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Infusions of physostigmine into the hippocampus or the entorhinal cortex attenuate avoidance retention deficits produced by intra-septal infusions of the GABA agonist muscimol
Brain Research 920 (2001) 10–18 www.elsevier.com / locate / bres
Research report
Infusions of physostigmine into the hippocampus or the entorhinal cortex attenuate avoidance retention deficits produced by intra-septal infusions of the GABA agonist muscimol Aldemar Degroot a , Marise B. Parent a,b , * b
a Department of Psychology, University of Alberta, Edmonton, Alberta, Canada T6 G 2 E9 Department of Psychology, Georgia State University, University Plaza, Atlanta, GA 30303 -3083, USA
Accepted 12 July 2001
Abstract Septal g-aminobutyric acid (GABA) receptor activation is known to disrupt memory formation, although the mechanisms underlying this impairment remain unclear. The present study explored the possibility that high levels of septal GABA receptor activity might impair memory by down-regulating acetylcholine (ACh) function in archicortex and entorhinal cortex. To test this possibility, rats were trained on an avoidance task 15 min after receiving intra-septal infusions of vehicle or muscimol (5 nmol / 0.5 ml) combined with unilateral intra-hippocampal (10 ml / 1 ml) or intra-entorhinal cortex (1.875 mg / 0.25 ml) infusions of vehicle or the acetylcholinesterase inhibitor physostigmine. We demonstrate that these infusions do not alter acquisition performance on a continuous multiple trial inhibitory avoidance task. However, intra-septal infusions of muscimol dramatically impair retention performance 48 h later. More importantly, infusions of physostigmine into the hippocampus or the entorhinal cortex, at doses that do not influence acquisition or retention performance when infused alone, attenuate the impairing effects of the muscimol infusions on retention. We suggest that high levels of septal GABA receptor activity might impair memory by down-regulating ACh levels in the hippocampal region, and that such memory impairments can be ameliorated by increasing ACh levels in the hippocampus or entorhinal cortex. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Entorhinal; Hippocampus; Septal area; Memory
1. Introduction Activation of g-aminobutyric acid (GABA) receptors in the medial septum is known to affect learning and memory. For example, intra-septal infusions of the GABA agonist muscimol impair learning and memory in a variety of tasks, including visual discrimination, spontaneous and rewarded alternation, inhibitory avoidance, and performance in the radial arm and water maze tasks
*Corresponding author. Tel.: 11-404-463-9795; fax: 11-404-6513929. E-mail address: [email protected] (M.B. Parent).
[3,4,6,15,16,32–36]. However, the process by which elevated septal GABA receptor activity impairs memory remains unknown. One possibility is that septal GABA receptor activation might indirectly affect learning and memory by modulating cholinergic function in the archicortex. In support of this hypothesis, intra-septal infusions of a variety of memory-modulating drugs, including infusions of muscimol, affect cholinergic function in the hippocampus [3,17,20,22,23]. In many cases, the actions of intra-septal infusions of drugs on memory parallel the effects of the drugs on hippocampal cholinergic function. For example, only those doses of muscimol that impair spatial water maze memory decrease hippocampal high affinity choline uptake [3]. Intra-septal infusions of muscimol also prevent training-induced increases in hippocam-
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02798-6
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pal ACh [7,31] and affect the dose–response characteristics of intra-hippocampal infusions of an ACh agonist on avoidance memory [9]. We recently explored the possibility that the impairing effects of septal GABA activation can be reversed by increasing ACh levels in the hippocampus. Specifically, we examined the effect of simultaneously manipulating septal GABA receptors and hippocampal ACh levels in a rat performing in a spontaneous alternation task [6]. The results indicate that intra-hippocampal infusions of the acetylcholinesterase (AChE) inhibitor physostigmine, at a dose that has no effect when infused alone, prevents the impairing effects of intra-septal infusions of muscimol on spontaneous alternation performance. The finding that concurrent infusions of physostigmine prevent the impairing effects of intra-septal infusions of muscimol on memory is currently limited to one memory task: spontaneous alternation. Evidence suggests that spontaneous alternation is a measure of spatial working memory. For example, alternation scores are lowered by the removal of directional cues [40] or by increasing the temporal interval between location choices [2,19,27,28,38,42]. In the present study, we aimed to determine whether the interaction between the effects of intra-septal infusions of muscimol and intra-hippocampal infusions of physostigmine reflects a general effect on memory rather than an effect on some other process that might influence performance. To address this question, we examined the effects of these manipulations on memory in a different paradigm: continuous multiple trial inhibitory avoidance (CMIA). In contrast to spontaneous alternation, CMIA involves the administration of footshock and is considered a measure of emotional, reference memory. In addition, unlike spontaneous alternation, CMIA provides a measure of both acquisition and retention performance, which allows for the effects of drugs on these two variables to be dissociated. The goal of experiment 1 was to determine whether any deficits in CMIA produced by intra-septal infusions of muscimol could be reversed by intra-hippocampal infusions of physostigmine. Limited evidence suggests that the medial septum may also influence memory via a process that involves septal cholinergic projections to the entorhinal cortex. Inactivation of the medial septum abolishes theta activity in the entorhinal cortex in freely moving rats [26] and alters the firing rate of entorhinal cells in rats performing in a spatial task [30]. Lesions of septal cholinergic efferents, which include damage to the cholinergic projection from the medial septum to the entorhinal cortex, impair radial arm maze performance [44]. As in the case of intra-hippocampal infusions, infusions of physostigmine into the entorhinal cortex prevent the impairing effects of intra-septal infusions of muscimol on spontaneous alternation performance [6]. The purpose of experiment 2 was to determine whether infusions of physostigmine into the
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entorhinal cortex would also attenuate the impairing effects of intra-septal infusions of muscimol on CMIA.
