5-ht4 receptors in the hippocampus modulate rat locomotor activity

5-HT4 Receptors in the Hippocampus Modulate RatLocomotor Activity

Hiroshi Takahashi,1 Yumiko Takada,2

Tetsumei Urano,1 and Akikazu Takada1*

1Department of Physiology, Hamamatsu University,School of Medicine, Shizuoka-ken, Japan2Department of Pathophysiology, Hamamatsu University,School of Medicine, Shizuoka-ken, Japan

ABSTRACT: In the present study, the ability of 5-hydroxytryptamine-4(5-HT4) receptors in the hippocampus to enhance locomotor activity inrats was investigated by local infusion via microdialysis probes. The localinfusion of 5-HT bilaterally into the striatum did not alter rat motoractivity. The local infusion of 1.0 mM 5-HT into the bilateral hippocam-pus, but not lower doses, significantly increased motor activity as com-pared with the baseline values or the control rats. During the day hours(0700–1900, light on), the local infusion of either 5-HT4 agonist, 5-MeOT(100 �M) or mosapride (10 �M), but not in their lower concentrations,into the bilateral hippocampus significantly increased motor activity ascompared with the baseline values or the control rats. Almost all in-creased motor activity was normal forward locomotion. This 5-MeOT-induced hyperlocomotion was completely reversed by the combined in-fusion of a 5-HT4 antagonist, either GR125487D (100 �M), SB204070(100 �M) or RS23597-190 (100 �M). During the night hours (1900–0700, light off), the local infusion of either SB204070 (100 �M) orRS23597-190 (100 �M), but not in their lower concentrations, into thebilateral hippocampus significantly decreased rat motor activity and in-hibited rat nocturnal hyperactivity. These hypoactivities during the nighthours induced by 5-HT4 antagonist were reversed by the combined infu-sion of a 5-HT4 agonist, 5-MeOT (100 �M). The present study demon-strates that the serotonergic neurons projecting to the hippocampus, butnot to the striatum, modulate rat locomotor activity by stimulating 5-HT4receptors in the hippocampus. Hippocampus 2002;12:304–310.© 2002 Wiley-Liss, Inc.

KEY WORDS: serotonin; 5-HT4 receptor; hippocampus; striatum; lo-comotor activity; microdialysis

INTRODUCTION

It is well known that the central serotonergic systems play important rolesin the regulation of a wide variety of physiologic, emotional, and behavioralfunctions (Kostwski, 1975; Geyer et al., 1976; Lorens et al., 1976; Hillegaartand Hjorth, 1989). It has been reported that serotonergic systems centrally

modulate various locomotor responses (Grahame-Smith,1971; Geyer et al., 1976; Kennett and Curzon, 1988;Kennett et al., 1997), while the reduced central seroto-nergic activity is closely related to aggressive behaviors(Soubrie, 1989; Lipska et al., 1992). These wide varietiesof serotonin-induced motor function may depend on thesubtypes of serotonergic receptors (5-HT1, 5-HT2,5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7). Regardingthe current study, m-chlorophenylpiperazine (mCPP), anonselective 5-HT2B/2C receptor agonist, is known tocause hypolocomotion in rats (Kennett and Curzon,1988; Bonhaus et al., 1997; Kennett et al., 1997), and ithas also been documented that 8-OH-DPAT, a 5-HT1

receptor agonist, induces 5-HT1 receptor-mediated5-HT behavioral syndrome (Tricklebank et al., 1984;Albinsson et al., 1994; Hajos-Korcsok and Sharp, 1996).Recent studies have shown that the 5-HT4 receptor ishighly expressed not only in the peripheral organs, butalso in many regions of the brain, such as the hippocam-pus, cerebral cortex, striatum, and limbic and basal gan-glia structures (Domenech et al., 1994; Vilaro et al.,1996; Waeber et al., 1996; Compan et al., 1996), and therole of the 5-HT4 receptor has been the subject of con-siderable investigation (Letty et al., 1997; Meneses andHong, 1997a; Panocka et al., 1995; Semenova and Tiku,1997). In particular, pharmacological studies have impli-cated the central 5-HT4 receptor in the control of emo-tional and psychological functions such as cognition andanxiety (Silvestre et al., 1996; Buhot, 1997; Letty et al.,1997). Little is known, however, of the effect of the5-HT4 receptor on locomotor activity (Semenova andTiku, 1997).

