Effect of stimulation of brain serotonergic system on mouse-killing behavior in rats

Physiology & Behavior,Vol. 25, pp. 161-165. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Effect of Stimulation of Brain Serotonergic System on Mouse-Killing Behavior in Rats WOJCIECH

KOSTOWSKI,

OLGIERD

PUCIILOWSKI 1 AND ADAM PLA~NIK

Department o f Pharmacology and Physiology o f the Nervous System, Psychoneurological Institute, 02-957 Warszawa, Sobieskiego 1/9 Poland R e c e i v e d 15 N o v e m b e r 1979 KOSTOWSKI, W., O. PUCILOWSKI AND A. PLA~NIK. Effect of stimulation of brain serotonergic system on mousekilling behavior in rats. PHYSIOL. BEHAV. 25(2) 161-165, 1980.--Mouse-killing behavior was induced in male Wistar rats due to 6 weeks isolation. The selective stimulation of the dorsal raphe nucleus markedly reduced muricide behavior. On the other hand rats with electrodes situated outside of the dorsal raphe (within the substantia grisea centralis) showed no changes in muricide behavior when compared with control (non-stimulated) animals. Pharmacological stimulation of whole serotonergic brain neurons by quipazine, an agonist of serotonin receptors, and compound CGP 6085-A, a selective inhibitor of serotonin uptake, strongly decreased mouse-killing behavior. Our data indicates that serotonergic neurons inhibit predatory aggression in rats. Raphe system

Killer rats

Aggressive behavior

Brain serotonin

Predatory aggression

fined to the DR produced muricide behavior in naturally non-killing rats whereas lesions of the median raphe nucleus (MR) were without effect. The second purpose of our study was to investigate the effect of drugs that stimulate 5-HT neurons: quipazine, a 5-HT receptor agonist [8] and compound CGP 6085-A, a selective inhibitor of 5-HT uptake [27].

T H E R E L A T I O N S H I P between central serotonergic neurons and aggressive behavior has been the focus of much research, p-Chlorophenylalanine (pCPA), which depletes brain serotonin (5-HT) through inhibition of tryptophan hydroxylase [11] induces the characteristic pattern of aggression in rats-mouse-killing or muricide behavior and facilitates the occurrence of muricide induced by ablation of the olfactory bulbus [2, 20, 22, 23]. Lesions of the midbrain raphe nuclei, an area containing 5-HT cell bodies, markedly reduce 5-HT concentrations in various brain areas [6, 12, 14, 17] and induce aggressive behavior including muricide [1, 2, 4, 9, 20, 22, 24, 25]. Thus, pharmacological or surgical manipulations leading to depletion of brain 5-HT concentrations increase muricide behavior in rats. There is, however, little evidence indicating that increased activity of 5-HT neurons reduces this aggressive pattern. Muricide behavior in raphelesioned rats is inhibited by 5-hydroxytryptophan (5-HTP) without suppressing hyperactivity [23]. Interestingly, muricide behavior o f " spontaneous" nature (i.e. not induced by brain lesions or isolation) is also inhibited by 5-HTP but much higher doses than those required for inhibition of muricide in the raphe lesioned rats are necessary [21,23]. Chloroimipramine, an inhibitor of 5-HT uptake, exerts a strong inhibition upon muricide in raphe lesioned rats [23]. The first purpose of this study was to determine the effect of electrical stimulation of the dorsal raphe nucleus (DR) on muricide behavior in rats. We decided to stimulate this 5-HT nucleus since stimulation of the DR has been reported to induce some inhibitory symptoms such as sedation and analgesia [5, 13, 18]. It was also reported that stimulation of the DR increases turnover and release of 5-HT in the forebrain [5]. Recently Waldbillig [26] showed that lesions con-

METHOD

Animals Experiments were carried out on male Wistar rats weighing approximately 180 g at the start of experimentation. Animals were housed in single cages 25×25x30 cm under normal laboratory conditions (temperature 20-22°C, 60 percent humidity, normal light condition) with ad lib access to standard lab diet and water. All experiments were performed from March to May, and animals were tested between 10:00 a.m. and 13:00 p.m.

Evaluation of Muricide Behavior Animals were isolated in single cages for 6 weeks. From the second week of isolation rats were tested every other day in order to develop a fixed behavioral pattern (killer or non-killer). Swiss albino male mice were used as prey. The mouse was put into the rat home cage for 30 min and rats killing their prey within this period were designated as killers whereas rats that did not develop such a behavioral pattern were considered as non-killers. Then the killers were divided into three groups: the first was stereotaxically implanted with electrodes in the dorsal raphe and the second and third were injected with quipazine and CGP 6085-A respectively.

qnstitute of Physiological Sciences, Medical Academy, Warszawa.

