Inhibition of G Protein-Activated Inwardly Rectifying K Channels by Phencyclidine

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244 Current Neuropharmacology, 2011, 9, 244-246 Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Phencyclidine

Toru Kobayashi1,2,*, Daisuke Nishizawa1 and Kazutaka Ikeda1

1Division of Psychobiology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan; 2Department of Project Programs, Center for Bioresource-based Researches, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata, Niigata 951-8585, Japan

Abstract: Addictive drugs, such as opioids, ethanol, cocaine, amphetamine, and phencyclidine (PCP), affect many functions of the nervous system and peripheral organs, resulting in severe health problems. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability through activation of various Gi/o protein-coupled receptors including opioid and CB1 cannabinoid receptors. Furthermore, the channels are directly activated by ethanol and inhibited by cocaine at toxic levels, but not affected by methylphenidate, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) at toxic levels. The primary pharmacological action of PCP is blockade of N-methyl-D-aspartate (NMDA) receptor channels that are associated with its psychotomimetic effects. PCP also interacts with several receptors and channels at relatively high concentrations. However, the molecular mechanisms underlying the various effects of PCP remain to be clarified. Here, we investigated the effects of PCP on GIRK channels using the Xenopus oocyte expression system. PCP weakly but significantly inhibited GIRK channels at micromolar concentrations, but not Kir1.1 and Kir2.1 channels. The PCP concentrations effective in inhibiting GIRK channels overlap clinically relevant brain concentrations in severe intoxication. The results suggest that partial inhibition of GIRK channels by PCP may contribute to some of the toxic effects after overdose. Keywords: Phencyclidine, GIRK channel, intoxication, Kir channel, Xenopus oocyte.

INTRODUCTION Phencyclidine (PCP) has been used as a drug of abuse, although it was originally developed as a general anesthetic G protein-activated inwardly rectifying K+ (GIRK) chan- in the 1950s [13, 14]. The primary pharmacological action of nels (also known as Kir3 channels) are members of a family PCP is blockade of N-methyl-D-aspartate (NMDA) receptor of inwardly rectifying K+ (Kir) channels that includes seven channels that are associated with its psychotomimetic effects subfamilies [1]. Four GIRK channel subunits have been [13, 14]. PCP at relatively high concentrations interacts with identified in mammals [1]. Neuronal GIRK channels are several receptors and channels, namely, , μ-opioid, nico- predominantly heteromultimers composed of GIRK1 and tinic- and muscarinic-acetylcholine receptors, voltage-gated GIRK2 subunits in most brain regions or homomultimers K+, Na+ and Ca2+ channels, and adenosine triphosphate composed of GIRK2 subunits in the substantia nigra, (ATP)-sensitive K+ channels [14, 15, 16]. However, the mo- whereas atrial GIRK channels are heteromultimers com- lecular mechanisms underlying the various effects of PCP posed of GIRK1 and GIRK4 subunits [2]. GIRK channels have not yet been sufficiently clarified. In the present study, play an important role in the inhibitory regulation of neu- we investigated the effects of PCP on GIRK channels ronal excitability in most brain regions and heart rate through and other Kir channels using the Xenopus oocyte expression activation of various G protein-coupled receptors, such as i/o system. opioid, CB1 cannabinoid, and D2 dopamine receptors [2]. Furthermore, the channels are modulated by various psy- METHODS choactive agents, such as ethanol, antipsychotics, antidepres- sants, anesthetics, and hormones [2-11]. Recently, we dem- For Xenopus oocyte experiments [4, 5], Xenopus laevis onstrated that cocaine at toxic levels inhibited GIRK chan- oocytes were injected with mRNA for GIRK1/GIRK2 or nels expressed in Xenopus oocytes. In contrast, methylpheni- GIRK1/GIRK4 combinations, GIRK2, Kir1.1, or Kir2.1. The date, methamphetamine, and 3,4-methylenedioxymetham- oocytes were incubated at 19°C in Barth's solution and defol- phetamine (MDMA) at toxic levels had little effect on GIRK liculated after collagenase treatment. Whole-cell currents of channels, although these drugs at higher concentrations in- the oocytes were recorded with a conventional two-electrode hibited the channels to a lesser extent than cocaine [12]. voltage clamp. Oocytes were superfused with a high- potassium solution containing 96 mM K+. The membrane potential was held at 70 mV. The values obtained are *Address correspondence to this author at the Department of Project Pro- expressed as mean ± SEM, with n indicating the number of grams, Center for Bioresource-based Researches, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata, Niigata 951-8585, oocytes tested. PCP was generously provided by Shionogi Japan; Tel: +81-25-227-0646; Fax: +81-25-227-0818; Pharmaceutical Co. Ltd. (Osaka, Japan). E-mail: [email protected]

