Brain Research, 591 (1992) 54-61 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
54
BRES 18082
Muscimol infused into the medial septal area impairs long-term memory but not short-term memory in inhibitory avoidance, water maze place learning and rewarded alternation tasks Alan H. N a g a h a r a and James L. M c G a u g h Center for the Neurobiology of Learning and Memory and Department of Psychobiology, Unicersityof California, lrcine, CA 92717 (USA) (Accepted 14 April 1992)
Key words: y-Aminobutyric acid; Hippocampus; Inhibitory avoidance; Long-term memory; Medial septal area:, Memory; Muscimol, Place-learning; Reinforced alternation; Septo-hippocampal system
These experiments investigated the effects of injections of muscimol (1 or 5 nmol), administered into the medial septal area prior to training, on memory tested at different retention delays after training in 3 tasks: an inhibitory avoidance task, a one-trial place learning task, and a rewarded alternation task. In all 3 tasks, intraseptal injections of muscimol did not impair memory performance at short retention delays, but impaired memory at the longer retention delays. These findings are consistent with the view that GABAergic regulation of the septohippocampal cholinergic system plays a selective role in the establishment of long-term memory.
INTRODUCTION
Extensive evidence suggests that the hippocampus participates in the formation of memory for some tasks 42'5t. Furthermore, the findings of many experiments suggest that the hippocampus is involved in the formation of long-term memory, but not short-term memory. Human patients with hippocampal or temporal lobe damage (e.g.H.M.) show a distinct pattern of relatively normal short-term memory for declarative information, but impaired formation of long-term memory 2~'5°, Furthermore, primates with damage to the hippocampus or hippocampus/amygdala show normal short-term retention, but impaired long-term retention when tested on a non-matching.to-sample task 37'65. In addition, several studies have shown that rodents with hippocampal lesions or other manipulations affecting hippocampal function also show normal memory performance at short retention delays, but are impaired at longer retention delays 2,25,4°,45,~2. The medial septal area (MSA) sends a major afferent projection consisting of a large population of
cholinergic and GABAergic fibers to the hippocampus 12'tx4s'49. In rodents, the septohippocampal projections appear to be important in the memory function of the hippocampus; lesions of the MSA or the septohippocampal interconnection produce learning impairments similar to those observed with hippocampus lesions~O,zs,4t,54, see rer. ts. The view that the septohippocampal cholinergic system is involved in regulating memory is supported by findings indicating that memory performance is correlated with cholinergic activity in the hippocampus ~,t4`15aT,36and that disruption of the cholinergic system disrupts memory retention 3,6,t4,4°,57. However, other components of the septohippocampal system may also play a significant role in memory functions. The septohippocampal cholinergic neurons in the medial septai nucleus and horizontal diagonal band are modulated by synaptic input from a GABAergic projection from the lateral septal nucleus 28'43. Infusion of muscimol, a GABAergic agonist, into the MSA inhibits the septohippocampal cholinergic system, as measured by high-affinity choline uptake and acetylcholine
Correspondence: A.H, Nagahara, c / o J,L, McGaugh, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, USA,
55 turnover in the hippocampus 4'63'~. In addition, intraseptal administration of muscimol decreases theta rhythmic activity in the hippocampus 1'33. Several studies have shown that the administration of GABAergic drugs into the MSA can affect memory in a variety of tasks4,6,16,39, 52.
The findings of previous experiments in our laboratory indicated that infusion of muscimol into the MSA impaired memory tested after long delays in two tasks, inhibitory avoidance (48 h) and spatial learning (24 h), when administered prior to training but not when administered after training 4'39. These results suggest that the treatment affected memory storage processes occurring at the time of training, but not memory processes occurring after training (e.g. consolidation). The previous experiments in our laboratory did not address the question of whether the intraseptal injections of muscimol differentially affected retention tested at different delay intervals following learning. Thus, it is not known whether the impairment is due to blocking of acquisition or to selective impairment of long-term memory. To investigate this issue, the present set of experiments examined the effects of intraseptal injection of muscimol on retention tested at different delays following training in 3 different tasks: an inhibitory avoidance task, a place learning task in the water maze, and a rewarded alternation task. MATERIALS AND METHODS
Subjects Male Sprague-Dawloy rats (60 days old, 200-250 g on arrival) from Charles River Laboratories were used. The animals were individually housed and maintained on a 12-h light-dark cycle (lightson at 07.00 h) with food and water available ad libitum. Animals were acclimatized to laboratory conditic~nsfor 1 week prior to surgery. Surgical procedures Animals were implanted with a cannula aimed at the MSA using stereotaxic surgical procedures. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and were given atropine sulfate (0.4 mg/kg, i.p.). The cannulae were constructed of 23 gauge stainless steel tubing (i.d. 0.305 ram, o.d, 0.635 mm) and were 15 mm in length. The tip of the cannula was implanted above the surface of the MSA region using the following stereotaxic coordinates: AP + 0.5 mm from bregma; ML 0.0 mm; DV -4.0 mm from dura; with the nose bar at -3.3 mm from the interaural lines44. The cannula was fixed to the skull using two screws and dental acrylic. A stylet was inserted in the cannula and remained there at all times except during intracranial injections. Immediately after surgery the animals received an intramuscular injection of penicillin (30,000 units) and were maintained in a temperature controlled chamber until recovery from anesthesia. The animals were allowed to recover from the surgery for a week prior to behavioral testing.