Hippocampus is known to be related to the modula-tion of locomotor activity. In previous articles, serotoner-gic neurons in the median raphe nuclei projecting to thehippocampus have been reported to be related to motoractivity (Jacobs et al., 1974; Jacobs and Cohen, 1976;Wirtshafter et al., 1986; Wing et al., 1984; Hillegaart andHjorth, 1989). A clear relationship between behavioralactivity and extracellular 5-HT concentrations in the hip-pocampus has been reported (Linthorst et al., 1994). We

*Correspondence to: Akikazu Takada, Department of Physiology,Hamamatsu University, School of Medicine, 1-20-1 Handa-yama,Hamamatsu-shi, Shizuoka-ken, 431-3192 Japan.E-mail: [email protected] for publication 3 August 2001DOI 10.1002/hipo.10012Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 12:304–310 (2002)

© 2002 WILEY-LISS, INC.

recently reported that local infusion of 5-HT2B/2C receptor agonistmCPP into the hippocampus caused hyperlocomotion in contrastwith the results of systemic administration, which was inhibited by5-HT2C antagonist SB 242084 (Takahashi et al., 2001). In thepresent study, we investigated the ability of the 5-HT4 receptor tomodulate locomotor activity by local microinfusions into the hip-pocampus, as compared with the effects in the striatum or nucleusaccumbens in which serotonergic neurons are terminated.

It is well known that rat behavior is regulated by circadianrhythms. Rats exhibited high activity during the night hours andlow activity during day hours (Takahashi et al., 1998, 2001). Mostrecent studies, however, have been performed during the dayhours, with the rats exhibiting low levels of activity. In the presentstudy, we examined the above effects not only during the dayhours, but also during the night hours, so as to clarify whether thenocturnal hyperlocomotion would be blocked by local infusion ofthe 5-HT4 receptor antagonists.

MATERIALS AND METHODS

Animals

Adult male Wistar rats (200–250 g) were used. They were in-dividually housed for 2 weeks before the experiments in a temper-ature-controlled room (24� 1°C) maintained on a 12:12-h light/dark cycle (0700–1900, light on: day hours, and 1900–0700,light off: night hours). Animals were given free access to food andwater.

Surgical Procedures

In an effort to monitor rat motor activity under free-movingconditions, to avoid volume-induced increases in intracranial pres-sure after the administration of drugs, and to exclude the effects ofinjector insertion on motor activity, we employed a microdialysistechnique for the local infusion.

Three or 5 days before the experiments, guide cannulas werestereotactically implanted bilaterally, with the rats under sodiumpentobarbital anesthetization (50 mg/kg, i.p.) to permit penetra-tion of a microdialysis probe. The coordinates were chosen accord-ing to the stereotactic atlas of Paxinos and Watson (1986). Thecoordinates were as follows. For the striatum, AP, 0.2; ML, 3.0;DV, 3.0 (mm). For the hippocampus, AP, �5.8; ML, 5.0; DV,3.0 (mm). For the nucleus accumbens (NAC), AP, 2.1; ML, 2.5;DV, 4.5 (mm); angled at 5° perpendicularly. The microdialysisprobes were implanted 4 mm deeper than the tips of the guidecannulas. For the intraventricular infusion, the coordinate of theguide cannula was as follows: AP, �1.4; ML, 2.0; and DV, 3.0.The other 5-HT4 receptor areas, including olfactory tubercle andhypothalamus, were too small to infuse drugs locally into the dis-crete structures. The study animals displayed no obvious signsof bleeding or infection around the wound, nor of postsurgicaldistress.

Microdialysis Procedures

Microdialysis probes were constructed according to the meth-ods of Nakahara et al. (1993), with an outer diameter of 220 �mand an exposed tip of 4.0 mm. These probes had an active dialyzinglength of 4 mm and a membrane with a cutoff value of molecularweight 50,000. Microdialysis probes were connected to a specialswivel (Eicom, Tokyo, Japan) via spiral micro silicone tube. Usingthese flexible tubes, rats were allowed to move freely. This swivelwas connected to the pump via micro silicone tube (inlet line). Thelactate Ringer solution used as the perfusate contained 0.1 MNaCl, 1.8 mM CaCl2, 4.0 mM KCl, and 0.03 M sodium lactate.The microdialysis tube was perfused continuously with Ringersolution at a rate of 2.0 �l/min, using a microinfusion pump, andthe experiments were started 3 h after probe insertion to allow forbasal-level stabilization.

