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Brain serotonergic neurons: Their role in a form of dominance-subordination behavior in rats
Physiology& Behavior,Vol. 33, pp. 365-371. Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/84 $3.00 + .00
Brain Serotonergic Neurons: Their Role in a Form of Dominance-Subordination Behavior in Rats WOJCIECH
KOSTOWSKI, MAILGORZATA PLEWAKO AND ANDRZEJ BIDZINSKI*
Department o f Pharmacology and Physiology o f the Nervous System and *Department o f Biochemistry, Psychoneurological Institute 02-957 Warszawa, Poland R e c e i v e d 31 M a r c h 1983 KOSTOWSKI, W., M. PLEWAKO AND A. BIDZINSKI. Brain serotonergic neurons: Their role in a form of dominance-subordination behavior in rats. PHYSIOL BEHAV 33(3) 365-371, 1984.---The present study evaluated the possible role of brain serotonergic neurons in dominant-subordinate (D-S) behavior in Wistar male rats competing for water. Treatment of D rat with drugs that stimulate serotonergic neurons of receptors (tryptophan, 5-hydroxytryptophan, quipazine, femoxetine) resulted in D-S reversal. A similar effect was observed when the S animal was treated with drugs that blocked serotonin synthesis (p-chlorophenylalanine) or receptors (metergoline). The D-S relationship was unchanged when serotonergic drugs were given to the S subject (tryptophan or qulpazine) or when D animal received p-chlorophenylalanine. None of the drugs tested influence the water intake and the general activity of rats. Rats with lesioned midbrain raphe nuclei were always dominant when paired with sham lesioned counterparts. Our results indicate that one form of dominance behavior can be inversely related to the activity of brain 5-HT system. Dominance behavior
Brain serotonin
Raphe neurons
BRAIN serotonergic (5-HT) neurons are supposed to play an inhibitory role in various behavioral processes such as locomotor activity [ 11], acquisition of conditioned tasks [14,18] and aggressive behavior [22]. Lesions of the raphe nuclei, the area containing 5-HT cell bodies, have been reported to produce locomotor excitation [5, 11, 15, 21], facilitation of avoidance acquisition [15, 16, 18] and increased irritability and aggressivity in laboratory animals [3, 6, 11, 24, 25]. On the other hand 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, reduces aggresiveness in a variety of experimental conditions [20,23]. There is not, however, sufficient current data on the potential role of the brain 5-HT system in social dominance hierarchies. This problem is of particular interest as it might demonstrate a potential relationship between mental disorders and the mode o f action of psychotropic drugs. Dominance and subordination involve a complex behavioral pattern displayed by animals competing for food, water, territory, etc. The present study was designed to test the effects o f drugs influencing serotonergic neurotransmission on dominant-subordinate behavior in rats competing for water. In addition, the effects of lesions of the raphe nuclei on this behavioral pattern were studied.
oratory conditions with 12 hr light/dark cycle (light on 7.0019.00). Granulated food was available ad lib, but the access to tap water was limited to one 60 min period in 24 hr. The experimental apparatus was a box (30x30x45 cm) containing the drinking tube constructed in such a manner that only one animal could drink water at the same time. During the pretraining period the rats were singly placed into the apparatus for 5 min in order to habituate them to the experimental procedure and eliminate the " p o o r drinkers" i.e., the animals that drunk water for less than 3 min during 5 min of observation. The animals were then randomly paired, placed into the apparatus and tested for 5--6 consecutive days (one 5 min observation was performed each day). The rats were paired during the 5 rain dominance test and then returned to their individual house cages. During this portion (the control period) of the experiment the dominance-subordination relationship in pairs of rats competing for water developed. That resulted subsequently in the dominant (D) rat drinking water for a longer period of time than its submissive (S) counterpart. In the experimental part (test period) groups of pairs of rats were given different treatment for 3-4 consecutive days (see Drug Testing Procedure). Actual treatment was preceded by one day of saline administration.
METHOD
Water Intake
Animals and Experimental Procedure
The effects of drugs on water consumption were evaluated in parallel experiments. The rats were kept in similar conditions as described before but with free access to water
Male Wistar rats with an initial body weight of 180___10 g were kept in separate cages (20×20x30 cm) in standard lab-
365
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K O S T O W S K I , P L E W A K O AND BIDZINSKI
and granulated food. The mean amount of water drunk (in ml) in 24 hr was measured during 5-6 days of treatment (for dosage see Drug Testing Procedure).
Open Field Test In order to assess the effects of drugs and the lesions alone on the animals' gross behavior, the open field test was conducted in a separate set of experiments. Rats with the lesioned raphe nuclei and sham lesioned animals were submitted to the procedure 10-11 days after surgery and then after 3-4 days were tested for dominance behavior. The open field was 60×60 cm arena divided into 16 squares (12 " p e ripheral" and 4 " c e n t r a l " ones) surrounded with a 30 cm high wall. The number of entries into peripheral squares and rearings during 5 min of observation was noted. All behavioral experiments were always performed between 10:00 and 15:00 hours.
s:pCPA OQO0
40
Brain Lesions The effects of lesions of the midbrain raphe nuclei on dominance behavior were studied in a separate experiment. Electrolytic lesions were produced stereotaxically in the median (MR) or dorsal (DR) raphe nuclei according to the coordinates of KCinig and Klippel's stereotaxic atlas [9]. The coordinates were: A = 0 . 4 mm, L = 0 . 0 mm, H = 1.8 mm (for MR) and 2.6 mm (for DR) above the interaural line. The rats were anaesthetized with chloral hydrate (400 mg/kg), their heads were positioned in the stereotaxic apparatus and two mA DC current was passed through the intracranial anode made from stainless steel wire (0.25 mm in dia) insulated except for the tip. Sham operated animals were treated in the same manner except that the electrode was not lowered into their brains. Two weeks after the operation, lesioned and shamlesioned rats were paired and tested for the dominancesubordination relationship.