2. Materials and methods
2.1. Experiment 1 2.1.1. Subjects Male Sprague–Dawley rats, weighing 250–300 g upon arrival, were used. They were individually housed and maintained on a 12-h light / dark cycle with food and water ad libitum. All rats were handled 2 days prior to surgery for 3 min each. During handling, the rats were given an oral administration of water in order to habituate them to the oral administration of an analgesic on the day of surgery. 2.1.2. Surgery Surgery was performed at least 1 week after the rats arrived. On the day of surgery, the rats were given an oral dose of the analgesic acetaminophen (Tylenol 120 mg / 1.5 cc). One hour later, they were given atropine sulfate (0.4 mg / kg, i.p.), anaesthetized with pentobarbitol (Nembutal 50 mg / kg, i.p.), hydrated with saline (3 cc, s.c.), and given the antibiotic penicillin (Crystiben, Rhone Merieux Canada Inc., 4300 U / kg, i.m.). Stereotaxic surgical procedures were used to implant one 22-gauge stainless-steel guide cannula (Plastics One, Inc. Roanoke, VA) aimed at the medial septum (0.5 mm anterior to bregma [AP], 4.9 mm ventral to dura [DV], 3.2 mm from the interaural line; 37) and one guide cannula aimed at the dorsal hippocampus (24.2 mm AP, 2.0 mm DV, 4.1 mm lateral [ML] to the midline). For half of the rats, the hippocampal cannula was implanted in the left hemisphere. The cannulae were attached to the skull with four jeweler’s screws and cranioplastic cement and a dummy cannula was inserted into each guide cannula to keep the cannula tract clear. Immediately after surgery, the rats were placed in a warm environment until they regained consciousness. Two days after surgery, each cannula was checked for obstructions and betadine was applied to the surgical wound. 2.1.3. Procedure and drugs Two days prior to behavioral testing, all rats were handled for 3 min and then 5 min the next day. All testing occurred at least 1 week following surgery between 09:00 and 19:00 h. Rats were given an infusion of vehicle (phosphate-buffered saline, pH 7.4) or physostigmine (10 mg / 1 ml / over 1 min) into the hippocampal cannula, followed immediately by an infusion of vehicle or muscimol (570 ng / 0.5 ml / over 1 min) into the medial septal cannula. The dose of muscimol was selected based on previously published work [32,33,35]. The dose of physostigmine was selected based on our previous findings that
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indicate that intra-hippocampal infusions of this dose reverse deficits in spontaneous alternation performance induced by intra-septal infusions of muscimol [6]. The solutions were infused through a 28-gauge injection needle that extended 1 mm beyond the guide cannula. The needle was connected to a 10-ml Hamilton syringe by polyethylene tubing and the infusions were delivered using an infusion pump (Harvard Apparatus 22). The injection needles were left in place for 1 min following the infusions in order to allow for diffusion. An experimenter that was blind to the drug treatment delivered the infusions.
2.1.4. Continuous multiple inhibitory avoidance Each rat was trained in the CMIA task 15 min after the last infusion. A trough-shaped straight alley (91 cm long, 20 cm wide at the top, 6.4 cm wide at the floor, and 15 cm deep), separated into two compartments by a retractable door, was used for the training. The starting compartment (30 cm long) was illuminated by a 15-W tensor lamp and the adjoining compartment (61 cm) where rats received a footshock, was dark. The apparatus was located in an unlit room. For the training, each rat was placed into the lighted compartment with its head facing away from the door. When the rat turned around, the door was opened and the latency to enter the dark compartment with all four paws was recorded (100 s maximum). When the rat entered the dark compartment, it received a footshock (0.5 mA) through the metal floor plates until it returned to the lighted compartment. This sequence of events constituted one training trial. The door remained open for the remainder of the training session, and whenever the rat entered the dark compartment with all four paws, it received a 0.5-mA footshock until it escaped back to the lighted compartment. Training continued until the rat remained in the lighted compartment for a criterion of 100 consecutive seconds or until a maximum of five footshocks had been given. The number of trials (i.e. footshocks) needed to achieve the criterion and the latency to enter the dark compartment on each trial were recorded and used as measures of acquisition. Retention of the CMIA training was assessed 48 h later. Each rat was placed in the lighted compartment with its head facing away from the door and the door was lowered 30 s later. Latency to enter the dark compartment with all four paws in the absence of footshock (600 s maximum) was recorded and used as an index of memory. 2.1.5. Histology After the completion of the behavioral tests, the rats were anesthetized with an overdose of chloral hydrate (800 mg / ml) and perfused intracardially with 0.9% saline followed by 10% formalin. The brains were removed and placed in a 10% formalin solution. At least 48 h later, the brains were frozen and sectioned (60 mm), mounted onto glass slides, and stained with thionin. The sections were
examined under a light microscope by an observer blind to the drug history and behavioral results of each rat. In the case of ambiguous placements, an additional experienced observer who was also blind to the treatment history and behavioral results examined the histology.
2.1.6. Statistics The CMIA data were expressed as means and standard errors of the mean (S.E.M.) and were analyzed using analysis of variance (ANOVA) and Fisher adjusted leastsignificant differences (LSD) post-hoc tests. An alpha level of 0.05 was used as the criterion for statistical significance. Due to heterogeneity of variance, the acquisition latencies were transformed to their natural logs prior to the statistical analyses. 2.2. Experiment 2 The procedure used in this experiment was the same as that used in experiment 1 with the following exceptions.
2.2.1. Surgery Stereotaxic surgical procedures were one guide cannula aimed at the medial aimed at the entorhinal cortex (27.5 mm 5.3 mm ML). The unilateral entorhinal were counterbalanced for hemisphere.
used to implant septum and one AP, 6.2 mm DV, cortex cannulae
2.2.2. Procedure The rats were given an infusion of vehicle or physostigmine (1.875 mg / 0.25 ml / over 1 min) into the entorhinal cortex cannula immediately followed by an infusion of vehicle or muscimol (570 ng / 0.5 ml / over 1 min) into the septal cannula. The dose of physostigmine was selected based on our previous work indicating that intra-entorhinal infusions of this dose effectively reversed spontaneous alternation deficits induced by intra-septal infusions of muscimol [6].
