaterally into the targeted brain region by means of a stereotaxic apparatus and with lambda and bregma in the same horizontal plane. The stereotaxic coordinates (in mm) were as follows. Amygdala: -1.8 P (posterior to bregma), 3.1 L (lateral to the midline) and 4.3 V (ventral to the skull). Hippocampus: 2.2 P, 2.2 L, and 2.4 V. Caudate-putamen: 0.5 A (anterior to bregma), 2.2 L, 2.7 V. The lesions were induced by delivering a 2.5-mA anodal current through the electrode using a cathode taped to the animal's tail. The procedures were the same for sham operations except that no current was delivered. The mice were allowed to recover from the surgery for one week prior to behavioral testing. At the completion of the behavioral experiments the lesioned and sham-operated animals were sacrificed under chloral anesthesia and their brains were fixed in formalin (10% solution) and sectioned coronally (60 gm) and stained with Toluidine blue according to the Nissl method. Figure 1 shows typical amygdala, hippocampal and caudate lesions.

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Behavioral procedures The animals were trained and tested in an inhibitory avoidance apparatus using procedures described previously ~. The apparatus, a 20 x 20 x 20 cm Lucite box with black walls and a grid floor was placed at the edge of a table. A platform (12 cm long and 7.5 cm wide) extended from a small door (4 × 3 cm) in the front wall of the box. A 40-W lamp was positioned 50 cm above the platform, The inside of the box was dark. Training and testing were conducted between 14.00 and 17.00 h. The mice received a single training trial and a single test trial. On the training trial the mouse was placed on the platform with its head away from the box. When the animal entered the box with all four feet the step-through latency was recorded, the entry door was closed with a sliding door, and a footshock (1.0 s, 0.7 mA, 50 Hz) was delivered. As is described below, drugs or control solutions were administered i.p. immediately following the training trial. The mouse was then returned to its home cage. On the retention test 24 h later, the mouse was placed on the platform and the step-through latency (maximum of 240 s) was recorded. Footshock was not administered on the retention test trial. Groups of 10 mice were used for all experimental conditions.

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Fig. 4. Dose-response effects of posttraining injections of (A) muscimol (MUSC) and (B) bicuculline (BIC) on retention in dorsal hippocampal-lesioned (LES) and sham-lesioned (SHAM) mice.

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4B) produced dose-dependent enhancement of retention (F2,27 = 28.23, P < 0.001). However, bicuculline and muscimol did not significantly affect the retention performance of the hippocampal-lesioned groups (F2,27 = 0.22 and 0.007, respectively, P > 0.05). As is shown in Fig. 5, posttraining injections of bicuculline and muscimol affected the retention performance of both caudate-lesioned and sham-lesioned mice. Muscimol (Fig. 5A) impaired retention in both lesioned and sham groups (F2,27 = 28.89 (lesioned), 101.14 (sham), P < 0.001 for both). Bicuculline (Fig. 5B) enhanced retention in both lesioned and sham groups (F2,27 = 8.62 (lesioned) 28.25 (sham), P < 0.001 for both). DISCUSSION

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:E LES SHAM Fig. 5. Dose-response effects of posttraining of (A) muscimol (MUSC) and (B) bicuculline(BIC) on retention in caudate-lesioned (LES) and sham-lesioned (SHAM) mice. P < 0.02, 0.01, 0.005 for the amygdala, hippocampal and caudate lesioned groups, respectively). There were no significant differences among the 3 lesioned control groups in retention latencies. The effects of immediate posttraining injections of bicuculline and muscimol on the retention performance of amygdala-lesioned and sham-lesioned mice are shown in Fig. 3. As can be seen, in the same groups, muscimol (Fig. 3A) produced dose-dependent impairment of retention (F3,36 = 27.84, P < 0.001), and bicuculline (Fig. 3B) produced dose-dependent enhancement of retention (F3,36 = 13.81 P < 0.001). In contrast, bicuculline and muscimol did not significantly affect the retention performance of the amygdala-lesioned groups ( F 3 , 3 6 = 2.03 and 0.20, respectively, P > 0.05). Figure 4 shows the effects of posttraining injections of bicuculline and muscimol on the retention performance of hippocampal-lesioned and sham-lesioned mice. In the sham-operated groups, muscimol (Fig. 4A) impaired retention (/'2,27 = 75.48, P < 0.001) and bicuculline (Fig.

