Blockade of 5-HT1A receptors compensates loss of hippocampal cholinergic neurotransmission involved in working memory of rats

BRAIN RESEARCH ELSEVIER

Brain Research 736 (1996) 180-188

Research report

Blockade of 5-HT1Areceptors compensates loss of hippocampal cholinergic neurotransmission involved in working memory of rats Masuo Ohno *, Shigenori Watanabe Department of'Pharmacology, Faculty of Pharma( eutical Sciences, l~vushu Unit,el:~it~,62, Fukuoka 812-82, Japan

Accepted 4 June 1996

Abstract

NAN-190, a selective 5-HTIA receptor antagonist, had no effect on the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task with a three-panel runway setup, when injected bilaterally at 0.32 or 1.0 txg/side into the dorsal hippocampus. Intrahippocampal administration of the muscarinic receptor antagonist scopolamine at 3.2 ixg/side or the competitive NMDA receptor antagonist (+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) at 32 ng/side significantly increased the number of working memory errors. The increase in working memory errors induced by intrahippocampal scopolamine (3.2 ixg/side) was reduced by concurrent infusion of 0.32 and 1.0 ~g/side NAN-190, an effect that reached significance only for the 1.0 txg/side dose. In contrast, NAN-190 at 1.0 ixg/side did not affect the increase in working memory errors when infused intrahippocampally together with 32 ng/side CPP. These results suggest that blockade of hippocampal 5-HTIA receptors does not affect impairment of working memory resulting from block of NMDA receptor-mediated neurotransmission, but that it can compensate deficiency of septohippocampal cholinergic activity involved in working memory performance of rats. Keywords: Hippocampus; Working memory; 5-HTIA receptor; NAN-190; Acetylcholine;Scopolamine; NMDA receptor; (+)-3-(2-Carboxypiperazin-4yl)propyl-1-phosphonic acid

1. Introduction

The hippocampus receives extensive innervation from 5-HT cell bodies in the raphe nuclei [4,46], and contains high densities of 5-HT 1 receptors, most of which belong to the 5-HTIA receptor subtype [50,68]. Since stimulation of hippocampal 5-HTIA receptors causes hyperpolarization of pyramidal neurons, it is likely that the 5-HTergic system functions to inhibit the activity of the hippocampus via postsynaptic 5-HTIA receptors [3,5,16]. The hippocampus is one of the brain structures that play a critical role in mnemonic processes [ 19,28,71 ]. These findings suggest a possible role of hippocampal 5-HTtA receptors in regulating memory functions. In fact, recent behavioral studies have demonstrated that administration of the 5-HT~A receptor agonists, such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and tandospirone, significantly impairs memory performance of rats and mice in various learning tasks, such as Morris water maze, 8-arm radial maze and passive avoidance tasks * Corresponding author. Fax: (81) (92) 632-2752.

[8,9,13,34,35,58,70]. It has been thought that postsynaptic 5-HTLA receptors in the hippocampus mediate the disruptive effect of 8-OH-DPAT on memory function, since the impairment of spatial learning in a water maze induced by systemically administered 8-OH-DPAT is attenuated by intrahippocampal infusion of the 5-HT~A receptor antagonists spiroxatrine or ( + ) W A Y 100135 [13], and is potentiated in rats with 5-HT depletion induced by intracerebroventricular injection of 5,7-dihydroxytryptamine [8]. Furthermore, Carli et al. [10,11] reported that 8-OH-DPAT disrupted spatial learning and passive avoidance learning when administered directly into the hippocampus, effects that were blocked by concurrent injection of spiroxatrine. We also showed that not only systemic but also intrahippocampal administration of 8-OH-DPAT impaired working memory performance of rats on a three-panel runway task, acquisition of new and variable information that was useful only within a session, and that these effects of 8-OH-DPAT were attenuated by the 5-HTIA receptor antagonist (--)-propranolol [41]. These results suggest the inhibitory role played by hippocampal 5-HTIA receptors in the regulation of mnemonic processes.

