Participation of Histamine in the Step-Through Active Avoidance Response and Its Inhibition by H1-Blockers

Chiaki Kamei and Kenji Tasaka*

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Japan

Received February 22, 1991 Accepted August 29, 1991

ABSTRACT-The effects of intravenous and intracerebroventricular administrations of certain H1-blockers on the active avoidance response in rats were studied. Among the classic Hi-blockers used in this study: pyrilamine, diphenhydramine, promethazine and chlorpheniramine, promethazine was the most effective and chlorpheniramine the least in inhibiting the active avoidance response; namely, a variation of prolongation in the response latency of the avoidance response. Meanwhile, ketotifen most potently inhibited the active avoidance response when the drugs were administered intracere broventricularly. Mequitazine, astemizole and oxatomide were weak depressants when administered by either route. Azelastine was less effective than the classic H1-blockers by intravenous injection, while by intracerebroventricular injection, the inhibition was almost identical to those induced by the classic H1-blockers. Intracerebroventricular injection of histamine was antagonized the prolonged latency in the avoidance re sponse induced by pyrilamine or diphenhydramine. A similar effect was also produced by 2-methylhistamine, but 4-methylhistamine had no effect. Intracerebroventricular in jection of acetylcholine was restored the retarded avoidance response induced by pyri lamine, but a dose 20 times greater than that of histamine was required. From these findings, it can be concluded that inhibition of the active avoidance response induced by Hl-blockers may be exerted through interaction with Hl-receptors in the brain.

It is well-known that the central cholinergic classical H1-blockers inhibited the active system is intimately involved in the modula avoidance response by prolonging the re tion of learning acquisition and in memory re sponse latency, even with a single administra tention in humans and animals (1). Recently, tion (6). Moreover, the chronic oral adminis it has also been reported that in rats, hista tration of these H1-blockers both remarkably mine takes part not only in learning but also retarded the acquisition of the active avoid in memory retention (2, 3). However, it has ance response and impaired the retention of long been emphasized that classic H1-blockers acquired learning for as long as chronic ad such as diphenhydramine, pyrilamine and ministration was continued (6). These findings promethazine provide potent depressant ac seem to support the data reported by de tions on the central nervous system (CNS) in Almeida and Izquierdo (2). However, a series many different situations (4, 5). We have pre of new H1-blockers such as oxatomide, astemi viously reported that oral administration of zole and mequitazine, which were developed exclusively for the treatment of allergic dis eases, were much less effective in inhibiting the response when administered orally than and unless the animals moved into the lighted the classic Hi-blockers (6). compartment within 5 sec, the sliding door The present study was performed to clarify was closed and an electric shock was delivered the effect of Hi-blockers on active avoidance for 5 sec. Acquisition training was repeated when injected either intravenously or intra once a day for 10 days, and rats showing posi cerebroventricularly, and also to determine tive avoidance response within 2 sec (moving whether 11-blockers exert their inhibitory from the dark to the lighted room) were used effect by competing with histamine for the for testing the drug effect. To evaluate the same (H1) receptor in the brain. drug effects on the avoidance response, the latency (sec) before moving into the lighted MATERIALS AND METHODS side was measured. A group of eight to ten rats was used for each dose. When the drugs Animals were administered both intravenously and in Male Wistar rats weighing 200-300g were tracerebroventricularly into the same animal, used. All animals were kept in an air-con the drug was injected into the tail vein under ditioned room at a controlled temperature the restraint conditions, and 10 min later, the (24-25°C) and humidity (50-60%). The ani other drugs were injected intracerebroventricu mals were given food and water ad libitum. larly via the implanted cannula under free movement conditions. Surgery Under sodium pentobarbital anesthesia (35 Drugs mg/kg, i.p.), the animals were placed on a The drugs used were astemizole (Janssen), stereotaxic apparatus (Narishige, Type SR-5). azelastine hydrochloride (Eisai), d-chlor A guide cannula made of stainless steel tub pheniramine maleate (Yoshitomi), diphenhy ing, 700,um in outside diameter and 14 mm in dramine hydrochloride (Wako), ketotifen length, was implanted in the right lateral ven fumarate (Sandoz), oxatomide (Janssen), tricle (A:5.4, L:1.5, H:3.0) according to the promethazine hydrochloride (Yoshitomi), atlas of de Groot (7) and fixed to the skull pyrilamine maleate (ICN Pharm), mequitazine with dental cement. The animals were allowed (Nippon Shoji), histamine dihydrochloride at least 10 days to recover from the surgery (Wako), 2-methylhistamine dihydrochloride (8). (SK&F), 4-methylhistamine dihydrochloride (SK&F) and acetylcholine chloride (Daiichi). Apparatus The drugs were dissolved in saline. In the in The apparatus was divided into two com tracerebroventricular injection, the drug solu partments, a 19 X 15 X 14 cm lighted room tions were adjusted to pH 7.2, and 5 ml of and a 33 X 20.5 X 14 cm dark room, linked drug solutions were injected into the lateral by a sliding door (5 X 5 cm). The lighted com ventricle through the injection cannula at a partment was painted white, had a flat floor constant speed of 60 sec. and was illuminated by a lamp (100 W). The floor of the dark compartment consisted of a Statistical analysis series of copper rods (3 mm in outside diam One way analysis of variance with Dunnett's eter), arranged side by side, 1.0 cm apart, test was used to determine statistical signifi through which electroshock was delivered (40 cance. ED50 was calculated according to the V, 0.2 mA). method of Litchfield-Wilcoxon (9) using those among the eight to ten tested animals that dis Experimental procedures played a latency more than twice as long as Immediately after the rats were placed in that of the pre-drug values (sec) for the active the dark room, the sliding door was opened, avoidance response. RESULTS prolonged the response latency at 15 min and a significant effect was observed even 120 min Effects of intravenous administration of certain after drug administration. At the same dosage, drugs on the active avoidance response chlorpheniramine was less potent than pyrila Figure 1 shows the effects of pyrilamine, mine. Ketotifen elicited a slight but significant chlorpheniramine, ketotifen, astemizole, oxato prolongation of the latency even at a dose of 2 mide and azelastine after intravenous adminis mg/kg. Because the LD50 of ketotifen by in tration. At a dose of 10 mg/kg, pyrilamine travenous injection was 5.3 mg/kg (10), doses

