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Effect of cholinergic activation by physostigmine on working memory failure caused in rats by pharmacological manipulation of hippocampal glutamatergic and 5-HTergic neurotransmission
ELSEVIER
Neuroscience Letters 217 (1996) 21-24
Effect of cholinergic activation by physostigmine on working memory failure caused in rats by pharmacological manipulation of hippocampal glutamatergic and 5HTergic neurotransmission Masuo Ohno*, Akihiro Kishi, Shigenori Watanabe Department
of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82, Japan
Received 8 July 1996; revised version received 16 August 1996; accepted 4 September 1996
Abstract The muscarinic receptor antagonist scopolamine significantly increased the number of errors in the working memory task with a threepanel runway setup, when injected bilaterally at 3.2 &side into the dorsal hippocampus. The increase in working memory errors induced by intrahippocampal3.2 &side scopolamine was reduced by concurrent injection of the cholinesterase inhibitor physostigmine (1.0 and 3.2 &side). However, physostigmine (3.2 pg/side) did not affect an increase in working memory errors induced by intrahippocampal injection of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist (f)-3-(2-carboxypiperazin-4-yl)propyl-lphosphonic acid (CPP) at 32 @side. Likewise, physostigmine (3.2 &side) was ineffective in reducing an increase in working memory errors caused by intrahippocampal administration of the 5-hydroxytryptamineIA (5HTIA) receptor agonist (f)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) at 10 &side. These results suggest that the septohippocampal cholinergic activity is necessary for normal working memory processes, but that cholinergic activation neither compensates loss of hippocampal NMDA receptor-mediated neurotransmission nor counteracts the overstimulation of hippocampal 5-HTIA receptors in terms of working memory function. Keywords:
Hippocampus; Working memory; Acetylcholine; Physostigmine; Scopolamine; N-Methyl-D-aspartate receptor; (_+)-3-(2Carboxypiperazin-4-yl)propyl-1-phosphonic acid; 5Hydroxytryptamine,, receptor
The septohippocampal cholinergic nervous system is known as excitatory input to the hippocampus that plays an important role in memory function [4]. Also, it is well documented that activation of N-methyl-D-aspartate (NMDA) receptors is prerequisite for induction of longterm potentiation (LTP) in the hippocampus which is hypothesized to be a neural basis of memory formation [3], and is involved in memory performances of rats in some learning tasks that depend on hippocampal functions [ 12,231. We previously reported that intrahippocampal administration of the muscarinic acetylcholine receptor antagonist scopolamine or the selective and competitive NMDA receptor antagonists such as (+)-3-(2-carboxypiperazin4-yl)propyl-1-phosphonic acid (CPP) caused a dose-dependent disruption of working memory performance of rats on a three-panel runway task, acquisition of new and variable information that was useful only within a session [16], indicating that this behavior depended on the excitatory neurotransmission via mus* Corresponding author. fax: +81 92 6322752.
carinic and NMDA receptors in the hippocampus. On the other hand, the hippocampus receives SHTergic innervation from the raphe nuclei, which functions to inhibit the activity of hippocampal neurons via postsynaptic ShydroxytryptaminelA (5HTIA) receptors [2]. It has been demonstrated that stimulation of hippocampal 5-HTlA receptors by the selective agonist (+)-Shydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) impairs spatial learning in a water maze and passive avoidance learning [5,7]. We also showed that intrahippocampal administration of 8OH-DPAT impaired working memory performance on the three-panel runway task, an effect that was reversed by the 5-HTIA receptor antagonist (-)-propranolol [17]. These results suggest the inhibitory role played by hippocampal 5-HT1* receptors in the regulation of mnemonic processes. In contrast to extensive evidence for the individual roles of the hippocampal neurotransmission in memory function, little is known about interactive regulations of memory processes through the multiple transmitter systems in this structure. In the present study, we investigated, using
0304-3940/96/$12.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO304-3940(96)13057-3