Biochemical and Histological Analyses After the completion of the experiment, the rats with the lesioned raphe nuclei were killed by decapitation, their brains were quickly removed and dissected by pre-collicular section caudally to the hypothalamus to remove the forebrain and brain stem. The brainstems were checked histologically after fixation in 10 percent formalin and the sections (10 ttm) were stained with haematoxylin and eosin. Biogenic amine levels were measured fluorimetrically in the forebrain according to Haubrich and Denzer [4] with the modification of Korf and Sebens [10]. In addition, the rats treated with either p-chiorophenylalanine or 5-HTP were checked for the whole brain 5-HT concentration.
Drug Testing Procecure All drugs were given intraperitoneally once daily usually 1 hr before the test session in selected pairs with a well established D-S relationship. The dosage was as follows: p-Chlorophenylalanine methyl ester (Koch Light Laboratories) 300 mg/kg (first day) and 100 mg/kg (second day), d-I 5-hydroxytryptophan (Reanal, Budapest) 50 mg/kg, d-I tryptophan (Serva, FRG) 100 mg/kg, quipazine maleate (Miles Laboratories) 2.5 mg/kg given 30 rain prior to test session, femoxetine HCI (Ferrosan) 4.0 mg/kg, metergoline HC! (Farmitalia) 2.0 mg/kg. Dominant and submissive rats were submitted to different treatments (see Figs. 1-3).
E'i
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FIG. 1. Effects of drugs on dominance-submission relationship in rats. Ordinate--drinking time (expressed as mean percentage drinking time for submissive animals); abscissa--day of experiment. Interrupted line--treatment with saline, solid line--treatment with drugs (d---dominant, s--submissive). TRY--trypthophan, pCPA-p-C hlorophenylalanine, 5-HTP--5-hydroxytryptophan, SAL-saline. Statistical analysis--d: TRY, s: PCPA (n=9), F-8.64, p<0.05; d: TRY, s: SAL, (n=8), F= I 1.94,p<0.01; d: SAL, s: pCPA (n=6) F=2.84, p<0.01 ; d: 5-HTP, s: SAL (n =8), F=26.45, p<0.01.
Statistical Analysis The D-S relationship was analysed using the analysis of variance (treatment x group x days). The biochemical data were analysed using a two-tailed Student's t-test while a two-tailed Mann-Whitney U test was employed for the analysis of activities in the open field test as well as for the evaluation of water intake. RESULTS The results showed that a well-established D-S relationship occurred during the test period in approximately 70 percent of pairs of rats. In the remaining pairs the social dominance hierarchy was not stable, both animals were either mutually coactive or strongly competitive and these pairs were excluded from the experiment. The D-S relationship was abolished by the treatment of the dominant rats with
BRAIN SEROTONERGIC N E U R O N S
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FIG. 3. Effect of drugs on dominance-submission behavior in rats (for explanations see Fig. 1 and Fig. 2). Statistical analysis: d: pCPA, s: SAL (n=8), F=2.09, p<0.1; d: SAL, s: QUIP (n=7), F=1.53, p<0.2; d: SAL, s: TRY (n=7), F= 1.65, p<0.2.
FIG. 2. Effects of drugs on dominance-submission relationship. QUIP quipazine, MET--metergoline, F--femoxetine (other explanation as in Fig. 1). Statistical analysis---d: QUIP, s: MET (n=8), F=45.06,p<0.01; d: QUIP, s: SAL (n=7), F=6.59,p<0.01; d: F, s: MET (n=6), F=2.22, p<0.05.
drugs that enhanced 5-HT neurotransmission (e.g., tryptophan, 5-HTP, quipazine, femoxetine). This effect occurred if the S animal received either drugs that reduced 5-HT neurotransmission (p-chlorophenylalanine, metergoline) or saline (see Fig. 1 and Fig. 2). The reversal of the D-S relationship occurred also when the S animals received p-chlorophenylalanine and the D rats were treated only with saline (Fig. 1). The "reversal" of the D-S relationship made the D rat drink water for a shorter period of time than the S animal. On the other hand, no change in the D-S relationship occurred when the S animals received drugs that enhanced 5-HT neurotransmission (tryptophan, quipazine) and the D rats received saline. Similarly, no change in D-S relationship was observed when the D rats received p-chlorophenylalanine and the S rats received saline (Fig. 3). None of the drugs used in this study influenced water consumption (Table 1). The results indicate also that no change in the animals' behavior in the open field occurred following the treatment with drugs administered in doses identical with those used for the dominance test. The only exception was p-chlorophenylalanine which produced a slight decrease in gross activity (Table 2). When raphe-lesioned and sham-lesioned rats were .l~aired the lesioned subject always became dominant. Lesions of the MR were more potent in producing dominance behavior than lesions involving the DR (Fig. 4). When tested in the open
TABLE 1 EFFECTS OF TREATMENTAND LESIONS ON WATER CONSUMPTION IN RATS Experimental group Saline (8) Metergoline (7) pCPA (7) Tryptophan (8) 5-HTP (6) Femoxetine (6) Sham-lesion (7) MR lesion (6) DR lesion (6)
Mean water intake in ml/24 hr 30.9 _+ 4.1 29.6 _+ 4.2 30.9 _+ 3.8 27.2 _+ 4.5 29.9 _+ 5.2 27.8 _+ 5.0 35.2 _+ 5.2 47.7 _+ 4.0* 36.0 _+ 3.8
Number of animals in parentheses. For daily doses of drugs see Drug Testing Procedure. Drugs were given once daily for 6 consecutive days, pCPA (p-chlorophenylalanine) was given at 300 mg/kg (lst day) and 100 mg/kg (2nd day) and water consumption was measured for 5 consecutive days starting from 2nd day of treatment. The water intake of lesioned rats was measured during 6 consecutive days starting from the 10th post-operative day. *p<0.05 in respect to sham-lesioned group (Mann and Whitney U test, two tailed). Data are expressed as mean _+ SEM.