Counts of Locomotor Activity

Spontaneous motor activities were measured by Supermex (Mu-romachi Kikai, Tokyo, Japan). The Supermex consists of a sensormonitor mounted above the cage to detect changes in heat acrossmultiple zones of the cage through an array of Fresnel lenses. In thisway, the system could monitor and count all spontaneous move-ments, both vertical and horizontal, including locomotion andhead movements (Takahashi et al., 1998). All counts were auto-matically added and recorded at 30-min intervals.

Materials

5-Hydroxytryptamine hydrochloride (5-HT) and 5-me-thoxytryptamine (5-MeOT), a 5-HT4 receptor agonist (Bockaertet al., 1992; Ge and Barnes, 1996; Ford and Clarke, 1993), werepurchased from Sigma Chemical Company (St Louis, MO).FR173240 (SB204070), a selective 5-HT4 antagonist (Wardle etal., 1994), was kindly presented by Fujisawa (Tokyo, Japan).GR125487D, a selective 5-HT4 antagonist (Gale et al., 1994), waskindly furnished by Dr. G.A. Kennett (SmithKline Beecham Phar-maceuticals: Harlow, UK). RS23597-190, a selective 5-HT4 an-tagonist (Eglen et al., 1993; Bonhaus et al., 1994), was purchasedfrom Tocris (Bristol, UK). Mosapride citrate, a selective 5-HT4

receptor agonist (Yoshikawa et al., 1998), was kindly presented byDainihon Pharmaceuticals Co. (Tokyo, Japan). The pharmacolog-ical specificity of these agents to the 5-HT4 receptor has been welldocumented (Bockaert et al., 1992; Bonhaus et al., 1994; Gale etal., 1994; Wardle et al., 1994; Yoshikawa et al., 1998).

Experimental Procedure

1. Effects of 5-HT4 agonist: 5-HT, 5-MeOT or mosapride citratewere dissolved in lactated Ringer solution adjusted to 1.0 �M,10�M, 100 �M, or 1.0 mM for local infusion. The pH was adjustedto 7.2–7.4 with sodium hydroxide. These drugs were infused for0.5 h during the day hours. When any doses proved ineffective, 10mM of the drugs was infused. Ringer solution was perfused con-tinuously throughout the experiments in the control group.2. Effects of 5-HT4 antagonist during the day hours: FR173240(SB204070), GR125487D, or RS23597-190 were infused in com-

_______________ 5-HT4 RECEPTORS IN HIPPOCAMPUS MODULATE RAT LOCOMOTOR ACTIVITY 305

bination with 5-MeOT for 0.5 h. In the control group, theseantagonist alone infused for 0.5 h.3. Effects of 5-HT4 agonist during the night hours: FR173240.(SB204070) or RS23597-190 was infused for 2 h during the nighthours, and after that, mCPP was infused in combination with5-MeOT for 1.5 h.

Lactated Ringer solution was perfused continuously throughoutthe experiments except during the drug infusion.

Histology

After the termination of each experiment, animals were anesthe-tized with pentobarbital and decapitated; brains were removed andstored in buffered formalin. The probe placement was verifiedmicroscopically, and data were discarded if any part of the dialysisprobe was not in the regions intended. (Three animals were dis-carded, because the dialysis probes were placed in the unwantedregions of the temporal or occipital cortex.)

Statistical Analysis

A statistical analysis of the results was performed by one- ortwo-way analysis of variance (ANOVA) with repeated measures,followed by a multicomparison test using Fisher’s protected leastsignificant difference (PLSD). The comparison was made in rela-tion to the basal values, which were defined as the average of the lastthree samples before drug infusion. P-values of �0.05 were con-sidered statistically significant.

Ethics

The study was submitted to the Ethical Committee of our Uni-versity. The committee judged the experimental designs to “theguiding principles for the care and use of animals in the field ofphysiological sciences” recommended by the Physiological Societyof Japan on December 19, 1988. The guidelines were prepared tomeet the requirements contained in the publications such as U.K.Animals Act, 1986, DHEW Publication No. (NIH) 85-23. 1985,and International Guiding Principle for Biomedical Research In-volving Animals, CIOM, 1983. The committee approved the ex-periments after careful examination.