22
M. Ohno et al.
I Neuroscience Letters 217 (1996) 21-24
the three-panel runway task, the effects of cholinergic activation by the cholinesterase inhibitor physostigmine on the working memory deficits resulting from blockade of hippocampal muscarinic or NMDA receptors and on those by hippocampal 5-HTiA receptor stimulation, and thereby clarified whether the functional interactions between hippocampal cholinergic and other neuronal activities are involved in regulating the memory process. Male Wistar-strain rats (Japan SLC) were placed on a deprivation schedule to maintain their weights at approximately 80% of the free-feeding level (230-250 g) before the experiment. Working memory was assessed with a three-panel runway apparatus, as described in our previous reports [13,16]. In brief, this apparatus (175 x 36 x 25 cm) was composed of a start box, a goal box and four consecutive choice points intervening between them. Each choice point consisted of a gate with three panels (12 x 25 cm). The rats were prevented from passing through two of the three panels in the gate by front stoppers, and were prevented from returning to the start box or to a previous choice point by rear stoppers affixed to each of the panels in all the gates. When the rats reached the goal box, they received two food pellets (about 50 mg each; Muromachi Kikai). The rats were made to run the task in six consecutive trials at 2-min intervals (defined as one session) per day with removal of the front stopper of only one of the three panel-gates (the correct panel-gate) at each choice point. The locations of the correct panel-gates were held constant within a session, but were changed from one session to the next (working memory procedure). The number of times an animal attempted to pass through an incorrect panel-gate (defined as errors) and the time required for the animal to obtain food pellets (defined as latency) were recorded for each rat during each trial of a session. The number of errors and latency recorded in the first trial were presented separately, and those parameters in the second to the sixth trial of a session were summed together for the evaluation of working memory function. The learning criterion was less than eight errors summed from the second to sixth trials (working memory errors). A rat was used in the experiment, if it achieved this criterion in three consecutive sessions. The significance of differences between the groups was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s test when F ratios reached significance (P < 0.05). The rats that achieved the learning criterion were anesthetized with sodium pentobarbital (40 mg/kg i.p.>, and were implanted bilaterally with guide cannulae for microinjection of drugs into the hippocampus, as described previously [ 13,161. The position of the injection cannula tip, which protruded 1.0 mm below the tip of the guide cannula, was aimed at the dorsal hippocampus (A, 3.8 mm; L, f2.2 mm; H, 3.2 mm) according to the atlas of Paxinos and Watson [ 181. The rats were allowed at least 5 days of postoperative recovery before runway sessions were resumed. CPP (Research Biochemicals Int.) was dis-
solved in saline, after which the pH was adjusted to approximately 7.4 with an appropriate amount of NaOH. Other drugs were purchased from Sigma Chemical Co. and were dissolved in saline. Two microliters of the drug solution or saline was injected at 0.5 pl/min into the dorsal hippocampus through the injection cannula. The injection cannula was left in place for 1 min after completion of the injection. Rats received the runway test 10 min after the drug injection was completed. When microinjections were made repeatedly into each rat, a minimum of 3 days were allowed between microinjections. After completion of behavioral testing, the injection site in the hippocampus was verified by Cresyl violet staining, as described previously [13,16]. In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. The number of errors made from the second to the sixth trial (working memory errors) markedly decreased with repeated training, whereas the errors in the first trial remained constant at approximately four. Approximately 20-30 training sessions were required for the rats to reach the criterion of less than eight working memory errors. Latency was also reduced during repeated sessions and was stable from the 10th session on. Scopolamine at 3.2 pg/side, administered bilaterally into the dorsal hippocampus, significantly increased the number of working memory errors (Fl,,,, = 25.81, P < O.Ol), although it had no effect on the errors in the first trial (Fig. 1). Intrahippocampal 3.2 pg/side scopolamine significantly prolonged the latency to obtain food pellets from the second to sixth trials of a session (Fl,lo = 13.33, P < 0.01). The increase in working memory errors induced by intrahippocampal3.2 &side scopolamine was attenuated by concurrent injection of 1.0 and 3.2 pglside physostigmine (Fz,,d = 12.95, P < 0.01). Physostigmine (1.0 and 3.2 &side) was also effective in reducing the prolonged latency resulting from administration of 3.2 pg/side scopolamine into the hippocampus (F2,14 = 7.31, P < 0.05). Intrahippocampal 3.2 ,&side physostigmine by itself did not affect the number of working memory errors (5.5 + 2.2; mean + SEM, IZ = 4) or the latency from the second to sixth trials (27.3 f 2.0 s). The number of working memory errors was significantly increased by intrahippocampal injection of 32 ng/ side CPP (F1,9 = 49.18, P < 0.01) or by that of 10 pg/side 8-OH-DPAT (F1,8 = 17.50, P < O.Ol), whereas the number of errors in the first trial was not changed (Fig. 2). The latency from the second to sixth trials was significantly prolonged when rats received intrahippocampal 32 ng/ side CPP (F1,9 = 7.18, P < 0.05) or 10 &side 8-OHDPAT (Fl,* = 18.65, P < 0.01). Concurrent administration of 3.2 pg/side physostigmine into the hippocampus had no effect on the CPP (32 @side)-induced increase in working memory errors or in the latency to obtain food pellets placed in the goal box. Physostigmine (3.2 pg/side) was also ineffective in reducing the increase in working
M. Ohno et al. I Neuroscience