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006-8993(96)00678-6

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The septohippocampal cholinergic system is known as excitatory input to the hippocampus that plays an important role in memory functions [6,7]. We previously reported that the muscarinic acetylcholine receptor antagonist scopolamine was effective in disrupting working memory in the three-panel runway task when injected into the dorsal hippocampus, indicating that this behavior depended on the septohippocampal cholinergic function [40]. In the present study, we investigated, using the three-panel runway task, the effect of intrahippocampal administration of the 5-HT1A receptor antagonist NAN-190 [60,66] on the impairment of working memory resulting from blockade of hippocampal muscarinic receptor-mediated neurotransmission to elucidate the functional interactions between hippocampal 5-HTergic and cholinergic systems in the regulation of the mnemonic process. The results were compared with the effect of NAN-190 on working memory deficit induced by intrahippocampal administration of the selective NMDA receptor antagonist (_+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP), since NMDA receptors mediated another excitatory neurotransmission in the hippocampus that was responsible for working memory function [40].

2. Materials and methods

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rat could pass through any one of the three panel-gates at each choice point. The rats were made to run the task repeatedly until the time that elapsed from leaving the start box to reaching the goal box was consistently below 20 s. Once this time was reached, the rats received six consecutive trials (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. Trials were run at 2-min intervals, and water was freely available between trials in the home cage. The locations of the correct panel-gates at four choice points were held constant within a session, but were changed from one session to the next (working memory procedure). Twelve different patterns of correct panel-gate locations were used, as described previously [39]. 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. Since repetitive attempts to enter the same incorrect panel-gate were counted as one error, the maximal level was two errors at each choice point, and thus eight errors per trial. The learning criterion was fewer than eight errors summed from the second to sixth trials (working memory errors). Individual rats were used in the experiment if they achieved this criterion throughout three consecutive sessions.

2.1. Animals 2.3. Surgery and intrahippocampal injection

Eight- to 10-week-old male rats of the Wistar strain were obtained from Japan SLC. The rats weighed between 230-250 g at the start of the experiment, and they were then placed on a deprivation schedule to maintain their weights at approximately 80% of the free-feeding level, supplemented by a normal rate of weight gain. The rats were housed in groups of four per cage under a constant temperature ( 2 3 _ 2°C) and a 12:12-h light/dark cycle (light period: 07.00-19.00 h), with water freely available. 2.2. Three-panel runway task

Working memory was assessed with a three-panel runway apparatus, as described in our previous reports [39,40]. In brief, this apparatus (175 × 36 × 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 × 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) as positive reinforcement. Initially, all the front stoppers were removed so that a

The rats that achieved the learning criterion underwent bilateral chronic cannula implantation for microinjection of drugs into the dorsal hippocampus, as described previously [39,40]. Each rat was anesthetized with sodium pentobarbital (40 m g / k g i.p.) and was fixed in a stereotaxic instrument. A stainless-steel guide cannula (external diameter: 0.7 mm) was placed at a site 1.0 mm above the dorsal hippocampus (3.8 mm posterior to the bregma, 2.2 mm lateral to the midline, 3.2 mm ventral to the surface of the skull measured at the bregma), according to the brain atlas of Paxinos and Watson [49]. The cannula was fixed to the skull with three screws and dental acrylic cement. Between injections, the guide cannulae were occluded with stainless-steel wire pins. The rats were allowed at least 5 days of postoperative recovery before runway sessions were resumed. The rats were used after it was confirmed that they met the learning criterion of working memory performance after the surgical manipulation. A stainless-steel injection cannula (external diameter: 0.35 mm) was used to infuse the drugs. The injection cannula was connected to a 5-pA Hamilton syringe via a polyethylene tube. Two Ixl of drug solution or saline was injected into the dorsal hippocampus through the injection cannula, the tip of which protruded 1.0 mm below the tip of the guide cannula. The rate of injection was 0.5 Ixl per

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min. The injection cannula was left in place for I min after completion of the injection, to facilitate diffusion of the drug. On the test day, rats received a single injection of drugs, and then they were given six consecutive trials of a test session at 2-min intervals, starting from 10 min after the drug injection was completed. Microinjections were never made more than five times into each rat, and a minimum of 3 days was allowed between microinjections. Performance on the three-panel runway task during non-injected sessions was not affected by repeated intrahippocampal injections and met the learning criterion.

