Introduction

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Serotonin (5-HT)₄ receptors are widely expressed in the central and peripheral neuronal systems (Hoyer et al., 1994), however, little is known about their physiological roles in the central nervous system. 5-HT₄ receptors are concentrated in the limbic structures, including the hippocampus, which plays an essential role in memory processes (Waeber et al., 1993; Ullmer et al., 1996). The activation of 5-HT₄ receptors is known to stimulate adenylyl cyclase activity in the hippocampus of rats (Torres et al., 1995; Markstein et al., 1999) and guinea pigs (Bockaert et al., 1990). Furthermore, increasing evidence indicates that cAMP plays an important role in the hippocampal long-term potentiation (LTP), which is thought to be a form of the basic cellular mechanisms underlying learning and memory (Frey et al., 1993; Abel et al., 1997; Otmakhova et al., 2000). These previous findings suggest a possible involvement of 5-HT₄ receptors in the modulation of cognitive functions.

Indeed, several behavioral studies have shown that 5-HT₄ receptors were associated with learning and memory processes. For instance, the mixed 5-HT₄ agonist/5-HT₃ antagonist BIMU 1 showed ameliorative effects in the rat olfactory association learning test (Letty et al., 1997; Marchetti-Gauthier et al., 1997). BIMU 1 also improved the scopolamine-induced amnesia in the mouse passive avoidance test (Galeotti et al., 1998). The 5-HT₄ receptor agonist RS 67333 prevented atropine-induced learning impairment in the Morris water maze test (Fontana et al., 1997). Terry et al. (1998) reported, using a delayed matching-to-sample task, that the 5-HT₄ receptor agonist RS 17017 improved memory processes in younger and older monkeys. They suggested that the 5-HT₄ receptor-mediated improvements in older monkeys, in particular, have implications for neurodegenerative conditions such as Alzheimer's disease. Furthermore, a binding study suggested that a marked loss of 5-HT₄ receptors was found in the hippocampus of patients with Alzheimer's disease (Reynolds et al., 1995). These findings lead us to speculate that 5-HT₄ receptors partially contributed to the cholinergic function associated with cognitive processes. However, to our knowledge, there is no evidence supporting the presence of the functional interaction between the serotonergic neuronal mechanisms mediated via 5-HT₄ receptors and cholinergic function based on behavioral alterations and physiological changes in these neuronal systems.

The present study was undertaken to elucidate the functional role of 5-HT₄ receptors with a view toward modulating the cholinergic neuronal system. For this purpose, behavioral, electrophysiological, and neurochemical studies were performed in rats. The behavioral study was carried out to evaluate the memory and learning function using a passive avoidance task. The electrophysiological approach was performed to examine the synaptic transmission by measuring the population spike amplitude in the hippocampal CA1 field. Neurochemically, an in vivo microdialysis method was used to elucidate the dynamic changes in hippocampal cholinergic neuronal activity by determining acetylcholine (ACh) release. Based on these differential paradigms, the physiological function of central 5-HT₄ receptors was investigated.

	Materials and Methods

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General Procedures. Male Wistar strain rats (11-14 weeks old) were used. Rats were housed in a room with a 12-h light (7:00 AM to 7:00 PM)/dark (7:00 PM to 7:00 AM) cycle under a constant temperature (21 ± 2°C). All handling of animals was performed in accordance with guidelines for the Care and Use of Laboratory Animals of the Animal Research Committee of the Hokkaido University School of Medicine. Drugs were administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) or perfused locally via a dialysis probe. A probe for i.c.v. injection was inserted at the following coordinates relative to bregma and dural surface, i.e., caudal, 0.8 mm; lateral, 1.4 mm; ventral, 3.3 mm through the guide cannula, and 10 µl of drug was administered at a flow rate of 2 µl/min. As controls, saline (0.9% NaCl) or artificial cerebrospinal fluid (in mM: KCl 2.7, NaCl 140, CaCl₂ 1.2, MgCl₂ 1.0, NaH₂PO₄ 0.3, Na₂HPO₄ 1.7) was administered i.p. or i.c.v., respectively.