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Fig. 1. Effects of concurrent injection of physostigmine on increases in working memory errors and latency induced by intrahippocampal scopolamine. Rats received the runway test 10 min after drugs were injected. Each column represents the mean f SEM of errors and latenties for 5-7 animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). **P < 0.01 (versus saline), ‘P < 0.05, #P < 0.01 (versus scopolamine). errors or in the latency induced by 10 &side 8OH-DPAT. Rats that received intrahippocampal 3.2 j.& side physostigmine together with 32 r&side CPP or 10 &side 8-OH-DPAT showed significantly more working memory errors than rats given 3.2 &side physostigmine alone (Fi,s = 8.83, P < 0.05;F,,, = 21.72, P < 0.01, respectively). The present study with the three-panel runway task showed that concurrent injection of the cholinesterase inhibitor physostigmine reversed working memory deficits induced by intrahippocampal administration of the muscarinic receptor antagonist scopolamine, indicating that the septohippocampal cholinergic activity plays a critical role in working memory performance, i.e. acquisition process of new information within a session. Some interactive mechanism between cholinergic and glutamatergic systems in memory processes has been evidenced by the fact that the behaviorally ineffective doses of scopolamine and the noncompetitive NMDA receptor antagonist MK801 significantly impair inhibitory avoidance learning of rats [14] and visual recognition memory of rhesus monkeys in a delayed non-matching-to-sample task [ 1 l] when both drugs are administered in combination, Furthermore, D-cycloserine, which acts as a positive modulator of NMDA receptors through the glycine binding site on the NMDA receptor/channel complex, has been reported to attenuate the scopolamine-induced deficits in spatial leaming of rats in a water maze task and those in memory performance of rats in a T-maze alternation paradigm [9,22]. Markram and Segal [lo] found, using hippocampal memory
23
Letters 217 (1996) 21-24
slices, that acetylcholine amplified NMDA receptormediated responses through acting on muscarinic receptors and thereby caused a long-lasting facilitation of excitatory postsynaptic potentials, suggesting the cholinergic activity functioning to modulate the glutamatergic transmission postsynaptically in the hippocampus. These findings suggest a link between the NMDA and muscarinic receptor-mediated neurotransmission involved in the regulation of memory processes. However, the present study showed that the cholinergic activation by physostigmine was ineffective in reversing the working memory failure caused by intrahippocampal administration of the competitive NMDA receptor antagonist CPP. It is, therefore, conceivable that although the septohippocampal excitatory muscarinic neurotransmission allows hippocampal neuronal activities to process working memory, the cholinergic activation is insufficient to support the memory processing through specific hippocampal glutamatergic pathway when NMDA receptor-mediated transmission is fully blocked. Although further study is necessary to clarify the precise mechanism underlying the differential effects of physostigmine on the memory impairments produced by blockade of hippocampal muscarinic and NMDA receptors. it is of interest to note that activation of NMDA receptors, but not that of muscarinic receptors, is essential for induction of hippocampal LTP, a neurobiological model of synaptic plasticity underlying learning and memory [1,3]. Abe et al. [I] demonstrated that the cholinergic system plays a modulatory role to function as a
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Fig. 2. Effects of concurrent injection of physostigmine on increases in working memory errors and latency induced by intrahippocampal CPP and I-OH-DPAT. Rats received the runway test 10 min after drugs were injected. Each column represents the mean rt SEM of errors and latencies for 5-6 animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). *P < 0.05, **P < 0.01 (versus saline).
24
M. Ohm et al. I Neuroscience Letters 217 (1996) 21-24
triggering amplifier for induction of hippocampal LTP by facilitating NMDA receptor-mediated responses. These findings may provide an explanation for the present result that cholinergic activation by physostigmine fails to compensate loss of hippocampal NMDA receptor-mediated neurotransmission responsible for working memory processing. It has been reported that stimulation of 5-HT1* receptors causes hyperpolarization of pyramidal neurons of the rat hippocampal CA1 and CA3 regions due to an increase in potassium conductance [2], in contrast to muscarinic receptor stimulation resulting in excitation of hippocampal pyramidal cells [8]. It has been found that hyperpolarization of hippocampal neurons through Kt channels opened by activation of 5-HTtA receptors functions to inhibit LTP [21], whereas acetylcholine facilitates LTP by regulating the threshold of LTP generation through a depolarizing action [l]. Some behavioral studies have demonstrated that the 5-HTiA receptor agonist and antagonist affect memory failure resulting from cholinergic deficiency. The behaviorally subthreshold dose of 8-OH-DPAT, a 5HTIA receptor agonist, exacerbates deficits in spatial learning in the water maze and passive avoidance learning induced by scopolamine or lesioning of the septohippocampal projection system [ 19,201. Furthermore, hippocampal 5-HTiA receptor blockade by WAY 100135 or NAN-190 is effective in preventing the scopolamine disruption of spatial learning in the water maze [6] and of working memory performance in the three-panel runway task [15]. It is, thus, likely that 5-HTiA and muscarinic receptors contribute jointly, but in the opposite direction, to the regulation of memory processes. In contrast, the present study showed that physostigmine, the dose of which was sufficient to reverse the memory failure by scopolamine, did not affect the working memory deficit resulting from hippocampal 5-HTiA receptor stimulation by 8-OH-DPAT. Taken together, these findings suggest that blockade of hippocampal 5-HTi* receptors can compensate loss of excitatory cholinergic input involved in working memory function, but that cholinergic activation, conversely, fails to counteract the interference with this memory function exerted by stimulation of inhibitory 5HTIA receptors in the hippocampus. VI Abe,
K., Nakata, A., Mizutani, A. and Saito, H., Facilitatory but nonessential role of the muscarinic cholinergic system in the generation of long-term potentiation of population spikes in the dentate gyms in vivo, Neurophatmacology, 33 (1994) 847-852. I21Beck, S.G., Choi, K.C. and List, T.J., Comparison of 5-hydroxytryptaminelA-mediated hyperpolarization in CA1 and CA3 hippocampal pyramidal cells, J. Pharmacol. Exp. Ther., 263 (1992) 350359. [31 Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 3139. [41 Brito, G.N.O., Davis, B.J., Stopp, L.C. and Stanton, M.E., Memory and the septo-hippocampal cholinergic system in the rat, Psychopharmacology, 81 (1983) 315-320.