RESULTS

Effects of Local Infusion of 5-HT into theVarious Serotonergic Terminal Areas

The local 30-min infusions of 1.0 mM 5-HT bilaterally into thestriatum (n � 6) did not alter the motor activity (Fig. 1A) at anydoses, but these infusions bilaterally into the hippocampus (n� 6)significantly increased motor activity when compared with thebaseline values (P � 0.05) or the control rats (F(1,11) � 41.27,P � 0.0001, Fig. 1B). This hyperactivity continued for a subse-quent 90 min after the infusions. During these hyperactive states,no increases in grooming, tremor, rearing, stereotyped behavior,

head shaking, or other involuntary movements were found bycontinuous observation. All growth in the motor activities con-sisted of an increase in normal forward locomotion. The lowerconcentration (0.01 mM, n� 6 or 0.1 mM, n� 6) of 5-HT didnot cause any changes when infused into the hippocampus. Thelocal infusions of 5-HT bilaterally into the nucleus accumbens(NAC) caused no significant changes in motor activity at any doses(Fig. 1C). Intraventricular infusion of either 1.0 mM or 0.1 mM,but not 0.01 mM, 5-HT significantly increased the motor activity,when compared with the baseline values (P� 0.005) or the controlrats (1.0 mM: F(1,10) � 16.68, P � 0.005; 0.1 mM: F(1, 8) �10.44, P� 0.05; see Fig. 4D). These increases in rat motor activityinduced by intraventricular infusion of 1.0 mM 5-HT were, how-ever, significantly lower than those induced by intrahippocampalinfusion (F(1, 10)� 9.73, P � 0.05).

Effects of Local Infusion of 5-HT4 Agonist orAntagonists into the Hippocampus During theDay Hours

During the day hours, the local infusion of 100 �M 5-MeOT(n� 6), but not lower concentrations (10 �M, n� 5), bilaterallyinto the hippocampus significantly increased rat motor activitywhen compared with the baseline values (P� 0.05) or the controlgroup (F(1, 10)� 6.86, P� 0.05; Fig. 2A). These increases were

FIGURE 1. Effects of local infusion of 1.0 mM 5-HT (bold lineswith open circles), 0.1 mM 5-HT (fine line with closed circles), orRinger solution (control; dotted lines with closed circles) into thestriatum (A), hippocampus (B), Nucleus accumbens (NAC; C), orlateral ventricle (D) on the motor activity of the rats (for lower con-centration of 5-HT infused into the striatum or NAC data not shown).Horizontal bars, term of 5-HT infusion. Values expressed as mean�SE. *P < 0.05, **P < 0.005 compared with the baseline value.

306 TAKAHASHI ET AL.

not observed in the control group. The local infusion of 100 �MGR125487D into the bil. hippocampus attenuated and reversedthe 5-MeOT-induced hyperactivity (n � 6, Fig. 3A). These in-creases in motor activity induced by 5-MeOT were also reversedcompletely by the combined infusion of either 100�M SB204070(n� 6, Fig. 3B) or 100 �M RS23597-190 (n� 6, Fig. 3C) intothe bilateral hippocampus. Local infusion of either 100 �M

GR125487D, 100 �M SB204070 or 100 �M RS23597-190alone did not cause any changes in rats motor activity (Fig.3A,B,C). The local infusion of mosapride (10�M, n� 6), but notlower concentrations (1.0 �M, n � 5), bilaterally into the hip-pocampus significantly increased the motor activity when com-pared with the baseline values (P � 0.005) or the control group(F(1, 9) � 45.3, P � 0.0005; Fig. 2B). During these hyperactivestates, no increases in grooming, tremor, rearing, stereotyped be-havior, head shaking, or other involuntary movements were foundby continuous observation. Almost all growth in the motor activ-ities was the increase in normal forward locomotion.