2.4. Drugs

The drugs used in the present study were 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190; Research Biochemicals International), ( - )-scopolamine hydrobromide (Sigma Chemical Co.) and ( _+)-3-(2-carboxypiperazin-4-yl)propyl- l-phosphonic acid (CPP; Tocris Cookson). NAN-190 was dissolved in warm saline and then cooled. Scopolamine and CPP were dissolved in saline. Thereafter, the pH of the CPP solution was adjusted to approximately 7.4 with an appropriate amount of NaOH.

2.5. Histology

After completion of the experiment, the rats were deeply anesthetized with ether and then perfused transcardially with saline, followed by 4% paraformaldehyde solution. The brains were removed from the skull and postfixed for 48 h in paraformaldehyde solution. Thereafter, 50-1xmthick sections were stained with cresyl violet to verify the injection site histologically, as described previously [39,40]. Fig. 1 shows an example of cannula tracks in the hippocampus. The stained sections showed that damage associated with the guide cannulae was restricted to the overlying cortex, and that all of the tips of the injection cannulae were successfully located in the dorsal hippocampus. In our previous experiments, we examined the diffusion of cresyl violet dye that was injected intrahippocampally at a volume of 2 ixl/side, and confirmed that the dye injections were confined to the dorsal hippocampus [39,40]. 2.6. Data analysis

In the working memory task, the rats tbund the correct panel-gates on trial 1 by chance, which did not involve any working memory functions. The number of errors and the

Fig. 1. Photomicrographof a coronal section stained with cresyl violet showing typical placements of cannulae in the hippocampus.

M. Ohno, S. Watanabe / Brain Research 736 (1996) 180-188

latency summed from the second to the 6th trial of each working memory session were important for evaluating the ability of rats to remember new correct panel-gate locations (working memory function). Thus, these parameters were presented separately from those recorded in the first trial. 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).

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2.5' :g:g

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NAN-t90 Scopolamine (3.2 pg/side)

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3. Results In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. In the working memory task, 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 fewer than eight working memory errors. Latency was also reduced during repeated sessions and was stable from the 10th session on. NAN-190 at 0.32 or 1.0 p~g/side, administered bilaterally into the dorsal hippocampus, did not affect the number of errors or the latency to obtain food pellets in the working memory task (Fig. 2). lntrahippocampal administration of 3.2 ~ g / s i d e scopolamine significantly increased working memory errors (F1,8= 113.06, P < 0 . 0 1 ) , although it had no effect on the number of errors made in the first trial. Intrahippocampal 3.2 p~g/side scopolamine had a tendency to prolong the latency from the second to sixth trials of a session (Fl, 8 = 4.84, P < 0.1), but this effect was not statistically significant. The increase in working memory errors induced by intrahippocampal injection of 3.2 ~zg/side scopolamine was attenuated by concurrent administration of 0.32 and 1.0 p~g/side NAN190 (F2,~2 = 7.28, P < 0.01), an effect that reached significance only for the 1.0 ~ g / s i d e dose ( P < 0.01). 25-

1;1 0.32 Saline

1.0

0.32

NAN-190

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NAN-lg0 Scopolamine (3.2 pg/side)

Fig. 2. Effects of concurrent injection of NAN-190 on increases in working memory errors and latency induced by intrahippocampal administration of scopolamine. The runway test was given 10 min after drugs were administered. Each column represents the mean ± S.E.M. of errors and latencies for five animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). The significance of differences from the saline-injected group (** P < 0.01) and from the scopolamine-injected group (## P < 0 . 0 1 ) was determined by a one-way ANOVA followed by Dunnett's test.

As shown in Fig. 3, intrahippocampal injection of 32 ng/side CPP caused significant increases in the number of working memory errors (FI, 8 = 45.10, P < 0.01), without affecting the number of errors in the first trial. The latency from the second to sixth trials was also significantly prolonged when rats received intrahippocampal administration of 32 ng/side CPP (F1, 8 = 12.70, P < 0.01). Concur80

70 20-

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10Z

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_¢_

5-

10 0

1.0 Saline

NAN-190 CPP (32 ng/side)

pg/side

1.0 Saline

pg/side

NAN-190 CPP (32 ng/side)

Fig. 3. Effects of concurrent injection of NAN-190 on increases in working memory errors and latency induced by intrahippocampal administration of CPP. The runway test was given 10 min after drugs were administered. Each column represents the mean ± S.E.M. of errors and latencies for five animals recorded in the first trial (open columns), and those summed from the second to the sixth trial within a session (hatched columns). The significance of differences from the saline-injected group was determined by a one-way ANOVA followed by Dunnett's test, * * P < 0.01.