lntraseptal injection procedures Each animal received an intraseptal injection of muscimol (Sigma) dissolved in phosphate buffer solution (0.!05 g PO4NaH2 and 0.029 g PO4Na2H, in 100 ml physiological saline; pH to 7.0) or the buffer solution 5 min prior to training. The drug and control solutions were infused through a 30-gauge injection needle connected with a 10 ~1
Hamilton syringe by polyethlene tubing. The injection needle was inserted into the guide cannula and protruded 2 mm beyond the tip of the cannula. The injections were delivered in a volume of 0.5 ~l over a 37-s period using a syringe pump (Sage Instruments: Model 341A). The injection needle was retained in the guide cannula for an additional 30 s after the injection before removal.
Histological procedures The animals were anesthetized and perfused intracardially with saline and 10% formalin. The brains were removed and placed in 10% formalin for at least 1 week. The brains were sectioned at 40 /~m on a freezing microtome and stained with Cresyi violet. Animals with improper cannula placement or brain lesions extending beyond the site of the cannula were not included in the statistical analyses.
EXPERIMENT 1. EFFECTS OF INTRASEPTAL MUSCIMOL TREATMENT ON IMMEDIATE AND DELAYED RETENTION OF INHIBITORY AVOIDANCE TRAINING Previously, we found that intraseptal injections of muscimol administered prior to training impaired memory in an inhibitory avoidance task when retention was tested 48 h after training 39. The present experiment was conducted to determine effects of such injections on inhibitory avoidance retention tested at shorter intervals following training. The animals received intraseptal injections of 5 nmol muscimol or buffer solutions 5 min prior to training on an inhibitory avoidance task. The trough-shaped inhibitory avoidance apparatus consisted of two compartments separated by a sliding door 29. The starting compartment was illuminated by a Tensor lamp (25 W). The floor of the the other compartment was constructed of stainless-steel plates through which footshock could be delivered. On the training trial, the rat was placed in start compartment facing away from the door. When the rat turned around, the door was opened, and a timer was started when the rat turned to face the dark compartment. When the rat entered the darkened compartment, the door was closed and a mild footshock (0.5 mA, 1.0 s, constant current)was administered. The rat's latency to enter the dark compartment was recorded. Animals in different groups were first given retention tests at inte~als of 15 s or 15 rain following the training trial. On the retention test, the animal was placed in the starting compartment as on the training trial. The timer recording the retention latency was started immediately after the animal turned to face the darkened compartment or after 10 s if the animal did not turn around within that interval. No footshock was administered on the retention tests. The latency to enter the darkened compartment (maximum of 600 s) was used as the index of retention of the footshock training. All animals were also given a second retention test 48 h later using the same test procedures.
56
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Fig. I. Effects of intraseptal infusion of 5 nmol of muscimol administered prior to trainingon an inhibitory avoidance task on retention tested at delays of 15 s and 48 h. Compared to the buffer group (n = I0). animals given muscimol (n = 9) were impaired on the 48-h retention lest. but not on the 15-s retention test. * P < 0.05 vs. respective buffer group.
Resultsand Discussion Fig. 1 shows the retention latencies of the group given the first retention test 15 s after training. The latencies of the muscimol group did not significantly differ from those of the buffer group on the 15-s retention test (Uq.,)= 44, P > 0.80). However, on the 48-h retention test the retention latencies of the 5 nmoi muscimol group were significantly lower than those of the buffer group (U,~.m. = 23, P < 0.05). Fig. 2 shows the retention latencies of the group given the first retention test 15 min after training. The retention latencies of the muscimol group were significantly lower than those of the buffer group on the 15-rain retention test (U,),q- 15, P
0.50). The present results are similar to those of Expt. 1: intraseptai administration of muscimol did not affect retention tested following a short delay but impaired retention tested at a longer delay. These results clearly indicate that intraseptal injections of muscimol do not impair the animals' ability to acquire and use spatial information in a familiar environment. Thus, the effect of pretraining intraseptal injection of muscimol on retention performance seen at longer training-retention intervals cannot be attributed to deficits in sensory, motor, or motivational processes. Consequently, these findings provide additional evidence supporting the view that the retention impairment seen at longer delays is due to selective interference with the formation of long-term memory.
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EXPERIMENT 3. THE EFFECT OF INTRASEPTAL MUSCIMOL ON REWARDED ALTERNATION
20, 10 Muscimol / 5 rain
Acquisi'tion Trial
Retention Trial
Fig. 3. Effects of pretraining intraseptal infusion of 1 nmol muscimol on a single-trial place learning task on performance on 15-s or 5-min retention tests. The performance of the muscimol group tested at the 5-min delay (n--14) was impaired, in comparison with that of the buffer group tested at the 5-min delay (n = 14). The performance of the muscimol group tested at the 15-s delay (n = 15) was not impaired, in comparison with that of the buffer group tested at the 15-s delay (n = 15). * P < 0.01 vs. respective buffer group.