Effects of Local Infusion of a 5-HT4 Antagonistinto the Hippocampus During the Night Hours

During the night hours, the rats exhibited a higher level ofactivity than during the day hours, and the motor activity changedover a wide range (900–4,000/30 min). The drugs were thereforelocally infused for a longer period of time so as to clarify the effectsof the 5-HT4 receptor antagonist on locomotion. The local infu-sion of 100 �M SB204070 into the bilateral hippocampus signif-icantly attenuated the rat motor activity when compared with thebasal values (P � 0.0005) or the control group (F(1, 8) � 15.7,P � 0.005, Fig. 4A). The rats exhibited again a higher level ofactivity after combined infusion of the 5-HT4 agonist, 5-MeOT(100 �M). The local infusion of 100 �M RS23597-190 into thebil. hippocampus (n � 6) significantly attenuated the rat motoractivity when compared with the basal values (P� 0.0005) or thecontrol group (F(1, 9) � 19.88, P � 0.005, Fig. 4B). The com-bined infusion of 5-MeOT into the bil. hippocampus again in-creased the rat motor activity. The lower concentration of eitherSB204070 (10 �M) or RS23597-190 (10 �M) did not cause anychanges in motor activity when infused into the hippocampus.

FIGURE 2. Effects of local infusion of 5-HT4 agonist into thehippocampus on the motor activity. A: Time course of motor activitylocally infused with 100�M 5-MeOT (bold line with open circles), 10�M 5-MeOT (fine line with closed circles), or Ringer solution (con-trol; dotted line with closed circles). B: Time course of motor activitylocally infused with 10 �M mosapride (bold line with open circles),1.0 �M mosapride (fine line with closed circles), or Ringer solution(control; dotted line with closed circles). Horizontal bars, term of5-MeOT or mosapride infusion. Values expressed as mean�SE. *P <0.05, **P < 0.005 compared with the control group.

FIGURE 3. Effects of combined infusion of 5-MeOT (100 �M)and 5-HT4 antagonist, GR125487 (100�M: A), SB204070 (100�M:B), or RS23597-190 (100�M: C) into the hippocampus on the motoractivity of the rats during the daytime. Black lines, changes in motoractivity of the experimental groups; dotted lines, those in the controlgroups. Horizontal black bar, term of 5-MeOT infusion in the exper-imental groups; gray bars, term of GR125487 (A), SB204070 (B), orRS23597-190 (C). Values are expressed as mean �SE.

FIGURE 4. Effects of combined infusion of 5-HT4 antagonist,SB204070 (A) or RS23597-190 (B) and 5-MeOT (100 �M) into thehippocampus on the motor activity of the rats during the night hours.Solid lines, changes of the experimental group injected with 100 �MSB204070 (A) or 100 �M RS23597-190 (B); dotted line, those of thecontrol group. Horizontal black bar, term of 5-MeOT infusion; dot-ted bar, term of 100�M SB204070 (A) or 100�M RS23597-190(B).Values expressed as mean �SE. *P < 0.05, **P < 0.0001 comparedwith the baseline value.


DISCUSSION

It is known 5-HT4 receptors have central cognition-enhancingeffects (Buhot, 1997; Letty et al., 1997), as related to the consoli-dation of learning (Meneses and Hong, 1997b), or the associationof memory (Marchetti et al., 1997; Letty et al., 1997); little isknown, however, of the effects of modulating locomotor activity(Semenova and Tiku, 1997; Reavill et al., 1998). It has been wellestablished that 5-HT4 receptors are located in various areas of thebrain such as the striatum, basal ganglia, nucleus accumbens, hip-pocampus, frontal cortex, or olfactory tubercle (Vilaro et al., 1996;Ullmer et al., 1996; Domenech et al., 1994).

Most up-to-date reports, however, utilized the systemic ratherthan local injections of substances related to 5-HT4 receptors, whatcan introduce much bias and can hardly give a closer look into therelationship between 5-HT4 receptors in discrete brain areas andlocomotor functions. To overcome these limitations, in the presentstudy, we locally infused substances into the bilateral striatum,nucleus accumbens and hippocampus into which serotonergicneurons were projected (Compan et al., 1996). The local infusionof 5-HT into the bilateral striatum had no effect on the motoractivity of the rats. This finding is in excellent agreement with ourprevious report, in which we found no relationship between motoractivity and extracellular 5-HT levels in the striatum either duringthe day hours or night hours (Takahashi et al., 1998). It is wellknown that the striatum is the main terminal of the nigrostriataldopaminergic pathway (Steward et al., 1996), which has somebehavioral functions such as amphetamine; morphine, or nicotine-induced hyperactivity (Reavill et al., 1998). Furthermore, 5-HT4