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rent administration of 1.0 ~,g/side NAN-190 with 32 ng/side CPP into the hippocampus had no significant effect on the CPP-induced increase in working memory errors or in the latency to obtain food pellets placed in the goal box.

4. Discussion

The three-panel runway task allows us to investigate hippocampal neural mechanisms underlying working memory performance, since this behavior is significantly disrupted by dorsal hippocampal lesions [29]. The hippocampus receives 5-HTergic and cholinergic innervation from the midbrain raphe nuclei and the medial septal region, respectively [4,36,46], and contains high concentrations of postsynaptic 5-HTIA and muscarinic (M~-subtype) receptors [24,31,67]. We previously found, using the three-panel runway task, that hippocampal 5-HT]A receptor stimulation by 8-OH-DPAT and muscarinic receptor blockade by scopolamine or by the Ml-selective antagonist pirenzepine were sufficient to disrupt working memory when each drug was locally infused into the hippocampus [40,41,43]. It has been reported that the 5-HTIA receptor agonist affects the memory performance of scopolamine-treated or medial septal-lesioned rats. For example, Riekkinen [53] and Riekkinen et al. [57] demonstrated that systemic administration of 8-OH-DPAT by itself significantly impaired spatial learning in the water maze and passive avoidance learning, and that the behaviorally subthreshold dose of 8-OH-DPAT aggravated the learning deficits induced by treatment with scopolamine or lesioning of the septohippocampal projection system. From these results, they suggested that the imbalance between 5-HT~A and muscarinic receptor-mediated neurotransmission, i.e., overstimulation of 5-HT1A receptors and understimulation of muscarinic receptors, resulted in more severe memory deficits, as compared with those induced by either of the treatment alone. The present study showed that the impairment of working memory induced by intrahippocampal scopolamine was attenuated by concurrent injection of the 5-HTIA receptor antagonist NAN-190, whereas intrahippocampal NAN-190 by itself did not affect working memory function. These results suggest that hippocampal 5HT~A receptors are not tonically activated to interfere with memory processing under normal conditions, but they participate in exerting an inhibitory regulation of working memory function, i.e., acquisition process of new information within a session, when septohippocampal cholinergic neurotransmission declines. Concerning the lack of effect of NAN-190 on normal memory function, we cannot rule out the possibility that the rapid acquisition performance of rats in the working memory task does not allow us to find the facilitatory influence of 5-HTIA receptor blockade on the memory processes.