368
KOSTOWSKI, PLEWAKO AND BIDZINSKi TABLE 2 EFFECTS OF DRUGS AND LESIONS TO T H E R A P H E N U C L E I ON RATS BEHAVIOR IN T H E OPEN F I E L D
Behavior in the open field Experimental group Saline (10) Metergoline (8) Quipazine (7) Tryptophan (8) pCPA (8) Sham-lesion (13) MR lesion (12) DR lesion (8)
A 4.6 4.5 6.1 3.2 2.8 2.5 45.5 6.7
B
± 1.8 + 2.0 ± 1.8 _+ 1.3 ± 1.0 ± 1.8 ± 12.3t ± 4.3
32.1 ± 28.0 ± 30.5 ± 25.2 ± 20.0 ÷ 22.7 ± 176.0 ± 36.3 ±
C 14.8 5.5 4.7 6.5 5.5* 6.9 26.5~ 11.0
15.4 ± 10.1 ± 10.5 ± 9.2 ± 9.1 ± 4.8 ± 8.7 + 7.8 +
7.2 4.1 5.1 3.1 2.8 2.5 1.6" 3.4
Number of anmals in parentheses, A--number of entries into central squares, B--number of entries into peripheral squares, C--number of rears. Data are expressed as mean _+ SEM. *p<0.05, tp<0.02 in respect to saline group or sham lesioned group, respectively.
field, the MR-lesioned animals showed increased locomotor activity (increased number of entries into peripheral and central squares) and increased number of rears. On the other hand rats with the lesioned DR did not differ from the shamlesioned group (Table 2). Lesions of the MR slightly but significantly increased water consumption while lesions of the DR produced no effect (Table 1). Histological examination showed that lesions involved mainly the MR or the DR and in some rats partially destroyed the superior cerebellar peduncle and the mesencephalic reticular formation (when the MR was lesioned) or small portions of the central gray substance (when the DR was destroyed). In four rats lesions were incorrectly situated and the results from pairs including these subjects were not further evaluated. Typical lesions are shown in Fig. 5. Biochemical analysis showed decreased forebrain 5-HT and 5-hydroxyindoleacetic acid concentrations in the raphe lesioned animals. No change in noradrenaline and dopamine concentrations were observed (Table 3). The whole brain 5-HT concentrations in the rats treated with p-chlorophenylalanine were markedly reduced. On the other hand, the whole brain 5-HT levels in the animals receiving 5-HTP were significantly increased. The mean value in control rats (receiving 0.1 ml of saline IP l hr prior to decapitation) was 512_+31 ng/g (n=9) while in rats treated with p-chlorophenylalanine (300 mg/kg IP 48 hr and 100 mg/kg 24 hr before testing) it was 66.0+-8.0 ng/g (n=9, p<0.01). The mean value in the rats receiving a single dose of 5-HTP (50 mg/kg IP 2 hr prior to testing) it was 885+-26 ng/g (n= 12, p<0.05). DISCUSSION Our results indicate that the D-S relationship in rats competing for water can be influenced by drugs acting upon serotonergic neurotransmission in the central nervous system. The reversal of dominance occurred when D-animal was treated with drugs enhancing 5-HT neurotransmission such as tryptophan and 5-HTP (precursors of brain 5-HT) and quipazine, an agonist of 5-HT receptors [7] as well as femoxetine, a selective inhibitor of 5-HT neuronal uptake
time
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FIG. 4. Effect of lesions of the midbrain raphe nuclei (R) on dominance-subordinationbehavior in rats. White columns--sham lesioned rats, black columns--raphe-lesioned rats (upper figure-MR lesions, lower figure--DR lesions), Ordinate,--time of drinking in seconds; abscissa--day of the experiment, mean values from 8-12 pairs of rats. The values of drinking time for lesioned animals differ from the corresponding controls at the level of p<0.01 (the only exception was the day 3 in DR lesioned rats, p=0.32). Mann and Whitney two tailed test.
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BRAIN SEROTONERGIC NEURONS
TABLE 3 EFFECT OF LESIONS OF THE MIDBRAIN RAPHE NUCLEI ON BIOGENIC AMINE FOREBRAIN CONCENTRATIONS Forebrain concentrations ng/g Experimental group Sham lesion (13) MR lesion (12) DR lesion (8)
5-HT
5-HIAA
NE
DA
470 _+ 25 331 -+ 42t 185 _+ 36~
365 -+ 41 299 _+ 35t 301 _+ 26*
385 -+ 65 334 +_ 32 447 _+ 67
2120 -+ 131 2132 -+ 245 2162 -+ 287
Number of rats in parentheses. 5-HT--serotonin, 5-HIAA--5-hydroxyindole acetic acid, NE--noradrenaline, DA---dopamine. *,o<0.05, tp<0.02, Cp<0.01 in respect to sham lesioned group. Data are expressed as mean _+ SEM.