[5] Carli, M., Lazarova, M., Tatarczynska, E. and Samanin, R., Stimulation of 5-HT,* receptors in the dorsal hippocampus impairs acquisition and performance of a spatial task in a water maze, Brain Res., 595 (1992) 50-56. [6] Carli, M., Luschi, R. and Samanin, R., (S)-WAY 100135, a 5-HTtA receptor antagonist, prevents the impairment of spatial learning caused by intrahippocampal scopolamine, Eur. J. Pharmacol., 283 (1995) 133-139. [7] Carli, M., Luschi, R., Garofalo, P. and Samanin, R., I-OH-DPAT impairs spatial but not visual learning in a water maze by stimulating 5-HTt* receptors in the hippocampus, Behav. Brain Res., 67 (1995) 67-74. [8] Cole, A.E. and Nicoll, R.A., The pharmacology of cholinergic excitatory responses in hippocampal pyramidal cells, Brain Res., 305 (1984) 283-290. [9] Fishkm, R.J., Ince, E.S., Carlezon, W.A. Jr. and DUM, R.W., DCycloserine attenuates scopolamine-induced learning and memory deficits in rats, Behav. Neural Biol., 59 (1993) 150-157. [lo] Markram, H. and Segal, M., Long-lasting facilitation of excitatory postsynaptic potentials in the rat hippocampus by acetylcholine, J. Physiol., 427 (1990) 381-393. [l l] Matsuoka, N. and Aigner, T.G., Cholinergic-glutamatergic interactions in visual recognition memory of rhesus monkeys, NeuroReport, 7 (1996) 565-568. [12] Morris, R.G.M., Synaptic plasticity and learning: selective impairment of learning in rats and blockade of long-term potentiation in vivo by the N-methyl-o-aspartate receptor antagonist AP5, J. Neurosci., 9 (1989) 3040-3057. [13] Ohno, M. and Watanabe, S., Concurrent blockade of hippocampal metabotropic glutamate and N-methyl-o-aspartate receptors disrupts working memory in the rat, Neuroscience, 70 (1996) 303311. [14] Ohno, M. and Watanabe, S., Interactive processing between glutamatergic and cholinergic systems involved in inhibitory avoidance learning of rats, Eur. J. Pharmacol., (1996) in press. [15] Ohno, M. and Watanabe, S., Blockade of 5Xft~ receptors compensates loss of hippocampal cholinergic neurotransmission involved in working memory of rats, Brain Res., (1996) in press. [16] Ohno, M., Yamamoto, T. and Watanabe, S., Effects of intrahippocampal injections of N-methyl-o-aspartate receptor antagonists and scopolamine on working memory and reference memory assessed in rats by a three-panel runway task, J. Pharmacol. Exp. Ther., 263 (1992) 943-950. [17] Ohno, M., Yamamoto, T. and Watanabe, S., Working memory deficits induced by intrahippocampal administration of g-OHDPAT, a 5-HT1* receptor agonist, in the rat, Eur. J. Pharmacol., 234 (1993) 29-34. [18] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. [19] Riekkinen, P. Jr., 5-HTtA and muscarinic acetylcholine receptors jointly regulate passive avoidance behavior, Eur. ,I. Pharmacol., 262 (1994) 77-90. [20] Riekkinen, M., Sin%, J., Toivanen, T. and Riekkinen, P. Jr., Combined treatment with a 5HTt* receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior, Psychopharmacology, 122 (1995) 137-146. [21] Sakai, N. and Tanaka, C., Inhibitory modulation of long-term potentiation via the 5-HT,* receptor in slices of the rat hippocampal dentate gyrus, Brain Res., 613 (1993) 326-330. [22] Sirviii, J., Ekonsalo, T., Riekkinen, P. Jr., Lahtinen, H. and Riekkinen, P. Sr., o-Cycloserine, a modulator of the N-methyl-oaspartate receptor, improves spatial learning in rats treated with muscarinic antagonist, Neurosci. Lett., 146 (1992) 215-218. [23] Ward, L., Mason, S.E. and Abraham, W.C., Effects of the NMDA antagonists CPP and MK-801 on radial arm maze performance in rats, Pharmacol. Biochem. Behav., 35 (1990) 785-790.