Considerable evidence suggests that the hippocampal system is involved in the learning of tasks requiring the use of spatial cues. In our laboratory, we found that pretraining injection of muscimol in the MSA impaired a rewarded alternation task at a delay of 15 s 3s. To examine further the effect of intraseptal injections of muscimol on this task, the present studied retention of rewarded alternation tested either immediately after each trial or after a 15 s intertrial interval.
58
Subjects Rats previously trained on an inhibitory avoidance task were used in the present experiment. The animals were given limited access to food in order to reduce their body weights by about 15% over a 7-10 day period. The animals were tfien allowed to gain about 5 g of additional body weight during each subsequent week. The animals were first habituated to a T-maze apparatus (starting arm and goal arms: 17 cm wide, 20 cm high and 30 cm long). During maze habituation, .pieces of Froot Loops were scattered on the floor of the T-maze, and the rats were allowed to explore the T-maze and eat the Froot Loops. The animals were then trained to retrieve pieces of Froot Loops from black food cups located at the end of each of the two goal arms. This was followed by 2 days of training using a rewarded alternation, i.e. 'win-shift' paradigm. On the first training trial, both goal arms were baited and the animal was allowed to enter only one arm. On the next 15 test trials, only the goal arm not entered in the immediately preceding trial was baited. On each trial, after the animal entered the goal arm and retrieved the food (or investigated the empty food well), the animal was removed from the maze. On the next 2 days, the animals were tested in the rewarded alternation task 5 min after receiving intraseptal injections of 1 nmol muscimol or buffer. The animals were assigned to either a buffer or musci. tool group for both days of testing and tested at the two different delay conditions. On the first day of testing, the animals were tested in either a 15-s delay condition or an immediate condition. In the 15-s delay condition, the animal was placed in a holding container for 15 s between test trials. In the immediate condition, the animal was immediately placed back into the start compartment after removal from the goal arm (a delay of 2-3 s). After the first test day, the animals were assigned to the other delay condition. Results attd Discussion The mean percent alternation for the 4 groups is shown in Fig. 4. in the immediate condition, the performance of the muscimol-treated animals did not differ significantly from that of the buffer-treated group (Fi,i, = 1.10, n.s,). However, in the 15-s delay condition, the alternation performance of the muscimoltreated animals was significantly impaired compared with that of the buffer-treated animals (Fl,lo= 20.1, P < 0.01). In addition, the alternation performance of the muscimol-treated animals was significantly poorer on the 15-s delay test than on the immediate test (Fl.~ = 32.1 P < 0.01).
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Fig. 4. Effects of pretraining intraseptal infusion of 1 nmol of muscimol on a rewarded alternation task tested with no intertrial interval (immediate condition) or with a 15-s intertrial interval. The performance of the muscimol group tested at the 15-s intertrial interval (n -- 7) was impaired, in comparison with that of the buffer group tested at the same intertrial interval (n = 7). The muscimol group tested in the immediate condition ( n - - 7 ) was not impaired compared to the buffer group tested in the immediate condition (n = 7). * P < 0.01 vs. respective buffer group.
These findings are consistent with the findings of Expts. 1 and 2 above indicating that intraseptal administration of muscimol does not impair memory performance when there is a short delay between training and retention but impairs retention tested at longer delays. In addition, the present findings are similar to those of experiments examining the effects, on alternation performance, of other manipulation of the hippocampal system I'~J°,4°. The present results also confirm previous findings that intraseptal administration of muscimol impairs rewarded alternation at a 15-s intertrial interval "~H. GENERAL DISCUSSION The present study showed that, in 3 different tasks, intraseptal injections of muscimol administered produced a selective pattern of memory impairment that is observed only after a retention delay. These results are similar to the findings of numerous studies showing that hippocampal lesions produce a memory deficit only after a certain retention delay, but not at a short retention delay 2-~.37,4s,-~,~'2.~'s.In addition, other studies showed that manipulations disrupting the septohippocamai system (e.g. fornix lesions) produce a similar pattern of selective memory deficit 10,23A0.Thus, in light of these studies, it seems likely that the effects of intraseptai injections of muscimol on memory are mediated by its action on the septohippocampal projection neurons. The effects of the intraseptal muscimol treatment on memory may be mediated, in part, by the cholinergic component of this system, since intraseptal
