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Post-training systemic and intra-amygdala administration of the GABA-B agonist baclofen impairs retention
BEHAVIORALAND NEURALBIOLOGY52, 170--179 (1989)
Post-training Systemic and Intra-amygdala Administration of the GABA-B Agonist Baclofen Impairs Retention 1 CLAUDIO CASTELLANO,* JORGE D . BRIONI,'~ ALAN H . NAGAHARA,'~'~ AND JAMES L . MCGAUGHt':~ '2
*Istituto di Psicobiologia e Psicofarmacologia del CNR, Via Reno 1 00198, Roma, ltaly, tCenter for the Neurobiology of Learning and Memory, University of California, lrvine, California 92717, and SDepartment of Psychobiology, University of California, Irvine, California 92717 The effects of the GABA-B receptor agonist baclofen-on memory storage were studied in two series of experiments. In the first series, CD-1 mice were trained in two aversively motivated tasks: A one-trial inhibitory avoidance task and a classical conditioning task (conditional emotional response). Immediate posttraining ip administration of ( + / - )baclofen (10 and 30 mg/kg) impaired retention of animals in both tasks. The effect was time-dependent: Retention was not affected by baclofen administered 120 min after training. In the second series of experiments, which used Sprague-Dawley rats, post-training intra-amygdala administration of baclofen impaired retention of an inhibitory avoidance response. These results support the view that the GABAergic system is involved in the modulation of memory storage and that the amygdaloid complex may be a critical site for effects of drugs affecting the GABAergic system. © 1989AcademicPress, Inc.
GABA (y-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian CNS (Krnjevic & Schwartz, 1967; Roberts, 1986). There is evidence that in the mammalian brain there are two different GABA receptor sites: GABA-A and GABA-B (see Bowery, 1982). Baclofen (fl-p-chlorophenyl GABA) mimics the effects of GABA on GABAB receptors (Hill & Bowery, 1981). The findings of a number of studies suggest that GABAergic mechanisms are involved in the regulation of memory storage. Experiments examining memory in rats and mice under a wide variety of experimental conditions have reported that post-training systemic injections of the GABA antagonists bicuculline and picrotoxin enhance the retention of recently acquired information (Breen & 1 This research was supported by Research Grant MH12526 from NIMH and NIDA and Office of Naval Research Contract N00014-87-k-0518. 2 Send correspondence and reprints requests to Dr. James L. McGaugh, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717. 170 0163-1047/89 $3.00 Copyright@ 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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McGaugh, 1961; Bovet, McGaugh & Oliverio, 1966; Garg & Holland, 1968; Garg, 1970; Yonkov & Georgiev, 1985; CasteUano & Pavone, 1988; Brioni & McGaugh, 1988; Castellano & McGaugh, 1989; Castellano, Introini-Collison, Pavone, & McGaugh, 1989). Other recent studies examining retention of an inhibitory avoidance response have reported that post-training systemic injections of GABAergic receptor agonists impair retention. Castellano and Pavone (1988) found that, in mice, retention is impaired by post-training injections of the GABA-A receptor agonist muscimol and Swartzwelder and his colleagues (Swartzwelder, Tilson, McLamb, & Wilson, 1987) reported that, in rats, retention is impaired by post-training injections of baclofen. It is known that post-training intra-amygdala administration of a number of drugs that influence transmitter and neuromodulatory systems can affect retention as tested in a variety of training tasks (see McGaugh, 1989). Such findings suggest that amygdala may play a critical role in the effects, on memory, of systemically administered drugs and hormones (Gallagher & Kapp, 1978; McGaugh, Introini-Collison, & Nagahara, 1988; Introini-Collison, Nagahara, & McGaugh, 1989). Recent findings from our laboratory suggest that the amygdala GABAergic system may be involved in the modulation of memory storage. We found that, in rats trained in an inhibitory avoidance task, retention was modulated by posttraining intra-amygdala injections of GABAergic drugs: Retention was enhanced by the GABA antagonist bicuculline methiodide and impaired by the GABA-A agonist muscimol (Brioni, Nagahara, & McGaugh, 1989). The present experiments examined further the effects of post-training baclofen on retention. A first experiment examined the effects of posttraining systemic injections of baclofen in two aversively motivated training tasks: A one-trial inhibitory avoidance and a classical conditioning task in which a conditioned emotional response (CER) is used to assess retention (Brioni & Izquierdo, 1988). A second experiment, which used rats as subjects, examined the effect of post-training intra-amygdala administration of baclofen on retention of a one-trial inhibitory avoidance task. METHODS Animals
Male CD1 mice (60 days old), supplied by Charles River Laboratories (Wilmington, MA), were caged in groups of six with food and water continuously available. The mice were acclimated to the laboratory conditions for one week prior to training and were maintained on a 12-h light-dark period (light on 7:00 aM). Male Sprague-Dawley rats (60 days old, 200-220 g on arrival) from Charles River Laboratories were individually housed upon arrival and maintained on a 12-h light-dark period
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(lights on at 7:00 AM) with food and water available ad libitum. They were acclimatized to laboratory conditions for one week before undergoing amygdala cannulae-implantation surgery. The animals were allowed one week of recovery after surgery.