RESULTS potent than those of cocaine, methylphenidate, methamphetamine, and MDMA (Fig. (2)). In other Kir channel sub- In Xenopus oocytes injected with GIRK1 and GIRK2 families, Kir1.1 and Kir2.1 channels were insensitive to mRNA, PCP reversibly reduced basal GIRK inward currents these psychostimulants [12] and PCP, whereas PCP inhibited (Fig. (1A)). Similar results were observed in oocytes injected + cardiac ATP-sensitive K channels, which comprise four with either GIRK1 and GIRK4 mRNA or GIRK2 mRNA. pore-forming Kir6 subunits and four regulatory sulfonylurea However, in oocytes expressing either Kir1.1 or Kir2.1 receptor subunits, with an IC value of approximately 20 channels, PCP caused no significant response even at 100 50 μM [16]. Further studies using GIRK/Kir1.1 and GIRK/ M (less than 5 nA, n = 4, Fig. (1A)). Additionally, in unin- μ Kir2.1 chimeric channels and mutant GIRK channels jected oocytes, 100 M PCP and 3 mM Ba2+, a Kir channel μ may clarify the critical sites mediating the effects of PCP on blocker, caused no significant response (Fig. (1A)). The results GIRK channels. suggest that PCP inhibits GIRK channels. The inhibition by PCP was concentration-dependent (n = 4, Fig. (1B)). The use of PCP as a drug of abuse is an important medi- cal problem. Serum PCP concentrations after overdose were DISCUSSION reported to reach up to approximately 10 μM in some post- We demonstrated that PCP at micromolar concentrations mortem cases, up to 45 μM in one massive overdose case inhibited brain-type GIRK1/2 and GIRK2 channels and [17], and up to approximately 3.3 μM in some nonfatal cases atrial-type GIRK1/4 channels expressed in Xenopus oocytes. [18], although serum concentrations in most intoxication At 100 μM or less, the inhibitory effects of PCP were more cases ranged widely from nanomolar to low micromolar con-

