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Involvement of the amygdala GABAergic system in the modulation of memory storage
Brain Research, 487 (1989) 105-112 Elsevier
105
BRE 14477
Involvement of the amygdala GABAergic system in the modulation of memory storage J o r g e D . B r i o n i 1, A l a n H . N a g a h a r a 1'2 a n d J a m e s
L. McGaugh
1'2
ICenter for the Neurobiology of Learning and Memory and 2Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted 18 October 1988) Key words: Amygdala; Bicuculline-methiodide; Caudate-putamen; y-Aminobutyric acid; Inhibitory avoidance; Memory; Muscimol
These experiments examined the involvement of the intrinsic GABAergic system of the amygdaloid complex in the modulation of memory storage. Rats were chronically implanted with bilateral cannulae in the amygdala, trained in an inhibitory avoidance task, and given post-training bilateral intra-amygdala injections of either the GABA receptor antagonist bicuculline methiodide (BMI) (0.1-1.0 nmol) or the GABAA receptor agonist muscimol (0.001-0.1 nmol). As indicated by performance on a 48 h retention test, BMI enhanced retention of the inhibitory avoidance conditioning, while muscimol impaired retention. The memory-enhancement obtained with BMI (0.1 nmol) was produced by a dose lower than that necessary to induce convulsions. Post-training injections of BMI did not affect retention when injected into the caudate-putamen dorsal to the amygdala. These results suggest that the amygdaloid GABAergic system is involved in the modulation of memory storage.
INTRODUCTION ~,-Aminobutyric acid ( G A B A ) is the main inhibitory neurotransmitter in the mammalian brain and is known to be involved in a variety of physiological functions 17'32'33'57. The G A B A receptor is a protein complex that also contains receptor sites for benzodiazepines, as well as picrotoxin/barbiturates, which are associated with a chloride channel ionophore 9"52. The characteristic response of the postsynaptic neuron to G A B A is electrical inhibition mediated by an increase in chloride conductance 32'33'42. At the behavioral level, there is extensive evidence indicating that retention of recently acquired information is influenced by post-training systemic injections of G A B A antagonists and agonists. A number of studies using a variety of training tasks have reported that memory is enhanced by posttraining systemic injections of the chloride channel
blocker, picrotoxin a-5'7'8'18. Post-training systemic injections of bicuculline, a G A B A receptor antagonist with central actions 1°'11'53'62, have also been reported to enhance retention in rats and mice 5'7'61. In contrast, retention is impaired by post-training systemic injections of either the G A B A A agonist muscimol or the GABA-transaminase inhibitor amino-oxyacetic acid 8"28. A number of recent studies have reported that retention can be modulated by post-training intraamygdala injections of drugs affecting transmitter and neuromodulatory systems aS. Opiate antagonists such as naloxone, for example, enhance retention when injected either systemically or intra-amygdally 14'21'24'47. Further, retention can be enhanced by post-training intra-amygdala injections of norepinephrine, and the memory-enhancing effects of systemically administered naloxone as well as epinephrine are blocked by intra-amygdala injections of
Correspondence: J.D. Brioni, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, U.S.A.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
106 propranolo121'39'47. Such findings suggest that systemically administered drugs and hormones may affect memory storage through influences involving the amygdaloid complex. The presence of GABAergic neurons in the amygdala has been extensively documented 2'6'3s' 43.49. The GABAergic cells project mainly within the amygdala 51. The findings cited above indicating that retention can be enhanced by intra-amygdala as well as systemic injections of a number of drugs, considered together with the evidence of GABAergic neurons in the amygdala, suggest the possibility that systemically administered G A B A agonists and antagonists may influence memory storage through influences involving the amygdaloid GABAergic system. To examine this possibility, the present experiments investigated the effects of post-training intra-amygdala injections of bicuculline methiodide (BMI), a GABAergic antagonist 11"25'27'53'56, and of muscimol, a GABAergic agonist 1'~2'2°'34, on the retention of an inhibitory avoidance response. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (60 days old, 200-220 g on arrival) from Charles River Laboratories were used. They were individually housed upon arrival and maintained on a 12-h light-dark period (lights on 07.00 h) with food and water available ad libitum. They were acclimatized to laboratory conditions for 1 week before undergoing surgery. The animals were allowed 1 week of recovery after surgery. At the time of the training rats' weights were 264 _+ 5 g.