Behavioral Study. The passive avoidance test was performed according to the step-through method (Jarvik and Kopp, 1967). The apparatus consisted of a light compartment connected to a dark box by a dividing wall containing a guillotine door. In the training session, an acquisition trial was performed as follows; rats were acclimated in the light compartment for 2 min, then the guillotine door was opened. Three seconds later, when the rats crossed over into the dark box, an inescapable electrical shock (0.8 mA, 3 s) was delivered. This trial was repeated until acquisition was established. Acquisition was regarded as being established when rats eventually remained in the light compartment for more than 300 s to avoid entering the dark box. The number of entries into the dark box to learn the "information for electrical shock" was counted until acquisition was established. After 24 h, the retention test was conducted: rats were placed in the light compartment and the time taken to enter the dark box, the latency time (seconds), was measured up to a maximum of 600 s. In this experiment, drugs were administered following three protocols: in protocol 1, the muscarinic receptor antagonist scopolamine was administered after acquisition. In protocol 2, scopolamine was injected before the training session, and the 5-HT₄ receptor agonist SC 53116 (Flynn et al., 1992) was administered after acquisition. In protocol 3, scopolamine and SC 53116 were administered before the training session.

Electrophysiological Study. The efficacy of synaptic transmission was evaluated by monitoring the population spike amplitude in the hippocampal CA1 field. Rats anesthetized with 1% halothane in a mixture of 20% O₂ and 80% N₂ were fixed in a stereotaxic frame according to bregma and lambda in the same horizontal plane. A bipolar stainless steel electrode was used to stimulate Schaffer collaterals (3.0 mm posterior, 1.5 mm lateral to the bregma, 2.8 mm ventral to the dura). A monopolar glass-coated recording electrode was placed in the ipsilateral pyramidal cell layer of CA1 (5.0 mm posterior, 3.0 mm lateral to the bregma, approximately 2.3 mm ventral to the dura). The evoked potential by test stimulation (frequency 0.1 Hz, pulse duration 250 µs, stimulus interval 30 s) was amplified and monitored with an oscilloscope. The integrated population spike amplitude obtained from five successive stimuli was recorded every 5 min with a data-analyzing system (ATAC-450, Nihon Kohden, Japan). The intensity of the test stimulation was adjusted for each rat to elicit a population spike amplitude of approximately 50% maximum amplitude. LTP, which is suggested as the electrophysiological model for learning and memory (Bliss and Collingridge, 1993), was induced by tetanic stimulation (5 trains at 1 Hz each, composed of eight pulses at 400 Hz) at the same intensity as the test stimulus. Tetanic stimulation was given 20 min after the drug administration.

Neurochemical Study. In vivo microdialysis technique was used to estimate the cholinergic neuronal activity in the hippocampus of freely moving rats. Rats were anesthetized with ketamine (100 mg/kg i.p.) and a 3-mm concentric guide cannula was stereotaxically implanted into the hippocampus (5.8 mm posterior, 4.8 mm lateral to the bregma, 4.0 mm ventral to the dura). Two days after surgery, a dialysis probe was inserted through the guide cannula and perfused at a flow rate of 1 µl/min with Ringer's solution (KCl 2.7, NaCl 147, CaCl₂ 2.3 mM) containing 10 µM physostigmine. Perfusion was performed to obtain the stable baseline for 120 to 180 min, and successive 20-µl samples were collected at 20-min intervals. Drugs were administered into the hippocampus via the dialysis probe. The extracellular levels of ACh were measured using high performance liquid chromatography (HPLC) with an electrochemical detector (Acetylcholine Analytical System, Eicom, Co. Ltd., Japan), as described previously (Hirokami et al., 1994). Briefly, ACh and choline were separated using an AC-Gel column attached to the enzyme reactor containing acetylcholinesterase and choline oxidase. ACh was assayed using the HPLC-electrochemical detector following its conversion to hydrogen peroxide, which was electrochemically detected at a platinum working electrode at 450 mV versus the Ag/AgCl reference electrode. The mobile phase contained 0.1 M sodium phosphate buffer (pH 8.5), 0.6 mM tetramethylammonium chloride, and 0.8 mM sodium 1-decanesulfonate. The HPLC separation and enzymatic reactions were performed at 33°C.