Surgery Amygdala cannulae implantation. The animals were anesthetized with sodium pentobarbital (50 mg/kg, ip), and given atropine sulfate (0.4 mg/kg, ip). The skull of the rat was fixed to a stereotaxic frame (Kopf Instruments) and permanent stainless-steel guide cannulae (23 gauge; 15 mm) were implanted bilaterally. The tips were aimed at the dorsal surface of the amygdala. The stereotaxic coordinates were A.P., - 2 . 3 from bregma; M.L., + / - 4 . 4 mm from midline; D.V., - 5 . 5 mm from dura; with the nosebar at - 3 . 3 mm from the interaural line (Paxinos & Watson, 1986). The cannulae were fixed to the skull using two screws and dental acrylic. A stylet was inserted into each cannula to keep the guide cannulae patent. Immediately after surgery, animals received an intramuscular injection of penicillin G and were maintained in a temperaturecontrolled chamber until recovery from anesthesia.
Apparatus and Procedures Inhibitory avoidance (mice). Mice were trained on a step-through inhibitory avoidance apparatus as previously described (McGaugh & Landfield, 1970). On the training day each mouse was placed in the lighted compartment, facing the dark side, and the door leading to the dark compartment was opened. When the mouse stepped with four paws into the dark side, the door was closed, a footshock (0.5 mA, 60 Hz, 2 s) was delivered and the latency to step-through was recorded. The mouse was then removed from the apparatus and injected intraperitoneally. Retention was tested 24 h later following a similar procedure. A maximum step-through latency of 300 s was recorded on the retention trial. The retention step-through latency minus training step-through latency was used as the retention score of each animal. Inhibitory avoidance (rats). The rats were trained on a step-through inhibitory avoidance apparatus as previously described (McGaugh et al., 1988). On the training day each animal was placed in the lighted compartment, facing the dark side, and the door leading to the dark compartment was opened. When the rat stepped with four paws into the dark side, the door was closed, a footshock (0.45 mA, 60 Hz, 1 s) was delivered and the latency to step-through was recorded. The rat was then removed from the apparatus and immediately injected intra-amygdally via the chronic cannulae. On the retention test, 48 h later, the rat was placed in the lighted compartment as on the training session and the step-through latency (maximum of 300 s) was recorded. The retention
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step-through latency minus training step-through latency was used as the retention score for each animal. CER conditioning. Mice were trained to acquire a conditioned emotional response using a tone cue repeatedly paired with a brief footshock in a closed compartment. The animals were placed in one of the arms of a trough-shaped radial Y-maze. The compartment was 13.5 cm high, 15.5 cm long, and 11.5 cm wide at the top and 2.5 cm wide at the base. Starting after 15 s, a 5-s, 1-kHz tone was delivered 20 times, at 20-s intervals, through a loudspeaker placed 20 cm away from the training apparatus. The last 2 s of each tone overlapped with a footshock delivered nominally at 0.75 mA to the floor of the closed compartment. Training therefore involved a classically conditioning procedure and lasted 400 s. In the test session 24 h later, the animals were again placed in one of the arms of the maze as in the training session, the doors of the three compartments were open, and the total number of the entries into the arms of the Y-maze were recorded. The tone was presented immediately after the animals were placed in the apparatus and remained on during the test session which lasted 120 s. Retention of the conditioned emotional response was measured by the decrease in the number of arm entries relative to that of the animals exposed only to the tone in the training session. Intra-amygdala injection. Immediately after the training session in the inhibitory avoidance task, rats received simultaneous bilateral injections via the amygdala cannulae. The rats were gently restrained by hand, the stylets were withdrawn from the guide cannulae, and 30-gauge injection needles were inserted. The injection needles were attached by a polyethylene tube to a 5-txl syringe driven by a minipump. The injection needles were slightly bent so that when they were inserted into the guide cannulae the bend would prevent further penetration and the tips would thus protrude 2 mm beyond the tip of the implanted guide cannulae. Baclofen was administered in a total volume of 0.5 txl at a rate of 0.75 ~l/min. The injection needles were retained in the guide cannulae for an additional 30 s after completion of the injection. Drug administration. For the experiments using mice, ( + / - )baclofen (Research Biochemical, Inc.) was dissolved in saline and injected ip in a volume of 10 ml/kg. Control animals received saline injections in the same volume. For the experiment using rats, baclofen was dissolved in a saline solution and administered bilaterally into the amygdala in doses of 0.01 and 0.1 nmole per cannula (2.13 and 21.3 ng, respectively). Control animals received saline injections (0.5/xl). Histology. Cannulae placements were verified histologically. The rats were anesthetized with an overdose of sodium pentobarbital and perfused through the heart with saline solution followed by 10% formaldehyde solution. Slices (40 /xm) revealing the position of the cannulae were
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stained with Cresyl violet. Rats with incorrect placement (fewer than 10%) of the cannulae were excluded from the analyses. Statistics. Data were analyzed by the Kruskal-Wallis one-way analysis of variance. Individual comparisons were carried out by means of MannWhitney U tests (two tailed). EXPERIMENT 1