3. Results
3.1. Experiment 1 3.1.1. Histology The behavioral data for animals with misplaced cannulae or extensive necrosis at the cannula site were discarded (n59). Fig. 1 shows the location of the infusion sites in the (A) medial septum and (B) dorsal hippocampus of the rats whose data were included in the statistical analysis. Five rats were given infusions of vehicle in both the hippocampus and medial septum (VEH-VEH), ten rats were given infusions of physostigmine in the hippocampus and vehicle in the medial septum (PHYSO-VEH), seven rats were given infusions of vehicle in the hippocampus and muscimol in the medial septum (VEH-MUSC), and
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Fig. 1. Schematic illustration of a coronal section of the rat brain showing the approximate location of (A) medial septal and (B) hippocampal infusion sites in experiment 1. Atlas plates adapted from Ref. [37].
six rats were given infusions of physostigmine in the hippocampus and muscimol in the medial septum (PHYSO-MUSC).
3.1.2. CMIA acquisition performance The pretraining drug infusions into the hippocampus and the medial septum did not affect CMIA acquisition performance. Although there was a non-significant trend, the pretraining infusions did not affect latency to enter the dark compartment on the first training trial [F(3, 24)5 2.59; P50. 077]. The infusions did not affect the total number of trials required to reach the acquisition criterion [F(3, 24)50.44; P.0.05; see Table 1]. 3.1.3. CMIA retention performance The pretraining infusions into the hippocampus and the septum significantly affected retention performance tested 48 h after CMIA training [F(3,24)53.79; P,0.05; see Fig. 2]. As expected, based on previous findings, intra-septal infusions of muscimol impaired retention performance. The retention latencies of VEH-MUSC rats were significantly shorter than those of VEH-VEH rats (P,0.05). Infusions of physostigmine into the hippocampus did not affect retention performance. The retention latencies of PHYSO-VEH rats were not significantly different from
those of VEH-VEH rats (P.0.05). Importantly, the pretraining intra-hippocampal infusions of physostigmine attenuated the impairing effects of the septal muscimol infusions. The retention latencies of PHYSO-MUSC rats were not significantly different from those of VEH-VEH rats (P.0.05).
3.2. Experiment 2 3.2.1. Histology As in experiment 1, the behavioral results of those rats with misplaced cannulae or extensive necrosis at the cannula site were discarded (n512). Fig. 3 shows the location of the infusion sites in the entorhinal cortex of the rats whose data were included in the statistical analyses. The septal placements were comparable to those shown in Fig. 1A. Ten rats were given infusions of vehicle in both the entorhinal cortex and medial septum (VEH-VEH), ten rats were given infusions of physostigmine in the entorhinal cortex and vehicle in the medial septum (PHYSOVEH), ten rats were given infusions of vehicle in the entorhinal cortex and muscimol in the medial septum (VEH-MUSC), and seven rats were given infusions of physostigmine in the entorhinal cortex and muscimol in the medial septum (PHYSO-MUSC).
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Table 1 The effects of intra-septal infusions of vehicle or muscimol combined with intra-hippocampal (experiment 1) or intra-entorhinal (experiment 2) infusions of vehicle or physostigmine on mean (6S.E.M.) latency to enter the dark compartment on the first training trial and the mean (6S.E.M.) number of trials needed to reach the training criterion Medial septum Vehicle
Experiment 1: Hippocampus n Latency Number of trials Experiment 2: Entorhinal cortex n Latency Number of trials
Muscimol
Vehicle
Physostigmine
Vehicle
Physostigmine
5 0.85 (0.20) 2.8 (0.37)
10 0.63 (0.12) 3.0 (0.30)
7 1.16 (0.25) 2.7 (0.29)
6 1.28 (0.22) 2.5 (0.34)
10 0.38 (0.04) 3.4 (0.34)
10 0.56 (0.10) 3.6 (0.22)
10 0.97 (0.17) 3.0 (0.26)
7 0.97 (0.23) 3.14 (0.34)
3.2.2. Acquisition performance The pretraining drug infusions into the entorhinal cortex and the medial septum affected latency to enter the dark compartment on the first training trial [F(3, 33)54.47; P,0.05; see Table 1]. Specifically, the acquisition latencies of PHYSO-MUSC and PBS-MUSC rats were significantly longer than those of VEH-VEH rats (P,0.01 for both comparisons). However, the infusions did not affect the total number of trials required to reach the acquisition criterion [F(3,33)50.89; P.0.05; see Table 1].
3.2.3. Retention performance The pretraining infusions into the entorhinal cortex and the medial septum significantly affected CMIA retention performance [F(3,33)53.57, P,0.05; see Fig. 4]. As in experiment 1, intra-septal infusions of muscimol impaired retention performance. The retention latencies of VEHMUSC rats were significantly shorter than the retention latencies of VEH-VEH rats (P,0.05). Infusions of physostigmine into the entorhinal cortex did not affect retention performance. The retention latencies of PHYSO-VEH rats
Fig. 2. Infusions of physostigmine (10 mg / 1 ml) into the dorsal hippocampus attenuate the impairing effects of intra-septal infusions of muscimol (570 ng / 0.5 ml) on continuous multiple inhibitory avoidance performance (* P,0.05 vs. VEH-VEH).
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Fig. 3. Schematic illustration of a coronal section of the rat brain showing the approximate location of infusion sites in the entorhinal cortex in experiment 2. Atlas plates adapted from Ref. [37].
were not significantly different from those of VEH-VEH rats (P.0.05). Notably, infusions of physostigmine into the entorhinal cortex attenuated the impairing effects of the intra-septal infusions of muscimol. The retention latencies of PHYSO-MUSC rats were not significantly different from those of VEH-VEH rats (P.0.05).