Surgery Amygdala cannulae implantation. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and given atropine sulfate (0.4 mg/kg, i.p.). The skull of the rat was fixed to a stereotaxic frame (Kopf Instruments) and permanent stainlesssteel guide cannulae (23 gauge; 15 mm) were implanted bilaterally. The tips were aimed at the dorsal surface of the amygdala. The stereotaxic coordinates were: AP -2.3 mm from bregma; ML _+4.4 mm from midline; DV -5.5 mm from dura; with the nose-bar at -3.3 mm from the interaural line 55. 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 and were maintained in a temperature-controlled chamber until recovery from anesthesia. Caudate-putamen cannulae implantation. Cannulae implantation was carried out using the same general procedures described above. The stereotaxic coordinates were: AP -2.3 from bregma; ML _+4.4 from midline; DV -3.5 from dura; with the nose-bar at -3.3 from the interaural line.
Apparatus and procedures Inhibitory avoidance. The rats were trained on a step-through inhibitory avoidance apparatus 47. On the training day each rat 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 4 paws into the dark side, the door was closed, a footshock (0.35-0.45 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 48-h retention test 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 step-through latency minus training step-through latency was used as the retention score for each animal. Intra-amygdala injection. Immediately after the training session, animals 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/~1 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. The drugs were administered in a total volume of 0.5 /~1 at a rate of 0.75 /A/min. The injection needles were kept in their position for an additional 30 s after the injection. The findings of previous research in our laboratory using this can-
107 nula implantation and injection procedure indicate that the retention performance of implanted animals is comparable to that of unoperated controls 47.
Behavioral effects of intra-amygdala BMI injections. To determine the behavioral effects of the intra-amygdala BMI injections different groups of rats were injected with saline (0.5 pl) or BMI (0.1, 0.3, 1.0, 3.0 or 5.0 nmol) and observed for 60 min. The incidences of salivation, oral stereotypy, jumps, 'wet dog shakes', and clonic/tonic seizures were recorded.
Drugs (-)-Bicuculline methiodide (BMI, Sigma Chemicals) was dissolved in saline solution immediately before use, and administered bilaterally in doses of 0.1 to 5.0 nmol per cannula. BMI is a GABAergic antagonist that displaces G A B A from GABA binding sites 11"27"53'56,and despite some confusion in the literature regarding the nature of the isomer of the methiodide of (+)-bicuculline, there is now agreement that the correct form is l e v o ]3'59. Muscimol (Sigma Chemicals) was dissolved in saline solution and injected bilaterally in doses of 0.001 to 0.1 nmol per cannula.
tions of BMI is shown in Fig. 1. For this experiment a training footshock intensity of 0.35 mA was used. As it can be seen, BMI produced a dose-dependent increase in the retention latencies (F3,47 = 5.1, P < 0.01), as there was a significant effect at the 0.1 nmol dose (P < 0.01) but higher doses were ineffective. The positions of the needle tips for these animals are shown in Fig. 2. Cannulae were aimed at the dorsal part of the amygdala and the tip of the injecting needle protruded 2 mm beyond the guide cannula. To evaluate the neuroanatomical specificity of the effect of BMI on retention, other groups of animals were implanted with chronic cannulae either in the amygdala or in a region of the caudate-putamen 2 mm dorsal to the amygdala at the same AP and ML coordinates. The animals were trained and given post-training injections as described above. As it can be seen in Fig. 3, there was a significant drug effect (F1,51 = 6.5, P < 0.025). Only the animals that received BMI injections intra-amygdally showed an increase in retention latencies in comparison with those of control rats (F1,51 = 7.5, P < 0.01; interaction F1,51 = 4.2, P < 0.05). Table I summarizes the behavioral observation of rats tested following intra-amygdala injections of
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/~m) revealing the position of the cannulae were stained with Cresyl violet. Rats with incorrect placement (fewer than 10%) of the cannulae were excluded from the analyses. Fig. 2 shows the position of the injection needle tip in 51 rats from the experiment reported in Fig. 1.
Z
Statistics
I,-
Behavioral data were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Newman-Keuls' tests for individual means. RESULTS
The mean training step-through latencies of the groups were comparable (overall mean = 4.6 + 0.4 s). The effect of post-training intra-amygdala injec-
300ttl
250
Z UJ
.1
3 _z <
1~
o saline o.1 0.3 BICUCULLINE METHIODIDE
1 .o nmoles
Fig. 1. Effect of intra-amygdala injections of bicucullinemethiodide on retention of an inhibitory avoidance response. Rats implanted bilaterally with chronic cannulae were trained on the inhibitory avoidance task (shock intensity 0.35 mA), and were injected immediately post-training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 12-14 rats. **P < 0.01 as compared to saline-injected rats.