The first set of experiments examined, in mice, the effects of posttraining intraperitoneal administration of baclofen on retention of the two tasks: Inhibitory avoidance and CER. In both experiments mice received ip injections of baclofen (3, 10, or 30 mg/kg) immediately after training. An additional group in each experiment was injected with baclofen (10 mg/kg) 120 min after training to determine whether the effect on subsequent retention varied with the training-testing interval. Groups of 24 animals were used for the inhibitory avoidance experiments, and for the classical conditioning task groups of 9-12 animals were employed. Finally, to assess possible nonspecific effects of the baclofen injection, groups of 10 animals for each experimental condition did not receive footshock but were injected with baclofen (10 and 30 mg/kg) immediately after training and tested 24 h later. The performance of each group was compared with that of saline-injected mice. Results. As is shown in Fig. l, the post-training injection of baclofen produced a significant impairment of retention in the IA task (H = 56.7; p < .0001) and in the CER task (H = 32.7; p < .0001), with significant effects at the 10 and 30 mg/kg doses. The memory-impairing effects of post-training injections of baclofen were also time-dependent: As shown in Fig. 2, the retention latencies of mice injected with the drug 120 min after training did not differ from those of the saline control group. Further, in both tasks, the step-through latencies of mice that did not receive footshock, but were injected with saline or baclofen immediately after training, did not differ significantly from each other (Table 1). EXPERIMENT 2
The second experiment examined the effects, in rats, of post-training intra-amygdala injections of baclofen. The aim of this study was to determine whether the GABAergic system in the amygdala could be the central site of action of peripherally injected baclofen. For that purpose, rats with chronically implanted cannulae in the amygdala were trained on the inhibitory avoidance task and given bilateral intra-amygdala injections immediately following training. Results. The effects of post-training intra-amygdala injections of baclofen are shown in Fig. 3. There was a significant effect of baclofen on retention of the IA task (H = 6.1 ; p < .05). The lower dose of baclofen
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(0.01 nmole) significantly impaired retention performance. However, the higher dose (0.1 nmole) was ineffective. DISCUSSION
The findings of Experiment 1 clearly indicate that, in mice, retention is impaired by post-training systemic injections of baclofen. Comparable effects were obtained with the two tasks: Inhibitory avoidance and classical conditioning of a CER. The effects were both dose-dependent and time-dependent: Retention was not affected by injections of baclofen
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Fro. 2. Time-dependency of the effect of post-training ip injections of baclofen on retention. (A) Inhibitory avoidance: Median (IQ) testing-training step-through latencies in seconds. (B) Conditioned emotional response: Median (IQ) number of arm entries. N = 18 mice per group for inhibitory avoidance and 12 per group for the CER. **p < .01 versus saline-injected mice (Mann-Whitney U test). TABLE 1 Effect of Baclofen on Performance of Mice not Given Footshock on the Training Day
Saline Baclofen (10 mg/kg) Baclofen (30 mg/kg)
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Classical conditioning
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administered 2 h following training. Furthermore, injections of baclofen did not affect retention test performance of animals that were not given footshock on the training. Thus, the effects of baclofen on retention are not attributable to nonspecific influences on response latencies. Considered together, these findings are consistent with the view that baclofen impairs retention performance by interfering with post-training processes involved in memory storage. These findings are consistent with those of studies of the effects of GABAergic antagonists on memory. There is now extensive evidence that picrotoxin, which blocks GABAergic synaptic transmission by interaction occurring at the level of the chloride ionophore (Olsen, 1981; Olsen, Ticku & Miller, 1978), enhances memory storage in rats (Breen & McGaugh, 1961; Garg & Holland, 1968; Yonkov & Georgiev, 1985) as well as mice (Bovet et al., 1966; Castellano & Pavone, 1988) tested under a variety of experimental conditions. Recent studies have shown that, in mice, retention of an inhibitory avoidance response is also enhanced by post-training administration of the specific GABA receptor antagonist bicuculline. (Castellano & Pavone, 1988; Brioni & McGaugh, 1988). Considered together, the findings strongly suggest that GABAergic mechanisms may play a role in the modulation of memory storage processes. The effects of baclofen on retention seen in the present experiments are highly comparable to those previously found with muscimol (Cas-