4. General discussion The major finding of this study is that impairments in emotional reference memory due to elevated septal GABA receptor activation can be prevented by up-regulating ACh function in the hippocampus and entorhinal cortex. Spe-
Fig. 4. Infusions of physostigmine (1.875 mg / 0.25 ml) into the entorhinal cortex attenuate the impairing effects of intra-septal infusions of muscimol (570 ng / 0.5 ml) on continuous multiple inhibitory avoidance performance (* P,0.05 vs. VEH-VEH).
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cifically, the present results demonstrate that infusions of the AChE inhibitor physostigmine into the dorsal hippocampus or the entorhinal cortex attenuate the impairing effect of septal muscimol infusions on CMIA retention performance. The present results are consistent with previous findings indicating that intra-septal infusions of muscimol impair avoidance retention performance tested 48 h after training [33,35] and increase the dose of intra-hippocampal infusions of an ACh agonist needed to enhance avoidance retention [9]. The present findings also concur with our previous results showing that the impairing effects of intra-septal infusions of muscimol on spontaneous alternation are reversed by infusions of physostigmine into the hippocampus or entorhinal cortex [6], and by infusions of glucose into the hippocampus [36], which would also presumably increase ACh levels [13,14,38,39]. Muscimol is a selective ligand for GABA-A receptors [29], which are present on both septal cholinergic and GABAergic projection neurons [11,18]. One way that muscimol may impair memory is by inhibiting the cholinergic projection neurons. According to this hypothesis, infusions of physostigmine into the hippocampus or entorhinal cortex reverse the muscimol-induced deficit by increasing ACh levels at these cholinergic terminals. However, the findings of a recent study suggest that muscimol may affect memory through an influence on both the cholinergic and GABAergic projection neurons. Specifically, infusions of lower doses of muscimol into the septum are sufficient to impair spatial working memory when the cholinergic projection is selectively lesioned [34]. Septal GABAergic projection neurons synapse onto GABAergic interneurons in the hippocampus, which in turn synapse onto hippocampal glutamatergic pyramidal cells [8,10,43]. Given this pattern of connections, muscimol-induced inhibition of the septo-hippocampal GABA projection would be expected to result in the inhibition of hippocampal pyramidal cells. The finding that cholinomimetic drugs depolarize hippocampal pyramidal cells [41] raises the possibility that physostigmine could prevent the memory-impairing effects of septal infusions of muscimol by preventing muscimol-induced inhibition of hippocampal pyramidal cells. Finally, infusions of glutamatergic antagonists into the hippocampus decrease hippocampal extracellular ACh levels [31], suggesting that muscimol-induced inhibition of pyramidal cells could lead to a decrease in acetylcholine levels. Thus, it is also possible that intra-hippocampal infusions of physostigmine reverse the memory-impairing effects of septal GABA receptor activation by counteracting this decrease in ACh. The present results also show that the effects of the pretraining infusions appeared to be specific to retention performance. In experiment 1, neither muscimol, physostigmine, nor the combination of the two affected acquisition performance. In experiment 2, muscimol significantly increased latency to enter the dark compartment on the first
training trial before shock was administered. However, this finding does not likely reflect an acquisition deficit because rats given muscimol required the same number of training trials to reach the acquisition criterion as did control rats. Prior experiments examining the effects of pretraining intra-septal infusions of muscimol on avoidance retention performance have typically employed a one trial avoidance paradigm and therefore did not provide any measures of acquisition [32,33,35]. The present findings indicating that the pretraining infusions of muscimol did not affect acquisition performance in a multiple trial paradigm have several implications. First, these findings suggest that the drug infusions did not affect sensorimotor or motivational processes that could have interfered with acquisition. Our findings suggest that all of the rats learned the task to a similar degree and that the deficits that were observed on the retention test likely reflect an effect of muscimol on memory, rather than on learning or performance. This interpretation is consistent with previous findings indicating that pretraining intra-septal infusions of muscimol do not impair retention performance tested 15 s after training, but do impair retention tested 15 min or 48 h later [32] and the finding that posttraining intra-septal infusions of muscimol also impair retention performance [24]. Importantly, the present finds show that physostigmine did not influence acquisition or retention performance on its own but did attenuate the impairing effect of muscimol on retention performance. This finding suggests that septal GABA receptors interact with cholinergic processes in the hippocampus and entorhinal cortex specifically during memory consolidation processes. It is well established that the medial septum interacts with the hippocampus to influence learning and memory [1,5,6,44]. However, the possibility that the medial septum may interact with the entorhinal cortex to affect mnemonic processes is less well understood. Our discovery that infusions of physostigmine into the entorhinal cortex reverse the impairing effects of septal GABA receptor stimulation on spontaneous alternation [6] and inhibitory avoidance (present findings) provide convincing evidence to support the hypothesis that the septum and entorhinal cortex interact to influence learning and memory. Future studies are needed to determine whether the medial septum interacts directly with the entorhinal cortex, or indirectly via the hippocampus [12,21,25]. In summary, the present findings demonstrate that infusions of the AChE inhibitor physostigmine into the hippocampus or the entorhinal cortex attenuate the impairing effects of intra-septal infusions of the GABA agonist muscimol on CMIA retention performance. In combination with our previous findings, the results of the present study indicate that increasing ACh levels in the hippocampus or the entorhinal cortex is sufficient to reverse the memory impairing effects of septal GABAergic receptor activation in two different memory tasks. These findings provide further support for the hypothesis that septal GABA
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receptors might impair memory by decreasing ACh levels in the hippocampus and the entorhinal cortex.
Acknowledgements This work was supported by NINDS, NIDDK, and JDF (1RO1 NS 41173-01), MRC G11821018 and NSERC OGP019453. We thank Justin Park for his invaluable technical assistance and Katherine Harter and Dr Benjamin Philpot for their comments on an earlier version of this manuscript.
