In this study, we investigated the effects of isofloxythepin on WDS induced by hip pocampal stimulation and examined whether neuroleptic in general have an inhibi tory action on WDS.

Effect of Isofloxythepin, a Novel Neuroleptic, on Hippocampal Stimulation-Induced Wet-Dog Shaking in the Rat

Masuo OHNO, Tsuneyuki YAMAMOTO and Showa UEKI

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812, Japan

Accepted September 19, 1988

Abstract (+)isofloxythepin (0.32-3.2 mg/kg, i.p.) significantly inhibited in a dose-dependent manner wet-dog shaking (WDS) induced by electrical stimulation of the rat hippocampus. In addition, both optical isomers of isofloxythepin inhibited WDS, with the (-)-isomer being almost 3 times more potent than the (+)-isomer. Other neuroleptics such as , , and also reduced significantly the number of WDS. The inhibitory potency of haloperidol was comparable to that of (±)isofloxythepin, which was approximately 3 times more potent than that of chlorpromazine or zotepine. Sulpiride suppressed sig nificantly WDS only at the high dose of 100 mg/kg. None of the drugs affected hippocampal afterdischarge. Inhibition of WDS produced by (±)isofloxythepin or haloperidol was antagonized by pretreatment with a receptor , lisuride. The present results indicate that isofloxythepin shares with other neuroleptics an inhibitory effect on WDS; blocking action appears to be important in the inhibition of WDS induced by hippocampal stimulation.