Fig. 1. Effects of intravenous injection of H1-blockers on the active avoidance response in rats. saline (0), 0.5 mg/kg (•), 1 mg/kg (o), 2 mg/kg (A), 5 mg/kg (D), 10 mg/kg (•), 20 mg/kg (o). *' **: Significant ly different from the control group with P < 0.05 and P < 0.01, respectively (error bars = S.E.M.). higher than 2 mg/kg were not depicted. The 28. 1) mg/kg and 21.4 (18.9-25.2) mg/kg, effect of astemizole was almost negligible at a respectively. dose of 10 mg/kg, but at 20 mg/kg, the drug prolonged the response latency significantly. Effects of intracerebroventricular administra Both oxatomide and azelastine showed a sig tion of certain drugs on the active avoidance re nificant effect at doses of 10 and 20 mg/kg. sponse Table 1 summarizes the ED50 values of H, Figure 2 shows the effects of the same blockers on the active avoidance response. group of drugs tested after intracerebroven Promethazine appeared to be the most potent tricular administration. At doses of 20 and 50 drug, with an ED50 of 2.50 (1.19-5.25) ,ug, pyrilamine caused a significant retardation mg/kg. Both astemizole and mequitazine had in the response latency. Chlorpheniramine low potencies; The ED50s were 25.6 (23.7 elicited a significant prolongation at doses of