59 injections of muscimoi depress cholinergic activity in the hippocampus 4't'3"~ and numerous studies implicate the hippocampal cholinergic system in memory a.-s.'~,~4.~5. However, since only 30-40% of the septohippocampal system is cholinergic, the effects of the intraseptal muscimol treatment on memory may also be mediated by an action on the non-cholinergic (e.g. GABAergic) component of the septohippocampal system ~2.~3. Although the present findings showed a similar pattern of memory deficits on 3 different tasks, the temporal gradient of the memory deficit differed between the tasks. More specifically, at a 15-s retention delay, intraseptal injection of muscimol impaired retention in the continuous alternation task, but not in the place learning task or the inhibitory avoidance task. The rapid decline in retention seen in the alternation task may be due to the training procedures used: in the alternation task the animals received 15 consecutive retention trials while in the inhibitory avoidance and the place learning task the animals received only a single training trial. Thus, in the alternation task, retention may have been influenced by proactive interference from the prior training trials. In studies of human memory, it has been long recognized that maintenance of information in short-term memory is not solely the function of a temporal gradient, but is affected by both proacti~,z interference 24 and retroactive interference t'~. In rodent studies, retroactive interference, in the form of interpolated activity, has been shown to affect the temporal gradient of memory performance of hippocampal-lesioned animals ''~'4~'. Of course, other task-specific factors may have contributcd to the differences in retention performance in these tasks. Additional research is needed to clarify this issue. The present results considered together with previous findings provide insight into the role of the septohippocampal system in the acquisition and consolidation of memory. In previous experiments we found that pretraining, but not posttraining, injection of muscimoi into the MSA impaired retention performance in an inhibitory avoidance task and a multiple-trial place learning task 4''~'J. The absence of a posttraining effect suggests that intraseptal injection of muscimol probably does not disrupt the memory consolidation process 32. Thus, these results suggest that intraseptal injections of muscimol impair memory by disrupting some underlying memory process that occurs at the time of training. In the present study, the absence of a memory deficit at short retention delays indicates that the intraseptal muscimol treatment did not block the acquisition of information. Furthermore, this result indicates that the retention impairments seen at the
longer delays cannot be simply attriouted to influences on sensory, motor, or motivational processes necessary for performance of these 3 tasks. Considered together, these findings suggest that the intraseptal muscimol treatment impairs long-term memory by acting on a memory-related process that occurs at the time of training rather than by acting on posttraining consolidation processes. Since the underlying mechanisms mediating longterm memory formation are not clearly understood, it is difficult to determine what memory process is affected by the intraseptal injections of muscimol. However, it is interesting to note that some evidence suggests that the administration of N-methyl-o-aspartate (NMDA) antagonists impairs memory when administered prior to training, but not after training ~'47,6°.bu~~c,~ =f. 34; we found that the intraseptal injection of muscimol produces a similar effect on memory on two tasks4,3,~, t,ut =~ =f. t,. These behavioral effects are significant in light of the findi~,~:s that NMDA antagonists block the formation of long-term potentiation (LTP) in hippocampus slices when applied prior to induction, but not after induction 7,~,35.55, and since LTP has been proposed as a basis for long-term memory e'g' 3~,.~. According to this view, the effects of NMDA antagonists on memory may be due to disruption of LTP formation. Interestingly, since induction of LTP is influenced by both rhythmic theta activity t'~'27''~'~ and cholinergic activity 2m':2, intraseptal injections of muscimol could disrupt LTP induction and thus impair long-term memory by depressing hippocampal theta activity ~'3"~ and cholinergic activity ~,t,3. According to this interpretation, the effects of pretraining intraseptal injections of muscimol on memory may reflect a selective action on one process underlying long-term memory formation (e.g. LTP induction), but not on another process (e.g. LTP development). Acklzowledgemems. This research was supported by USPHSMH1252t~
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61 cortex, and olfactory bulb: a combined wheatgerm agglutininapohoreradish peroxidase-goid immunohistochemical study, Neuroscience, 30 (1989) 385-403. 50 Squire, L.R., Memory and Brain, Oxford University Press, New York, 1987, p. 315. 51 Squire, L.R. and Cohen, N.J., Human memory and amnesia. In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Learning and Memory, The Guilford Press, New York, pp. 3-77. 52 Stackman, R.W., Emerich, D.F., Taylor, L.A. and Walsh, T.J., lntraseptal administration of GABA & benzodiazepine agonists & antagonists: alteration in hippocampal choline uptake and cognitive behavior, So~. Neurosci. Abstr., 15 (1989) 699. 53 Staubli, U. and Lyr.,:h, G., Stable hippocampai long-term potentiation elicited by "theta' pattern stimulation, Brain Res., 435 (1987) 227-234. 54 Sutherland, R.J. and Rodriguez, A.J., The role of the fornix/fimbria and some related subcortical structures in place learning and memory, Behm'. Brain Res., 32 (1989) 265-277. 55 Swartzwelder, H.S., Ferrari, C., Anderson, W.W. and Wilson, W.A., The drug MK-801 attenuates the development, but not the expression, of long-term potentiation and stimulus train-induced bursting in hippocampal slices, Neuropharmacology, 28 (1989) 441-445. 56 Symons, J.P., Davis, R.E. and Marriott, J.G., Water-maze learning and effects of cholinergic drugs in mouse strains with high and low hippocampal pyramidal cell counts, Life ScL, 42 (1988) 375-383. 57 Ta:eishi, M., Takano, Y., Honda, K., Yamada, K., Kamiya, Y.