Fig. (1). Inhibitory effects of PCP on GIRK channels expressed in Xenopus oocytes. (A) Top, in an oocyte injected with GIRK1 and GIRK2 mRNA, current responses to 10 μM and 100 μM PCP and to 3 mM Ba2+, a Kir channel blocker. Middle, in an oocyte injected with Kir1.1 mRNA, current responses to 100 μM PCP and to 3 mM Ba2+. Bottom, in an uninjected oocyte, no significant current responses to 100 μM PCP or 3 mM Ba2+. Asterisks show the zero current level. Bars show the duration of application. (B) Concentration-dependent inhibition of GIRK channels by PCP. The magnitudes of inhibition of GIRK currents by PCP were compared with the current components sensitive to 3 mM Ba2+. 246 Current Neuropharmacology, 2011, Vol. 9, No. 1 Kobayashi et al. concentrations [17, 18]. Furthermore, the concentrations in [2] Kobayashi, T.; Ikeda, K. G protein-activated inwardly rectifying the brain were reported to be from 3 to 14-fold higher than potassium channels as potential therapeutic targets. Curr. Pharm. Des., 2006, 12, 4513-4523. those in serum [19]. Additionally, because the early phase [3] Kobayashi, T.; Ikeda, K.; Kojima, H.; Niki, H.; Yano, R.; Yoshi- elimination rate after administration of PCP is relatively high oka, T.; Kumanishi, T. Ethanol opens G-protein-activated inwardly [19], the peak concentrations would be higher than the con- rectifying K+ channels. Nat. Neurosci., 1999, 2, 1091-1097. centrations measured. Therefore, the PCP concentrations [4] Kobayashi, T.; Ikeda, K.; Kumanishi, T. Inhibition by various antipsychotic drugs of the G-protein-activated inwardly rectifying effective in inhibiting GIRK channels overlap the clinically + K (GIRK) channels expressed in Xenopus oocytes. Br. J. Pharma- relevant brain concentrations in severe intoxication or fatal col., 2000, 129, 1716-1722. cases. [5] Kobayashi, T.; Washiyama, K.; Ikeda, K. Inhibition of G protein- activated inwardly rectifying K+ channels by fluoxetine (Prozac). Br. J. Pharmacol., 2003, 138, 1119-1128. [6] Kobayashi, T.; Washiyama, K.; Ikeda, K. Inhibition of G protein- activated inwardly rectifying K+ channels by various antidepressant drugs. Neuropsychopharmacology, 2004, 29, 1841-1851. [7] Kobayashi, T.; Washiyama, K.; Ikeda, K. Inhibition of G protein- + activated inwardly rectifying K channels by the antidepressant paroxetine. J. Pharmacol. Sci., 2006, 102, 278-287. [8] Yamakura, T.; Lewohl, J.M.; Harris, R.A. Differential effects of general anesthetics on G protein-coupled inwardly rectifying and other potassium channels. Anesthesiology, 2001, 95, 144-153. [9] Zhou, W.; Arrabit, C.; Choe, S.; Slesinger, P.A. Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. USA, 2001, 98, 6482- 6487. [10] Kelly, M.J.; Ronnekleiv, O.K.; Ibrahim, N.; Langrange, A.H.; Wager, E.J. Estrogen modulation of K+ channel activity in hypotha- Fig. (2). Comparison of the effects of five addictive drugs: PCP, lamic neurons involved in the control of the reproductive axis. cocaine, methylphenidate (MPH), methamphetamine (MAP) and Steroids, 2002, 67, 447-456. [11] Evaul, K.; Jamnongjit, M.; Bhagavath, B.; Hammes, S.R. Testos- MDMA, on GIRK channels. Drug concentration was 100 M. The μ terone and progesterone rapidly attenuate plasma membrane G- magnitudes of inhibition of GIRK currents by the drugs were com- mediated signaling in Xenopus laevis oocytes by signaling through pared with the 3 mM Ba2+-sensitive current components. Data ex- classical steroid receptors. Mol. Endocrinol., 2007, 21, 186-196. cept for PCP are from our previous study [12]. [12] Kobayashi, T.; Nishizawa, D.; Iwamura, T.; Ikeda, K. Inhibition by cocaine of G protein-activated inwardly rectifying K+ channels ex- PCP concentrations in severe intoxication cases are asso- pressed in Xenopus oocytes. Toxicol. In Vitro, 2007, 21, 656-664. [13] O'Brien, C.P. Drug addiction and drug abuse. In: Goodman & ciated with coma, seizures, muscle rigidity, and respiratory Gilman's The Pharmacological Basis of Therapeutics, 10th ed.; arrest [20]. GIRK2 knockout mice show spontaneous sei- Hardman, J.G., Limbird, L.E., Gilman, A.G., Eds.; McGraw-Hill: zures and are more susceptible to seizures induced by penty- New York, 2001, pp. 621-642. lenetetrazol than wild type mice [21]. Furthermore, GIRK [14] Javitt, D.C.; Zukin, S.R. Recent advances in the phencyclidine inhibitors can depolarize the membrane potential and induce model of schizophrenia. Am. J. Psychiatry, 1991, 148, 1301-1308. [15] Ffrench-Mullen, J.M.H.; Rogawski, M.A. Phencyclidine block of action potentials [22]. Because GIRK channels play an im- calcium current in isolated guinea-pig hippocampal neurones. J. portant role in regulating cell excitability, even partial inhibi- Physiol., 1992, 456, 85-105. tion of neuronal GIRK channels by PCP might contribute to [16] Kokoz, Y.M.; Alekseev, A.E.; Povzun, A.A.; Korystova, A.F.; the incidence of seizures and some of neuropsychiatric com- Peres-Saad, H. Anesthetic phencyclidine, blocker of the ATP- sensitive potassium channels. FEBS Lett., 1994, 337, 277-280. plications observed in severe or fatal cases after overdose. [17] Budd, R.D.; Liu, Y. Phencyclidine concentrations in postmortem GIRK channels may be considered as important molecules body fluids and tissues. J. Toxicol. Clin. Toxicol., 1982, 19, 843-850. mediating the effects of PCP, cocaine, opioids, cannabinoids, [18] Bailey, D.N. Phencyclidine abuse. Clinical findings and concentra- and ethanol among addictive drugs. tions in biological fluids after nonfatal intoxication. Am. J. Clin. Pathol., 1979, 72, 795-799. ACKNOWLEDGEMENTS [19] Proksch, J.W.; Gentry, W.B.; Owens, S.M. The effect of rate of drug administration on the extent and time course of phencyclidine We thank Dr. Kansaku Baba for his cooperation. We also distribution in rat brain, testis, and serum. Drug Metab. Dispos., thank Dr. Steven C. Hebert (Yale University) and Dr. Lily Y. 2000, 28, 742-747. Jan (University of California, San Francisco) for generously [20] Liden, C.B.; Lovejoy, F.H.Jr.; Costello, C.E. Phencyclidine. Nine cases of poisoning. JAMA, 1975, 234, 513-516. providing Kir1.1 cDNA and Kir2.1 cDNA, respectively. This [21] Signorini, S.; Liao, Y.J.; Duncan, S.A.; Jan, L.Y.; Stoffel, M. Nor- work was supported by research grants from the Ministry of mal cerebellar development but susceptibility to seizures in mice Education, Culture, Sports, Science and Technology of Japan, lacking G protein-coupled, inwardly rectifying K+ channel GIRK2. and the Ministry of Health, Labour and Welfare of Japan. Proc. Natl. Acad. Sci. USA, 1997, 94, 923-927. [22] Kuzhikandathil, E.V.; Oxford, G.S. 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Received: October 01, 2009 Revised: April 17, 2010 Accepted: May 26, 2010