receptors are known to be located in dopamine-rich areas of thestriatum (Reavill et al., 1998) and to play a part in increases in therelease of striatal dopamine (Steward et al., 1996; Bonhomme etal., 1995; De Deurwaerdre et al., 1997). However, the selective5-HT4 antagonist, SB-204070A had no effect on amphetamine,morphine; or nicotine-induced hyperactivity (Reavill et al., 1998),and it has been reported that morphine-induced hyperactivity isnot mediated by serotonergic systems (Klitenick and Wirtshafter,1995). These previous reports and the present results exclude thepossibility that the serotonergic neurons projecting to the striatummodulate rat locomotor activity.

By contrast, in the present study, the local infusion of 1 mM5-HT into the hippocampus caused a significant increase in ratmotor activity. Almost growth in the motor activities in this part ofthe present study consisted of increased normal forward locomo-tion. These results are thus in good agreement with previous re-ports that serotonergic neurons projecting to the hippocampus areinvolved in increasing locomotor activity (Jacobs et al., 1974; Ja-cobs and Cohen, 1976; Wing et al., 1984; Wirtshafter et al., 1986;Hillegaart and Hjorth, 1989). The local infusion of 5-HT into thenucleus accumbens produced no significant changes in motor ac-tivity. The other areas in which 5-HT4 receptors were located, suchas the olfactory tubercle, were too small to study the effects by localinfusion. It is not clear, based on the findings of this study, that thehippocampus really is the site of action of 5-HT rather than nearby

structures by diffusion across the dialysis membrane. The occipitaland temporal cortex are located in the lateral side of the caudalCA1 region. These areas are not known as a motor or supplemen-tary motor cortex. The subarachnoid space is located in the medialside of the CA1 region. When drugs are diffused to the subarach-noid space or lateral ventricle, drugs may reach several brain motorareas via cerebrospinal fluid. In the present study, rat motor activitywas significantly increased when 5-HT was infused to the lateralventricle. These increases in rats motor activity were, however,significantly lower than those induced by the local infusion of thesame dose 5-HT to the hippocampus. These results indicate thathippocampus is the site of action of 5-HT to activate rat locomo-tion. Furthermore, if the drugs (5-HT) were diffused to somemotor areas, rat locomotion should be increased with a time delayby local infusion to the striatum or nucleus accumbens. In thepresent results, however, local infusion of 5-HT into the striatumor nucleus accumbens did not cause any changes. Therefore, wefocused on the hippocampus to elucidate the effect of the 5-HT4

receptor on locomotor activity. The local infusion of 100 �M5-MeOT into the hippocampus significantly increased rat motoractivity. Almost all the increased activity induced by 5-MeOTinvolved forward locomotion and was different from those of5-HT1 receptor-mediated 5-HT syndrome, in which a flat bodyposture, hindlimb abduction, head weaving, and forepaw treadingare generally observed (Grahame-Smith, 1971; Tricklebank et al.,1984; Albinsson et al., 1994; Hajos-Korcsok and Sharp, 1996).We used 5-MeOT as the 5-HT4 receptor agonist because it hasbeen used with success in previous reports (Ge and Barnes, 1996).5-MeOT, however, is known to have efficacy in a number of other5-HT receptor subtypes that may influence the release of 5-HT(Bockaert et al., 1992) or induce hyperlocomotion. However, this5-MeOT-induced hyperlocomotion was completely reversed byintrahippocampal infusion of a highly potent and selective 5-HT4

receptor antagonist, either SB204070, GR125487D, or RS23597-190. These three drugs are suitable for 5-HT4 antagonists in thepresent study. Because GR125487D was reported previously toinhibit the effects of 5-MeOT in the hippocampus (Ge and Barnes,1996), SB204070 is the most potent and selective 5-HT4 receptorantagonist (5,000-fold selective over all other sites, Wardle et al.,1994), and RS23597-190 is known to have higher affinity for5-HT4 receptor than D2 or 5-HT3 receptor.