and Kamiya, H., Effects of intrahippocampal injections of the cholinergic effects of the cholinergic neurotoxin AF64A on presynaptic cholinergic markers and on passive avoidance response in the rat, Ciin. Exp. Pharmacol. Physiol., 88 (1987) 300-323. 58 Teyler, T.J. and Discenna, P., Long-term potentiation as a candidate mnemonic device, Brain Res. Re~'., 7 (1984) 15-28. 59 Thompson, R., Rapid forgetting of a spatial habit in rats with hippocampal lesions, Science, 212 ( 1981) 959-960. 60 Walker, D.L and Gold, P.E., Effects of the novel NMDA antagonist, NPC 12626, on long-term potentiation, learning and memory, Brain Res., 549 (1991) 213-221. 61 Waugh, N.C. and Norman, D.A., Primary memory, Psychol. Ret'., 72 (1965) 89-104. 62 Winocur, G., The hipopcampus and thalamus: the roles in shortterm and long-term memory and the ~ffects of interference, Behat'. Brain Res., 16 (1985) 135-152. 63 Wood, P.L., Pharmacological evaluation of GABAergic and glutamatergic inputs to the nucleus basalis-cortical and the septalhippocampal cholinergic projections, Can. J. Physiol. Phannacol., 64 (1986) 325-328. 64 Wood, P.L., Cheney, D.L. and Costa, E., An investigation of whether septal gamma-aminobutyrate-containinginterneurons are involved in the reduction in the turnover rate of acetylcholine elicited by substance P and fl-endorphin in the hippocampus, Neuroscience, 4 (1979) 1479-1484. 65 Zola-Morgan, S., Squire, L.R. and Mishkin, M., The neuroanatomy of amnesia: amygdala-hippocampus versus temporal stem, Science, 218 (1982) 1337-1339.
54
BRES 18082
Muscimol infused into the medial septal area impairs long-term memory but not short-term memory in inhibitory avoidance, water maze place learning and rewarded alternation tasks Alan H. N a g a h a r a and James L. M c G a u g h Center for the Neurobiology of Learning and Memory and Department of Psychobiology, Unicersityof California, lrcine, CA 92717 (USA) (Accepted 14 April 1992)
Key words: y-Aminobutyric acid; Hippocampus; Inhibitory avoidance; Long-term memory; Medial septal area:, Memory; Muscimol, Place-learning; Reinforced alternation; Septo-hippocampal system
These experiments investigated the effects of injections of muscimol (1 or 5 nmol), administered into the medial septal area prior to training, on memory tested at different retention delays after training in 3 tasks: an inhibitory avoidance task, a one-trial place learning task, and a rewarded alternation task. In all 3 tasks, intraseptal injections of muscimol did not impair memory performance at short retention delays, but impaired memory at the longer retention delays. These findings are consistent with the view that GABAergic regulation of the septohippocampal cholinergic system plays a selective role in the establishment of long-term memory.
INTRODUCTION
Extensive evidence suggests that the hippocampus participates in the formation of memory for some tasks 42'5t. Furthermore, the findings of many experiments suggest that the hippocampus is involved in the formation of long-term memory, but not short-term memory. Human patients with hippocampal or temporal lobe damage (e.g.H.M.) show a distinct pattern of relatively normal short-term memory for declarative information, but impaired formation of long-term memory 2~'5°, Furthermore, primates with damage to the hippocampus or hippocampus/amygdala show normal short-term retention, but impaired long-term retention when tested on a non-matching.to-sample task 37'65. In addition, several studies have shown that rodents with hippocampal lesions or other manipulations affecting hippocampal function also show normal memory performance at short retention delays, but are impaired at longer retention delays 2,25,4°,45,~2. The medial septal area (MSA) sends a major afferent projection consisting of a large population of
cholinergic and GABAergic fibers to the hippocampus 12'tx4s'49. In rodents, the septohippocampal projections appear to be important in the memory function of the hippocampus; lesions of the MSA or the septohippocampal interconnection produce learning impairments similar to those observed with hippocampus lesions~O,zs,4t,54, see rer. ts. The view that the septohippocampal cholinergic system is involved in regulating memory is supported by findings indicating that memory performance is correlated with cholinergic activity in the hippocampus ~,t4`15aT,36and that disruption of the cholinergic system disrupts memory retention 3,6,t4,4°,57. However, other components of the septohippocampal system may also play a significant role in memory functions. The septohippocampal cholinergic neurons in the medial septai nucleus and horizontal diagonal band are modulated by synaptic input from a GABAergic projection from the lateral septal nucleus 28'43. Infusion of muscimol, a GABAergic agonist, into the MSA inhibits the septohippocampal cholinergic system, as measured by high-affinity choline uptake and acetylcholine
Correspondence: A.H, Nagahara, c / o J,L, McGaugh, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, USA,
55 turnover in the hippocampus 4'63'~. In addition, intraseptal administration of muscimol decreases theta rhythmic activity in the hippocampus 1'33. Several studies have shown that the administration of GABAergic drugs into the MSA can affect memory in a variety of tasks4,6,16,39, 52.