Current Neuropharmacology, 2011, 9, 247-250 247 Effects of Gastrodia Elata Bl on Phencyclidine-Induced Schizophrenia-Like Psychosis in Mice

E.-J. Shin1, J.-M. Kim1, X.-K. T. Nguyen1, T.-T. L. Nguyen1, S. Y. Lee1, J.-H. Jung1, M. J. Kim2, W. K. Whang3, K. Yamada4, T. Nabeshima5 and H.-C. Kim1,*

1Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea; 2Oriental Bio-Herb Research Institute, Kangwon National University, Chunchon 200-701, South Korea; 3Phamaceutical Resources Botany, College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea; 4Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan; 5Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Meijo University, Nagoya 468-8503, Japan

Abstract: It has been demonstrated that 5-HT1A receptors play an important role in the pathophysiology of schizophrenia. Because Gastrodia elata Bl (GE) modulates the serotonergic system, we examined whether GE could affect phencyclidine (PCP)-induced abnormal behavior in mice. Repeated treatment with PCP increased immobility time, while it decreased social interaction time and recognition memory. PCP-induced abnormal behaviors were significantly attenuated by GE, and these effects were comparable to those of 8-OH-DPAT, a 5-HT1A receptor agonist. Furthermore, GE-mediated effects were counteracted by WAY 100635, a 5-HT1A receptor antagonist. Our results suggest that the antipsychotic effects of GE are, at least in part, mediated via activation of 5-HT1A in mice.

Keywords: Gastrodia elata Bl, phencyclidine, schizophrenia, 5-HT1A receptors.