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tellano and Pavone, 1988). The findings are also consistent with previous evidence indicating that, in rats, retention of an inhibitory avoidance response is impaired by post-training systemic injections of baclofen (Swartzwelder et al., 1987). Our experiments extend the findings to mice as well as to a CER task. Thus, in experiments using different tasks and species there does not appear to be receptor specificity with respect to the effects of GABAergic agonists on memory. There is extensive evidence demonstrating the presence of GABAergic neurons in the amygdaloid area (Ben-Ari, Kanazawa, & Zigmond, 1976; Carlsen, 1988; McDonald, 1985; Nagai, McGeer & McGeer, 1983). Furthermore, autoradiographic techniques that allow the determination of the quantitative distribution of the two GABAergic subtypes, GABA-A and GABA-B, have demonstrated that the concentration of GABA-B sites in the amygdala is even higher than the concentration of GABAA sites (Bowery, Hudson, & Price, 1987). Considering the high concentration of GABA-B sites in the amygdala, which is a structure critically involved in the modulation of memory processes (Brioni et al., 1989; McGaugh et al., 1988; McGaugh, 1989), we also evaluated the effect of baclofen injections in the amygdala after training in an inhibitory avoidance paradigm. The findings of Experiment 2 show that intra-amygdala injections of baclofen induced a significant impairment of retention. Thus, our findings indicating that retention is impaired by intra-amygdala as well as systemic injections of baclofen provide additional evidence suggesting that the GABAergic system within the amygdaloid complex may be involved in the modulating influence of drugs affecting the GABAergic system. REFERENCES Ben-Ari, Y., Kanazawa, I., & Zigmond, R. E. (1976). Regional distribution of glutamate decarboxylase and GABA within the amygdaloid complex and stria terminalis system of the rat. Journal of Neurochemistry, 26, 1279-1283. Bovet, D., McGaugh, J. L., & Oliverio, A. (1966). Effects of post-trial administration of drugs on avoidance learning in mice. Life Sciences, 5, 1309-1315. Bowery, N. G. (1982). Baclofen: 10 years on. Trends in Pharmacological Sciences, 3, 400-403. Bowery, N. G., Hudson, A. L., & Price, G. W. (1987). GABA-A and GABA-B receptor site distribution in the rat central nervous system. Neuroscience, 20, 365-383. B reen, R. A., & McGaugh, J. L. (1961). Facilitation of maze learning with posttrial injection of picrotoxin. Journal of Comparative and Physiological Psychology, 54, 498-501. Brioni, J. D., & Izquierdo, I. (1988). Retention enhancement by pre-test/3-endorphin and oxotremorine and its reversal by scopolamine. Behavioral and Neural Biology, 50, 251-254. Brioni, J. D., & McGaugh, J. L. (1988). Post-training administration of GABAergic antagonists enhances retention of aversively motivated tasks. Psychopharmacology, 96, 505-510. Brioni, J. D., Nagahara, A. H., & McGaugh, J. L. (1989). Involvement of the amygdala GABAergic system in the modulation of memory storage. Brain Research, 487, 105112.
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Carlsen, J. (1988). Immunocytochemical localization of glutamate decarboxylase in the rat basolateral amygdala nuclei, with special reference to GABAergic innervation of amygdalo-striatal projection neurons. Journal of Comparative Neurology, 273, 513-526. Castellano, C., & McGaugh, J. L. (1989). Retention enhancement with post-training picrotoxin: Lack of state-dependency. Behavioral and Neural Biology, 51, 165-170. Castellano, C., & Pavone, F. (1988). Effects of ethanol on passive avoidance behavior in the mouse: Involvement of GABAergic mechanisms. Pharmacology, Biochemistry and Behavior, 29, 321-324. Castellano, C., Introini-Collison, I. B., Pavone, F., & McGaugh, J. L. (1989). Effects of naloxone and naltrexone on memory consolidation in CD1 mice: Involvement of GABAergic mechanisms. Pharmacology, Biochemistry and Behavior, 32, 563-567. Gallagher, M., & Kapp, B. S. (1978). Manipulation of opiate activity in the amygdala alters memory processes. Life Sciences, 23, 1973-1978. Garg, M. (1970). Combined effect of drug and drive on the consolidation process. Psychopharmacologia, 18, 172-179. Garg, M., & Holland, H. C. (1968). Consolidation and maze learning: The effects of posttrial injections of a stimulant drug (picrotoxin). Psychopharmacologia, 12, 96-103. Hill, D. R., & Bowery, N. G. (1981). 3H Baclofen and 3H-GABA bind to bicuculline insensitive GABA-B sites in rat brain. Nature (London), 290, 149-152. Introini-Collison, I. B., Nagahara, A. H., & McGaugh, J. L. (1989). Memory enhancement with intra-amygdala post-training naloxone is blocked by concurrent administration of propranolol. Brain Research, 476, 94-101. Krnjevic, K., and Schwartz, S. (1967). The action of gamma-aminobutyric acid on cortical neurons. Experimental Brain Research, 3, 320-336. McDonald, A. J. (1985). Immunohistochemical identification of 3,-aminobutyric acid-containing neurons in the rat basolateral amygdala. Neuroscience Letters, 53, 203-207. McGaugh, J. L. (1989). Involvement of neuromodulatory and memory systems in the regulation of memory storage. Annual Review of Neuroscience, 12, 255-287. McGaugh, J. L., Introini-Collison, I. B., & Nagahara, A. H. (1988). Memory-enhancing effects of post-training naloxone: Involvement of/3-noradrenergic influences in the amygdaloid complex. Brain Research, 446, 37-49. McGaugh, J. L., & Landfield, P. W. (1970). Delayed development of amnesia following electroconvulsive shock. Physiology and Behavior, 5, 1109-1113. Nagai, T., McGeer, P. L., & McGeer, E, G. (1983). Distribution of GABA-T intensive neurons in the rat forebrain and midbrain. Journal of Comparative Neurology, 218, 220-238. Olsen, R. W. (1981). GABA-benzodiazepine-barbiturate receptor interactions. Journal of Neurochemistry, 37, 1-13. Olsen, R. W., Ticku, M. K., & Miller, T. (1978). Dihydropicrotoxin in binding to crayfish muscle sites probably related to gamma-aminobutyric acid receptor ionophores. Molecular Pharmacology, 14, 381-390. Paxinos, G., & Watson, C. (1986). The rat brain in stereotaxic coordinates. New York: Academic Press. Roberts, E. (1986). GABA: The road to neurotransmitter status. In R. V. Olsen & J. C. Venter (Eds.), Benzodiazepine/Gaba receptors and chloride channels: Structural and functional properties (pp. 1-39). New York: Liss. Swartzwelder, H. S., Tilson, H. A., McLamb, R. L., & Wilson, W. A. (1987). Baclofen disrupts passive avoidance retention in rats. Psychopharmacology, 92, 398-401. Yonkov, D. I., & Georgiev, V. P. (1985). Memory effects of GABAergic antagonists in rats trained with two-way active avoidance tasks. Acta Physiologica and Pharmacologica Bulgarica, 11, 44-49.