161

KOSTOWSKI ET AL.

162

TABLE I EXPERIMENTALPROCEDUREAND GROUPS Group 1 Stimulation ~of the brain Isolation for 6 weeks

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FIG. 1. Photograph showing chronically implanted killer rat during stimulation of the dorsal raphe. Note sedation and crouched posture of stimulated animal,

Electrode Implantation The animals were anesthetized with chloral hydrate (400 mg/kg IP) and bipolar stainless steel electrodes 0.25 mm in diameter, insulated except for 0.5 mm tips, were stereotaxically implanted into the DR according to K r n i g and Klippel [16] coordinates: A=0.4 mm, L=0.0 mm and H = - 0 . 6 ram. Details of surgery were described elsewhere [13]

Electrical Stimulation of the DR The animals were stimulated in their home cages using a Grass S 88 impulse generator (5-6 V, 0.2 msec, 10 cps) for 45 rain. The muricide behavior was evaluated during the course of stimulation (starting from the 15th rain of stimulation) and again 1 hr and 24 hr after the stimulation. One week later the rats were again tested for muricide behavior under similar conditions i.e. with the stimulation wire on the head, however, without being stimulated (control period). The details of the experimental procedure are shown in Fig. 1 and Table 1.

Histology At the end of the experiments rat were killed, the electrode tips were marked by passage of a small DC current and the brain stems were sectioned after fixation with 10 percent Formalin and stained with haematoxyllin and eosin.

lt!iections of Drugs Rats were isolated for 6 weeks and tested for muricide

FIG. 2. Coronal section (plate 350 ~ according to K6nig and Klippel atlas, [131) showing electrode tips locations within the dorsal raphe (black circles) and substantia grisea centralis (open circles). A--aqueductus, SGC--substantia grisea centralis, CS--colluculus superior, DR~nucleus raphe dorsalis, FR--formatio reticularis, MR--nucleus raphe medianus, PCS---pedunculus cerebellaris superior.

behavior as described above. During the last week of isolation animals were handled 3-4 times and then were subjected to different treatments of the following sequence: (a) rats received saline and muricide behavior was tested 1 hr, 24 hr and 48 hr after injection, (b) 5-7 days later rats received quipazine (2.5, 5.0 and 10.0 mg/kg IP) or compound CGP 6085-A (1.25, 2.5 and 5.0 mg/kg IP) and were tested for muricide 1 hr, 24 hr and 48 hr after injection. CGP 6085-A was synthetized in the Research Department, Pharmaceutical Division, Ciba-Geigy AG, and quipazine maleate was obtained from Miles Laboratories. RESULTS

Stimulation of the Dorsal Raphe Histological examination showed in 8 rats electrode tips located within the dorsal raphe (Fig. 2 and 3). In 7 rats, however, electrode tips were found dorsolaterally

SEROTONIN AND MURICIDE

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FIG. 4. Effect of stimulation of the dorsal raphe and substantia grisea centralis on muricide behavior in rats. White columns indicate muricide behavior on non-stimulated animals (control period), black columns indicate muricide behavior of rats with stimulated substantia grisea centralis (SGC) and dorsal raphe (DR). Vertical scale indicates the percent of rats showing mouse-killing behavior (mean values from 8-15 rats). PRE--muricide behavior 1 day before stimulation, STIM--muricide behavior in the course of stimulation. One hour and 24 hr indicate 1 hr and 24 hr after stimulation respectively. *=p<0.05 with respect to non-stimulated group, §=p<0.025 with respect to SGC-stimulated group, &&=p<0.025 and AAA=p<0.005 with respect to pre-stimulation value (according to x2test).

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FIG. 5. Effects of quipazine (Q) and CGP 6085-A (CGP) on muricide behavior in rats. Ordinate-percent of animals showing inhibition of muricide (1 hr after injection of drugs), abscissa--log dose mg/kg. Injection of saline failed to reduce the fixed muricide pattern. The values obtained with doses 2.5-10.0 mg/kg differ significantly from saline-treated animals (.o<0.05 according to x2test).

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shifted--within the substantia grisea centralis (Fig. 2). Stimulation of the DR produced sedation of animals (see Fig. 1) and almost completely blocked muricide behavior. This effect occurred during the stimulation period as well as 1 hr after stimulation. Twenty four hours after stimulation all animals again became aggressive (Fig. 4). Rats with stimulated substantia grisea centralis showed only a slight and insignificant decrease in muricide behavior. It should be, however, noted that muricide behavior was significantly reduced even when the animals had undergone only the control procedure, i.e. were treated similarly to stimulated groups except for stimulation (see methods). This effect was observed only during the sham-stimulation procedure but not 1 hr later (Fig. 4).