Neuroscience Letters 217 (1996) 21-24
Effect of cholinergic activation by physostigmine on working memory failure caused in rats by pharmacological manipulation of hippocampal glutamatergic and 5HTergic neurotransmission Masuo Ohno*, Akihiro Kishi, Shigenori Watanabe Department
of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82, Japan
Received 8 July 1996; revised version received 16 August 1996; accepted 4 September 1996
Abstract The muscarinic receptor antagonist scopolamine significantly increased the number of errors in the working memory task with a threepanel runway setup, when injected bilaterally at 3.2 &side into the dorsal hippocampus. The increase in working memory errors induced by intrahippocampal3.2 &side scopolamine was reduced by concurrent injection of the cholinesterase inhibitor physostigmine (1.0 and 3.2 &side). However, physostigmine (3.2 pg/side) did not affect an increase in working memory errors induced by intrahippocampal injection of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist (f)-3-(2-carboxypiperazin-4-yl)propyl-lphosphonic acid (CPP) at 32 @side. Likewise, physostigmine (3.2 &side) was ineffective in reducing an increase in working memory errors caused by intrahippocampal administration of the 5-hydroxytryptamineIA (5HTIA) receptor agonist (f)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) at 10 &side. These results suggest that the septohippocampal cholinergic activity is necessary for normal working memory processes, but that cholinergic activation neither compensates loss of hippocampal NMDA receptor-mediated neurotransmission nor counteracts the overstimulation of hippocampal 5-HTIA receptors in terms of working memory function. Keywords:
Hippocampus; Working memory; Acetylcholine; Physostigmine; Scopolamine; N-Methyl-D-aspartate receptor; (_+)-3-(2Carboxypiperazin-4-yl)propyl-1-phosphonic acid; 5Hydroxytryptamine,, receptor
The septohippocampal cholinergic nervous system is known as excitatory input to the hippocampus that plays an important role in memory function [4]. Also, it is well documented that activation of N-methyl-D-aspartate (NMDA) receptors is prerequisite for induction of longterm potentiation (LTP) in the hippocampus which is hypothesized to be a neural basis of memory formation [3], and is involved in memory performances of rats in some learning tasks that depend on hippocampal functions [ 12,231. We previously reported that intrahippocampal administration of the muscarinic acetylcholine receptor antagonist scopolamine or the selective and competitive NMDA receptor antagonists such as (+)-3-(2-carboxypiperazin4-yl)propyl-1-phosphonic acid (CPP) caused a dose-dependent disruption of working memory performance of rats on a three-panel runway task, acquisition of new and variable information that was useful only within a session [16], indicating that this behavior depended on the excitatory neurotransmission via mus* Corresponding author. fax: +81 92 6322752.
carinic and NMDA receptors in the hippocampus. On the other hand, the hippocampus receives SHTergic innervation from the raphe nuclei, which functions to inhibit the activity of hippocampal neurons via postsynaptic ShydroxytryptaminelA (5HTIA) receptors [2]. It has been demonstrated that stimulation of hippocampal 5-HTlA receptors by the selective agonist (+)-Shydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) impairs spatial learning in a water maze and passive avoidance learning [5,7]. We also showed that intrahippocampal administration of 8OH-DPAT impaired working memory performance on the three-panel runway task, an effect that was reversed by the 5-HTIA receptor antagonist (-)-propranolol [17]. These results suggest the inhibitory role played by hippocampal 5-HT1* receptors in the regulation of mnemonic processes. In contrast to extensive evidence for the individual roles of the hippocampal neurotransmission in memory function, little is known about interactive regulations of memory processes through the multiple transmitter systems in this structure. In the present study, we investigated, using
0304-3940/96/$12.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO304-3940(96)13057-3