The findings of previous experiments in our laboratory indicated that infusion of muscimol into the MSA impaired memory tested after long delays in two tasks, inhibitory avoidance (48 h) and spatial learning (24 h), when administered prior to training but not when administered after training 4'39. These results suggest that the treatment affected memory storage processes occurring at the time of training, but not memory processes occurring after training (e.g. consolidation). The previous experiments in our laboratory did not address the question of whether the intraseptal injections of muscimol differentially affected retention tested at different delay intervals following learning. Thus, it is not known whether the impairment is due to blocking of acquisition or to selective impairment of long-term memory. To investigate this issue, the present set of experiments examined the effects of intraseptal injection of muscimol on retention tested at different delays following training in 3 different tasks: an inhibitory avoidance task, a place learning task in the water maze, and a rewarded alternation task. MATERIALS AND METHODS
Subjects Male Sprague-Dawloy rats (60 days old, 200-250 g on arrival) from Charles River Laboratories were used. The animals were individually housed and maintained on a 12-h light-dark cycle (lightson at 07.00 h) with food and water available ad libitum. Animals were acclimatized to laboratory conditic~nsfor 1 week prior to surgery. Surgical procedures Animals were implanted with a cannula aimed at the MSA using stereotaxic surgical procedures. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and were given atropine sulfate (0.4 mg/kg, i.p.). The cannulae were constructed of 23 gauge stainless steel tubing (i.d. 0.305 ram, o.d, 0.635 mm) and were 15 mm in length. The tip of the cannula was implanted above the surface of the MSA region using the following stereotaxic coordinates: AP + 0.5 mm from bregma; ML 0.0 mm; DV -4.0 mm from dura; with the nose bar at -3.3 mm from the interaural lines44. The cannula was fixed to the skull using two screws and dental acrylic. A stylet was inserted in the cannula and remained there at all times except during intracranial injections. Immediately after surgery the animals received an intramuscular injection of penicillin (30,000 units) and were maintained in a temperature controlled chamber until recovery from anesthesia. The animals were allowed to recover from the surgery for a week prior to behavioral testing.
lntraseptal injection procedures Each animal received an intraseptal injection of muscimol (Sigma) dissolved in phosphate buffer solution (0.!05 g PO4NaH2 and 0.029 g PO4Na2H, in 100 ml physiological saline; pH to 7.0) or the buffer solution 5 min prior to training. The drug and control solutions were infused through a 30-gauge injection needle connected with a 10 ~1
Hamilton syringe by polyethlene tubing. The injection needle was inserted into the guide cannula and protruded 2 mm beyond the tip of the cannula. The injections were delivered in a volume of 0.5 ~l over a 37-s period using a syringe pump (Sage Instruments: Model 341A). The injection needle was retained in the guide cannula for an additional 30 s after the injection before removal.
Histological procedures The animals were anesthetized and perfused intracardially with saline and 10% formalin. The brains were removed and placed in 10% formalin for at least 1 week. The brains were sectioned at 40 /~m on a freezing microtome and stained with Cresyi violet. Animals with improper cannula placement or brain lesions extending beyond the site of the cannula were not included in the statistical analyses.
EXPERIMENT 1. EFFECTS OF INTRASEPTAL MUSCIMOL TREATMENT ON IMMEDIATE AND DELAYED RETENTION OF INHIBITORY AVOIDANCE TRAINING Previously, we found that intraseptal injections of muscimol administered prior to training impaired memory in an inhibitory avoidance task when retention was tested 48 h after training 39. The present experiment was conducted to determine effects of such injections on inhibitory avoidance retention tested at shorter intervals following training. The animals received intraseptal injections of 5 nmol muscimol or buffer solutions 5 min prior to training on an inhibitory avoidance task. The trough-shaped inhibitory avoidance apparatus consisted of two compartments separated by a sliding door 29. The starting compartment was illuminated by a Tensor lamp (25 W). The floor of the the other compartment was constructed of stainless-steel plates through which footshock could be delivered. On the training trial, the rat was placed in start compartment facing away from the door. When the rat turned around, the door was opened, and a timer was started when the rat turned to face the dark compartment. When the rat entered the darkened compartment, the door was closed and a mild footshock (0.5 mA, 1.0 s, constant current)was administered. The rat's latency to enter the dark compartment was recorded. Animals in different groups were first given retention tests at inte~als of 15 s or 15 rain following the training trial. On the retention test, the animal was placed in the starting compartment as on the training trial. The timer recording the retention latency was started immediately after the animal turned to face the darkened compartment or after 10 s if the animal did not turn around within that interval. No footshock was administered on the retention tests. The latency to enter the darkened compartment (maximum of 600 s) was used as the index of retention of the footshock training. All animals were also given a second retention test 48 h later using the same test procedures.
56
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48-hr Retest
Fig. I. Effects of intraseptal infusion of 5 nmol of muscimol administered prior to trainingon an inhibitory avoidance task on retention tested at delays of 15 s and 48 h. Compared to the buffer group (n = I0). animals given muscimol (n = 9) were impaired on the 48-h retention lest. but not on the 15-s retention test. * P < 0.05 vs. respective buffer group.