FIG. 5. Typical lesion involving the median raphe nucleus (upper) and the dorsal raphe nucleus (lower), frontal sections stained with H-E.
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KOSTOWSKI, PLEWAK() AND BIDZINSKI
[2]. This effect occurred when the S-animal was treated either with saline or drugs reducing 5-HT neurotransmission such as p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase 181 and metergoline, a relatively selective and long-acting 5-HT receptor blocking agent [1]. Even when only the S animal received p-chlorophenylalanine and D subject received saline, the reversal of dominance was observed. The D-S relationship remained unchanged when serotonergic agents were administered to the S subject or when the D animal received p-chlorophenylalanine. The result indicates that dominance behavior is inversely related to the activity of the brain 5-HT system. In other words, the animals with pharmacologically increased 5-HT neurotransmission dominate over those with normal (saline treated) or enhanced activity of 5-HT neurotransmission (treated with serotoninomimetics). It is of particular importance that none of the drugs tested (at least at the doses used in this study) influenced the water intake. This indicates that simple thirst could not account for the action of drugs upon the D-S relationship. This is probably true also for locomotion and general activity of animals since most of the drugs used in our experiments failed to influence the animals' behavior in the open field. Neither metergoline nor quipazine changed the locomotor activity and orienting behavior (rearing). Similarly, tryptophan failed to change animals behavior while p-chlorophenylalanine produced slight sedation. Rats with the lesioned brain 5-HT neurons always become dominant when paired with sham-lesioned subjects. Lesion of the MR induced stronger effect than lesion of the DR. However, lesions involving the MR produced behavioral excitation as shown in the open field test and slightly but significantly increased water consumption. It is, therefore, possible that these mechanisms account for the change in the dominance produced by MR lesion. By contrast, lesions of the DR which also produced a significant decrease in the forebrain 5-HT concentration failed to influence water consumption and only slightly stimulated activity of rats in the open field. Interestingly enough, dominance was markedly increased by p-chlorophenylalanine, a compound that failed to stimulate general activity in the open field and water consumption in the rats. Moreover, biochemical analysis showed dramatically reduced brain 5-HT concentrations in
animals treated with p-chlorophenylalanine. It seems, therefore, unlikely that an impairment of central serotonergic mechanisms enhances dominance simply due to stimulation of general activity and for an increase in the motivation to drink. The precise mechanisms underlying the stimulatory effect upon dominance which occurs following both pharmacological and surgical impairment of the 5-HT neurons remain to be identified. The data obtained in the present study should be discussed together with other results showing increased irritability and aggressiveness in animals with destroyed 5-HT neurons [3, 6, 12, 23, 25] and pharmacologically-induced suppression of 5-HT synthesis [19,20]. It must be, however, pointed out that dominance is not simply connected with increased aggressivity. Both terms denote a variety of forms of aggression and domination which occur in different situations. In the extention to the findings mentioned above, the result of this study indicates that the D-S relationship in rats competing for water seems to be also closely associated with the brain 5-HT neurons. Obviously, the mechanism of dominance is not simply related merely to serotonergic neurons and other transmitter systems are involved in the mechanism of this behavioral phenomenon. It is commonly accepted that changes in one transmitter system lead to the alterations in other transmitter systems. For example, lesions of the 5-HT neurons have been reported to increase the activity of noradrenergic neurons and modulate response to drugs acting upon dopaminergic neurotransmission (see 112]). The results of the study have also implication for research on mechanism of depression and should be also discussed in terms of "antidepressive'" efficacy of antiserotonergic drugs and other treatments leading to the reduced brain 5-HT neurotransmission [13,17]. Recently we have found that social dominance produced in rats competing for food may also provide a suitable method for the selection of antidepressive drugs [ 14]. ACKNOWLEDGMENTS This paper was supported by Polish Academy of Sciences, Grant Nr 10.4. The authors are grateful to Ms. Anna Reszka and Ms. Elzbieta Zawadzka for technical assistance.
REFERENCES 1. Baenninger, L. P. Social dominance orders in the rat. J Comp Physiol Psychol 71: 202-209, 1970. 2. Beretta, C. R., R. Ferrini and A. H. Glasser. lMethyl-8-carbobenzyloxyaminomethyl-10-ergoline, a potent and long lasting 5-hydroxytryptamine antagonist. Nature 207: 421-422, 1965. 3. Buss-Lassen, J., R. F. Suires, J. A. Christensen and L. Molander. Neurochemical and pharmacological studies on a new 5-HT uptake inhibitor, FG 4963 with potential antidepressant properties. Psychopharmacologia 42: 21-26, 1976. 4. Haubrich, D. and J. Denzer. Simultaneous extraction and fluorimetric measurement of brain serotonin, catecholamines, 5-hydroxyindoleacetic acid and homovanillic acid. Anal Biochem 55: 306-312, 1974. 5. Jacobs, B. L., W. D. Wise and K. M. Taylor. Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats. Brain Res 79: 353-362, 1974. 6. Jacobs, B. L. and A. Cohere. Differential behavioral effects of lesions of the median dorsal raphe nuclei in rats: open field and pain-elicited aggression. J Comp Physiol Psychol 46: 102112, 1976.