Table 1. ED50 values of H,-blockers for the prolongation of the response latency in the active avoidance response in rats 20 and 50 pg. Ketotifen extended the response summarized in Table 1. Ketotifen and prom latency significantly at doses of 10 and 20 jig. ethazine were effective in inhibiting the avoid However, astemizole elicited no significant ance response (ED50 values of 14.3 (14.0 effect even at a dose of 50,ug. Both oxato 14.5) ,ug and 19.8 (17.8-22.0) ,ug, respective mide and azelastine showed significant effects ly). Mequitazine, astemizole and oxatomide at 50 or 100 ,u g and 10, 20 or 50 ,u g, respec were extremely weak compared with the clas tively. The ED50 values determined after in sic Hi-blocking agents. tracerebroventricular administration are also

Fig. 2. Effects of intracerebroventricular injection of H,-blockers on the active avoidance response in rats. saline (0), 5 gg (0), 10,ug (o), 20 jig (A), 50,ug (D), 100,ug (•). *' **: Significantly different from the control group with P < 0.05 and P < 0.01, respectively (error bars = S.E.M.). Effect of histamine administered intracerebro Intracerebroventricular injections of hista ventricularly on the active avoidance response mine at doses of 0.1-1 ug dose-dependently inhibited by intravenous injection of pyrilamine suppressed the prolonged latency in the avoid or diphenhydramine ance response produced by intravenous injec

Fig. 3. Effects of intracerebroventricular injection of histamine, 2-methylhistamine, 4-methylhistamine and acetylcholine on the prolonged latency in the active avoidance response induced by pyrilamine or diphenhydramine or atropine. saline (0), 0.1,ug (s), 0.5,ug (o), 1,ug (A), 2,ug (LI), 5,ug (•), 10,ug (o). Pyrilamine, diphenhydramine and atropine were injected intravenously at doses of 9.7 mg/kg, 7.9 mg/kg and 5.2 mg/kg, respectively. Arrows indicate the injection time of saline, histamine, 2-methylhista mine, 4-methylhistamine or acetylcholine (error bars = S.E.M.). *' **: Significantly different from the con trol group with P. < 0.05 and P < 0.01, respectively. tion of pyrilamine (9.7 mg/kg, double dose of As shown in Fig. 3, intracerebroventricular ED50) (Fig. 3). A significant effect appeared injection of 2-methylhistamine, an H1 agonist, almost instantaneously after histamine injec antagonized the pyrilamine-induced inhibition tion at doses higher than 0.5,ug. The protract of the active avoidance response in the same ed latency in the avoidance response induced way as histamine; significant shortening of the by diphenhydramine (7.9 mg/kg, double dose response latency was observed at doses greater of ED50) was also shortened after a brief than 1 pg. In contrast, 4-methylhistamine, an period by intracerebroventricular administra H2 agonist, did not affect the response laten tion of histamine as shown in Fig. 3. ID50 cy, even at a dose of 5,ug (Fig. 3, Table 2). values of histamine on pyrilamine or diphen When these two agonists were injected sepa hydramine-induced inhibitions of active avoid rately, without being combined with H1-block ance were 0.52 (0.22 2.99) ,ug and 0.79 (0.44 ers, neither of them had any effect at doses of -3 .32) u g, respectively (Table 2). Histamine 2 and 5 ,ug, respectively (Table 3). alone at a dose of 1 ug caused no significant effect on the active avoidance response (Table Effect of acetylcholine administered intracer 3). ebroventricularly on the active avoidance re sponse inhibited by intravenous injection of Influence of intracerebroventricular administra pyrilamine or atropine tion of 2-methylhistamine and 4-methylhista Intracerebroventricular administration of mine on the inhibition induced by intravenous acetylcholine at doses of 1 and 5 u g did not injection of pyrilamine influence the prolonged latency in the active