Resultsand Discussion Fig. 1 shows the retention latencies of the group given the first retention test 15 s after training. The latencies of the muscimol group did not significantly differ from those of the buffer group on the 15-s retention test (Uq.,)= 44, P > 0.80). However, on the 48-h retention test the retention latencies of the 5 nmoi muscimol group were significantly lower than those of the buffer group (U,~.m. = 23, P < 0.05). Fig. 2 shows the retention latencies of the group given the first retention test 15 min after training. The retention latencies of the muscimol group were significantly lower than those of the buffer group on the 15-rain retention test (U,),q- 15, P
0.50). The present results are similar to those of Expt. 1: intraseptai administration of muscimol did not affect retention tested following a short delay but impaired retention tested at a longer delay. These results clearly indicate that intraseptal injections of muscimol do not impair the animals' ability to acquire and use spatial information in a familiar environment. Thus, the effect of pretraining intraseptal injection of muscimol on retention performance seen at longer training-retention intervals cannot be attributed to deficits in sensory, motor, or motivational processes. Consequently, these findings provide additional evidence supporting the view that the retention impairment seen at longer delays is due to selective interference with the formation of long-term memory.
3o,
8 ~
EXPERIMENT 3. THE EFFECT OF INTRASEPTAL MUSCIMOL ON REWARDED ALTERNATION
20, 10 Muscimol / 5 rain
Acquisi'tion Trial
Retention Trial
Fig. 3. Effects of pretraining intraseptal infusion of 1 nmol muscimol on a single-trial place learning task on performance on 15-s or 5-min retention tests. The performance of the muscimol group tested at the 5-min delay (n--14) was impaired, in comparison with that of the buffer group tested at the 5-min delay (n = 14). The performance of the muscimol group tested at the 15-s delay (n = 15) was not impaired, in comparison with that of the buffer group tested at the 15-s delay (n = 15). * P < 0.01 vs. respective buffer group.
Considerable evidence suggests that the hippocampal system is involved in the learning of tasks requiring the use of spatial cues. In our laboratory, we found that pretraining injection of muscimol in the MSA impaired a rewarded alternation task at a delay of 15 s 3s. To examine further the effect of intraseptal injections of muscimol on this task, the present studied retention of rewarded alternation tested either immediately after each trial or after a 15 s intertrial interval.
58
Subjects Rats previously trained on an inhibitory avoidance task were used in the present experiment. The animals were given limited access to food in order to reduce their body weights by about 15% over a 7-10 day period. The animals were tfien allowed to gain about 5 g of additional body weight during each subsequent week. The animals were first habituated to a T-maze apparatus (starting arm and goal arms: 17 cm wide, 20 cm high and 30 cm long). During maze habituation, .pieces of Froot Loops were scattered on the floor of the T-maze, and the rats were allowed to explore the T-maze and eat the Froot Loops. The animals were then trained to retrieve pieces of Froot Loops from black food cups located at the end of each of the two goal arms. This was followed by 2 days of training using a rewarded alternation, i.e. 'win-shift' paradigm. On the first training trial, both goal arms were baited and the animal was allowed to enter only one arm. On the next 15 test trials, only the goal arm not entered in the immediately preceding trial was baited. On each trial, after the animal entered the goal arm and retrieved the food (or investigated the empty food well), the animal was removed from the maze. On the next 2 days, the animals were tested in the rewarded alternation task 5 min after receiving intraseptal injections of 1 nmol muscimol or buffer. The animals were assigned to either a buffer or musci. tool group for both days of testing and tested at the two different delay conditions. On the first day of testing, the animals were tested in either a 15-s delay condition or an immediate condition. In the 15-s delay condition, the animal was placed in a holding container for 15 s between test trials. In the immediate condition, the animal was immediately placed back into the start compartment after removal from the goal arm (a delay of 2-3 s). After the first test day, the animals were assigned to the other delay condition. Results attd Discussion The mean percent alternation for the 4 groups is shown in Fig. 4. in the immediate condition, the performance of the muscimol-treated animals did not differ significantly from that of the buffer-treated group (Fi,i, = 1.10, n.s,). However, in the 15-s delay condition, the alternation performance of the muscimoltreated animals was significantly impaired compared with that of the buffer-treated animals (Fl,lo= 20.1, P < 0.01). In addition, the alternation performance of the muscimol-treated animals was significantly poorer on the 15-s delay test than on the immediate test (Fl.~ = 32.1 P < 0.01).
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Fig. 4. Effects of pretraining intraseptal infusion of 1 nmol of muscimol on a rewarded alternation task tested with no intertrial interval (immediate condition) or with a 15-s intertrial interval. The performance of the muscimol group tested at the 15-s intertrial interval (n -- 7) was impaired, in comparison with that of the buffer group tested at the same intertrial interval (n = 7). The muscimol group tested in the immediate condition ( n - - 7 ) was not impaired compared to the buffer group tested in the immediate condition (n = 7). * P < 0.01 vs. respective buffer group.