7. Jacoby, J. H., R. A. Howd, M. S. Levin and R. J. Wurtman. Mechanism by which quipazine, a putative serotonin receptor agonist alters brain 5-hydroxyindole metabolism. Neuropharmacology 15: 52%534, 1976. 8. Koe, B. and A. Weissman. p-Chlorophenylalanine: A specific depletor of brain serotonin. J Pharmaeol Exp Ther 154:49%516, 1966. 9. K6nig, J. T. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Part o f the Brain Stem. Baltimore: Williams and Wilkins, 1976. 10. Korf, J. and B. J. Sebens. Determination of o-conjugated of 5-hydroxytryptamine in human urine. Clin Chem Aeta 27: 149153, 1970. 11. Kostowski, W., E. Giacalone, S. Garattini and L. Valzelli. Studies on behavioral and biochemical changes in rats after lesion of midbrain raphe. Eur J Pharmacol 4: 371-376, 1968. 12. Kostowski, W. Brain serotonergic and catecholamine systems: Facts and hypothesis. In: Current Deveh)pments in Psychopharmacology, vol 1, edited by W. B. Essman and L. Valzelli. New York: Spectrum Publications Inc., 1975, pp. 37-64.
BRAIN SEROTONERGIC NEURONS 13. Kostowski, W. Brain noradrenaiine, depression and antidepressive drugs: Facts and hypothesis. Trends Pharmacol Sci 2: 314-317, 1981. 14. Kostowski, W. and E. Malatyfiska. Clonidine model of depression. In: Second Joint Symposium, Soc o f Pharmacol o f GDR and Polish Pharmacological Society. Abstracts, Ahrenshoop 27-28 Oct. 1981, Humboldt University Berlin 1981. 15. Lorens, S. A., H. C. Guldberg, K. Hole, Ch. K6chler and B. Srebro. Activity, avoidance learning and regional 5-hydroxytryptamine following intra-brain stem 5,7-dihydroxytryptamine and electrolytic midbrain raphe lesions in the rat. Brain Res 108: 97-113, 1976. 16. Lorens, S. A. and L. M. Yunger. Morphine analgesia, two-way avoidance and consumatory behavior after lesions of the midbrain raphe nuclei of the rat. Pharmacol Biochem Behav 2: 215-221, 1973. 17. Maj, J. Antidepressant drugs: Will new findings change the present theories of their action? Trends Pharmacol Sei 2: 80-82, 1981. 18. Pla~nik, A., W. Kostowski, A. Bidzinski and M. Hauptmann. Effects of lesions of the midbrain raphe nuclei on avoidance in rats. Physiol Behav 24: 257-262, 1980.
371 19. Sheard, M. H. The effect of p-chlorophenylalanine on behavior in rats: Relation to brain serotonin and 5-hydroxyindoleacetic acid. Brain Res 15: 524--528, 1969. 20. Sheard, M. H. and M. Davies. Shock elicited fighting in rats: Importance of intershock interval upon the effect of p-chlorophenylalanine (pCPA). Brain Res 111: 433-437, 1976. 21. Srebro, B. and S. A. Lorens. Behavioral effects of selective midbrain raphe lesions in the cat. Brain Res 89: 303-325, 1975. 22. Valzelli, L. Psychobiology o f Aggression and Violence. New York: Raven Press, 1981, pp. 98-103. 23. Vergnes, M., G. Mack and E. Kempf. L~sions du raph~ et r~action d'agression interspecifique rat-souries. Effets comportmentaoux et biochimiques. Brain Res 57: 67-74, 1973. 24. Welch, B. L. and A. S. Welch. Aggression and the biogenic amine neurohumors. In: Aggressive Behavior, edited by S. Garattini and B. Sigg. Amsterdam: Excerpta Medica, 1969, pp. 188--202. 25. Yamamoto, T. and S. Ueki. Characteristics in aggressive behavior induced by midbrain raphe lesions in rats. Physiol Behav 19: 103-110, 1977.
0031-9384/84 $3.00 + .00
Brain Serotonergic Neurons: Their Role in a Form of Dominance-Subordination Behavior in Rats WOJCIECH
KOSTOWSKI, MAILGORZATA PLEWAKO AND ANDRZEJ BIDZINSKI*
Department o f Pharmacology and Physiology o f the Nervous System and *Department o f Biochemistry, Psychoneurological Institute 02-957 Warszawa, Poland R e c e i v e d 31 M a r c h 1983 KOSTOWSKI, W., M. PLEWAKO AND A. BIDZINSKI. Brain serotonergic neurons: Their role in a form of dominance-subordination behavior in rats. PHYSIOL BEHAV 33(3) 365-371, 1984.---The present study evaluated the possible role of brain serotonergic neurons in dominant-subordinate (D-S) behavior in Wistar male rats competing for water. Treatment of D rat with drugs that stimulate serotonergic neurons of receptors (tryptophan, 5-hydroxytryptophan, quipazine, femoxetine) resulted in D-S reversal. A similar effect was observed when the S animal was treated with drugs that blocked serotonin synthesis (p-chlorophenylalanine) or receptors (metergoline). The D-S relationship was unchanged when serotonergic drugs were given to the S subject (tryptophan or qulpazine) or when D animal received p-chlorophenylalanine. None of the drugs tested influence the water intake and the general activity of rats. Rats with lesioned midbrain raphe nuclei were always dominant when paired with sham lesioned counterparts. Our results indicate that one form of dominance behavior can be inversely related to the activity of brain 5-HT system. Dominance behavior
Brain serotonin
Raphe neurons
BRAIN serotonergic (5-HT) neurons are supposed to play an inhibitory role in various behavioral processes such as locomotor activity [ 11], acquisition of conditioned tasks [14,18] and aggressive behavior [22]. Lesions of the raphe nuclei, the area containing 5-HT cell bodies, have been reported to produce locomotor excitation [5, 11, 15, 21], facilitation of avoidance acquisition [15, 16, 18] and increased irritability and aggressivity in laboratory animals [3, 6, 11, 24, 25]. On the other hand 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, reduces aggresiveness in a variety of experimental conditions [20,23]. There is not, however, sufficient current data on the potential role of the brain 5-HT system in social dominance hierarchies. This problem is of particular interest as it might demonstrate a potential relationship between mental disorders and the mode o f action of psychotropic drugs. Dominance and subordination involve a complex behavioral pattern displayed by animals competing for food, water, territory, etc. The present study was designed to test the effects o f drugs influencing serotonergic neurotransmission on dominant-subordinate behavior in rats competing for water. In addition, the effects of lesions of the raphe nuclei on this behavioral pattern were studied.