Isofloxythepin, 3-fluoro-8-isopropyl-10 Recently, it has been shown that the hip [4-(2-hydroxyethyl) piperazino]-10,11-dihy pocampal stimulation-induced WDS is drodibenzo(b,f)thiepin, is a newly synthe significantly and dose-dependently inhibited sized neuroleptic , that has been shown by morphine (7, 9) and by representative to inhibit -induced stereotyped neurolepic drugs such as chlorpromazine and behavior (1, 2). Isofloxythepin as well as haloperidol (7, 8). These results indicate that other neuroleptics such as haloperidol and central dopaminergic mechanisms may be zotepine causes the elevation of serum important in WDS elicited by hippocampal level in rats (3, 4), which might be stimulation because neuroleptics directly and due to the blockade of dopamine receptors in morphine indirectly inhibit the dopaminergic the pituitary. Isofloxythepin has also been function. shown to increase striatal dopamine turnover in rats (3, 5), which presumably reflects the blockade of dopaminergic transmission in the striatum. Recently, Lau and Runice (1) in dicated that isofloxythepin has a blocking action on the rat striatal dopamine receptors, using the [3H] binding technique. Materials and Methods Wet-dog shaking (WDS) is a characteris Animals: The animals used were male tic behavior observed in morphine-dependent Wistar rats (body weight: 260-300 g at the rats after abrupt morphine withdrawal or time of surgery) obtained from Shizuoka after the administration of a narcotic anta Laboratory Animal Center. The rats were gonist (6). WDS can also be induced by housed in a room maintained at a temperature electrical stimulation of the hippocampus (7 of 22±1 'C with a 12 hr light-dark cycle (light 10), the septum (11) or the fornix (12). period: 07:00-19:00). Food and water were available ad libitum throughout the experi N HCI, and the pH was adjusted to 4-5 with mental period. NaOH. Drugs were administered 1.p. in a Electrode implantation and experimental volume of 0.1 ml per 100 g of body weight, procedures: Rats were anesthetized with 1 hr before hippocampal stimulation. The pre sodium pentobarbital (40 mg/kg, i.p.) and treatment with lisuride in the antagonism fixed in a stereotaxic instrument. Stainless test was performed 10 min prior to drug in steel bipolar electrodes (diameter: 0.2 mm, jection. uninsulated length: 0.5 mm, interelectrode Statistical analysis: The significance of the distance: 0.5 mm) were chronically implanted drug effect on the number of WDS and AD bilaterally in the dorsal hippocampus (an duration was determined using Student's t terior: 3.2 mm, lateral: 2.8 mm, horizontal: test or the Cochran-Cox test. 3.0 mm) according to the rat brain atlas of Paxinos and Watson (13). Following im Results plantation of the chronic electrodes, at least Electrical stimulation of the dorsal hip one week was allowed for recovery from pocampus with an intensity above the AD surgery before starting the experiment. threshold (mean+S.E.: 51.4±5.0 /,,A) induced Rats were placed in a transparent plastic a high frequency of WDS (31.8+2.6/3 min), box (30x25x30 cm) containing wood shav which was associated with the occurrence of ings and allowed to adapt to the new environ hippocampal AD. WDS was never induced by ment for 5-10 min prior to brain stimulation. stimulation below this threshold. As shown in The hippocampus was stimulated for 5 sec Fig. 1, (±)isofloxythepin (0.32-3.2 mg/kg, with a square wave pulse (60 Hz in frequency, i.p.) caused a dose-dependent reduction in 1.0 msec in duration, bipolar). One hip the number of WDS induced by hippocampal pocampal electrode was used for stimulation stimulation. The appearance of WDS was and the other, for recording afterdischarge almost completely abolished when 3.2 mg/kg (AD). First, the threshold for inducing AD of (±)isofloxythepin was injected. However, was determined, starting from an ineffective at this dose, all animals showed severe seda current of 30 ,eA. The stimulus intensity was tion and ataxia. The (-)-isomer of isofloxy increased in 10 pA increments until AD was thepin, at doses of 0.32 and 1.0 mg/kg, also induced. The initial stimulation intensity markedly inhibited hippocampal stimulation which induced AD was regarded as the induced WDS. WDS was also significantly threshold for AD. After the AD threshold was inhibited by (+)isofloxythepin at 1.0 mg/kg, determined, electrical stimulation was per but not by 0.32 mg/kg. The inhibitory effect formed at this intensity. The number of WDS of (-)isofloxythepin on WDS was almost 3 was counted by an observer and simul times more potent than that of its (+)-isomer. taneously electrical responses from the dorsal The duration of hippocampal AD was not hippocampus were recorded on a polygraph significantly affected either by the racemic (Nihon Kohden) during the 180 sec period isofloxythepin or by its optical isomers (Table following the stimulation. The drug tests were 1). carried out 5-6 hr after the measurement of Figure 2 shows the effects of other neuro control values. leptic drugs on hippocampal stimulation Drugs: The following drugs were used in induced WDS. Ail four neuroleptics used in this study: (±)isofloxythepin methanesul this experiment produced a significant and fonate and its optical isomers, (+) and (-) dose-dependent inhibition of WDS. Haloper isofloxythepin methanesulfonate (Showa idol was almost equipotent to (+)isofloxy Denko), haloperidol (Serenace Injection, thepin, which was approximately 3 times Dainippon), chlorpromazine (Contomin In more potent than chlorpromazine and jection, Yoshitomi), zotepine (Fujisawa), zotepine. Haloperidol, at a dose of 1.8 mg/kg , sulpiride (Dogmatyl Injection, Fujisawa) and caused severe sedation and ataxia. Sulpiride lisuride hydrogen maleate (Nihon Schering). suppressed significantly the appearance of Isofloxythepin and lisuride were dissolved in WDS only at a high dose of 100 mg/kg. None distilled water. Zotepine was dissolved in 0.1 of the drugs had an effect on hippocampal AD Fig. 1. Effects of (--±)isofloxythepin (IFT) and its optical isomers on wet-dog shaking (WDS) induced by electrical stimulation of the rat hippocampus. IFT was injected i.p. 1 hr before hippocampal stimulation for 5 sec with a square wave pulse (60 Hz, 1.0 msec). The number of WDS was expressed as a percen tage of the control values (Pre-drug) for each animal. Each value is the mean±S.E. for 5-10 animals. The number of WDS in the saline-treated group was 29.4±3.0/3 min. Significant differences from the saline group or between optical isomers were determined using Student's t-test or the Cochran-Cox test. *P<0 .05, **P<0.01, ***P<0.001.

Table 1. Effects of isofloxythepin, haloperidol, chlorpromazine, zotepine, sulpiride and lisuride or afterdischarge induced by hippocampal stimulation in rats Fig. 2. Effects of various neuroleptic drugs on hippocampal stimulation-induced wet-dog shaking (WDS). Each drug was injected i.p. 1 hr before hippocampal stimulation. Each value is the mean±S.E. for 5-10 animals. Significant differences from the saline group (**P<0.01, ***P<0.001) were deter mined using Student's t-test or the Cochran-Cox test.

was blocked by pretreatment with lisuride.