INTRODUCTION psychiatric diseases, including schizophrenia, and that 5- HT1A receptors might be an important target for emotion and Schizophrenia is a chronic, devastating, and costly men- cognition [4]. tal illness, affecting about 1% of the world population. It develops progressively, and is often undetected during child- Gastrodia elata Blume (GE) is a well-known herbal hood and adolescence in a premorbid phase, leading to the agent that has long been used to treat headache, paralysis, onset of psychosis in early adulthood [1]. migraine, and other neurological disorders in traditional oriental medicine. Major components of GE include Phencyclidine [1-(1-phenylcyclohexyl)piperidine hydro- gastrodin, p-hydroxybenzyl aldehyde, p-hydroxybenzyl chloride (PCP)], a non-competitive N-methyl-D-aspartate alcohol, vanillyl alcohol, and vanillin. Earlier reports indi- (NMDA) antagonist, has been shown to induce schizophrenia- cated that GE has various biological properties, including like psychosis, with positive symptoms, negative symptoms, anti-convulsant, anti-oxidant, cognitive enhancing, anx- and cognitive deficits in humans [2], which persist for iolytic, and anti-depressant effects [5]. Recently, it was dem- several weeks after withdrawal from chronic PCP use [3]. onstrated that GE significantly decreased immobility dura- To understand the pathophysiology of schizophrenia, an tion in a forced-swimming test in rats, primarily by modulat- animal model of schizophrenia was established using PCP ing the serotonergic system [6]. [3]. Nabeshima and colleagues previously demonstrated that Thus, to extend the pharmacological investigation of GE, repeated treatment with PCP induces several behavioral we examined whether GE affected PCP-induced changes in abnormalities, such as increased immobility in a forced immobility, social interaction, and cognitive function in mice. swimming test, social deficits on a social interaction test, We also examined whether the 5-HT1A receptor is involved in impairment of latent learning in a water finding test, and GE-mediated pharmacological actions in response to PCP. associative learning impairment in cue and contextual fear conditional tests in mice [3]. Thus, PCP-treated mice might METHODS be a useful animal model of schizophrenia. All animals were treated in accordance with the NIH Several lines of evidence have suggested that serotonin 5- Guide for the care and use of laboratory animals (NIH Pub- HT1A receptors may play a role in the pathophysiology of lication No. 85-23, 1985; www.dels.nas.edu/ila). This study was performed in accordance with the Institute for Labora- tory Animal Research (ILAR) guidelines for the care and use

*Address correspondence to this author at Neuropsychopharmacology and of laboratory animals. Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea; Tel: +82-33-250-6917; Male C57BL/6J mice or male ICR mice (Bio Genomic Fax: +82-33-255-7865; E-mail: [email protected] Inc., Charles River Technology, Gapyung-Gun, Gyeonggi-

Do, South Korea), weighing 25±3 g, were maintained on a in immobility time [GE (500 mg/kg) + PCP vs. saline + PCP, 12:12 h light:dark cycle and fed ad libitum. Male ICR mice P < 0.05; GE (1000 mg/kg) + PCP vs. saline + PCP, P < 0.01] were only used as the “target” in the social interaction test, (Fig. 2A), sociability deficit [GE (1000 mg/kg) + PCP vs. with no drug treatment. saline + PCP, P < 0.01] (Fig. 2B), and impaired visual recognition memory [GE (1000 mg/kg) + PCP vs. saline + PCP, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2- P < 0.01] (Fig. 2C), in a dose-dependent manner. The effects pyridinyl)cyclohexane carboxamide trihydrochloride (WAY of GE (8-OH-DPAT + PCP vs. saline + PCP, P < 0.05 in all 100635; Sigma-Aldrich, St. Louis, MO, USA), PCP hydro- behaviors) were comparable to those of 8-OH-DPAT (0.05 chloride (Tocris Bioscience, Ellisville, MO, USA), and (+)- mg/kg, i.p.), a 5-HT receptor agonist. Consistently, WAY 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; Sigma- 1A 100635 (0.5 mg/kg, i.p.), a 5-HT receptor antagonist, sig- Aldrich) were dissolved in 0.9% sterile saline. The GE was 1A nificantly inhibited GE-mediated pharmacological actions obtained from Samsung Herb Medicine, Co. (Chunchon, [forced swimming test: WAY 100635 + GE (1000 mg/kg) + South Korea) and was suspended in 0.5% carboxymethylcel- PCP vs. saline + GE (1000 mg/kg) + PCP, P < 0.05; social lulose. All solutions were prepared immediately before use. interaction test and novel object recognition test: WAY Experimental schedules are shown in Fig. (1). 100635 + GE (1000 mg/kg) + PCP vs. saline + GE (1000 The novel object recognition, forced swimming, and so- mg/kg) + PCP, P < 0.01] in response to PCP (Fig. 2). These cial interaction tests were performed as described previously results suggest that GE attenuated PCP-induced changes in [6,7,8]. An automated video-tracking system (Noldus Infor- immobility time, social interaction, and cognitive function mation Technology, Wageningen, The Netherlands) was via modulation of 5-HT1A receptors. used to record and analyze the movements of mice in all Our results are consistent with earlier findings that re- three tests. peated treatment with PCP showed significant increases in Statistical analyses were performed using one-way analy- immobility time and significant decreases in social interac- sis of variance (ANOVA) or repeated measure one-way tion and recognition memory in mice [3]. Prolonged expo- ANOVA. A post-hoc Fisher’s PLSD test was then applied. A sure to GE significantly blocked PCP-induced behavioral P value < 0.05 was deemed to indicate statistical significance. effects, in a dose-related manner. The protective effects of GE in response to PCP were about equipotent to those of the RESULTS AND DISCUSSION 5-HT1A receptor agonist 8-OH-DPAT (0.05 mg/kg, i.p.). Repeated treatment with PCP resulted in significant Furthermore, the 5-HT1A receptor antagonist WAY 100635 increases (P < 0.01) in immobility time in the forced swim- (WAY; 0.5 mg/kg, i.p.) significantly counteracted GE- ming test (Fig. 2A), while PCP resulted in significant mediated pharmacological effects in response to PCP. Thus, decreases (P < 0.01) in the interaction time in the social we believe that GE-mediated activation of 5-HT1A receptors interaction test (Fig. 2B) and exploration rate for a novel may be important in antipsychotic effects in response to object (P < 0.01) in the novel object recognition test (Fig. 2C). PCP, although this remains to be explored further in other GE treatment significantly attenuated PCP-induced increase GE-mediated neuropharmacological activities.