Post-training Systemic and Intra-amygdala Administration of the GABA-B Agonist Baclofen Impairs Retention 1 CLAUDIO CASTELLANO,* JORGE D . BRIONI,'~ ALAN H . NAGAHARA,'~'~ AND JAMES L . MCGAUGHt':~ '2
*Istituto di Psicobiologia e Psicofarmacologia del CNR, Via Reno 1 00198, Roma, ltaly, tCenter for the Neurobiology of Learning and Memory, University of California, lrvine, California 92717, and SDepartment of Psychobiology, University of California, Irvine, California 92717 The effects of the GABA-B receptor agonist baclofen-on memory storage were studied in two series of experiments. In the first series, CD-1 mice were trained in two aversively motivated tasks: A one-trial inhibitory avoidance task and a classical conditioning task (conditional emotional response). Immediate posttraining ip administration of ( + / - )baclofen (10 and 30 mg/kg) impaired retention of animals in both tasks. The effect was time-dependent: Retention was not affected by baclofen administered 120 min after training. In the second series of experiments, which used Sprague-Dawley rats, post-training intra-amygdala administration of baclofen impaired retention of an inhibitory avoidance response. These results support the view that the GABAergic system is involved in the modulation of memory storage and that the amygdaloid complex may be a critical site for effects of drugs affecting the GABAergic system. © 1989AcademicPress, Inc.
GABA (y-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian CNS (Krnjevic & Schwartz, 1967; Roberts, 1986). There is evidence that in the mammalian brain there are two different GABA receptor sites: GABA-A and GABA-B (see Bowery, 1982). Baclofen (fl-p-chlorophenyl GABA) mimics the effects of GABA on GABAB receptors (Hill & Bowery, 1981). The findings of a number of studies suggest that GABAergic mechanisms are involved in the regulation of memory storage. Experiments examining memory in rats and mice under a wide variety of experimental conditions have reported that post-training systemic injections of the GABA antagonists bicuculline and picrotoxin enhance the retention of recently acquired information (Breen & 1 This research was supported by Research Grant MH12526 from NIMH and NIDA and Office of Naval Research Contract N00014-87-k-0518. 2 Send correspondence and reprints requests to Dr. James L. McGaugh, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717. 170 0163-1047/89 $3.00 Copyright@ 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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McGaugh, 1961; Bovet, McGaugh & Oliverio, 1966; Garg & Holland, 1968; Garg, 1970; Yonkov & Georgiev, 1985; CasteUano & Pavone, 1988; Brioni & McGaugh, 1988; Castellano & McGaugh, 1989; Castellano, Introini-Collison, Pavone, & McGaugh, 1989). Other recent studies examining retention of an inhibitory avoidance response have reported that post-training systemic injections of GABAergic receptor agonists impair retention. Castellano and Pavone (1988) found that, in mice, retention is impaired by post-training injections of the GABA-A receptor agonist muscimol and Swartzwelder and his colleagues (Swartzwelder, Tilson, McLamb, & Wilson, 1987) reported that, in rats, retention is impaired by post-training injections of baclofen. It is known that post-training intra-amygdala administration of a number of drugs that influence transmitter and neuromodulatory systems can affect retention as tested in a variety of training tasks (see McGaugh, 1989). Such findings suggest that amygdala may play a critical role in the effects, on memory, of systemically administered drugs and hormones (Gallagher & Kapp, 1978; McGaugh, Introini-Collison, & Nagahara, 1988; Introini-Collison, Nagahara, & McGaugh, 1989). Recent findings from our laboratory suggest that the amygdala GABAergic system may be involved in the modulation of memory storage. We found that, in rats trained in an inhibitory avoidance task, retention was modulated by posttraining intra-amygdala injections of GABAergic drugs: Retention was enhanced by the GABA antagonist bicuculline methiodide and impaired by the GABA-A agonist muscimol (Brioni, Nagahara, & McGaugh, 1989). The present experiments examined further the effects of post-training baclofen on retention. A first experiment examined the effects of posttraining systemic injections of baclofen in two aversively motivated training tasks: A one-trial inhibitory avoidance and a classical conditioning task in which a conditioned emotional response (CER) is used to assess retention (Brioni & Izquierdo, 1988). A second experiment, which used rats as subjects, examined the effect of post-training intra-amygdala administration of baclofen on retention of a one-trial inhibitory avoidance task. METHODS Animals
Male CD1 mice (60 days old), supplied by Charles River Laboratories (Wilmington, MA), were caged in groups of six with food and water continuously available. The mice were acclimated to the laboratory conditions for one week prior to training and were maintained on a 12-h light-dark period (light on 7:00 aM). Male Sprague-Dawley rats (60 days old, 200-220 g on arrival) from Charles River Laboratories were individually housed upon arrival and maintained on a 12-h light-dark period
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(lights on at 7:00 AM) with food and water available ad libitum. They were acclimatized to laboratory conditions for one week before undergoing amygdala cannulae-implantation surgery. The animals were allowed one week of recovery after surgery.