Discussion In the present study, WDS induced by hippocampal stimulation was markedly and dose-dependently inhibited by the novel neuroleptic drug isofloxythepin, which is a dibenzothiepin derivative. Furthermore, by investigating the effects of both optical isomers of isofloxythepin, the WDS inhibition caused by (-)isofloxythepin was found to be approximately 3 times more potent than that of its (+)-isomer. Metysova and Protiva (14) reported that (-)isofloxythepin is 6-20 times more potent than its (+)-isomer with respect to antagonism of apomorphine-induced Fig. 3. Effect of lisuride on the inhibitory action of chewing and agitation, cataleptic activity in (±)isofloxythepin (IFT) or haloperidol (HPD) on rats and anti- activity in mice. .Net-dog shaking (WDS). IFT or HPD was injected From these findings, they concluded that the i.p. 1 hr before hippocampal stimulation. The pre central anti-dopaminergic activity of isofloxy treatment with lisuride was performed 10 min prior thepin was attributable almost exclusively to to the drug injection. Each value is the mean±S.E. the (-)-isomer. In the present experiment, for 6-10 animals. The significance of the drug effect significant differences between both optical on WDS was determined using Student's t-test or the isomers of isofloxythepin were also shown to Cochran-Cox test. **P<0.01. exist in the inhibitory potency of WDS in duced by hippocampal stimulation. (Table 1). Another derivative of dibenzothiepin, When administered alone at a dose of 0.32 zotepine, also caused a significant reduction mg/kg, the agonist lisuride in the number of WDS induced by hippo had no significant effect on either the number campal stimulation. Furthermore, WDS was of WDS or AD duration (Fig. 3 and Table 1 also significantly suppressed by the injection The inhibition of WDS caused by a dose of 1.0 of other neuroleptic drugs such as haloperidol, mg/kg of (+)isofloxythepin or haloperidol chlorpromazine and sulpiride. Isofloxythepin

isofloxythepin. Psychopharmacology (Berlin) 76, 381-384 (1982)