GE (500 or 1000 mg/kg/day, p.o. x 26) or 8OH (0.05 mg/kg/day, i.p. x 26)

PCP WAY (10 mg/kg/day, s.c. x 14) (0.5 mg/kg/day, i.p. x 7)

Day 1 5 18 19 24 25 26

Training Test

Novel object recognition test

Forced swimming test

Social interaction test Fig. (1). Experimental schedules. PCP = phencyclidine, GE = Gastrodia elata Blume, 8OH = 8-OH-DPAT, WAY = WAY 100635. 8-OH- DPAT was used as a reference drug. Mice were treated with PCP (10 mg/kg/day, s.c.) for 14 consecutive days. After a 7- or 8-day withdrawal period, the novel object recognition, forced swimming, and social interaction tests were performed using independent sets of mice; each set of mice was used for one of the three behavioral tests. Treatment with GE (500 or 1000 mg/kg/day, p.o.) or 8-OH-DPAT (0.05 mg/kg/day, i.p.) was started from 4 days before the first PCP injection, and continued throughout the experimental period. WAY 100635 (0.5 mg/kg/day, i.p.) was administered during the PCP withdrawal period. GE was injected 1 h prior to PCP or WAY 100635, and WAY 100635 was injected 30 min prior to the behavior test. Gastrodia Elata Bl and Phencyclidine Current Neuropharmacology, 2011, Vol. 9, No. 1 249

depression. Recent reports have indicated that potential antipsychotic effects on PCP require combined modulation of

5-HT1A and dopamine receptors [8]. Interestingly, the GE- A. Forced swimming test mediated anti-depressant effects are exerted, at least in part, 80 * by dopaminergic modulation in the rat brain [6]; however, & the interaction between 5-HT1A and specific dopamine recep- 60 # # ## tors remains to be determined. 40 Similar to GE, various 5HT1A receptor agonists, such as buspirone, 8-OH-DPAT, and ipsapirione, have been shown 20 (sec) duration bility to enhance social interaction [13]. Furthermore, various pre- o clinical data strengthen the notion that targeting the 5HT1A 0 Imm Sal WAY Sal Sal WAY Sal WAY Sal Sal Sal WAY receptor system should result in beneficial effects on dys- Sal 8OH GE Sal 8OH GE GE functional social behavior, possibly not only in schizo- (1000) (500) (1000) phrenic patients but also in the population suffering from Sal PCP social withdrawal of other etiologies.