Surgery Amygdala cannulae implantation. The animals were anesthetized with sodium pentobarbital (50 mg/kg, ip), and given atropine sulfate (0.4 mg/kg, ip). The skull of the rat was fixed to a stereotaxic frame (Kopf Instruments) and permanent stainless-steel guide cannulae (23 gauge; 15 mm) were implanted bilaterally. The tips were aimed at the dorsal surface of the amygdala. The stereotaxic coordinates were A.P., - 2 . 3 from bregma; M.L., + / - 4 . 4 mm from midline; D.V., - 5 . 5 mm from dura; with the nosebar at - 3 . 3 mm from the interaural line (Paxinos & Watson, 1986). The cannulae were fixed to the skull using two screws and dental acrylic. A stylet was inserted into each cannula to keep the guide cannulae patent. Immediately after surgery, animals received an intramuscular injection of penicillin G and were maintained in a temperaturecontrolled chamber until recovery from anesthesia.
Apparatus and Procedures Inhibitory avoidance (mice). Mice were trained on a step-through inhibitory avoidance apparatus as previously described (McGaugh & Landfield, 1970). On the training day each mouse was placed in the lighted compartment, facing the dark side, and the door leading to the dark compartment was opened. When the mouse stepped with four paws into the dark side, the door was closed, a footshock (0.5 mA, 60 Hz, 2 s) was delivered and the latency to step-through was recorded. The mouse was then removed from the apparatus and injected intraperitoneally. Retention was tested 24 h later following a similar procedure. A maximum step-through latency of 300 s was recorded on the retention trial. The retention step-through latency minus training step-through latency was used as the retention score of each animal. Inhibitory avoidance (rats). The rats were trained on a step-through inhibitory avoidance apparatus as previously described (McGaugh et al., 1988). On the training day each animal was placed in the lighted compartment, facing the dark side, and the door leading to the dark compartment was opened. When the rat stepped with four paws into the dark side, the door was closed, a footshock (0.45 mA, 60 Hz, 1 s) was delivered and the latency to step-through was recorded. The rat was then removed from the apparatus and immediately injected intra-amygdally via the chronic cannulae. On the retention test, 48 h later, the rat was placed in the lighted compartment as on the training session and the step-through latency (maximum of 300 s) was recorded. The retention
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step-through latency minus training step-through latency was used as the retention score for each animal. CER conditioning. Mice were trained to acquire a conditioned emotional response using a tone cue repeatedly paired with a brief footshock in a closed compartment. The animals were placed in one of the arms of a trough-shaped radial Y-maze. The compartment was 13.5 cm high, 15.5 cm long, and 11.5 cm wide at the top and 2.5 cm wide at the base. Starting after 15 s, a 5-s, 1-kHz tone was delivered 20 times, at 20-s intervals, through a loudspeaker placed 20 cm away from the training apparatus. The last 2 s of each tone overlapped with a footshock delivered nominally at 0.75 mA to the floor of the closed compartment. Training therefore involved a classically conditioning procedure and lasted 400 s. In the test session 24 h later, the animals were again placed in one of the arms of the maze as in the training session, the doors of the three compartments were open, and the total number of the entries into the arms of the Y-maze were recorded. The tone was presented immediately after the animals were placed in the apparatus and remained on during the test session which lasted 120 s. Retention of the conditioned emotional response was measured by the decrease in the number of arm entries relative to that of the animals exposed only to the tone in the training session. Intra-amygdala injection. Immediately after the training session in the inhibitory avoidance task, rats received simultaneous bilateral injections via the amygdala cannulae. The rats were gently restrained by hand, the stylets were withdrawn from the guide cannulae, and 30-gauge injection needles were inserted. The injection needles were attached by a polyethylene tube to a 5-txl syringe driven by a minipump. The injection needles were slightly bent so that when they were inserted into the guide cannulae the bend would prevent further penetration and the tips would thus protrude 2 mm beyond the tip of the implanted guide cannulae. Baclofen was administered in a total volume of 0.5 txl at a rate of 0.75 ~l/min. The injection needles were retained in the guide cannulae for an additional 30 s after completion of the injection. Drug administration. For the experiments using mice, ( + / - )baclofen (Research Biochemical, Inc.) was dissolved in saline and injected ip in a volume of 10 ml/kg. Control animals received saline injections in the same volume. For the experiment using rats, baclofen was dissolved in a saline solution and administered bilaterally into the amygdala in doses of 0.01 and 0.1 nmole per cannula (2.13 and 21.3 ng, respectively). Control animals received saline injections (0.5/xl). Histology. Cannulae placements were verified histologically. The rats were anesthetized with an overdose of sodium pentobarbital and perfused through the heart with saline solution followed by 10% formaldehyde solution. Slices (40 /xm) revealing the position of the cannulae were
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stained with Cresyl violet. Rats with incorrect placement (fewer than 10%) of the cannulae were excluded from the analyses. Statistics. Data were analyzed by the Kruskal-Wallis one-way analysis of variance. Individual comparisons were carried out by means of MannWhitney U tests (two tailed). EXPERIMENT 1
The first set of experiments examined, in mice, the effects of posttraining intraperitoneal administration of baclofen on retention of the two tasks: Inhibitory avoidance and CER. In both experiments mice received ip injections of baclofen (3, 10, or 30 mg/kg) immediately after training. An additional group in each experiment was injected with baclofen (10 mg/kg) 120 min after training to determine whether the effect on subsequent retention varied with the training-testing interval. Groups of 24 animals were used for the inhibitory avoidance experiments, and for the classical conditioning task groups of 9-12 animals were employed. Finally, to assess possible nonspecific effects of the baclofen injection, groups of 10 animals for each experimental condition did not receive footshock but were injected with baclofen (10 and 30 mg/kg) immediately after training and tested 24 h later. The performance of each group was compared with that of saline-injected mice. Results. As is shown in Fig. l, the post-training injection of baclofen produced a significant impairment of retention in the IA task (H = 56.7; p < .0001) and in the CER task (H = 32.7; p < .0001), with significant effects at the 10 and 30 mg/kg doses. The memory-impairing effects of post-training injections of baclofen were also time-dependent: As shown in Fig. 2, the retention latencies of mice injected with the drug 120 min after training did not differ from those of the saline control group. Further, in both tasks, the step-through latencies of mice that did not receive footshock, but were injected with saline or baclofen immediately after training, did not differ significantly from each other (Table 1). EXPERIMENT 2