108
- 1.80 mm
-2.12 mm
-2.30
mm
- 2.56 mm
- 2.80 mm
-3.14 mm
Fig. 2. Distribution of injection needle tips in the amygdala of 51 brains corresponding
to the experiment
in Fig. 1.
109
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3
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100 ¸
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so,
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0.1
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BMI
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0.1
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Fig. 3. Effect of bicuculline-methiodide (0.1 nmol) injections in the amygdala and in the caudate-putamen on the retention of an inhibitory avoidance task. Rats with chronic implanted cannulae were trained in the inhibitory avoidance (shock intensity 0.35 mA), and were injected immediately after training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 12-14 rats. **P < 0.01 as compared to saline-injected animals.
Fig. 4. Testing-training latencies to step-through after muscimol injections in the amygdala. Rats with chronic implanted cannulae were trained in the inhibitory avoidance task (shock intensity 0.45 mA), and were injected immediately after training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 14-15 rats. *P < 0.05 as compared to saline-injected rats.
saline solution or BMI. Bilateral injections of saline induced oral stereotypies, mainly teeth chattering,
m e m o r y - e n h a n c i n g dose. The injections of 3 and 5 nmol per cannula in the amygdala resulted in
that occurred immediately after the injection and later disappeared. The lowest dose of BMI (0.1 nmol per cannula) also induced teeth chattering, and at a dose of 0.3 nmol induced 'wet dog shakes'. Clonic/ tonic convulsions were seen only following BMI doses of 1 nmol per cannula or higher. Thus, the convulsive dose of BMI was considerably above the
convulsive behavior in all animals immediately after the injection, with upper extremity clonus that finally developed into status epilepticus. O n e animal
TABLE I
in the 5 nmol group died within 30 min after the injection. The effect of post-training intra-amygdala injections of muscimol on retention of inhibitory avoidance task was examined in other groups of animals that were trained with a higher footshock (0.45 mA). As Fig. 4 shows, the post-training injections of
Behavioral observation o f rats after BMI injection into the amygdala
Bicuculline methiodide (BMI) was injected bilaterally into the amygdala and animals were observed for a period of 60 min. Five animals were evaluated at each dose level. Results express the number of rats showing each behavior. Sal
B M I (nmol) 0.1
Salivation Oral stereotypy Jumps Wet dog shakes Clonic/tonic seizures
MUSCIMOL nmoles
3/5 . . -
0.3
1.0
3.0
5.0
3/5 3/5 . . 2/5 -
1/5 3/5
2/5 5/5 1/5 5/5 5/5
2/5 4/5 5/5 5/5
3/5 1/5
muscimol decreased 48-h retention latencies (F3.50 = 4.1, P < 0.02), with a significant effect (P < 0.05) at 0.001 nmol. DISCUSSION The findings of these experiments indicate that rats' retention of an inhibitory avoidance response is enhanced by post-training intra-amygdala injections of low doses of the G A B A e r g i c antagonist bicuculline methiodide (BMI). Since the cannulae tips were in all cases located in the amygdala, it seems likely that the m e m o r y - e n h a n c i n g effects resulted from
110 influences on GABA receptors within the amygdala. The finding that BMI did not affect retention when injected into the caudate-putamen dorsal to the amygdala suggest that the amygdala is the critical area, although diffusion to other surrounding areas, such as the parahippocampal region, cannot be ruled out. The behavioral observations of the animals immediately following intra-amygdala BMI injections revealed that only doses of 1 nmol or higher induced convulsions. These observations are consistent with previous findings 6°. Thus, the dose of BMI that was most effective in enhancing memory (0.1 nmol) was well below the convulsive dose. Our findings also indicated that retention of an inhibitory avoidance response is impaired by posttraining intra-amygdala injections of the GABA A agonist muscimol. Since muscimol is a poor substrate for presynaptic uptake 35, the effect is most likely due to activation of postsynaptic receptors. It is interesting to note that the muscimol effect on memory was observed at a dose much lower than that found with BMI. This is in agreement with the evidence that muscimol is more potent than BMI in the displacement of [3H]GABA binding 12"27"53. The findings of our experiments using intraamygdala injections are generally consistent with those of previous studies using systemic injections of drugs affecting GABAergic systems. As we noted above, there is extensive evidence that, in rats and mice, retention of a variety of training tasks is enhanced by post-training systemic injections of either picrotoxin (a chloride channel blocker), or bicuculline (a specific GABAergic antagonist) and impaired by muscimol (a specific GABAA agonist) 35,8,61.