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Research report
Infusions of physostigmine into the hippocampus or the entorhinal cortex attenuate avoidance retention deficits produced by intra-septal infusions of the GABA agonist muscimol Aldemar Degroot a , Marise B. Parent a,b , * b
a Department of Psychology, University of Alberta, Edmonton, Alberta, Canada T6 G 2 E9 Department of Psychology, Georgia State University, University Plaza, Atlanta, GA 30303 -3083, USA
Accepted 12 July 2001
Abstract Septal g-aminobutyric acid (GABA) receptor activation is known to disrupt memory formation, although the mechanisms underlying this impairment remain unclear. The present study explored the possibility that high levels of septal GABA receptor activity might impair memory by down-regulating acetylcholine (ACh) function in archicortex and entorhinal cortex. To test this possibility, rats were trained on an avoidance task 15 min after receiving intra-septal infusions of vehicle or muscimol (5 nmol / 0.5 ml) combined with unilateral intra-hippocampal (10 ml / 1 ml) or intra-entorhinal cortex (1.875 mg / 0.25 ml) infusions of vehicle or the acetylcholinesterase inhibitor physostigmine. We demonstrate that these infusions do not alter acquisition performance on a continuous multiple trial inhibitory avoidance task. However, intra-septal infusions of muscimol dramatically impair retention performance 48 h later. More importantly, infusions of physostigmine into the hippocampus or the entorhinal cortex, at doses that do not influence acquisition or retention performance when infused alone, attenuate the impairing effects of the muscimol infusions on retention. We suggest that high levels of septal GABA receptor activity might impair memory by down-regulating ACh levels in the hippocampal region, and that such memory impairments can be ameliorated by increasing ACh levels in the hippocampus or entorhinal cortex. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Entorhinal; Hippocampus; Septal area; Memory
1. Introduction Activation of g-aminobutyric acid (GABA) receptors in the medial septum is known to affect learning and memory. For example, intra-septal infusions of the GABA agonist muscimol impair learning and memory in a variety of tasks, including visual discrimination, spontaneous and rewarded alternation, inhibitory avoidance, and performance in the radial arm and water maze tasks
*Corresponding author. Tel.: 11-404-463-9795; fax: 11-404-6513929. E-mail address: [email protected] (M.B. Parent).
[3,4,6,15,16,32–36]. However, the process by which elevated septal GABA receptor activity impairs memory remains unknown. One possibility is that septal GABA receptor activation might indirectly affect learning and memory by modulating cholinergic function in the archicortex. In support of this hypothesis, intra-septal infusions of a variety of memory-modulating drugs, including infusions of muscimol, affect cholinergic function in the hippocampus [3,17,20,22,23]. In many cases, the actions of intra-septal infusions of drugs on memory parallel the effects of the drugs on hippocampal cholinergic function. For example, only those doses of muscimol that impair spatial water maze memory decrease hippocampal high affinity choline uptake [3]. Intra-septal infusions of muscimol also prevent training-induced increases in hippocam-
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02798-6
A. Degroot, M.B. Parent / Brain Research 920 (2001) 10 – 18
pal ACh [7,31] and affect the dose–response characteristics of intra-hippocampal infusions of an ACh agonist on avoidance memory [9]. We recently explored the possibility that the impairing effects of septal GABA activation can be reversed by increasing ACh levels in the hippocampus. Specifically, we examined the effect of simultaneously manipulating septal GABA receptors and hippocampal ACh levels in a rat performing in a spontaneous alternation task [6]. The results indicate that intra-hippocampal infusions of the acetylcholinesterase (AChE) inhibitor physostigmine, at a dose that has no effect when infused alone, prevents the impairing effects of intra-septal infusions of muscimol on spontaneous alternation performance. The finding that concurrent infusions of physostigmine prevent the impairing effects of intra-septal infusions of muscimol on memory is currently limited to one memory task: spontaneous alternation. Evidence suggests that spontaneous alternation is a measure of spatial working memory. For example, alternation scores are lowered by the removal of directional cues [40] or by increasing the temporal interval between location choices [2,19,27,28,38,42]. In the present study, we aimed to determine whether the interaction between the effects of intra-septal infusions of muscimol and intra-hippocampal infusions of physostigmine reflects a general effect on memory rather than an effect on some other process that might influence performance. To address this question, we examined the effects of these manipulations on memory in a different paradigm: continuous multiple trial inhibitory avoidance (CMIA). In contrast to spontaneous alternation, CMIA involves the administration of footshock and is considered a measure of emotional, reference memory. In addition, unlike spontaneous alternation, CMIA provides a measure of both acquisition and retention performance, which allows for the effects of drugs on these two variables to be dissociated. The goal of experiment 1 was to determine whether any deficits in CMIA produced by intra-septal infusions of muscimol could be reversed by intra-hippocampal infusions of physostigmine. Limited evidence suggests that the medial septum may also influence memory via a process that involves septal cholinergic projections to the entorhinal cortex. Inactivation of the medial septum abolishes theta activity in the entorhinal cortex in freely moving rats [26] and alters the firing rate of entorhinal cells in rats performing in a spatial task [30]. Lesions of septal cholinergic efferents, which include damage to the cholinergic projection from the medial septum to the entorhinal cortex, impair radial arm maze performance [44]. As in the case of intra-hippocampal infusions, infusions of physostigmine into the entorhinal cortex prevent the impairing effects of intra-septal infusions of muscimol on spontaneous alternation performance [6]. The purpose of experiment 2 was to determine whether infusions of physostigmine into the
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entorhinal cortex would also attenuate the impairing effects of intra-septal infusions of muscimol on CMIA.