Although NAN-190 antagonizes several behavioral effects of the 5-HTIA receptor agonist 8-OH-DPAT [22,26], this compound by itself also reduces the release of 5-HT in the hippocampus as measured by in vivo microdialysis and decreases neuronal activity in the dorsal raphe nucleus, as does 8-OH-DPAT [14,23,26]. These findings provide evidence that NAN-190 acts as an antagonist at postsynaptic 5-HT~A receptors, but as a partial agonist at somatodendritic 5-HT~A receptors. Most of hippocampal 5-HTIA receptors appear to be located postsynaptic to 5-HTergic afferents from the raphe nuclei [24,67]. In fact, NAN-190 prevents the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in the hippocampus and shows no effect on the forskolin response by itself [14,63]. Thus, we can exclude the possibility that the agonistic activity of NAN-190 at presynaptic 5-HT~A receptors is involved in the reversal of scopolamine-induced memory failure by this compound applied directly into the hippocampus in the present study. Recently, the drugs that are highly selective antagonists at both presynaptic and postsynaptic 5-HTIA receptors, such as WAY 100135, have been described [20]. In accordance with the present findings, Carli et al. [12] recently reported, using (S)-WAY 100135, that blockade of 5-HTIA receptors was effective in preventing the impairment of spatial learning in the water maze produced by intrahippocampal scopolamine, whether the 5-HTtA receptor antagonist was administered systemically or was infused into the hippocampus together with scopolamine. Taken together, it is conceivable that postsynaptic 5-HT~A and muscarinic receptors in the hippocampus function jointly, but in the opposite direction, to regulate memory processes. NAN-190 has been reported to possess high affinity for c~-adrenoceptors as well as for 5-HTtA receptors, and shares the in vivo action of the selective C~l-receptor antagonist prazosin [14,37,66]. Interactive mechanisms between adrenergic and cholinergic systems have been known to play a role in regulating memory processes. However, such functional interactions are evidenced by the fact that noradrenergic depletion or receptor blockade, which by itself has no effect, exacerbates the scopolamine disruption of memory performance of rats on some learning tasks including the three-panel runway task, possibly by lowering [3-adrenergic neurotransmission [17,18,30,42,44]. Concerning the possibility of c~-adrenergic/cholinergic interactions, Ohta et al. [45] showed that scopolamine, but not prazosin, disrupted performance of rats on a radial maze task, a behavior related to spatial working memory, and that prazosin was not e f f e c t i v e in attenuating the scopolamine disruption of this mnemonic behavior. In the threepanel runway task, we also reported that the c~-adrenoceptor antagonist phentolamine did not affect the impairment of working memory caused by systemic administration of scopolamine [30]. Therefore, it appears unlikely that the a l-antagonist property of NAN-190 contributes to mediating the ameliorative effect of this compound on working

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memory disruption produced by intrahippocampal scopolamine. Serotonergic regulation of hippocampal acetylcholine release may be a possible explanation for neural mechanisms by which 5-HTLA receptor antagonist alleviates the intrahippocampal scopolamine-induced deficit in working memory. However, biochemical studies have shown that 5-HT receptors on cholinergic nerve terminals in the hippocampus that exert inhibitory regulation of acetylcholine release are of the 5-HT m receptor subtype, and that 8OH-DPAT does not reduce the high K ÷- or electrical stimulation-evoked [3H]acetylcholine efflux from hippocampal slices or synaptosomes, respectively [32,33]. In fact, Izumi et al. [27] reported, using a microdialysis technique, that local application of 8-OH-DPAT through dialysis probe increased acetylcholine release in the hippocampus of freely moving rats, an effect that was abolished by NAN-190, whereas the 5-HT1B receptor agonist CGS-12066B decreased hippocampal acetylcholine release. It is, therefore, unlikely that hippocampal 5-HTIA receptor blockade by NAN-190 attenuates the disruptive effect of scopolamine on working memory, by increasing acetylcholine release in the hippocampus. Alternatively, postsynaptic mechanisms with regard to the septohippocampal cholinergic input are likely to account for the behavioral interaction between intrahippocampal NAN-190 and scopolamine in regulating working memory function. It has been demonstrated that activation of 5-HTtA receptors causes hyperpolarization of pyramidal neurons of the rat hippocampal CA1 and CA3 regions due to an increase in potassium conductance [3,5,16], in contrast to stimulation of muscarinic receptors resulting in excitation of hippocampal pyramidal cells [15,64]. Concerning the phenomenon of long-term potentiation (LTP), which models synaptic plasticity and is a neural basis of learning and memory, it has been reported that the induction of hippocampal LTP recorded as an increase in populhtion spike amplitude is potentiated by NAN-190, and is suppressed by scopolamine, through blockade of 5-HTIA and muscarinic receptors, respectively [1,25,61]. These findings suggest that hyperpolarization of hippocampal neurons through K ÷ channels opened by activation of 5-HT~A receptors functions to inhibit LTP, whereas acetylcholine facilitates LTP by regulating the threshold of LTP generation through a depolarizing action. Taken together, it is conceivable that NAN-190 blocks the hyperpolarizing action of endogenous 5-HT via 5-HTIA receptors in the hippocampus and compensates loss of cholinergic excitatory input induced by scopolamine, leading to the prevention of impairment of working memory function. Some earlier studies have shown that depletion of brain 5-HT aggravates the deficits in acquisition processes in the water maze task that result from pharmacological blockade of muscarinic neurotransmission by scopolamine or atropine [52,55,65] and from lesions of the forebrain cholin-