Our findings provide additional support for the general view that the amygdala is involved in memory storage 15'16'23'36"37"4°'41'48'58. Mishkin and his colleagues 48 have argued that the amygdala may play a specific role in the acquisition of affective information and in integrating cross modal information. These suggestions are consistent with evidence from many studies of the effects of amygdala lesions on learning and memory as well as with the evidence that the amygdala is involved in a variety of somatic, autonomic, endocrine and behavioral responses 26'31. Other evidence suggests that the amygdala is involved in modulating the storage of information. For
example, there is extensive evidence indicating memory storage can be modulated by post-training electrical stimulation of the amygdala 29'46. Further, many recent studies have reported that retention is modulated by post-training intra-amygdala injections of drugs that influence noradrenergic, cholinergic, and opioid peptidergic systems. Since the treatments are effective when administered immediately post-training the findings are consistent with the view that they act by modulating the storage of the training experiences 45. And, more generally, the findings suggest that these neurochemical systems within the amygdala are involved in the endogenous regulation of memory storage. The present findings suggest the GABAergic system within the amygdala may also play a role in this process. Pascoe and Kapp 54 have shown that in classically conditioned rabbits, neurons in the central nucleus of the amygdala respond differentially to the CS + and CS . Other recent studies have reported that the conditional emotional response to a tone, which was previously associated with a footshock, is blocked by selective destruction of intrinsic neurons in the amygdala 23'37. If the processing of the emotional significance of external events depends upon inputs to intrinsic neurons in the amygdala 36, studies examining the participation of the intrinsic GABAergic system in the amygdala should contribute to an understanding of the role of the amygdala in learning and memory processes. Several previous studies have reported that learning and memory can be influenced by injections of GABAergic agonists and antagonists into brain regions other than the amygdala. Retention impairment has been obtained with picrotoxin administered to the substantia nigra 3° and hippocampus TM and with muscimol injected into the basal forebrain 5°. Other studies have reported enhancement of learning and memory with intraventricular injections of G A B A and intrahippocampal injections of valproic acid 19'22. As it is well known that many memory-modulating drugs may either impair or enhance memory storage, depending upon the specific drug doses and training conditions used 44, it cannot be concluded on the basis of available findings that the specific memory modulating influence - - i.e. impairment or enhancement - - varies with the site of the brain injection. But, the findings
111 suggest that G A B A e r g i c systems in several brain regions may participate in the modulation of memory storage. Further, while systemically adminis-
ACKNOWLEDGEMENTS This research was supported by U S P H S G r a n t
tered G A B A e r g i c antagonists may, as our findings suggest, influence m e m o r y through effects involving the amygdala, the possibility that the effects involve
M H 12526 and contract N00014-87-k-0518 from the
concurrent influences on G A B A e r g i c systems in other brain regions cannot be excluded.
technical assistance, and Mrs. Nan Collett for her
Office of Naval Research. We thank E m m a n u e l Fajardo, T h i n h Luu and Lynette G a m b o a for their invaluable assistance in the preparation of the manuscript.
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dulatory systems in the regulation of memory storage, Annu. Rev. Neurosci., in press. McGaugh, J.L. and Gold, P.E., Modulation of memory by electrical stimulation of the brain. In M.R. Rosenzweig and E.L. Bennett (Eds.), Neural Mechanisms of Learning and Memory, MIT, Cambridge, MA, 1976, pp. 549-560. McGaugh, J.L., Introini-Collison, I.B. and Nagahara, A.H., Memory-enhancing effects of posttraining naloxone: involvement of fl-noradrenergic influences in the amygdaloid complex, Brain Research, 446 (1988) 37-49. Murray, E.A. and Mishkin, M., Amygdalectomy impairs crossmodal association in monkeys, Science, 228 (1985) 604-606. Nagai, T., McGeer, P.L. and McGeer, E.G., Distribution of GABA-T-intensive neurons in the rat forebrain and midbrain, J. Comp. Neurol., 218 (1983) 220-238. Nagel, J.A. and Huston, J.P., Enhanced inhibitory avoidance learning produced by post-trial injections of substance P into the basal forebrain, Behav. Neural Biol., 49 (1988) 374-385. Nitecka, L. and Ben-Ari, Y., Distribution of GABA-Iike immunoreactivity in the rat amygdaloid complex, J. Comp. Neurol., 266 (1987) 45-55. Olsen, R.W., GABA-benzodiazepine-barbiturate receptor interactions, J. Neurochem., 37 (1981) 1-13. Olsen, R.W., Ticku, M.K., Van Ness, P.C. and Greenlee, D., Effects of drugs on y-aminobutyric acid receptors, uptake, release and synthesis in vitro, Brain Research, 139 (1978) 277-294. Pascoe, J.P. and Kapp, B.S., Electrophysiological characteristics of amygdaloid central nucleus neurons during Pavlovian fear conditioning in the rabbit, Behav. Brain Res., 16 (1985) 117-133. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1986. Pong, S.E and Graham, L.T., N-methyl bicuculline, a convulsant, more potent than bicuculline, Brain Research, 42 (1972) 486-490. Roberts, E., GABA: the toad to neurotransmitter status. In R.W. Olsen and J.C. Venter (Eds.), Benzodiazepine/ GABA Receptors and Chloride Channels: Structural and Functional Properties, Liss, New York, 1986, pp. 1-39. Sarter, M. and Markowitsch, H.J., Involvement of the amygdala in learning and memory: a critical review, with emphasis on anatomical relations, Behav. Neurosci., 99 (1985) 342-380. Simonyi, M., Blasko, G. and Kardos, J., Methiodide of the GABA antagonist (+)-bicuculline is laevorotatory, Chirality, in press. Turski, W.A., Cavalheiro, E.A., Calderazzo-Filho, L.S., Kleinrok, Z., Czuczwar, S.J. and Turski, L., Injections of picrotoxin and bicuculline in the amygdaloid complex of the rat: an electroencephalographic, behavioral and morphological analysis, Neuroscience, 14 (1985) 37-53. Yonkov, D.I. and Georgiev, V.P., Memory effects of GABAergic antagonists in rats trained with two-way active avoidance tasks, Acta Physiol. Pharrnacol. Bulg., 11 (1985) 44-49. Zukin, S.R., Young, A. and Snyder, S.H., Gammaaminobutyric acid binding to receptor sites in the rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 4802-4807.
105
BRE 14477
Involvement of the amygdala GABAergic system in the modulation of memory storage J o r g e D . B r i o n i 1, A l a n H . N a g a h a r a 1'2 a n d J a m e s
L. McGaugh
1'2
ICenter for the Neurobiology of Learning and Memory and 2Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted 18 October 1988) Key words: Amygdala; Bicuculline-methiodide; Caudate-putamen; y-Aminobutyric acid; Inhibitory avoidance; Memory; Muscimol
These experiments examined the involvement of the intrinsic GABAergic system of the amygdaloid complex in the modulation of memory storage. Rats were chronically implanted with bilateral cannulae in the amygdala, trained in an inhibitory avoidance task, and given post-training bilateral intra-amygdala injections of either the GABA receptor antagonist bicuculline methiodide (BMI) (0.1-1.0 nmol) or the GABAA receptor agonist muscimol (0.001-0.1 nmol). As indicated by performance on a 48 h retention test, BMI enhanced retention of the inhibitory avoidance conditioning, while muscimol impaired retention. The memory-enhancement obtained with BMI (0.1 nmol) was produced by a dose lower than that necessary to induce convulsions. Post-training injections of BMI did not affect retention when injected into the caudate-putamen dorsal to the amygdala. These results suggest that the amygdaloid GABAergic system is involved in the modulation of memory storage.
INTRODUCTION ~,-Aminobutyric acid ( G A B A ) is the main inhibitory neurotransmitter in the mammalian brain and is known to be involved in a variety of physiological functions 17'32'33'57. The G A B A receptor is a protein complex that also contains receptor sites for benzodiazepines, as well as picrotoxin/barbiturates, which are associated with a chloride channel ionophore 9"52. The characteristic response of the postsynaptic neuron to G A B A is electrical inhibition mediated by an increase in chloride conductance 32'33'42. At the behavioral level, there is extensive evidence indicating that retention of recently acquired information is influenced by post-training systemic injections of G A B A antagonists and agonists. A number of studies using a variety of training tasks have reported that memory is enhanced by posttraining systemic injections of the chloride channel
blocker, picrotoxin a-5'7'8'18. Post-training systemic injections of bicuculline, a G A B A receptor antagonist with central actions 1°'11'53'62, have also been reported to enhance retention in rats and mice 5'7'61. In contrast, retention is impaired by post-training systemic injections of either the G A B A A agonist muscimol or the GABA-transaminase inhibitor amino-oxyacetic acid 8"28. A number of recent studies have reported that retention can be modulated by post-training intraamygdala injections of drugs affecting transmitter and neuromodulatory systems aS. Opiate antagonists such as naloxone, for example, enhance retention when injected either systemically or intra-amygdally 14'21'24'47. Further, retention can be enhanced by post-training intra-amygdala injections of norepinephrine, and the memory-enhancing effects of systemically administered naloxone as well as epinephrine are blocked by intra-amygdala injections of
Correspondence: J.D. Brioni, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, U.S.A.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
106 propranolo121'39'47. Such findings suggest that systemically administered drugs and hormones may affect memory storage through influences involving the amygdaloid complex. The presence of GABAergic neurons in the amygdala has been extensively documented 2'6'3s' 43.49. The GABAergic cells project mainly within the amygdala 51. The findings cited above indicating that retention can be enhanced by intra-amygdala as well as systemic injections of a number of drugs, considered together with the evidence of GABAergic neurons in the amygdala, suggest the possibility that systemically administered G A B A agonists and antagonists may influence memory storage through influences involving the amygdaloid GABAergic system. To examine this possibility, the present experiments investigated the effects of post-training intra-amygdala injections of bicuculline methiodide (BMI), a GABAergic antagonist 11"25'27'53'56, and of muscimol, a GABAergic agonist 1'~2'2°'34, on the retention of an inhibitory avoidance response. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (60 days old, 200-220 g on arrival) from Charles River Laboratories were used. They were individually housed upon arrival and maintained on a 12-h light-dark period (lights on 07.00 h) with food and water available ad libitum. They were acclimatized to laboratory conditions for 1 week before undergoing surgery. The animals were allowed 1 week of recovery after surgery. At the time of the training rats' weights were 264 _+ 5 g.