2. Materials and methods
2.1. Experiment 1 2.1.1. Subjects Male Sprague–Dawley rats, weighing 250–300 g upon arrival, were used. They were individually housed and maintained on a 12-h light / dark cycle with food and water ad libitum. All rats were handled 2 days prior to surgery for 3 min each. During handling, the rats were given an oral administration of water in order to habituate them to the oral administration of an analgesic on the day of surgery. 2.1.2. Surgery Surgery was performed at least 1 week after the rats arrived. On the day of surgery, the rats were given an oral dose of the analgesic acetaminophen (Tylenol 120 mg / 1.5 cc). One hour later, they were given atropine sulfate (0.4 mg / kg, i.p.), anaesthetized with pentobarbitol (Nembutal 50 mg / kg, i.p.), hydrated with saline (3 cc, s.c.), and given the antibiotic penicillin (Crystiben, Rhone Merieux Canada Inc., 4300 U / kg, i.m.). Stereotaxic surgical procedures were used to implant one 22-gauge stainless-steel guide cannula (Plastics One, Inc. Roanoke, VA) aimed at the medial septum (0.5 mm anterior to bregma [AP], 4.9 mm ventral to dura [DV], 3.2 mm from the interaural line; 37) and one guide cannula aimed at the dorsal hippocampus (24.2 mm AP, 2.0 mm DV, 4.1 mm lateral [ML] to the midline). For half of the rats, the hippocampal cannula was implanted in the left hemisphere. The cannulae were attached to the skull with four jeweler’s screws and cranioplastic cement and a dummy cannula was inserted into each guide cannula to keep the cannula tract clear. Immediately after surgery, the rats were placed in a warm environment until they regained consciousness. Two days after surgery, each cannula was checked for obstructions and betadine was applied to the surgical wound. 2.1.3. Procedure and drugs Two days prior to behavioral testing, all rats were handled for 3 min and then 5 min the next day. All testing occurred at least 1 week following surgery between 09:00 and 19:00 h. Rats were given an infusion of vehicle (phosphate-buffered saline, pH 7.4) or physostigmine (10 mg / 1 ml / over 1 min) into the hippocampal cannula, followed immediately by an infusion of vehicle or muscimol (570 ng / 0.5 ml / over 1 min) into the medial septal cannula. The dose of muscimol was selected based on previously published work [32,33,35]. The dose of physostigmine was selected based on our previous findings that
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indicate that intra-hippocampal infusions of this dose reverse deficits in spontaneous alternation performance induced by intra-septal infusions of muscimol [6]. The solutions were infused through a 28-gauge injection needle that extended 1 mm beyond the guide cannula. The needle was connected to a 10-ml Hamilton syringe by polyethylene tubing and the infusions were delivered using an infusion pump (Harvard Apparatus 22). The injection needles were left in place for 1 min following the infusions in order to allow for diffusion. An experimenter that was blind to the drug treatment delivered the infusions.
2.1.4. Continuous multiple inhibitory avoidance Each rat was trained in the CMIA task 15 min after the last infusion. A trough-shaped straight alley (91 cm long, 20 cm wide at the top, 6.4 cm wide at the floor, and 15 cm deep), separated into two compartments by a retractable door, was used for the training. The starting compartment (30 cm long) was illuminated by a 15-W tensor lamp and the adjoining compartment (61 cm) where rats received a footshock, was dark. The apparatus was located in an unlit room. For the training, each rat was placed into the lighted compartment with its head facing away from the door. When the rat turned around, the door was opened and the latency to enter the dark compartment with all four paws was recorded (100 s maximum). When the rat entered the dark compartment, it received a footshock (0.5 mA) through the metal floor plates until it returned to the lighted compartment. This sequence of events constituted one training trial. The door remained open for the remainder of the training session, and whenever the rat entered the dark compartment with all four paws, it received a 0.5-mA footshock until it escaped back to the lighted compartment. Training continued until the rat remained in the lighted compartment for a criterion of 100 consecutive seconds or until a maximum of five footshocks had been given. The number of trials (i.e. footshocks) needed to achieve the criterion and the latency to enter the dark compartment on each trial were recorded and used as measures of acquisition. Retention of the CMIA training was assessed 48 h later. Each rat was placed in the lighted compartment with its head facing away from the door and the door was lowered 30 s later. Latency to enter the dark compartment with all four paws in the absence of footshock (600 s maximum) was recorded and used as an index of memory. 2.1.5. Histology After the completion of the behavioral tests, the rats were anesthetized with an overdose of chloral hydrate (800 mg / ml) and perfused intracardially with 0.9% saline followed by 10% formalin. The brains were removed and placed in a 10% formalin solution. At least 48 h later, the brains were frozen and sectioned (60 mm), mounted onto glass slides, and stained with thionin. The sections were
examined under a light microscope by an observer blind to the drug history and behavioral results of each rat. In the case of ambiguous placements, an additional experienced observer who was also blind to the treatment history and behavioral results examined the histology.
2.1.6. Statistics The CMIA data were expressed as means and standard errors of the mean (S.E.M.) and were analyzed using analysis of variance (ANOVA) and Fisher adjusted leastsignificant differences (LSD) post-hoc tests. An alpha level of 0.05 was used as the criterion for statistical significance. Due to heterogeneity of variance, the acquisition latencies were transformed to their natural logs prior to the statistical analyses. 2.2. Experiment 2 The procedure used in this experiment was the same as that used in experiment 1 with the following exceptions.
2.2.1. Surgery Stereotaxic surgical procedures were one guide cannula aimed at the medial aimed at the entorhinal cortex (27.5 mm 5.3 mm ML). The unilateral entorhinal were counterbalanced for hemisphere.
used to implant septum and one AP, 6.2 mm DV, cortex cannulae
2.2.2. Procedure The rats were given an infusion of vehicle or physostigmine (1.875 mg / 0.25 ml / over 1 min) into the entorhinal cortex cannula immediately followed by an infusion of vehicle or muscimol (570 ng / 0.5 ml / over 1 min) into the septal cannula. The dose of physostigmine was selected based on our previous work indicating that intra-entorhinal infusions of this dose effectively reversed spontaneous alternation deficits induced by intra-septal infusions of muscimol [6].