These findings are consistent with the findings of Expts. 1 and 2 above indicating that intraseptal administration of muscimol does not impair memory performance when there is a short delay between training and retention but impairs retention tested at longer delays. In addition, the present findings are similar to those of experiments examining the effects, on alternation performance, of other manipulation of the hippocampal system I'~J°,4°. The present results also confirm previous findings that intraseptal administration of muscimol impairs rewarded alternation at a 15-s intertrial interval "~H. GENERAL DISCUSSION The present study showed that, in 3 different tasks, intraseptal injections of muscimol administered produced a selective pattern of memory impairment that is observed only after a retention delay. These results are similar to the findings of numerous studies showing that hippocampal lesions produce a memory deficit only after a certain retention delay, but not at a short retention delay 2-~.37,4s,-~,~'2.~'s.In addition, other studies showed that manipulations disrupting the septohippocamai system (e.g. fornix lesions) produce a similar pattern of selective memory deficit 10,23A0.Thus, in light of these studies, it seems likely that the effects of intraseptai injections of muscimol on memory are mediated by its action on the septohippocampal projection neurons. The effects of the intraseptal muscimol treatment on memory may be mediated, in part, by the cholinergic component of this system, since intraseptal
59 injections of muscimoi depress cholinergic activity in the hippocampus 4't'3"~ and numerous studies implicate the hippocampal cholinergic system in memory a.-s.'~,~4.~5. However, since only 30-40% of the septohippocampal system is cholinergic, the effects of the intraseptal muscimol treatment on memory may also be mediated by an action on the non-cholinergic (e.g. GABAergic) component of the septohippocampal system ~2.~3. Although the present findings showed a similar pattern of memory deficits on 3 different tasks, the temporal gradient of the memory deficit differed between the tasks. More specifically, at a 15-s retention delay, intraseptal injection of muscimol impaired retention in the continuous alternation task, but not in the place learning task or the inhibitory avoidance task. The rapid decline in retention seen in the alternation task may be due to the training procedures used: in the alternation task the animals received 15 consecutive retention trials while in the inhibitory avoidance and the place learning task the animals received only a single training trial. Thus, in the alternation task, retention may have been influenced by proactive interference from the prior training trials. In studies of human memory, it has been long recognized that maintenance of information in short-term memory is not solely the function of a temporal gradient, but is affected by both proacti~,z interference 24 and retroactive interference t'~. In rodent studies, retroactive interference, in the form of interpolated activity, has been shown to affect the temporal gradient of memory performance of hippocampal-lesioned animals ''~'4~'. Of course, other task-specific factors may have contributcd to the differences in retention performance in these tasks. Additional research is needed to clarify this issue. The present results considered together with previous findings provide insight into the role of the septohippocampal system in the acquisition and consolidation of memory. In previous experiments we found that pretraining, but not posttraining, injection of muscimoi into the MSA impaired retention performance in an inhibitory avoidance task and a multiple-trial place learning task 4''~'J. The absence of a posttraining effect suggests that intraseptal injection of muscimol probably does not disrupt the memory consolidation process 32. Thus, these results suggest that intraseptal injections of muscimol impair memory by disrupting some underlying memory process that occurs at the time of training. In the present study, the absence of a memory deficit at short retention delays indicates that the intraseptal muscimol treatment did not block the acquisition of information. Furthermore, this result indicates that the retention impairments seen at the
longer delays cannot be simply attriouted to influences on sensory, motor, or motivational processes necessary for performance of these 3 tasks. Considered together, these findings suggest that the intraseptal muscimol treatment impairs long-term memory by acting on a memory-related process that occurs at the time of training rather than by acting on posttraining consolidation processes. Since the underlying mechanisms mediating longterm memory formation are not clearly understood, it is difficult to determine what memory process is affected by the intraseptal injections of muscimol. However, it is interesting to note that some evidence suggests that the administration of N-methyl-o-aspartate (NMDA) antagonists impairs memory when administered prior to training, but not after training ~'47,6°.bu~~c,~ =f. 34; we found that the intraseptal injection of muscimol produces a similar effect on memory on two tasks4,3,~, t,ut =~ =f. t,. These behavioral effects are significant in light of the findi~,~:s that NMDA antagonists block the formation of long-term potentiation (LTP) in hippocampus slices when applied prior to induction, but not after induction 7,~,35.55, and since LTP has been proposed as a basis for long-term memory e'g' 3~,.~. According to this view, the effects of NMDA antagonists on memory may be due to disruption of LTP formation. Interestingly, since induction of LTP is influenced by both rhythmic theta activity t'~'27''~'~ and cholinergic activity 2m':2, intraseptal injections of muscimol could disrupt LTP induction and thus impair long-term memory by depressing hippocampal theta activity ~'3"~ and cholinergic activity ~,t,3. According to this interpretation, the effects of pretraining intraseptal injections of muscimol on memory may reflect a selective action on one process underlying long-term memory formation (e.g. LTP induction), but not on another process (e.g. LTP development). Acklzowledgemems. This research was supported by USPHSMH1252t~
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