Drugs. SC 53116 (1-(S)-1-exo-4-amino-5-chloro-N-[(hexahydro-1H-pyrrolizin-1-yl)methyl]-2-methoxybenzamide, hydrocholoride, monohydrate) was donated by G.D. Searle and Co. Ltd (Chicago, IL). GR 113808 (1-[2-(methylsulphonylamino)ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate, maleate salt was donated by Glaxo Wellcome Co. Ltd. (Greedfold, UK). Scopolamine hydrobromide was purchased from Sigma (St. Louis, MO).

Statistics. All experimental results were represented as mean ± S.E.M. Values obtained by neurochemical studies using in vivo microdialysis and electrophysiological techniques were expressed as a percentage of the baseline levels before drug administration. For comparison with the experimental groups, Student's unpaired t test or Dunnett's test was conducted after assessment by analysis of variance (ANOVA). Probability values less than 5% were considered significant.

	Results

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Behavioral Study: Effects of SC 53116 on Cholinergic Function in the Passive Avoidance Test. To evaluate the involvement of 5-HT₄ receptors in the cholinergic function of cognitive processes, the muscarinic receptor antagonist, scopolamine was used as an amnesic agent. Scopolamine (0.3 and 1 mg/kg i.p.), when administered before the training session, reduced the latency for entering the dark box during the retention test in a dose-dependent manner. Scopolamine-induced (1 mg/kg i.p.) amnesic effects, however, were not observed when administered immediately after acquisition (Fig. 1). The 5-HT₄ receptor agonist, SC 53116 (10 µg/rat i.c.v.) tended to ameliorate the scopolamine-induced (1 mg/kg i.p.) reduction of entrance latency, but not significantly. SC 53116 (10 µg/rat i.c.v.), when administered alone, showed no influence on the latency during the retention test compared with controls (Fig. 2). Scopolamine (1 mg/kg i.p.), administered before the training session significantly increased the total number of entries into the dark box for acquisition. Co-administration of SC 53116 (10 µg/rat i.c.v) significantly attenuated increases in the number of entries induced by scopolamine (Fig. 3). Thus, SC 53116 improved the scopolamine-induced deficit in the learning processes. SC 53116 (10 µg/rat i.c.v.) by itself did not alter the entrance counts compared with controls (Fig. 3).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Effects of scopolamine on entrance latency in the retention of the passive avoidance test. Experiments were performed as follows: protocol 1; scopolamine (1 mg/kg i.p.) was administered immediately after acquisition of the training session. Protocol 2; scopolamine (0.3 and 1 mg/kg i.p.) was injected 30 min before the training session. The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 versus saline-treated controls.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Effects of SC 53116 on scopolamine-induced entrance latency in the retention of the passive avoidance test. Experiments were performed as follows. Protocol 2: scopolamine (1 mg/kg i.p.) was injected 30 min before the training session. SC 53116 (10 µg/rat i.c.v.) was administered immediately after the acquisition of the training session. Protocol 3: scopolamine (1 mg/kg i.p.) and SC 53116 (10 µg/rat i.c.v.) were administered 30 and 20 min, respectively, before the training session. The number of rats is shown inside each column. Values represents the mean ± S.E.M. *p < 0.05 versus saline-treated controls.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of SC 53116 on the scopolamine-induced number of entries (counts) during the training session of the passive avoidance test. Scopolamine (1 mg/kg i.p.) and SC 53116 (10 µg/rat i.c.v.) were administered 30 and 20 min, respectively, before the training session (protocol 3). The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 and #p < 0.05 versus saline-treated controls and scopolamine-administered rats, respectively.