22
M. Ohno et al.
I Neuroscience Letters 217 (1996) 21-24
the three-panel runway task, the effects of cholinergic activation by the cholinesterase inhibitor physostigmine on the working memory deficits resulting from blockade of hippocampal muscarinic or NMDA receptors and on those by hippocampal 5-HTiA receptor stimulation, and thereby clarified whether the functional interactions between hippocampal cholinergic and other neuronal activities are involved in regulating the memory process. Male Wistar-strain rats (Japan SLC) were placed on a deprivation schedule to maintain their weights at approximately 80% of the free-feeding level (230-250 g) before the experiment. Working memory was assessed with a three-panel runway apparatus, as described in our previous reports [13,16]. In brief, this apparatus (175 x 36 x 25 cm) was composed of a start box, a goal box and four consecutive choice points intervening between them. Each choice point consisted of a gate with three panels (12 x 25 cm). The rats were prevented from passing through two of the three panels in the gate by front stoppers, and were prevented from returning to the start box or to a previous choice point by rear stoppers affixed to each of the panels in all the gates. When the rats reached the goal box, they received two food pellets (about 50 mg each; Muromachi Kikai). The rats were made to run the task in six consecutive trials at 2-min intervals (defined as one session) per day with removal of the front stopper of only one of the three panel-gates (the correct panel-gate) at each choice point. The locations of the correct panel-gates were held constant within a session, but were changed from one session to the next (working memory procedure). The number of times an animal attempted to pass through an incorrect panel-gate (defined as errors) and the time required for the animal to obtain food pellets (defined as latency) were recorded for each rat during each trial of a session. The number of errors and latency recorded in the first trial were presented separately, and those parameters in the second to the sixth trial of a session were summed together for the evaluation of working memory function. The learning criterion was less than eight errors summed from the second to sixth trials (working memory errors). A rat was used in the experiment, if it achieved this criterion in three consecutive sessions. The significance of differences between the groups was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s test when F ratios reached significance (P < 0.05). The rats that achieved the learning criterion were anesthetized with sodium pentobarbital (40 mg/kg i.p.>, and were implanted bilaterally with guide cannulae for microinjection of drugs into the hippocampus, as described previously [ 13,161. The position of the injection cannula tip, which protruded 1.0 mm below the tip of the guide cannula, was aimed at the dorsal hippocampus (A, 3.8 mm; L, f2.2 mm; H, 3.2 mm) according to the atlas of Paxinos and Watson [ 181. The rats were allowed at least 5 days of postoperative recovery before runway sessions were resumed. CPP (Research Biochemicals Int.) was dis-
solved in saline, after which the pH was adjusted to approximately 7.4 with an appropriate amount of NaOH. Other drugs were purchased from Sigma Chemical Co. and were dissolved in saline. Two microliters of the drug solution or saline was injected at 0.5 pl/min into the dorsal hippocampus through the injection cannula. The injection cannula was left in place for 1 min after completion of the injection. Rats received the runway test 10 min after the drug injection was completed. When microinjections were made repeatedly into each rat, a minimum of 3 days were allowed between microinjections. After completion of behavioral testing, the injection site in the hippocampus was verified by Cresyl violet staining, as described previously [13,16]. In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. The number of errors made from the second to the sixth trial (working memory errors) markedly decreased with repeated training, whereas the errors in the first trial remained constant at approximately four. Approximately 20-30 training sessions were required for the rats to reach the criterion of less than eight working memory errors. Latency was also reduced during repeated sessions and was stable from the 10th session on. Scopolamine at 3.2 pg/side, administered bilaterally into the dorsal hippocampus, significantly increased the number of working memory errors (Fl,,,, = 25.81, P < O.Ol), although it had no effect on the errors in the first trial (Fig. 1). Intrahippocampal 3.2 pg/side scopolamine significantly prolonged the latency to obtain food pellets from the second to sixth trials of a session (Fl,lo = 13.33, P < 0.01). The increase in working memory errors induced by intrahippocampal3.2 &side scopolamine was attenuated by concurrent injection of 1.0 and 3.2 pglside physostigmine (Fz,,d = 12.95, P < 0.01). Physostigmine (1.0 and 3.2 &side) was also effective in reducing the prolonged latency resulting from administration of 3.2 pg/side scopolamine into the hippocampus (F2,14 = 7.31, P < 0.05). Intrahippocampal 3.2 ,&side physostigmine by itself did not affect the number of working memory errors (5.5 + 2.2; mean + SEM, IZ = 4) or the latency from the second to sixth trials (27.3 f 2.0 s). The number of working memory errors was significantly increased by intrahippocampal injection of 32 ng/ side CPP (F1,9 = 49.18, P < 0.01) or by that of 10 pg/side 8-OH-DPAT (F1,8 = 17.50, P < O.Ol), whereas the number of errors in the first trial was not changed (Fig. 2). The latency from the second to sixth trials was significantly prolonged when rats received intrahippocampal 32 ng/ side CPP (F1,9 = 7.18, P < 0.05) or 10 &side 8-OHDPAT (Fl,* = 18.65, P < 0.01). Concurrent administration of 3.2 pg/side physostigmine into the hippocampus had no effect on the CPP (32 @side)-induced increase in working memory errors or in the latency to obtain food pellets placed in the goal box. Physostigmine (3.2 pg/side) was also ineffective in reducing the increase in working
M. Ohno et al. I Neuroscience
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32