3. Results
3.1. Experiment 1 3.1.1. Histology The behavioral data for animals with misplaced cannulae or extensive necrosis at the cannula site were discarded (n59). Fig. 1 shows the location of the infusion sites in the (A) medial septum and (B) dorsal hippocampus of the rats whose data were included in the statistical analysis. Five rats were given infusions of vehicle in both the hippocampus and medial septum (VEH-VEH), ten rats were given infusions of physostigmine in the hippocampus and vehicle in the medial septum (PHYSO-VEH), seven rats were given infusions of vehicle in the hippocampus and muscimol in the medial septum (VEH-MUSC), and
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Fig. 1. Schematic illustration of a coronal section of the rat brain showing the approximate location of (A) medial septal and (B) hippocampal infusion sites in experiment 1. Atlas plates adapted from Ref. [37].
six rats were given infusions of physostigmine in the hippocampus and muscimol in the medial septum (PHYSO-MUSC).
3.1.2. CMIA acquisition performance The pretraining drug infusions into the hippocampus and the medial septum did not affect CMIA acquisition performance. Although there was a non-significant trend, the pretraining infusions did not affect latency to enter the dark compartment on the first training trial [F(3, 24)5 2.59; P50. 077]. The infusions did not affect the total number of trials required to reach the acquisition criterion [F(3, 24)50.44; P.0.05; see Table 1]. 3.1.3. CMIA retention performance The pretraining infusions into the hippocampus and the septum significantly affected retention performance tested 48 h after CMIA training [F(3,24)53.79; P,0.05; see Fig. 2]. As expected, based on previous findings, intra-septal infusions of muscimol impaired retention performance. The retention latencies of VEH-MUSC rats were significantly shorter than those of VEH-VEH rats (P,0.05). Infusions of physostigmine into the hippocampus did not affect retention performance. The retention latencies of PHYSO-VEH rats were not significantly different from
those of VEH-VEH rats (P.0.05). Importantly, the pretraining intra-hippocampal infusions of physostigmine attenuated the impairing effects of the septal muscimol infusions. The retention latencies of PHYSO-MUSC rats were not significantly different from those of VEH-VEH rats (P.0.05).
3.2. Experiment 2 3.2.1. Histology As in experiment 1, the behavioral results of those rats with misplaced cannulae or extensive necrosis at the cannula site were discarded (n512). Fig. 3 shows the location of the infusion sites in the entorhinal cortex of the rats whose data were included in the statistical analyses. The septal placements were comparable to those shown in Fig. 1A. Ten rats were given infusions of vehicle in both the entorhinal cortex and medial septum (VEH-VEH), ten rats were given infusions of physostigmine in the entorhinal cortex and vehicle in the medial septum (PHYSOVEH), ten rats were given infusions of vehicle in the entorhinal cortex and muscimol in the medial septum (VEH-MUSC), and seven rats were given infusions of physostigmine in the entorhinal cortex and muscimol in the medial septum (PHYSO-MUSC).
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Table 1 The effects of intra-septal infusions of vehicle or muscimol combined with intra-hippocampal (experiment 1) or intra-entorhinal (experiment 2) infusions of vehicle or physostigmine on mean (6S.E.M.) latency to enter the dark compartment on the first training trial and the mean (6S.E.M.) number of trials needed to reach the training criterion Medial septum Vehicle
Experiment 1: Hippocampus n Latency Number of trials Experiment 2: Entorhinal cortex n Latency Number of trials
Muscimol
Vehicle
Physostigmine
Vehicle
Physostigmine
5 0.85 (0.20) 2.8 (0.37)
10 0.63 (0.12) 3.0 (0.30)
7 1.16 (0.25) 2.7 (0.29)
6 1.28 (0.22) 2.5 (0.34)
10 0.38 (0.04) 3.4 (0.34)
10 0.56 (0.10) 3.6 (0.22)
10 0.97 (0.17) 3.0 (0.26)
7 0.97 (0.23) 3.14 (0.34)
3.2.2. Acquisition performance The pretraining drug infusions into the entorhinal cortex and the medial septum affected latency to enter the dark compartment on the first training trial [F(3, 33)54.47; P,0.05; see Table 1]. Specifically, the acquisition latencies of PHYSO-MUSC and PBS-MUSC rats were significantly longer than those of VEH-VEH rats (P,0.01 for both comparisons). However, the infusions did not affect the total number of trials required to reach the acquisition criterion [F(3,33)50.89; P.0.05; see Table 1].
3.2.3. Retention performance The pretraining infusions into the entorhinal cortex and the medial septum significantly affected CMIA retention performance [F(3,33)53.57, P,0.05; see Fig. 4]. As in experiment 1, intra-septal infusions of muscimol impaired retention performance. The retention latencies of VEHMUSC rats were significantly shorter than the retention latencies of VEH-VEH rats (P,0.05). Infusions of physostigmine into the entorhinal cortex did not affect retention performance. The retention latencies of PHYSO-VEH rats
Fig. 2. Infusions of physostigmine (10 mg / 1 ml) into the dorsal hippocampus attenuate the impairing effects of intra-septal infusions of muscimol (570 ng / 0.5 ml) on continuous multiple inhibitory avoidance performance (* P,0.05 vs. VEH-VEH).
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Fig. 3. Schematic illustration of a coronal section of the rat brain showing the approximate location of infusion sites in the entorhinal cortex in experiment 2. Atlas plates adapted from Ref. [37].
were not significantly different from those of VEH-VEH rats (P.0.05). Notably, infusions of physostigmine into the entorhinal cortex attenuated the impairing effects of the intra-septal infusions of muscimol. The retention latencies of PHYSO-MUSC rats were not significantly different from those of VEH-VEH rats (P.0.05).