Effects of Drugs Both quipazine and compound CGP 6085-A reduced muricide behavior. The effect was dose-dependent (Fig. 5). The maximal inhibition of muricide behavior occurred I hr

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FIG. 6. Time-course effect of quipazine (10.0 mg/kg) and CGP (5.0 mg/kg) on muricide behavior in rats. Ordinate-percent of animals showing inhibition of muricide, abscissa--time after injection in hr. Injection of saline failed to reduce muricide. The values obtained 1 hr after injection differ from saline-treated animals (p<0.05 according to x2test).

after injection whereas 48 hr later all animals again became killers (Fig. 6). Injection of saline failed to produce any change in fixed muricide pattern. DISCUSSION

The results of this study indicate that serotonergic inhibition of predatory aggression in rats is at least partially related to the DR nucleus. The 5-HT fibres arising from cells located in the DR form the system that innervates cortical areas, the amygdala, the striatum as well as some hypothalamic and thalamic areas and is called the mesostriatal 5-HT system

164

KOSTOWSKI E T A L .

[3,6]. The second 5-HT ascending pathway is called the mesolimbic pathway and originates in the median raphe nucleus (MR). This system innervates mostly the hippocampus and the septal area [3,6]. Several findings indicate that DR and MR function differentally in regulation of various behavioral patterns including aggression. Jacobs and Cohen [7] found that lesions of the DR produced a long-lasting increase in pain-elicited aggression in rats whereas lesions of the MR were without effect. Moreover, lesions confined to the DR have been reported to produce muricide behavior in naturally non-killers whereas lesions to the MR did not produce attack (Waldbillig, [26]). Our preliminary data (Pucilowski and Kostowski, to be published) indicate that electrical stimulation of the MR was insufficient to produce inhibition of muricide behavior. It seems, therefore, that 5-HT-induced inhibition of aggressive behavior is related to the DR system but not to the MR system. The neurophysiological mechanism of the antiaggressive effect induced by DR stimulation is unknown. It is commonly believed that brain 5-HT neurons play an important role in mediating various inhibitory influences. Lesions of the raphe nuclei, particularly the MR induce a persistent locomotor hyperactivity and EEG desynchronization in rats and cats [6, 7, 10, 12, 21]. Stimulation of the MR as well as the DR has been found to induce clear sedation in rats accompanied by an increase in turnover of forebrain 5-HT [5,12]. There is evidence that thte 5-HT raphe system might have an inhibitory control over the performance of conditioned avoidance reflex [15]. It has also been reported that stimulation of the DR produces analgesia and inhibition of afferent input in the spinal cord [18]. The inhibitory role of the DR has also been found in the relay neurons of the spinal trigeminal nucleus [19]. Wang and Aghajanian found an in-

hibition of neurons in the amygdala by DR stimulation [28]. It is possible that this mechanism may have an important inhibitory influence on aggressive behavior. According to Waldbling [26] the occurrence of the muricide effect following destruction of the dorsal raphe in rats may be due to the loss of 5-HT input to the amygdala. Our results indicate that stimulation of total brain 5-HT neurons by pharmacological methods produces significant suppression of muricide behavior. It should be mentioned that quipazine has been shown to block 5-HT uptake and activate central 5-HT receptors [8]. CGP 6085-A (4-5,6dimethyl-2-benzofuranyl-piperidine)is a potent and selective inhibitor of 5-HT uptake with no effect on noradrenaline uptake [27]. In conclusion, our results confirm the hypothesis that 5-HT is preponderantly inhibitory to predatory aggression. Most of the data supporting this hypothesis are based on experiments in which the 5-HT action is blocked pharmacologically or due to lesions of brain 5-HT neurons [1, 2, 4, 7, 9, 22-26]. Up to date there was practically no evidence that electrical stimulation of discrete brain regions containing 5-HT cell bodies may suppress this type of aggression. Our data provide direct evidence for 5-HT mediated inhibitory action upon predatory aggression and indicate that this inhibition may derive from the DR. ACKNOWLEDGMENTS This study was supported by Polish Academy of Sciences, Grant No. 10.4. We would like to thank Mrs. Katarzyna Staszewska for excellent technical assistance. We are indebted to Ciba-Geigy AG, Research Department, Pharmaceutical Division, Basle for the generous gift of CGP 6085-A.

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