The central finding of the present experiment is that bilateral lesions of the amygdala and dorsal hippocampus blocked the memory-enhancing effect of posttraining injections of the GABAergic antagonist bicuculline as well as the memory-impairing effect of the GABAergic agonist muscimol, on retention in an inhibitory avoidance task. In contrast, lesions of the caudate did not block the retention-modulating effects of posttraining injections of either bicuculline or muscimol. The lack of effect of the caudate lesions in blocking the drug effects on retention was not due to a 'baseline' or 'ceiling' effect. Lesions of the 3 brain regions produced highly similar impairment in retention performance in the inhibitory avoidance task. The retention latencies of each lesioned group were significantly lower than those of their sham controls. It is unlikely that the effects were due to lesion effects on locomotor activity as the training step-latencies of the lesion and control groups were highly comparable. These findings are consistent with those of previous experiments6,17,22,29,3]" The close-dependent effects of bicuculline and muscimol on the retention performance of the unoperated and sham-lesioned controls seen in the present study are comparable to those found in many previous experiments examining the effects of posttraining injections of GABAergic drugs on memory. Generally, GABAergic agonists impair retention and GABAergic antagonists improve retention 2,4,7,9. Findings indicating that the GABAergic drug effects on retention are time-dependent (i.e. are effective only when administered shortly after training) 4 and are not state-dependent 1°,11 have provided strong evidence for the view that the drugs affect retention by modulating memory storage processes 7. The present findings are also consistent with evidence from other recent studies suggesting that GABAergic drugs affect memory, at least in part, through effects

108 involving the amygdaloid complex and hippocampus. Posttraining intra-amygdala micro-injections of G A B A ergic drugs produce retention-modulating effects comparable to those produced by systemic injections: Retention is enhanced by bicuculline and impaired by muscimol as well as the G A B A - B agonist baclofen 5,8. In contrast, the G A B A e r g i c antagonist picrotoxin produces memoryenhancing effects comparable to those of bicuculline when administered systemically 4.9 but impairs retention when microinjected into the hippocampus after training TM. On the assumption that G A B A e r g i c drugs affect m e m o r y through influences involving the hippocampus it is not clear why different effects are produced by systemic and intra-hippocampal administration. It might be that the difference is due simply to the specific doses used for the intra-hippocampal injections, as it is well documented that drugs that impair memory when administered in low or moderate doses impair retention at higher doses 26. It is also possible that the different actions of bicuculline and picrotoxin on G A B A e r g i c transmission might account for the differential effects of systemic and central injections of picrotoxin on memory. Moreover, it is not as yet clear whether the memorymodulating effects of systemically administered drugs involve activation of G A B A receptors within the hippocampus. The hippocampus might be involved in mediating G A B A e r g i c influences on memory that are initiated in other brain regions. For example, recent studies have reported that, in rats, muscimol injected into the medial septum impairs spatial learning by altering hippocampal cholinergic functioning 3,12,13. Our findings indicating that lesions of the caudate nucleus do not block the memory-modulating effects of G A B A e r g i c drugs are consistent with previous evidence that retention is not influenced by posttraining micro-

injections of bicuculline administered into the caudatc nucleus 5. These findings, considered together, strongly suggest that the effects of G A B A e r g i c drugs on memory are not mediated by activation of G A B A receptors in the caudate nucleus. The findings indicating that lesions of the amygdala and hippocampus block the memory-modulating effects of G A B A e r g i c drugs are consistent with extensive evidence indicating that these limbic structures are involved in mediating neuromodulatory influences on memory storage. Lesions of the stria terminalis, a major amygdala pathway, block the memory modulating effects of treatments affecting adrenergic 24"25, opioid peptidergic 27 and cholinergic 2° systems. Moreover, numerous studies have reported that retention can be influenced by posttraining intra-amygdala and intra-hippocampal injections of drugs affecting several neurotransmitter systems. There is extensive evidence that retention is modulated by treatments affecting noradrenergic and opioid peptidergic receptors within the amygdala 14,~5,21,23,2~, as well as treatments affecting cholinergic and dopaminergic receptors in the hippocampus 18"~9"3°. The present findings are consistent with previous findings of studies using intra-amygdala injections suggesting that the effects of administration of G A B A e r g i c drugs on memory storage are mediated, at least in part, by the amygdala and the hippocampus. Thus, the findings provide additional support for the view that these two brain systems may play complementary roles in integrating neuromodulatory influences on memory storage. Acknowledgements. This research was supported in part by Research Grant MH-12526 from NIMH and NIDA and ONR Contract N00014-87-K-0518 (to J.L.M.). We thank Ines IntroiniCollison, Nancy Collett and Joanne Motoike for their assistance in the preparation of this manuscript.

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