Surgery Amygdala cannulae implantation. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and given atropine sulfate (0.4 mg/kg, i.p.). The skull of the rat was fixed to a stereotaxic frame (Kopf Instruments) and permanent stainlesssteel guide cannulae (23 gauge; 15 mm) were implanted bilaterally. The tips were aimed at the dorsal surface of the amygdala. The stereotaxic coordinates were: AP -2.3 mm from bregma; ML _+4.4 mm from midline; DV -5.5 mm from dura; with the nose-bar at -3.3 mm from the interaural line 55. 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 and were maintained in a temperature-controlled chamber until recovery from anesthesia. Caudate-putamen cannulae implantation. Cannulae implantation was carried out using the same general procedures described above. The stereotaxic coordinates were: AP -2.3 from bregma; ML _+4.4 from midline; DV -3.5 from dura; with the nose-bar at -3.3 from the interaural line.
Apparatus and procedures Inhibitory avoidance. The rats were trained on a step-through inhibitory avoidance apparatus 47. On the training day each rat 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 4 paws into the dark side, the door was closed, a footshock (0.35-0.45 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 48-h retention test 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 step-through latency minus training step-through latency was used as the retention score for each animal. Intra-amygdala injection. Immediately after the training session, animals 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/~1 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. The drugs were administered in a total volume of 0.5 /~1 at a rate of 0.75 /A/min. The injection needles were kept in their position for an additional 30 s after the injection. The findings of previous research in our laboratory using this can-
107 nula implantation and injection procedure indicate that the retention performance of implanted animals is comparable to that of unoperated controls 47.
Behavioral effects of intra-amygdala BMI injections. To determine the behavioral effects of the intra-amygdala BMI injections different groups of rats were injected with saline (0.5 pl) or BMI (0.1, 0.3, 1.0, 3.0 or 5.0 nmol) and observed for 60 min. The incidences of salivation, oral stereotypy, jumps, 'wet dog shakes', and clonic/tonic seizures were recorded.
Drugs (-)-Bicuculline methiodide (BMI, Sigma Chemicals) was dissolved in saline solution immediately before use, and administered bilaterally in doses of 0.1 to 5.0 nmol per cannula. BMI is a GABAergic antagonist that displaces G A B A from GABA binding sites 11"27"53'56,and despite some confusion in the literature regarding the nature of the isomer of the methiodide of (+)-bicuculline, there is now agreement that the correct form is l e v o ]3'59. Muscimol (Sigma Chemicals) was dissolved in saline solution and injected bilaterally in doses of 0.001 to 0.1 nmol per cannula.
tions of BMI is shown in Fig. 1. For this experiment a training footshock intensity of 0.35 mA was used. As it can be seen, BMI produced a dose-dependent increase in the retention latencies (F3,47 = 5.1, P < 0.01), as there was a significant effect at the 0.1 nmol dose (P < 0.01) but higher doses were ineffective. The positions of the needle tips for these animals are shown in Fig. 2. Cannulae were aimed at the dorsal part of the amygdala and the tip of the injecting needle protruded 2 mm beyond the guide cannula. To evaluate the neuroanatomical specificity of the effect of BMI on retention, other groups of animals were implanted with chronic cannulae either in the amygdala or in a region of the caudate-putamen 2 mm dorsal to the amygdala at the same AP and ML coordinates. The animals were trained and given post-training injections as described above. As it can be seen in Fig. 3, there was a significant drug effect (F1,51 = 6.5, P < 0.025). Only the animals that received BMI injections intra-amygdally showed an increase in retention latencies in comparison with those of control rats (F1,51 = 7.5, P < 0.01; interaction F1,51 = 4.2, P < 0.05). Table I summarizes the behavioral observation of rats tested following intra-amygdala injections of
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/~m) revealing the position of the cannulae were stained with Cresyl violet. Rats with incorrect placement (fewer than 10%) of the cannulae were excluded from the analyses. Fig. 2 shows the position of the injection needle tip in 51 rats from the experiment reported in Fig. 1.