Electrophysiological Study: Effects of SC 53116 on Synaptic Transmission in the Hippocampal CA1 Field. The efficacy of synaptic transmission was estimated by monitoring the population spike amplitude in the CA1 field. SC 53116 (1 and 10 µg/rat i.c.v.) enhanced the population spike amplitude evoked by test stimulation of Schaffer collaterals in a concentration-dependent manner (Fig. 4A). SC 53116-induced (10 µg/rat i.c.v.) facilitation was significantly prevented by pretreatment with the selective 5-HT₄ receptor antagonist GR 113808 (20 µg/rat i.c.v.). GR 113808 alone did not affect the synaptic transmission (Fig. 4B). As shown in Fig. 5, tetanic stimulation evoked long-lasting increases in the population spike amplitude with a maximum effect of 210%, i.e., LTP was induced. Application of SC 53116 (10 µg/rat i.c.v.) caused an enhanced amplitude of LTP (maximum effect; 307.0 ± 27.8%, n = 6). SC 53116-induced augmentation of LTP was significantly prevented by scopolamine (1 mg/kg i.p.). Scopolamine (1 mg/kg i.p.), when administered alone, significantly suppressed LTP formation compared with controls (Fig. 5). SC 53116-induced (10 µg/rat i.c.v.) enhancement of LTP was also abolished by pretreatment with GR 113808 (20 µg/rat i.c.v.) (maximum effects; 172.1 ± 23.4%, n = 4, p < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of SC 53116 on the population spike amplitude evoked by the test stimulation in the hippocampal CA1 field of anesthetized rats. A, time course responses of the population spike amplitude after SC 53116 (1 and 10 µg/rat i.c.v.) administration. Specimen recordings show superimposed traces of the population spike amplitude before and after SC 53116 (10 µg/rat i.c.v.) administration. Data were expressed as a percentage of the baseline levels before SC 53116 administration. B, maximum response of SC 53116-induced (10 µg/rat i.c.v.) changes in the population spike amplitude in the presence or absence of GR 113808 (20 µg/rat i.c.v.). GR 113808 was injected i.c.v. 10 min before SC 53116 administration. The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 and #p < 0.05 versus controls and SC 53116-administered rats, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of SC 53116 on the population spike amplitude evoked by tetanic stimulation in the hippocampal CA1 field of anesthetized rats. A, specimen recordings show superimposed traces of the population spike amplitude before and after tetanic stimulation in the presence of SC 53116 (10 µg/rat i.c.v.). Time course responses (A) and maximum responses (B) of LTP induced by SC 53116 (10 µg/rat i.c.v.) in the presence or absence of scopolamine (1 mg/kg i.p.). Scopolamine (1 mg/kg i.p.) was injected 10 min before SC 53116 administration. Data were expressed as a percentage of the baseline levels before SC 53116 administration. The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 and #p < 0.05 versus controls and SC 53116-administered rats, respectively.