oratory conditions with 12 hr light/dark cycle (light on 7.0019.00). Granulated food was available ad lib, but the access to tap water was limited to one 60 min period in 24 hr. The experimental apparatus was a box (30x30x45 cm) containing the drinking tube constructed in such a manner that only one animal could drink water at the same time. During the pretraining period the rats were singly placed into the apparatus for 5 min in order to habituate them to the experimental procedure and eliminate the " p o o r drinkers" i.e., the animals that drunk water for less than 3 min during 5 min of observation. The animals were then randomly paired, placed into the apparatus and tested for 5--6 consecutive days (one 5 min observation was performed each day). The rats were paired during the 5 rain dominance test and then returned to their individual house cages. During this portion (the control period) of the experiment the dominance-subordination relationship in pairs of rats competing for water developed. That resulted subsequently in the dominant (D) rat drinking water for a longer period of time than its submissive (S) counterpart. In the experimental part (test period) groups of pairs of rats were given different treatment for 3-4 consecutive days (see Drug Testing Procedure). Actual treatment was preceded by one day of saline administration.
METHOD
Water Intake
Animals and Experimental Procedure
The effects of drugs on water consumption were evaluated in parallel experiments. The rats were kept in similar conditions as described before but with free access to water
Male Wistar rats with an initial body weight of 180___10 g were kept in separate cages (20×20x30 cm) in standard lab-
365
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K O S T O W S K I , P L E W A K O AND BIDZINSKI
and granulated food. The mean amount of water drunk (in ml) in 24 hr was measured during 5-6 days of treatment (for dosage see Drug Testing Procedure).
Open Field Test In order to assess the effects of drugs and the lesions alone on the animals' gross behavior, the open field test was conducted in a separate set of experiments. Rats with the lesioned raphe nuclei and sham lesioned animals were submitted to the procedure 10-11 days after surgery and then after 3-4 days were tested for dominance behavior. The open field was 60×60 cm arena divided into 16 squares (12 " p e ripheral" and 4 " c e n t r a l " ones) surrounded with a 30 cm high wall. The number of entries into peripheral squares and rearings during 5 min of observation was noted. All behavioral experiments were always performed between 10:00 and 15:00 hours.
s:pCPA OQO0
40
Brain Lesions The effects of lesions of the midbrain raphe nuclei on dominance behavior were studied in a separate experiment. Electrolytic lesions were produced stereotaxically in the median (MR) or dorsal (DR) raphe nuclei according to the coordinates of KCinig and Klippel's stereotaxic atlas [9]. The coordinates were: A = 0 . 4 mm, L = 0 . 0 mm, H = 1.8 mm (for MR) and 2.6 mm (for DR) above the interaural line. The rats were anaesthetized with chloral hydrate (400 mg/kg), their heads were positioned in the stereotaxic apparatus and two mA DC current was passed through the intracranial anode made from stainless steel wire (0.25 mm in dia) insulated except for the tip. Sham operated animals were treated in the same manner except that the electrode was not lowered into their brains. Two weeks after the operation, lesioned and shamlesioned rats were paired and tested for the dominancesubordination relationship.
Biochemical and Histological Analyses After the completion of the experiment, the rats with the lesioned raphe nuclei were killed by decapitation, their brains were quickly removed and dissected by pre-collicular section caudally to the hypothalamus to remove the forebrain and brain stem. The brainstems were checked histologically after fixation in 10 percent formalin and the sections (10 ttm) were stained with haematoxylin and eosin. Biogenic amine levels were measured fluorimetrically in the forebrain according to Haubrich and Denzer [4] with the modification of Korf and Sebens [10]. In addition, the rats treated with either p-chiorophenylalanine or 5-HTP were checked for the whole brain 5-HT concentration.
Drug Testing Procecure All drugs were given intraperitoneally once daily usually 1 hr before the test session in selected pairs with a well established D-S relationship. The dosage was as follows: p-Chlorophenylalanine methyl ester (Koch Light Laboratories) 300 mg/kg (first day) and 100 mg/kg (second day), d-I 5-hydroxytryptophan (Reanal, Budapest) 50 mg/kg, d-I tryptophan (Serva, FRG) 100 mg/kg, quipazine maleate (Miles Laboratories) 2.5 mg/kg given 30 rain prior to test session, femoxetine HCI (Ferrosan) 4.0 mg/kg, metergoline HC! (Farmitalia) 2.0 mg/kg. Dominant and submissive rats were submitted to different treatments (see Figs. 1-3).