Z
Statistics
I,-
Behavioral data were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Newman-Keuls' tests for individual means. RESULTS
The mean training step-through latencies of the groups were comparable (overall mean = 4.6 + 0.4 s). The effect of post-training intra-amygdala injec-
300ttl
250
Z UJ
.1
3 _z <
1~
o saline o.1 0.3 BICUCULLINE METHIODIDE
1 .o nmoles
Fig. 1. Effect of intra-amygdala injections of bicucullinemethiodide on retention of an inhibitory avoidance response. Rats implanted bilaterally with chronic cannulae were trained on the inhibitory avoidance task (shock intensity 0.35 mA), and were injected immediately post-training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 12-14 rats. **P < 0.01 as compared to saline-injected rats.
108
- 1.80 mm
-2.12 mm
-2.30
mm
- 2.56 mm
- 2.80 mm
-3.14 mm
Fig. 2. Distribution of injection needle tips in the amygdala of 51 brains corresponding
to the experiment
in Fig. 1.
109
6maVal~a.6
eAUDATE Z gl I-
0 3
3
Z
15o
< E I-
100 ¸
Z gl I-
.1 I,-
so,
0
~ SAL.
BMI
0.1
SAL.
BMI
0
0.1
SAL.
0.001
0.01
0.1
Fig. 3. Effect of bicuculline-methiodide (0.1 nmol) injections in the amygdala and in the caudate-putamen on the retention of an inhibitory avoidance task. Rats with chronic implanted cannulae were trained in the inhibitory avoidance (shock intensity 0.35 mA), and were injected immediately after training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 12-14 rats. **P < 0.01 as compared to saline-injected animals.
Fig. 4. Testing-training latencies to step-through after muscimol injections in the amygdala. Rats with chronic implanted cannulae were trained in the inhibitory avoidance task (shock intensity 0.45 mA), and were injected immediately after training. Retention was evaluated 48 h later. Each bar represents the mean + S.E.M. testing minus training latency in seconds of 14-15 rats. *P < 0.05 as compared to saline-injected rats.
saline solution or BMI. Bilateral injections of saline induced oral stereotypies, mainly teeth chattering,
m e m o r y - e n h a n c i n g dose. The injections of 3 and 5 nmol per cannula in the amygdala resulted in
that occurred immediately after the injection and later disappeared. The lowest dose of BMI (0.1 nmol per cannula) also induced teeth chattering, and at a dose of 0.3 nmol induced 'wet dog shakes'. Clonic/ tonic convulsions were seen only following BMI doses of 1 nmol per cannula or higher. Thus, the convulsive dose of BMI was considerably above the
convulsive behavior in all animals immediately after the injection, with upper extremity clonus that finally developed into status epilepticus. O n e animal
TABLE I
in the 5 nmol group died within 30 min after the injection. The effect of post-training intra-amygdala injections of muscimol on retention of inhibitory avoidance task was examined in other groups of animals that were trained with a higher footshock (0.45 mA). As Fig. 4 shows, the post-training injections of
Behavioral observation o f rats after BMI injection into the amygdala
Bicuculline methiodide (BMI) was injected bilaterally into the amygdala and animals were observed for a period of 60 min. Five animals were evaluated at each dose level. Results express the number of rats showing each behavior. Sal
B M I (nmol) 0.1
Salivation Oral stereotypy Jumps Wet dog shakes Clonic/tonic seizures
MUSCIMOL nmoles
3/5 . . -
0.3
1.0
3.0
5.0
3/5 3/5 . . 2/5 -
1/5 3/5
2/5 5/5 1/5 5/5 5/5
2/5 4/5 5/5 5/5
3/5 1/5
muscimol decreased 48-h retention latencies (F3.50 = 4.1, P < 0.02), with a significant effect (P < 0.05) at 0.001 nmol. DISCUSSION The findings of these experiments indicate that rats' retention of an inhibitory avoidance response is enhanced by post-training intra-amygdala injections of low doses of the G A B A e r g i c antagonist bicuculline methiodide (BMI). Since the cannulae tips were in all cases located in the amygdala, it seems likely that the m e m o r y - e n h a n c i n g effects resulted from
110 influences on GABA receptors within the amygdala. The finding that BMI did not affect retention when injected into the caudate-putamen dorsal to the amygdala suggest that the amygdala is the critical area, although diffusion to other surrounding areas, such as the parahippocampal region, cannot be ruled out. The behavioral observations of the animals immediately following intra-amygdala BMI injections revealed that only doses of 1 nmol or higher induced convulsions. These observations are consistent with previous findings 6°. Thus, the dose of BMI that was most effective in enhancing memory (0.1 nmol) was well below the convulsive dose. Our findings also indicated that retention of an inhibitory avoidance response is impaired by posttraining intra-amygdala injections of the