These results suggest that the 5-MeOT-induced hyperlocomo-tion is caused by 5-HT4 receptor-mediated responses in the hip-pocampus. Furthermore, 10 �M mosapride, a selective 5-HT4

receptor agonist (Yoshikawa et al., 1998), significantly increasedthe rat locomotor activity during and after the local infusion to thehippocampus. Mosapride is one of the most potent and selective5-HT4 receptor agonist same as BIMU-8 or Cisapride. BIMU-8was characterized, however, by higher affinity for the 5-HT3 re-ceptor. Cisapride, but not mosapride, also has high affinity for the5-HT2 or D2 receptor (Yoshikawa et al., 1998). Therefore, in thepresent study, mosapride was used for the selective 5-HT4 receptoragonist.

These results suggest that 5-HT4 receptors in the hippocampusmodulate rat locomotor activity.


It is well known that rats exhibit high levels of activity during thenight hours (Takahashi et al., 1998). Almost all previous behav-ioral experiments, however, have been carried out during the dayhours, when rats exhibit low levels of activity. In the present study,the night hours, local infusion of either SB204070 (100 �M), orRS23597-190 (100 �M) into the hippocampus significantly at-tenuated rat motor activity and completely inhibited nocturnalhyperactivity, which was reversed by the combined infusion of 100�M 5-MeOT. These results verify the hypothesis that 5-HT4 re-ceptors in the hippocampus play a key role in modulating locomo-tor activity and also suggest that 5-HT4 receptors in the hippocam-pus are involved in rat nocturnal hyperactivity.

Mechanisms of hyperactivity mediated by hippocampal neu-rons remain to be elucidated. In the present study, microdialysisprobes were inserted into the caudal side of the hippocampus,which contained mainly a dorsal part of hippocampus (Moser etal., 1993). In previous reports, the dorsal but not the ventral half ofthe hippocampus is involved in spatial learning. There is a func-tional dissociation between the dorsal and the ventral hippocam-pus, which may be related to different connection. Little is known,however, to relate locomotor function in either the dorsal or theventral hippocampus. The CA1 field is known to project to anumber of cortical regions. Therefore, some neural networks,whether direct or indirect, between the hippocampus and motor orsupplementary motor cortex, may be involved in the regulation ofmotility. In the previous reports, the hippocampal–accumbenspathway was shown to initiate locomotor responses (Yang andMogenson, 1987). These responses were thought to be regulatedby D2 receptors in the nucleus accumbens (NAC). It is known thata neural network from NAC to the pedunculopontine nucleus(PPN), a major component of the mesencephalic locomotor re-gion, activate locomotor activity (Tsai et al., 1989). Furthermore,B7 cells in the dorsal raphe nucleus also innervate to PPN. Themost plausible explanation is, therefore, that the locomotor activitymay be regulated by neural networks between the hippocampusincluding 5-HT4 receptors and some locomotor regions such asPPN or motor cortex.

In the present study, we employed microdialysis probes for localinfusion. We ascertained beforehand that either direct injection tothe brain or local injection without microdialysis probes, evenwhen injected with saline, strongly increased the motor activity ofrats; this increase did not, however, appear to be mediated by5-HT4 receptors and masked 5-HT4 receptor-mediated behavioralresponses. Thus, the use of microdialysis probes appears to be abetter method of analyzing the behavioral responses of rats inducedby local infusion under free-moving conditions.

However, the use of microdialysis probes required the use ofrelatively high concentrations. For example, when 1 mM 5-HTwas infused through the microdialysis probes over 30 min at a rateof 2.0 �l/ min, the recovery or permeation rate of 5-HT was10–and 20%; thus, 5.0 and 10.0 nmol of 5-HT was administered.These amounts could have been lowered still further by washingout after perfusion. Because the administered drugs arrived at theneurons by diffusion, higher concentrations of drugs might benecessary for diffusion from a small microdialysis probe and forcausing increases in locomotor activity.

In summary, during the day hours, the local infusion of the5-HT4 receptor agonists, mosapride or 5-MeOT to the hippocam-pus increases rat locomotor activity, an increase that can be re-versed by either SB204070, GR125487D, or RS23597-190; inaddition, either SB204070 or RS23597-190 locally infused intothe hippocampus attenuates rat nocturnal hyperlocomotion. Theserotonergic neurons projecting to the hippocampus, but not tothe striatum, modulate rat locomotor activity by stimulating5-HT4 receptors in the hippocampus.

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5-ht4 receptors in the hippocampus modulate rat locomotor activity

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