The second experiment examined the effects, in rats, of post-training intra-amygdala injections of baclofen. The aim of this study was to determine whether the GABAergic system in the amygdala could be the central site of action of peripherally injected baclofen. For that purpose, rats with chronically implanted cannulae in the amygdala were trained on the inhibitory avoidance task and given bilateral intra-amygdala injections immediately following training. Results. The effects of post-training intra-amygdala injections of baclofen are shown in Fig. 3. There was a significant effect of baclofen on retention of the IA task (H = 6.1 ; p < .05). The lower dose of baclofen
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(0.01 nmole) significantly impaired retention performance. However, the higher dose (0.1 nmole) was ineffective. DISCUSSION
The findings of Experiment 1 clearly indicate that, in mice, retention is impaired by post-training systemic injections of baclofen. Comparable effects were obtained with the two tasks: Inhibitory avoidance and classical conditioning of a CER. The effects were both dose-dependent and time-dependent: Retention was not affected by injections of baclofen
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Classical conditioning
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administered 2 h following training. Furthermore, injections of baclofen did not affect retention test performance of animals that were not given footshock on the training. Thus, the effects of baclofen on retention are not attributable to nonspecific influences on response latencies. Considered together, these findings are consistent with the view that baclofen impairs retention performance by interfering with post-training processes involved in memory storage. These findings are consistent with those of studies of the effects of GABAergic antagonists on memory. There is now extensive evidence that picrotoxin, which blocks GABAergic synaptic transmission by interaction occurring at the level of the chloride ionophore (Olsen, 1981; Olsen, Ticku & Miller, 1978), enhances memory storage in rats (Breen & McGaugh, 1961; Garg & Holland, 1968; Yonkov & Georgiev, 1985) as well as mice (Bovet et al., 1966; Castellano & Pavone, 1988) tested under a variety of experimental conditions. Recent studies have shown that, in mice, retention of an inhibitory avoidance response is also enhanced by post-training administration of the specific GABA receptor antagonist bicuculline. (Castellano & Pavone, 1988; Brioni & McGaugh, 1988). Considered together, the findings strongly suggest that GABAergic mechanisms may play a role in the modulation of memory storage processes. The effects of baclofen on retention seen in the present experiments are highly comparable to those previously found with muscimol (Cas-
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tellano and Pavone, 1988). The findings are also consistent with previous evidence indicating that, in rats, retention of an inhibitory avoidance response is impaired by post-training systemic injections of baclofen (Swartzwelder et al., 1987). Our experiments extend the findings to mice as well as to a CER task. Thus, in experiments using different tasks and species there does not appear to be receptor specificity with respect to the effects of GABAergic agonists on memory. There is extensive evidence demonstrating the presence of GABAergic neurons in the amygdaloid area (Ben-Ari, Kanazawa, & Zigmond, 1976; Carlsen, 1988; McDonald, 1985; Nagai, McGeer & McGeer, 1983). Furthermore, autoradiographic techniques that allow the determination of the quantitative distribution of the two GABAergic subtypes, GABA-A and GABA-B, have demonstrated that the concentration of GABA-B sites in the amygdala is even higher than the concentration of GABAA sites (Bowery, Hudson, & Price, 1987). Considering the high concentration of GABA-B sites in the amygdala, which is a structure critically involved in the modulation of memory processes (Brioni et al., 1989; McGaugh et al., 1988; McGaugh, 1989), we also evaluated the effect of baclofen injections in the amygdala after training in an inhibitory avoidance paradigm. The findings of Experiment 2 show that intra-amygdala injections of baclofen induced a significant impairment of retention. Thus, our findings indicating that retention is impaired by intra-amygdala as well as systemic injections of baclofen provide additional evidence suggesting that the GABAergic system within the amygdaloid complex may be involved in the modulating influence of drugs affecting the GABAergic system. REFERENCES Ben-Ari, Y., Kanazawa, I., & Zigmond, R. E. (1976). Regional distribution of glutamate decarboxylase and GABA within the amygdaloid complex and stria terminalis system of the rat. Journal of Neurochemistry, 26, 1279-1283. Bovet, D., McGaugh, J. L., & Oliverio, A. (1966). Effects of post-trial administration of drugs on avoidance learning in mice. Life Sciences, 5, 1309-1315. Bowery, N. G. (1982). Baclofen: 10 years on. Trends in Pharmacological Sciences, 3, 400-403. Bowery, N. G., Hudson, A. L., & Price, G. W. (1987). GABA-A and GABA-B receptor site distribution in the rat central nervous system. Neuroscience, 20, 365-383. B reen, R. A., & McGaugh, J. L. (1961). Facilitation of maze learning with posttrial injection of picrotoxin. Journal of Comparative and Physiological Psychology, 54, 498-501. Brioni, J. D., & Izquierdo, I. (1988). Retention enhancement by pre-test/3-endorphin and oxotremorine and its reversal by scopolamine. Behavioral and Neural Biology, 50, 251-254. Brioni, J. D., & McGaugh, J. L. (1988). Post-training administration of GABAergic antagonists enhances retention of aversively motivated tasks. Psychopharmacology, 96, 505-510. Brioni, J. D., Nagahara, A. H., & McGaugh, J. L. (1989). Involvement of the amygdala GABAergic system in the modulation of memory storage. Brain Research, 487, 105112.