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FIG. 1. Effects of drugs on dominance-submission relationship in rats. Ordinate--drinking time (expressed as mean percentage drinking time for submissive animals); abscissa--day of experiment. Interrupted line--treatment with saline, solid line--treatment with drugs (d---dominant, s--submissive). TRY--trypthophan, pCPA-p-C hlorophenylalanine, 5-HTP--5-hydroxytryptophan, SAL-saline. Statistical analysis--d: TRY, s: PCPA (n=9), F-8.64, p<0.05; d: TRY, s: SAL, (n=8), F= I 1.94,p<0.01; d: SAL, s: pCPA (n=6) F=2.84, p<0.01 ; d: 5-HTP, s: SAL (n =8), F=26.45, p<0.01.
Statistical Analysis The D-S relationship was analysed using the analysis of variance (treatment x group x days). The biochemical data were analysed using a two-tailed Student's t-test while a two-tailed Mann-Whitney U test was employed for the analysis of activities in the open field test as well as for the evaluation of water intake. RESULTS The results showed that a well-established D-S relationship occurred during the test period in approximately 70 percent of pairs of rats. In the remaining pairs the social dominance hierarchy was not stable, both animals were either mutually coactive or strongly competitive and these pairs were excluded from the experiment. The D-S relationship was abolished by the treatment of the dominant rats with
BRAIN SEROTONERGIC N E U R O N S
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FIG. 3. Effect of drugs on dominance-submission behavior in rats (for explanations see Fig. 1 and Fig. 2). Statistical analysis: d: pCPA, s: SAL (n=8), F=2.09, p<0.1; d: SAL, s: QUIP (n=7), F=1.53, p<0.2; d: SAL, s: TRY (n=7), F= 1.65, p<0.2.
FIG. 2. Effects of drugs on dominance-submission relationship. QUIP quipazine, MET--metergoline, F--femoxetine (other explanation as in Fig. 1). Statistical analysis---d: QUIP, s: MET (n=8), F=45.06,p<0.01; d: QUIP, s: SAL (n=7), F=6.59,p<0.01; d: F, s: MET (n=6), F=2.22, p<0.05.
drugs that enhanced 5-HT neurotransmission (e.g., tryptophan, 5-HTP, quipazine, femoxetine). This effect occurred if the S animal received either drugs that reduced 5-HT neurotransmission (p-chlorophenylalanine, metergoline) or saline (see Fig. 1 and Fig. 2). The reversal of the D-S relationship occurred also when the S animals received p-chlorophenylalanine and the D rats were treated only with saline (Fig. 1). The "reversal" of the D-S relationship made the D rat drink water for a shorter period of time than the S animal. On the other hand, no change in the D-S relationship occurred when the S animals received drugs that enhanced 5-HT neurotransmission (tryptophan, quipazine) and the D rats received saline. Similarly, no change in D-S relationship was observed when the D rats received p-chlorophenylalanine and the S rats received saline (Fig. 3). None of the drugs used in this study influenced water consumption (Table 1). The results indicate also that no change in the animals' behavior in the open field occurred following the treatment with drugs administered in doses identical with those used for the dominance test. The only exception was p-chlorophenylalanine which produced a slight decrease in gross activity (Table 2). When raphe-lesioned and sham-lesioned rats were .l~aired the lesioned subject always became dominant. Lesions of the MR were more potent in producing dominance behavior than lesions involving the DR (Fig. 4). When tested in the open
TABLE 1 EFFECTS OF TREATMENTAND LESIONS ON WATER CONSUMPTION IN RATS Experimental group Saline (8) Metergoline (7) pCPA (7) Tryptophan (8) 5-HTP (6) Femoxetine (6) Sham-lesion (7) MR lesion (6) DR lesion (6)
Mean water intake in ml/24 hr 30.9 _+ 4.1 29.6 _+ 4.2 30.9 _+ 3.8 27.2 _+ 4.5 29.9 _+ 5.2 27.8 _+ 5.0 35.2 _+ 5.2 47.7 _+ 4.0* 36.0 _+ 3.8
Number of animals in parentheses. For daily doses of drugs see Drug Testing Procedure. Drugs were given once daily for 6 consecutive days, pCPA (p-chlorophenylalanine) was given at 300 mg/kg (lst day) and 100 mg/kg (2nd day) and water consumption was measured for 5 consecutive days starting from 2nd day of treatment. The water intake of lesioned rats was measured during 6 consecutive days starting from the 10th post-operative day. *p<0.05 in respect to sham-lesioned group (Mann and Whitney U test, two tailed). Data are expressed as mean _+ SEM.
368
KOSTOWSKI, PLEWAKO AND BIDZINSKi TABLE 2 EFFECTS OF DRUGS AND LESIONS TO T H E R A P H E N U C L E I ON RATS BEHAVIOR IN T H E OPEN F I E L D
Behavior in the open field Experimental group Saline (10) Metergoline (8) Quipazine (7) Tryptophan (8) pCPA (8) Sham-lesion (13) MR lesion (12) DR lesion (8)
A 4.6 4.5 6.1 3.2 2.8 2.5 45.5 6.7
B
± 1.8 + 2.0 ± 1.8 _+ 1.3 ± 1.0 ± 1.8 ± 12.3t ± 4.3
32.1 ± 28.0 ± 30.5 ± 25.2 ± 20.0 ÷ 22.7 ± 176.0 ± 36.3 ±
C 14.8 5.5 4.7 6.5 5.5* 6.9 26.5~ 11.0
15.4 ± 10.1 ± 10.5 ± 9.2 ± 9.1 ± 4.8 ± 8.7 + 7.8 +
7.2 4.1 5.1 3.1 2.8 2.5 1.6" 3.4
Number of anmals in parentheses, A--number of entries into central squares, B--number of entries into peripheral squares, C--number of rears. Data are expressed as mean _+ SEM. *p<0.05, tp<0.02 in respect to saline group or sham lesioned group, respectively.