Table 2. ID50 values of histamine, 2-methylhistamine, 4-methylhistamine or acetylcholine administered intracerebroventricularly on the response latency in the active avoidance response induced by H1-blockers and atropine Table 3. Effect of intracerebroventricular injection of histamine, 2-methylhistamine, 4-methylhistamine and acetylcholine on active avoidance response

avoidance response induced by pyrilamine. At ketotifen caused a relatively potent inhibition the dose of 10,ug, however, acetylcholine pro of the avoidance response in the present ex duced a transient, contradictory effect; a sig periment, it exerted only a weak influence on nificant shortening was observed 2 -10 min this response after oral administration when after acetylcholine administration (Fig. 3). In the ED50 values were compared (6). There contrast, the inhibition of active avoidance in fore, it seems likely that either 1) ketotifen duced by atropine (5.2 mg/kg, twice of ED50 may not be easily absorbed from the gastro value) (11) was strongly antagonized by acetyl intestinal tract or 2) the drug may go through choline injection; a significant suppression was the first-pass effect. Martin and Romer (12) observed at 1,ug of acetylcholine. ID50 values reported that an inhibition of passive for acetylcholine on pyrilamine and atropine cutaneous anaphylaxis (PCA) in rats induced induced inhibitions of the avoidance response by intravenous injection of ketotifen was 17 were 11.6 (5.11 75.4) ,ug and 1.23 (0.75 times more potent than that induced by oral 4.48) ,ug, respectively (Table 2). No influence administrations (ED50: 0.3 mg/kg, i.v.; 5.1 on the response latency was found following a mg/kg, p.o.). The discrepancy due to the dif single injection of acetylcholine at a dose of 10 ference in administration routes was also u g (Table 3). found in the other drugs used in the present study. The potency of azelastine exerted by DISCUSSION intracerebroventricular injection was slightly stronger than that of chlorpheniramine, while These studies demonstrated that newly de in intravenous injections, azelastine was veloped H1-blockers which are used as anti evidently less effective than chlorpheniramine. allergic agents (such as oxatomide, astemizole This suggests that the penetrating amount of and mequitazine) were less potent than classic azelastine into the CNS may be much less to Hi-blockers (pyrilamine, diphenhydramine, compare with that of chlorpheniramine. promethazine, chlorpheniramine) in inhibiting Actually, Tatsumi et al. (13) reported that the the active avoidance response when the drugs radioactivity derived from azelastine was ex were administered either intravenously or in tremely low in the brain when the drug was tracerebroventricularly. We found that while intravenously administered in rats, and the concentrations of the drug in the brain tissue two drugs are typical H1-blockers and the were lower than the concentration in the drugs showed a marked prolongation of re blood. Ohmori et al. (14) found that azelas sponse latency, pyrilamine and diphenhydra tine gave rise to a marked inhibition of [3H] mine were selected in this experiment. In simi pyrilamine binding to guinea pig cerebellum, lar experiments, 2-methylhistamine, an H1 indicating that the drug is able to bind the agonist, produced the same effect as hista CNS efficiently. Therefore, it is reasonable to mine, whereas 4-methylhistamine, an H2 ago assume that the relatively weak depressant nist, was totally ineffective. This may indicate effect on the active avoidance response caused that the inhibitory effect of H1-blockers on the by intravenous injection of azelastine may not avoidance response may be exerted through be due to its feeble CNS activity but simply to the H1-receptor. In accordance with this, its low capacity for penetrating the CNS. acetylcholine administered at a dose 20 times In the cases of mequitazine, oxatomide and greater than that of histamine was required to astemizole, relatively high ED50s were suppress the prolongation of the response obtained compared with those of classical H1 latency in the avoidance response induced by blockers when administered intravenously or the same dose of pyrilamine. However, the in intracerebroventricularly. It has been reported hibitory effect of atropine on active avoidance that the concentration of mequitazine in the was blocked by intracerebroventricular admin brain after intravenous administration was not istration of acetylcholine at a dose of 1.23,ug, lower than that determined in the blood (15). which is one-tenth of the dose used to com Similar results were obtained in the case of pete with the pyrilamine effect. From these oxatomide after oral administration (16). findings, it can be assumed that an inhibition Therefore, it was assumed that mequitazine of active avoidance due to H1-blockers may be may have almost negligible affinity for the exerted via H1-receptors in the brain. brain. Uzan et al. 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