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Fig. 1. Effects of concurrent injection of physostigmine on increases in working memory errors and latency induced by intrahippocampal scopolamine. Rats received the runway test 10 min after drugs were injected. Each column represents the mean f SEM of errors and latenties for 5-7 animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). **P < 0.01 (versus saline), ‘P < 0.05, #P < 0.01 (versus scopolamine). errors or in the latency induced by 10 &side 8OH-DPAT. Rats that received intrahippocampal 3.2 j.& side physostigmine together with 32 r&side CPP or 10 &side 8-OH-DPAT showed significantly more working memory errors than rats given 3.2 &side physostigmine alone (Fi,s = 8.83, P < 0.05;F,,, = 21.72, P < 0.01, respectively). The present study with the three-panel runway task showed that concurrent injection of the cholinesterase inhibitor physostigmine reversed working memory deficits induced by intrahippocampal administration of the muscarinic receptor antagonist scopolamine, indicating that the septohippocampal cholinergic activity plays a critical role in working memory performance, i.e. acquisition process of new information within a session. Some interactive mechanism between cholinergic and glutamatergic systems in memory processes has been evidenced by the fact that the behaviorally ineffective doses of scopolamine and the noncompetitive NMDA receptor antagonist MK801 significantly impair inhibitory avoidance learning of rats [14] and visual recognition memory of rhesus monkeys in a delayed non-matching-to-sample task [ 1 l] when both drugs are administered in combination, Furthermore, D-cycloserine, which acts as a positive modulator of NMDA receptors through the glycine binding site on the NMDA receptor/channel complex, has been reported to attenuate the scopolamine-induced deficits in spatial leaming of rats in a water maze task and those in memory performance of rats in a T-maze alternation paradigm [9,22]. Markram and Segal [lo] found, using hippocampal memory
23
Letters 217 (1996) 21-24
slices, that acetylcholine amplified NMDA receptormediated responses through acting on muscarinic receptors and thereby caused a long-lasting facilitation of excitatory postsynaptic potentials, suggesting the cholinergic activity functioning to modulate the glutamatergic transmission postsynaptically in the hippocampus. These findings suggest a link between the NMDA and muscarinic receptor-mediated neurotransmission involved in the regulation of memory processes. However, the present study showed that the cholinergic activation by physostigmine was ineffective in reversing the working memory failure caused by intrahippocampal administration of the competitive NMDA receptor antagonist CPP. It is, therefore, conceivable that although the septohippocampal excitatory muscarinic neurotransmission allows hippocampal neuronal activities to process working memory, the cholinergic activation is insufficient to support the memory processing through specific hippocampal glutamatergic pathway when NMDA receptor-mediated transmission is fully blocked. Although further study is necessary to clarify the precise mechanism underlying the differential effects of physostigmine on the memory impairments produced by blockade of hippocampal muscarinic and NMDA receptors. it is of interest to note that activation of NMDA receptors, but not that of muscarinic receptors, is essential for induction of hippocampal LTP, a neurobiological model of synaptic plasticity underlying learning and memory [1,3]. Abe et al. [I] demonstrated that the cholinergic system plays a modulatory role to function as a
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Fig. 2. Effects of concurrent injection of physostigmine on increases in working memory errors and latency induced by intrahippocampal CPP and I-OH-DPAT. Rats received the runway test 10 min after drugs were injected. Each column represents the mean rt SEM of errors and latencies for 5-6 animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). *P < 0.05, **P < 0.01 (versus saline).
24
M. Ohm et al. I Neuroscience Letters 217 (1996) 21-24
triggering amplifier for induction of hippocampal LTP by facilitating NMDA receptor-mediated responses. These findings may provide an explanation for the present result that cholinergic activation by physostigmine fails to compensate loss of hippocampal NMDA receptor-mediated neurotransmission responsible for working memory processing. It has been reported that stimulation of 5-HT1* receptors causes hyperpolarization of pyramidal neurons of the rat hippocampal CA1 and CA3 regions due to an increase in potassium conductance [2], in contrast to muscarinic receptor stimulation resulting in excitation of hippocampal pyramidal cells [8]. It has been found that hyperpolarization of hippocampal neurons through Kt channels opened by activation of 5-HTtA receptors functions to inhibit LTP [21], whereas acetylcholine facilitates LTP by regulating the threshold of LTP generation through a depolarizing action [l]. Some behavioral studies have demonstrated that the 5-HTiA receptor agonist and antagonist affect memory failure resulting from cholinergic deficiency. The behaviorally subthreshold dose of 8-OH-DPAT, a 5HTIA receptor agonist, exacerbates deficits in spatial learning in the water maze and passive avoidance learning induced by scopolamine or lesioning of the septohippocampal projection system [ 19,201. Furthermore, hippocampal 5-HTiA receptor blockade by WAY 100135 or NAN-190 is effective in preventing the scopolamine disruption of spatial learning in the water maze [6] and of working memory performance in the three-panel runway task [15]. It is, thus, likely that 5-HTiA and muscarinic receptors contribute jointly, but in the opposite direction, to the regulation of memory processes. In contrast, the present study showed that physostigmine, the dose of which was sufficient to reverse the memory failure by scopolamine, did not affect the working memory deficit resulting from hippocampal 5-HTiA receptor stimulation by 8-OH-DPAT. Taken together, these findings suggest that blockade of hippocampal 5-HTi* receptors can compensate loss of excitatory cholinergic input involved in working memory function, but that cholinergic activation, conversely, fails to counteract the interference with this memory function exerted by stimulation of inhibitory 5HTIA receptors in the hippocampus. VI Abe,
K., Nakata, A., Mizutani, A. and Saito, H., Facilitatory but nonessential role of the muscarinic cholinergic system in the generation of long-term potentiation of population spikes in the dentate gyms in vivo, Neurophatmacology, 33 (1994) 847-852. I21Beck, S.G., Choi, K.C. and List, T.J., Comparison of 5-hydroxytryptaminelA-mediated hyperpolarization in CA1 and CA3 hippocampal pyramidal cells, J. Pharmacol. Exp. Ther., 263 (1992) 350359. [31 Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 3139. [41 Brito, G.N.O., Davis, B.J., Stopp, L.C. and Stanton, M.E., Memory and the septo-hippocampal cholinergic system in the rat, Psychopharmacology, 81 (1983) 315-320.