GABA A agonist muscimol. Since muscimol is a poor substrate for presynaptic uptake 35, the effect is most likely due to activation of postsynaptic receptors. It is interesting to note that the muscimol effect on memory was observed at a dose much lower than that found with BMI. This is in agreement with the evidence that muscimol is more potent than BMI in the displacement of [3H]GABA binding 12"27"53. The findings of our experiments using intraamygdala injections are generally consistent with those of previous studies using systemic injections of drugs affecting GABAergic systems. As we noted above, there is extensive evidence that, in rats and mice, retention of a variety of training tasks is enhanced by post-training systemic injections of either picrotoxin (a chloride channel blocker), or bicuculline (a specific GABAergic antagonist) and impaired by muscimol (a specific GABAA agonist) 35,8,61.
Our findings provide additional support for the general view that the amygdala is involved in memory storage 15'16'23'36"37"4°'41'48'58. Mishkin and his colleagues 48 have argued that the amygdala may play a specific role in the acquisition of affective information and in integrating cross modal information. These suggestions are consistent with evidence from many studies of the effects of amygdala lesions on learning and memory as well as with the evidence that the amygdala is involved in a variety of somatic, autonomic, endocrine and behavioral responses 26'31. Other evidence suggests that the amygdala is involved in modulating the storage of information. For
example, there is extensive evidence indicating memory storage can be modulated by post-training electrical stimulation of the amygdala 29'46. Further, many recent studies have reported that retention is modulated by post-training intra-amygdala injections of drugs that influence noradrenergic, cholinergic, and opioid peptidergic systems. Since the treatments are effective when administered immediately post-training the findings are consistent with the view that they act by modulating the storage of the training experiences 45. And, more generally, the findings suggest that these neurochemical systems within the amygdala are involved in the endogenous regulation of memory storage. The present findings suggest the GABAergic system within the amygdala may also play a role in this process. Pascoe and Kapp 54 have shown that in classically conditioned rabbits, neurons in the central nucleus of the amygdala respond differentially to the CS + and CS . Other recent studies have reported that the conditional emotional response to a tone, which was previously associated with a footshock, is blocked by selective destruction of intrinsic neurons in the amygdala 23'37. If the processing of the emotional significance of external events depends upon inputs to intrinsic neurons in the amygdala 36, studies examining the participation of the intrinsic GABAergic system in the amygdala should contribute to an understanding of the role of the amygdala in learning and memory processes. Several previous studies have reported that learning and memory can be influenced by injections of GABAergic agonists and antagonists into brain regions other than the amygdala. Retention impairment has been obtained with picrotoxin administered to the substantia nigra 3° and hippocampus TM and with muscimol injected into the basal forebrain 5°. Other studies have reported enhancement of learning and memory with intraventricular injections of G A B A and intrahippocampal injections of valproic acid 19'22. As it is well known that many memory-modulating drugs may either impair or enhance memory storage, depending upon the specific drug doses and training conditions used 44, it cannot be concluded on the basis of available findings that the specific memory modulating influence - - i.e. impairment or enhancement - - varies with the site of the brain injection. But, the findings
111 suggest that G A B A e r g i c systems in several brain regions may participate in the modulation of memory storage. Further, while systemically adminis-
ACKNOWLEDGEMENTS This research was supported by U S P H S G r a n t
tered G A B A e r g i c antagonists may, as our findings suggest, influence m e m o r y through effects involving the amygdala, the possibility that the effects involve
M H 12526 and contract N00014-87-k-0518 from the
concurrent influences on G A B A e r g i c systems in other brain regions cannot be excluded.
technical assistance, and Mrs. Nan Collett for her
Office of Naval Research. We thank E m m a n u e l Fajardo, T h i n h Luu and Lynette G a m b o a for their invaluable assistance in the preparation of the manuscript.
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