B. Social interaction test Hagiwara et al. [9] demonstrated that the hippocampal 100 density of 5-HT1A receptor is much higher than the frontal ## (%) # cortical density of 5-HT receptor in mice, and that repeated 80 treatment with PCP did not significantly alter the frontal * && 60 cortical density of 5-HT, but did change the hippocampal density of 5-HT receptors, and that perospirone, a 5-HT 40 1A receptor agonist, ameliorated PCP-induced cognitive deficits, Time spent in 20 as measured by a novel object recognition test. Thus, the interaction zone 0 cognitive enhancing effect of GE or 8-OH-DPAT may be Sal WAY Sal Sal WAY Sal WAY Sal Sal Sal WAY similar to that of perospirone. It remains to be determined Sal 8OH GE Sal 8OH GE GE (1000) (500) (1000) whether GE also modulates hippocampal 5-HT1A receptors in our experimental system. Sal PCP Atypical antipsychotic drugs, such as clozapine, ziprai- C. Novel object recognition test 100 done, aripiprazole, and quetiapine, are all 5-HT1A receptor ## (partial) agonists, which may be relevant for their actions in # 80 treating schizophrenia [14]. While current antipsychotic * 60 && treatments are effective against positive symptoms, they have significant side effects and have little effect on negative 40 or cognitive symptoms [15]. tion percentage (%)

a 20 In conclusion, our finding suggests that 5-HT1A receptor 0 agonistic properties of GE offer potential therapeutic advan- Explor Sal WAY Sal Sal WAY Sal WAY Sal Sal Sal WAY tages in response to PCP-induced schizophrenia-like psycho-

Sal 8OH GE Sal 8OH GE GE sis, although many details of the GE-mediated effect(s) re- (1000) (500) (1000) main to be determined. Sal PCP ACKNOWLEDGEMENTS Fig. (2). Effect of WAY 100635 (WAY) on the GE-mediated This study was supported by a grant (#2010K000812) pharmacological actions in response to PCP-induced changes in the from the Brain Research Center from 21st Century Frontier immobility time (A), social interaction time (B), and recognition Research Program funded by the Ministry of Science and memory (C). Sal = saline, GE (500) = GE 500 mg/kg, p.o., GE Technology, Republic of Korea. Jin-Man Kim and Jae- (1000) = GE 1000 mg/kg, p.o., 8OH = 8-OH-DPAT 0.5 mg/kg. i.p., Hyung Bach were supported by BK 21 program. Equipment WAY = WAY 100635 0.5 mg/kg, i.p. Each value is the mean ± at the Institute of Pharmaceutical Science (Kangwon Na- S.E.M. of 12 mice. * P < 0.01 vs. Saline + Saline + Saline, # P < tional University) was used for this study. 0.05, ## P < 0.01 vs. Saline + Saline + PCP, & P < 0.05, && P < 0.01 REFERENCES vs. Saline + GE (1000) + PCP (One-way ANOVA followed by Fisher’s PLSD test). [1] Andreasen, N.C.; Flaum, M.; Swayze, B.V. II; Tyrrell, G.; Arndt, S. Positive and negative symptoms in schizophrenia. A critical re- Recent finding have suggested that repeated PCP treat- appraisal. Arch. Gen. Psychiatry, 1990, 47(7), 615-621. ment significantly decreases the density of 5-HT receptors [2] Javitt, D.C.; Zukin, S.R. Recent advances in the phencyclidine 1A model of schizophrenia. Am. J. Psychiatry, 1991, 148(10), 1301- in the mouse brain [9]. 5-HT1A agonist properties are thought 1308. to improve negative symptoms and cognitive deficits by [3] Mouri, A.; Noda, Y.; Enomoto, T.; Nabeshima, T. Phencyclidine stimulating the release of dopamine in the prefrontal cortex animal models of schizophrenia: approaches from abnormality of [10]. Consistent with this, Sumiyoshi et al. [11] reported that glutamatergic neurotransmission and neurodevelopment. Neuro- chem. Int., 2007, 51(2-4), 173-184. the 5-HT1A agonists exert anti-depressant-like effects [12], of [4] Meltzer, H.Y. The role of serotonin in antipsychotic drug action. particular interest for schizophrenic patients suffering from Neuropsychopharmacology, 1999, 21(2 Suppl), 106S-115S. 250 Current Neuropharmacology, 2011, Vol. 9, No. 1 Shin et al.

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Received: October 01, 2009 Revised: April 17, 2010 Accepted: May 26, 2010