Neurochemical Studies: Effects of SC 53116 on ACh Release in the Rat Hippocampus. To evaluate the dynamic changes of cholinergic neuronal activity, in vivo microdialysis was used by determining ACh release from the hippocampus. Extracellular levels of ACh were significantly and concentration-dependently increased by local application of SC 53116 (10 and 100 µM). SC 53116 (1 µM) did not affect the ACh release (Fig. 6). SC 53116-induced (10 µM) increases in ACh release were significantly abolished by coperfusion with GR 113808 (10 µM). GR 113808 (10 µM), when perfused alone, did not influence the ACh levels (Fig. 7). The mean basal level of ACh was 2.5 ± 0.2 pmol/sample (n = 25). There were no significant differences in the basal ACh levels among the groups.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effects of SC 53116 on the extracellular levels of ACh in the hippocampus of freely moving rats. Time course responses (A) and maximum responses (B) of SC 53116-induced (1, 10, and 100 µM) changes in ACh release. SC 53116 was administered locally via dialysis probe over a period of 60 min. Data were expressed as the percentage of baseline levels before SC 53116 perfusion. Basal levels, determined immediately before perfusion of SC 53116, were 1 µM, 2.04 ± 0.20 pmol/20 min; 10 µM, 2.69 ± 0.38 pmol/20 min; 100 µM, 2.11± 0.48 pmol/20 min. These values did not differ significantly from that of controls (2.61 ± 0.27 pmol/20 min). The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 versus controls.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effects of GR 113808 on SC 53116-induced changes in ACh release in the hippocampus of freely moving rats. Time course responses (A) and maximum responses (B) of SC 53116-induced (10 µM) changes in ACh release in the presence or absence of GR 113808 (10 µM). GR 113808 (10 µM) was administered locally via dialysis probe over a period of 60 min and coperfused with SC 53116 (10 µM). Data were expressed as a percentage of the baseline levels before GR 113808 perfusion. Basal levels, determined immediately before perfusion of drugs, were SC 53116 (10 µM), 2.69 ± 0.38 pmol/20 min; SC 53116 (10 µM) + GR 113808 (10 µM), 2.51 ± 0.19 pmol/20 min; and GR 113808 (10 µM), 2.11 ± 0.24 pmol/20 min. The number of rats is shown inside each column. Values represent the mean ± S.E.M. *p < 0.05 and #p < 0.05 versus controls and SC 53116-treated rats, respectively.

	Discussion

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Based on the findings using the behavioral tests and electrochemical techniques, the present study demonstrated that the 5-HT₄ receptors were involved in the modulation of the cholinergic function associated with learning and memory. This physiological interaction between the serotonergic system mediated via 5-HT₄ receptors and the cholinergic system was further confirmed by neurochemical evidence, i.e., hippocampal ACh release was enhanced by activation of 5-HT₄ receptors.

A previous study using in vivo microdialysis reported that i.c.v. injection of the nonselective 5-HT₄ receptor agonist, BIMU 1, caused enhanced ACh release in the rat frontal cortex, but its facilitation did not appear in the hippocampus (Consolo et al., 1994). In the present study, ACh release was increased by local application of the 5-HT₄ receptor agonist, SC 53116, and this facilitation was blocked by the selective 5-HT₄ receptor antagonist GR 113808. These findings strongly indicate that the 5-HT₄ receptor-mediated regulation of cholinergic neurons exists not only in the frontal cortex (Consolo et al., 1994; Yamaguchi et al., 1997) but also in the hippocampus. In agreement with the observation in the frontal cortex (Consolo et al., 1994), GR 113808 alone showed no effect on the ACh release, suggesting that the 5-HT₄ receptors did not tonically influence the cholinergic neuronal system in the rat hippocampus.

It is conceivable that 5-HT₄ receptors are located on cholinergic nerve terminals in the hippocampus as well as in the enteric nervous system (Tonini et al., 1991) and thus stimulate ACh release. Although the precise mechanisms of the neuronal interactions are unclear, one possibility is that the reduction in slow afterhyperpolarization, mediated by 5-HT₄ receptors, causes neuronal excitability (Torres et al., 1996) and ultimately contributes to the facilitation of ACh release. This assumption was strengthened by the present electrophysiological findings that the synaptic transmission was facilitated by the stimulation of 5-HT₄ receptors: SC 53116 enhanced the population spike amplitude in the hippocampal CA1 field. Furthermore, SC 53116 potentiated the population spike amplitude not only by the test stimulation but also by tetanization. It could not be excluded that the tetanus-induced LTP was augmented by activation of 5-HT₄ receptors, however, this facilitation appears to reflect the increased synaptic transmission, i.e., LTP-like effects produced by SC 53116 alone. The augmentation of LTP formation induced by SC 53116, on the other hand, was prevented not only by GR 113808 but also by scopolamine. These findings suggest that LTP formation mediated by 5-HT₄ receptors was, in part, regulated by muscarinic receptors. In turn, the cooperative mechanism via 5-HT₄ receptors and muscarinic receptors may contribute to LTP induction.