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Carlsen, J. (1988). Immunocytochemical localization of glutamate decarboxylase in the rat basolateral amygdala nuclei, with special reference to GABAergic innervation of amygdalo-striatal projection neurons. Journal of Comparative Neurology, 273, 513-526. Castellano, C., & McGaugh, J. L. (1989). Retention enhancement with post-training picrotoxin: Lack of state-dependency. Behavioral and Neural Biology, 51, 165-170. Castellano, C., & Pavone, F. (1988). Effects of ethanol on passive avoidance behavior in the mouse: Involvement of GABAergic mechanisms. Pharmacology, Biochemistry and Behavior, 29, 321-324. Castellano, C., Introini-Collison, I. B., Pavone, F., & McGaugh, J. L. (1989). Effects of naloxone and naltrexone on memory consolidation in CD1 mice: Involvement of GABAergic mechanisms. Pharmacology, Biochemistry and Behavior, 32, 563-567. Gallagher, M., & Kapp, B. S. (1978). Manipulation of opiate activity in the amygdala alters memory processes. Life Sciences, 23, 1973-1978. Garg, M. (1970). Combined effect of drug and drive on the consolidation process. Psychopharmacologia, 18, 172-179. Garg, M., & Holland, H. C. (1968). Consolidation and maze learning: The effects of posttrial injections of a stimulant drug (picrotoxin). Psychopharmacologia, 12, 96-103. Hill, D. R., & Bowery, N. G. (1981). 3H Baclofen and 3H-GABA bind to bicuculline insensitive GABA-B sites in rat brain. Nature (London), 290, 149-152. Introini-Collison, I. B., Nagahara, A. H., & McGaugh, J. L. (1989). Memory enhancement with intra-amygdala post-training naloxone is blocked by concurrent administration of propranolol. Brain Research, 476, 94-101. Krnjevic, K., and Schwartz, S. (1967). The action of gamma-aminobutyric acid on cortical neurons. Experimental Brain Research, 3, 320-336. McDonald, A. J. (1985). Immunohistochemical identification of 3,-aminobutyric acid-containing neurons in the rat basolateral amygdala. Neuroscience Letters, 53, 203-207. McGaugh, J. L. (1989). Involvement of neuromodulatory and memory systems in the regulation of memory storage. Annual Review of Neuroscience, 12, 255-287. McGaugh, J. L., Introini-Collison, I. B., & Nagahara, A. H. (1988). Memory-enhancing effects of post-training naloxone: Involvement of/3-noradrenergic influences in the amygdaloid complex. Brain Research, 446, 37-49. McGaugh, J. L., & Landfield, P. W. (1970). Delayed development of amnesia following electroconvulsive shock. Physiology and Behavior, 5, 1109-1113. Nagai, T., McGeer, P. L., & McGeer, E, G. (1983). Distribution of GABA-T intensive neurons in the rat forebrain and midbrain. Journal of Comparative Neurology, 218, 220-238. Olsen, R. W. (1981). GABA-benzodiazepine-barbiturate receptor interactions. Journal of Neurochemistry, 37, 1-13. Olsen, R. W., Ticku, M. K., & Miller, T. (1978). Dihydropicrotoxin in binding to crayfish muscle sites probably related to gamma-aminobutyric acid receptor ionophores. Molecular Pharmacology, 14, 381-390. Paxinos, G., & Watson, C. (1986). The rat brain in stereotaxic coordinates. New York: Academic Press. Roberts, E. (1986). GABA: The road to neurotransmitter status. In R. V. Olsen & J. C. Venter (Eds.), Benzodiazepine/Gaba receptors and chloride channels: Structural and functional properties (pp. 1-39). New York: Liss. Swartzwelder, H. S., Tilson, H. A., McLamb, R. L., & Wilson, W. A. (1987). Baclofen disrupts passive avoidance retention in rats. Psychopharmacology, 92, 398-401. Yonkov, D. I., & Georgiev, V. P. (1985). Memory effects of GABAergic antagonists in rats trained with two-way active avoidance tasks. Acta Physiologica and Pharmacologica Bulgarica, 11, 44-49.