has a higher affinity for dopamine D2 recep thepin. Eur. J. Pharmacol. 116, 25-31 (1985) tors than haloperidol (1). It was shown that 2 Metysova, J., Metys, J., Valch6f, M. and Dlabac, the affinity of haloperidol for D2 receptors was A.: Kinetics of central actions of isofloxythepin, almost 3 times greater than the affinity of an oral neuroleptic drug with prolonged action in chlorpromazine, which was similar to the af rodents. Act. Nerv. Super. 22, 164-165 (1980) finity of zotepine (15) . The affinity of sulpiride 3 Valchai•, M., Metysova, J., Chlebounova, J. and for D2 receptors was much lower than other Dlabac, A.: Induction of dopaminegic super neuroleptic drugs (1) . Based on these find sensitivity after a single dose of the neuroleptic ings, the relative potencies of neuroleptic in the inhibition of WDS were similar to the 4 Yamada, K. Matsuo N., Matsuda, T., Tanaka, M., affinity of these compounds for D2 receptors , although isofloxythepin was not as potent as Furukawa, T., Koja, T. and Fukuda, T.: Effects of new neuroleptics, isofloxythepin and zotepine, predicted from the binding studies. Therefore, on post-decapitation convulsions and prolactin it is suggested that the D2 receptor blocking secretion in rats. Pharmacol. Biochem. Behav. action is important in the inhibition of WDS 24, 1445-1449 (1986) induced by hippocampal stimulation . This 5 Valchar, M., Metysova, J. and Dlabac, A.: The conjecture is supported by the present results effect of a new fluorinated tricyclic neuroleptic showing that pretreatment with a direct drug isofloxythepin on the rat striatal dopamine dopaminergic agonist , lisuride (16-18), metabolism. Act. Nerv. Super. 21, 130-131 reversed the WDS suppression caused by (1979) isofloxythepin or haloperidol and that a 6 Wei, E., Loh, H.H. and Way, E.L.: Quantitative selective D2 , sulpiride aspects of precipitated abstinence in morphine (19), exerted the inhibitory effect on WDS. dependent rats. J. Pharmacol. Exp. Ther. 184, Some neuroleptics (e.g ., zotepine) have 398-403(1973) been reported to possess a relatively potent 7 Araki, H. and Aihara, H.: Effects of morphine and anti- action in addition to a dopa neuroleptics on wet-dog shaking behavior minergic blocking action (20) . However, elicited by hippocampal stimulation in rats. since the serotonin antagonist , at Psychopharmacology (Berlin) 85, 301-305 doses of 3.2-32 mg/kg, i.p., had no significant (1985) effect on hippocampal stimulation-induced 8 Araki, H. and Aihara, H.: Effects of neruoleptics WDS (10), it seems unlikely that the anti on hippocampal stimulation-induced 'wet-dog serotonin action of zotepine accounts for its shaking' in rats. Physiol. Behav. 36, 69-74 inhibitory effect on WDS . (1986) In conclusion, it is suggested that isofloxy 9 Ohno, M., Yamamoto, T., Araki, H. and Ueki, S.: The effect of z-opioid agonist U-50 thepin and zotepine, which are structually ,488H on wet-dog shaking behavior induced by hip novel neuroleptics that are dibenzothiepin pocampal stimulation in rats. Psychopharma derivatives, share an inhibitory action on WDS cology (Berlin) 91, 189-192 (1987) with other known neuroleptic drugs , and the 10 Ohno, M., Yamamoto, T. and Ueki, S.: dopamine D2 receptor blocking action plays attenuates wet-dog shaking induced by hip a crucial role in the inhibition of WDS induced pocarrrpal stimulation in rats. Eur. J. Pharmacol. by electrical stimulation of the rat hippocam 137, 161-166 (1987) pus. 11 Le Gal La Salle, G., Cavalheiro, E.A., Feldblum, Acknowledgments: The authors wish to express S. and Maresova, D.: Studies of wet-dog shake behavior induced by septohippocampal stimu their appreciation to Showa Denko Co., Ltd. (Tokyo, Japan) for the generous supply of isofloxythepin and lation in the rat. Can . J. Physiol. Pharmacol. 61, 1299-1304 (1983) its optical isomers and to Nihon Schering Co., Ltd. (Osaka, Japan) for lisuride. We also thank Miss H. 12 Slater, P.: Pharmacological profile of wct-dog Hitaka for typing the manuscript . shaking induced by electrical stimulation of the fornix in rats. Pharmacol. Res. Commun . 16, 273-280 (1984) References 13 Paxinos, G. and Watson , C.: The Rat Brain in 1 Lau, Y.-S. and Runice, C.: Prolonged inhibition of Stereotaxic Coordinates. New York, Academic striatal dopamine receptor sites by isofloxy Press (1982) 14 Metysova, J. and Protiva, M.: Stereoselectivity of 18 Horowski, R. and Wachtel, H.: Direct dopamin neuroleptic effects of isofloxythepin in rodents. ergic action of lisuride hydrogen maleate, an Act. Nerv. Super. 24, 233-234 (1982) derivative, in mice. Eur. J. Pharmacol. 36, 15 Harada, T., Ebara, T. and Otsuki, S.: Possible 373-383 (1976) relationship between antimanic effect and 19 Memo, M., Govoni, S., Carboni, E., Trabucchi, M. activity of zotepine to 5HT, receptor. Folia and Spano, P.F.: Characterization of stereo Psychiat. Neurol. Japon. 38, 473-480 (1984) specific binding of 3H-(-)sulpiride, a selective 16 Cunningham, K.A., Callahan, P.M. and Appel, antagonist at dopamine-D2 receptors, in rat CNS. J.B.: Discriminative stimulus properties of Pharracol. Res. Commun. 15, 191-199 (1983) lisuride revisited: Involvement of dopamine D2 20 Lai, H., Carino, M.A. and Horita, A.: Anti receptors. J. Pharmacol. Exp. Ther. 241, 147 serotonin properties of neuroleptic drugs. In 151 (1987) Psychopharmacology and Biochemistry of 17 Fujita, N., Saito, K., Yonehara, N. and Yoshida, Neurotransmitter Receptors, Edited by Yama H.: Lisuride inhibits 3H-spiroperidol binding to mura, H., Olsen, R.M. and Usdin, E., p. 347-353, membranes isolated from striatum. Neurophar Elsevier, Amsterdam (1980) macology 17, 1089-1091 (1978)