[5] Carli, M., Lazarova, M., Tatarczynska, E. and Samanin, R., Stimulation of 5-HT,* receptors in the dorsal hippocampus impairs acquisition and performance of a spatial task in a water maze, Brain Res., 595 (1992) 50-56. [6] Carli, M., Luschi, R. and Samanin, R., (S)-WAY 100135, a 5-HTtA receptor antagonist, prevents the impairment of spatial learning caused by intrahippocampal scopolamine, Eur. J. Pharmacol., 283 (1995) 133-139. [7] Carli, M., Luschi, R., Garofalo, P. and Samanin, R., I-OH-DPAT impairs spatial but not visual learning in a water maze by stimulating 5-HTt* receptors in the hippocampus, Behav. Brain Res., 67 (1995) 67-74. [8] Cole, A.E. and Nicoll, R.A., The pharmacology of cholinergic excitatory responses in hippocampal pyramidal cells, Brain Res., 305 (1984) 283-290. [9] Fishkm, R.J., Ince, E.S., Carlezon, W.A. Jr. and DUM, R.W., DCycloserine attenuates scopolamine-induced learning and memory deficits in rats, Behav. Neural Biol., 59 (1993) 150-157. [lo] Markram, H. and Segal, M., Long-lasting facilitation of excitatory postsynaptic potentials in the rat hippocampus by acetylcholine, J. Physiol., 427 (1990) 381-393. [l l] Matsuoka, N. and Aigner, T.G., Cholinergic-glutamatergic interactions in visual recognition memory of rhesus monkeys, NeuroReport, 7 (1996) 565-568. [12] Morris, R.G.M., Synaptic plasticity and learning: selective impairment of learning in rats and blockade of long-term potentiation in vivo by the N-methyl-o-aspartate receptor antagonist AP5, J. Neurosci., 9 (1989) 3040-3057. [13] Ohno, M. and Watanabe, S., Concurrent blockade of hippocampal metabotropic glutamate and N-methyl-o-aspartate receptors disrupts working memory in the rat, Neuroscience, 70 (1996) 303311. [14] Ohno, M. and Watanabe, S., Interactive processing between glutamatergic and cholinergic systems involved in inhibitory avoidance learning of rats, Eur. J. Pharmacol., (1996) in press. [15] Ohno, M. and Watanabe, S., Blockade of 5Xft~ receptors compensates loss of hippocampal cholinergic neurotransmission involved in working memory of rats, Brain Res., (1996) in press. [16] Ohno, M., Yamamoto, T. and Watanabe, S., Effects of intrahippocampal injections of N-methyl-o-aspartate receptor antagonists and scopolamine on working memory and reference memory assessed in rats by a three-panel runway task, J. Pharmacol. Exp. Ther., 263 (1992) 943-950. [17] Ohno, M., Yamamoto, T. and Watanabe, S., Working memory deficits induced by intrahippocampal administration of g-OHDPAT, a 5-HT1* receptor agonist, in the rat, Eur. J. Pharmacol., 234 (1993) 29-34. [18] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. [19] Riekkinen, P. Jr., 5-HTtA and muscarinic acetylcholine receptors jointly regulate passive avoidance behavior, Eur. ,I. Pharmacol., 262 (1994) 77-90. [20] Riekkinen, M., Sin%, J., Toivanen, T. and Riekkinen, P. Jr., Combined treatment with a 5HTt* receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior, Psychopharmacology, 122 (1995) 137-146. [21] Sakai, N. and Tanaka, C., Inhibitory modulation of long-term potentiation via the 5-HT,* receptor in slices of the rat hippocampal dentate gyrus, Brain Res., 613 (1993) 326-330. [22] Sirviii, J., Ekonsalo, T., Riekkinen, P. Jr., Lahtinen, H. and Riekkinen, P. Sr., o-Cycloserine, a modulator of the N-methyl-oaspartate receptor, improves spatial learning in rats treated with muscarinic antagonist, Neurosci. Lett., 146 (1992) 215-218. [23] Ward, L., Mason, S.E. and Abraham, W.C., Effects of the NMDA antagonists CPP and MK-801 on radial arm maze performance in rats, Pharmacol. Biochem. Behav., 35 (1990) 785-790.