The regulation of synaptic plasticity is considered to involve the long-term modulation of ion-channel activity, therefore, the stimulation of 5-HT₄ receptors may initiate this process by activation of adenylyl cyclase. Thus, accumulated cAMP formation stimulated by 5-HT₄ receptors leads to modulation of the activity of protein kinase A and results in closure of the potassium channels (Fagni et al., 1992). These signaling mechanisms contribute to stimulate ACh release and may result in induction of LTP. Indeed, several lines of evidence indicate that the cholinergic neuronal system plays an important role in plastic changes in the CA1 field in accordance with memory and learning. For instance, the cholinergic agonist carbachol caused a long-lasting increase in the efficacy of synaptic transmission (Blitzer et al., 1990). The cholinesterase inhibitor physostigmine, which prevents the degradation of endogenous ACh, enhanced the magnitude of LTP (Ito et al., 1987). Consistent with previous studies (Ito et al., 1987; Hirotsu et al., 1989), the scopolamine-induced attenuation of LTP was observed in this study. These findings indicate that the cholinergic system may play a significant role in the mechanism for the induction of long-lasting synaptic enhancement, i.e., LTP formation.

In the present study, pretreatment with SC 53116 tended to enhance the magnitude of LTP inhibited by scopolamine, but not significantly. Although no correlation was established between synaptic plasticity and behavioral alteration, these phenomena suggest that the stimulation of 5-HT₄ receptors are not contributed to improve the memory deficit induced by scopolamine. Indeed, the passive avoidance test showed that SC 53116 ameliorated the scopolamine-induced impairment in the training session, however, this drug failed to improve the amnesic effects induced by scopolamine in the retention test. These findings suggest that the 5-HT₄ receptors may participate in the learning processes when cholinergic function was impaired, but these receptors may have little influence on the memory processes. The present findings did not always agree with previous findings, which showed that BIMU 1 and BIMU 8 improve the scopolamine-induced amnesic effect in the mouse passive avoidance test (Galeotti et al., 1998). We cannot sufficiently explain this discrepancy, however, it may be related to the differences in species or the selectivity of the drugs used.

Interestingly, scopolamine did not cause amnesia when administered after acquisition. These findings were in agreement with a previous study (Flrod and Buccafusco, 1988). Farr et al. (2000) also reported that intrahippocampal infusion of scopolamine following footshock avoidance training in a T-maze did not impair retention. In the present electrophysiological study, the suppression of LTP induced by scopolamine was not observed when this drug was administered after tetanization (data not shown). An in vitro study using the rat hippocamal slices also showed that scopolamine, when applied for 20 min before and during the tetanus, caused suppression of LTP, whereas LTP was induced when scopolamine applied after tetanus stimulation (Hirotsu et al., 1989). These findings indicate that the memory formation and LTP induction may be associated with a common neuronal mechanism, at least in the cholinergic neuronal system. Indeed, Auerbach and Segal (1994) provided evidence of a linkage between the cholinergic neuronal system and synaptic plasticity in the hippocampus: the carbachol-induced slow onset and long-lasting increases in the synaptic transmission in the CA1 hippocampal cells, which was named muscarinic long-term potentiation (LTPm), was blocked by atropine when administered before and during application of carbachol. Atropine, when added after LTPm has already been induced, did not affect the population spikes. Thus, the activation of a muscarinic receptor may be necessary for the induction of LTPm but not for the expression of this synaptic plasticity. In addition, they proposed that LTPm shared several properties with tetanic-stimulated LTP and was an important link between the cholinergic action and hippocampal function (Segal and Auerbach, 1997). In other words, the cholinergic neuronal system may play an important role in LTP induction but may be of little importance in the maintenance of LTP.

In summary, the findings of the present study revealed that 5-HT₄ receptors played a significant role in the physiological relationship between the serotonergic and cholinergic systems associated with learning and memory. Thus, the functional role of 5-HT₄ receptors in cognitive processes appears to require interaction with cholinergic neurotransmission. The 5-HT₄ receptor agonists, therefore, could be useful in the treatment of cognitive deficits by enhancement of cholinergic neurotransmission.