185

ergic projection systems [38,54]. Application of 5-HT to hippocampal pyramidal neurons is known to produce hyperpolarization, but it increases the excitability after 5-HTIA receptors are blocked [2]. Hippocampal 5-HT receptors other than 5-HT~A receptors, for example, 5-HT4 receptors colocalized with 5-HT1A receptors on the same pyramidal neurons, are responsible for mediating the depolarizing response [59]. In contrast to the case of the 5-HTjA receptor blockade by (S)-WAY 100135 [12], the 5-HT 2 receptor antagonist methysergide, which by itself has no effect, augments the scopolamine-induced impairment of spatial learning in the water maze [56]. It is, thus, reasonable to speculate that deficits in neurotransmission via such excitatory 5-HT receptors rather than those via inhibitory 5-HT1A receptors are responsible for the potentiation of spatial learning impairment due to cholinergic dysfunction in 5-HT-depleted rats. This may explain the discrepancy with the results obtained by the present study and by Carli et al. [12], both of which show that the selective block of hippocampal 5-HTjA receptors leads to the reversal of memory loss induced by scopolamine. On the other hand, it is also important to note the differences in the behavioral paradigm used to assess memory function. Profound deficits in memory performance following concurrent deficiencies of 5-HTergic and cholinergic neurotransmission has been observed with the water maze task [38,52,54,55,65], where the rats were required to learn a constant platform location, i.e., acquisition processes of trial-independent information through trials (reference memory). Sakurai and Wenk [62] demonstrated, using a two-lever operant apparatus, that 5-HTergic destruction with p-chloroamphetamine reversed the scopolamine disruption of working memory of rats on a nonmatching-tosample task. Consistently, the present study showed that the pharmacological block of 5-HTIA receptors also attenuated the disruptive effect of scopolamine on working memory performance in the three-panel runway task, i.e., acquisition processes of trial-specific information. At any rate, selective blockade of hippocampal 5-HTjA receptors appears effective in reversing the scopolamine disruption of memory function, irrespective of whether the memory performance was measured by a working memory procedure (in the present study) or by acquisition processes of reference memory in the water maze [12]. The present study showed that concurrent injection of NAN-190 failed to reverse the working memory deficit induced by intrahippocampal administration of the NMDA receptor antagonist CPP, unlike the efficacy of NAN-190 in preventing scopolamine-induced memory impairment. At present, precise mechanisms underlying the differential effects of NAN-190 on the memory impairments are unclear. It is, however, apparent that excitation resulting from blockade of inhibitory 5-HTIA receptors by NAN-190 can compensate the deficiency in septohippocampal cholinergic activity, but it does not contribute to supporting memory processing through specific hippocampal glutamatergic

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pathway when NMDA receptor-mediated neurotransmission is fully blocked. The degree of loss of cholinergic function in the brain is known to correlate with the severity of cognitive deficits associated with Alzheimer's disease (AD) [51,69]. As compared with the substantial decline of cholinergic activity in the brains of patients with AD, the decrease in central 5-HT function is somewhat limited [21,47,48]. In the autopsy AD brain, half of the many regions examined showed no evidence of a reduction in presynaptic 5HTergic activity [48]. The remaining 5-HT neurons exhibit a compensatory increase in the turnover rate in the terminals, which is accompanied by the preservation of the concentration of 5-hyroxyindoleacetic acid, a major metabolite of 5-HT [21,47]. It is, therefore, reasonable to speculate that the imbalance related to 5-HTergic activity in preference to cholinergic neurotransmission may interfere with the functioning of hippocampal neurons by causing hyperpolarization via 5-HT~a receptors, making a contribution to the memory impairment in AD patients. Taken together, the present results of the efficacy of NAN-190 in alleviating scopolamine-induced memory impairment may provide evidence that the 5-HTIA receptor antagonist prevents the 5-HTIA receptor-mediated inhibitory influence on memory processes that is revealed by cholinergic deficits in the AD brain and deserves consideration as a new therapeutic approach for the treatment of memory decline in AD patients.

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