4. General discussion The major finding of this study is that impairments in emotional reference memory due to elevated septal GABA receptor activation can be prevented by up-regulating ACh function in the hippocampus and entorhinal cortex. Spe-
Fig. 4. Infusions of physostigmine (1.875 mg / 0.25 ml) into the entorhinal cortex attenuate the impairing effects of intra-septal infusions of muscimol (570 ng / 0.5 ml) on continuous multiple inhibitory avoidance performance (* P,0.05 vs. VEH-VEH).
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cifically, the present results demonstrate that infusions of the AChE inhibitor physostigmine into the dorsal hippocampus or the entorhinal cortex attenuate the impairing effect of septal muscimol infusions on CMIA retention performance. The present results are consistent with previous findings indicating that intra-septal infusions of muscimol impair avoidance retention performance tested 48 h after training [33,35] and increase the dose of intra-hippocampal infusions of an ACh agonist needed to enhance avoidance retention [9]. The present findings also concur with our previous results showing that the impairing effects of intra-septal infusions of muscimol on spontaneous alternation are reversed by infusions of physostigmine into the hippocampus or entorhinal cortex [6], and by infusions of glucose into the hippocampus [36], which would also presumably increase ACh levels [13,14,38,39]. Muscimol is a selective ligand for GABA-A receptors [29], which are present on both septal cholinergic and GABAergic projection neurons [11,18]. One way that muscimol may impair memory is by inhibiting the cholinergic projection neurons. According to this hypothesis, infusions of physostigmine into the hippocampus or entorhinal cortex reverse the muscimol-induced deficit by increasing ACh levels at these cholinergic terminals. However, the findings of a recent study suggest that muscimol may affect memory through an influence on both the cholinergic and GABAergic projection neurons. Specifically, infusions of lower doses of muscimol into the septum are sufficient to impair spatial working memory when the cholinergic projection is selectively lesioned [34]. Septal GABAergic projection neurons synapse onto GABAergic interneurons in the hippocampus, which in turn synapse onto hippocampal glutamatergic pyramidal cells [8,10,43]. Given this pattern of connections, muscimol-induced inhibition of the septo-hippocampal GABA projection would be expected to result in the inhibition of hippocampal pyramidal cells. The finding that cholinomimetic drugs depolarize hippocampal pyramidal cells [41] raises the possibility that physostigmine could prevent the memory-impairing effects of septal infusions of muscimol by preventing muscimol-induced inhibition of hippocampal pyramidal cells. Finally, infusions of glutamatergic antagonists into the hippocampus decrease hippocampal extracellular ACh levels [31], suggesting that muscimol-induced inhibition of pyramidal cells could lead to a decrease in acetylcholine levels. Thus, it is also possible that intra-hippocampal infusions of physostigmine reverse the memory-impairing effects of septal GABA receptor activation by counteracting this decrease in ACh. The present results also show that the effects of the pretraining infusions appeared to be specific to retention performance. In experiment 1, neither muscimol, physostigmine, nor the combination of the two affected acquisition performance. In experiment 2, muscimol significantly increased latency to enter the dark compartment on the first
training trial before shock was administered. However, this finding does not likely reflect an acquisition deficit because rats given muscimol required the same number of training trials to reach the acquisition criterion as did control rats. Prior experiments examining the effects of pretraining intra-septal infusions of muscimol on avoidance retention performance have typically employed a one trial avoidance paradigm and therefore did not provide any measures of acquisition [32,33,35]. The present findings indicating that the pretraining infusions of muscimol did not affect acquisition performance in a multiple trial paradigm have several implications. First, these findings suggest that the drug infusions did not affect sensorimotor or motivational processes that could have interfered with acquisition. Our findings suggest that all of the rats learned the task to a similar degree and that the deficits that were observed on the retention test likely reflect an effect of muscimol on memory, rather than on learning or performance. This interpretation is consistent with previous findings indicating that pretraining intra-septal infusions of muscimol do not impair retention performance tested 15 s after training, but do impair retention tested 15 min or 48 h later [32] and the finding that posttraining intra-septal infusions of muscimol also impair retention performance [24]. Importantly, the present finds show that physostigmine did not influence acquisition or retention performance on its own but did attenuate the impairing effect of muscimol on retention performance. This finding suggests that septal GABA receptors interact with cholinergic processes in the hippocampus and entorhinal cortex specifically during memory consolidation processes. It is well established that the medial septum interacts with the hippocampus to influence learning and memory [1,5,6,44]. However, the possibility that the medial septum may interact with the entorhinal cortex to affect mnemonic processes is less well understood. Our discovery that infusions of physostigmine into the entorhinal cortex reverse the impairing effects of septal GABA receptor stimulation on spontaneous alternation [6] and inhibitory avoidance (present findings) provide convincing evidence to support the hypothesis that the septum and entorhinal cortex interact to influence learning and memory. Future studies are needed to determine whether the medial septum interacts directly with the entorhinal cortex, or indirectly via the hippocampus [12,21,25]. In summary, the present findings demonstrate that infusions of the AChE inhibitor physostigmine into the hippocampus or the entorhinal cortex attenuate the impairing effects of intra-septal infusions of the GABA agonist muscimol on CMIA retention performance. In combination with our previous findings, the results of the present study indicate that increasing ACh levels in the hippocampus or the entorhinal cortex is sufficient to reverse the memory impairing effects of septal GABAergic receptor activation in two different memory tasks. These findings provide further support for the hypothesis that septal GABA
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receptors might impair memory by decreasing ACh levels in the hippocampus and the entorhinal cortex.
Acknowledgements This work was supported by NINDS, NIDDK, and JDF (1RO1 NS 41173-01), MRC G11821018 and NSERC OGP019453. We thank Justin Park for his invaluable technical assistance and Katherine Harter and Dr Benjamin Philpot for their comments on an earlier version of this manuscript.
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