field, the MR-lesioned animals showed increased locomotor activity (increased number of entries into peripheral and central squares) and increased number of rears. On the other hand rats with the lesioned DR did not differ from the shamlesioned group (Table 2). Lesions of the MR slightly but significantly increased water consumption while lesions of the DR produced no effect (Table 1). Histological examination showed that lesions involved mainly the MR or the DR and in some rats partially destroyed the superior cerebellar peduncle and the mesencephalic reticular formation (when the MR was lesioned) or small portions of the central gray substance (when the DR was destroyed). In four rats lesions were incorrectly situated and the results from pairs including these subjects were not further evaluated. Typical lesions are shown in Fig. 5. Biochemical analysis showed decreased forebrain 5-HT and 5-hydroxyindoleacetic acid concentrations in the raphe lesioned animals. No change in noradrenaline and dopamine concentrations were observed (Table 3). The whole brain 5-HT concentrations in the rats treated with p-chlorophenylalanine were markedly reduced. On the other hand, the whole brain 5-HT levels in the animals receiving 5-HTP were significantly increased. The mean value in control rats (receiving 0.1 ml of saline IP l hr prior to decapitation) was 512_+31 ng/g (n=9) while in rats treated with p-chlorophenylalanine (300 mg/kg IP 48 hr and 100 mg/kg 24 hr before testing) it was 66.0+-8.0 ng/g (n=9, p<0.01). The mean value in the rats receiving a single dose of 5-HTP (50 mg/kg IP 2 hr prior to testing) it was 885+-26 ng/g (n= 12, p<0.05). DISCUSSION Our results indicate that the D-S relationship in rats competing for water can be influenced by drugs acting upon serotonergic neurotransmission in the central nervous system. The reversal of dominance occurred when D-animal was treated with drugs enhancing 5-HT neurotransmission such as tryptophan and 5-HTP (precursors of brain 5-HT) and quipazine, an agonist of 5-HT receptors [7] as well as femoxetine, a selective inhibitor of 5-HT neuronal uptake
time
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0
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2
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FIG. 4. Effect of lesions of the midbrain raphe nuclei (R) on dominance-subordinationbehavior in rats. White columns--sham lesioned rats, black columns--raphe-lesioned rats (upper figure-MR lesions, lower figure--DR lesions), Ordinate,--time of drinking in seconds; abscissa--day of the experiment, mean values from 8-12 pairs of rats. The values of drinking time for lesioned animals differ from the corresponding controls at the level of p<0.01 (the only exception was the day 3 in DR lesioned rats, p=0.32). Mann and Whitney two tailed test.
369
BRAIN SEROTONERGIC NEURONS
TABLE 3 EFFECT OF LESIONS OF THE MIDBRAIN RAPHE NUCLEI ON BIOGENIC AMINE FOREBRAIN CONCENTRATIONS Forebrain concentrations ng/g Experimental group Sham lesion (13) MR lesion (12) DR lesion (8)
5-HT
5-HIAA
NE
DA
470 _+ 25 331 -+ 42t 185 _+ 36~
365 -+ 41 299 _+ 35t 301 _+ 26*
385 -+ 65 334 +_ 32 447 _+ 67
2120 -+ 131 2132 -+ 245 2162 -+ 287
Number of rats in parentheses. 5-HT--serotonin, 5-HIAA--5-hydroxyindole acetic acid, NE--noradrenaline, DA---dopamine. *,o<0.05, tp<0.02, Cp<0.01 in respect to sham lesioned group. Data are expressed as mean _+ SEM.
FIG. 5. Typical lesion involving the median raphe nucleus (upper) and the dorsal raphe nucleus (lower), frontal sections stained with H-E.
370
KOSTOWSKI, PLEWAK() AND BIDZINSKI
[2]. This effect occurred when the S-animal was treated either with saline or drugs reducing 5-HT neurotransmission such as p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase 181 and metergoline, a relatively selective and long-acting 5-HT receptor blocking agent [1]. Even when only the S animal received p-chlorophenylalanine and D subject received saline, the reversal of dominance was observed. The D-S relationship remained unchanged when serotonergic agents were administered to the S subject or when the D animal received p-chlorophenylalanine. The result indicates that dominance behavior is inversely related to the activity of the brain 5-HT system. In other words, the animals with pharmacologically increased 5-HT neurotransmission dominate over those with normal (saline treated) or enhanced activity of 5-HT neurotransmission (treated with serotoninomimetics). It is of particular importance that none of the drugs tested (at least at the doses used in this study) influenced the water intake. This indicates that simple thirst could not account for the action of drugs upon the D-S relationship. This is probably true also for locomotion and general activity of animals since most of the drugs used in our experiments failed to influence the animals' behavior in the open field. Neither metergoline nor quipazine changed the locomotor activity and orienting behavior (rearing). Similarly, tryptophan failed to change animals behavior while p-chlorophenylalanine produced slight sedation. Rats with the lesioned brain 5-HT neurons always become dominant when paired with sham-lesioned subjects. Lesion of the MR induced stronger effect than lesion of the DR. However, lesions involving the MR produced behavioral excitation as shown in the open field test and slightly but significantly increased water consumption. It is, therefore, possible that these mechanisms account for the change in the dominance produced by MR lesion. By contrast, lesions of the DR which also produced a significant decrease in the forebrain 5-HT concentration failed to influence water consumption and only slightly stimulated activity of rats in the open field. Interestingly enough, dominance was markedly increased by p-chlorophenylalanine, a compound that failed to stimulate general activity in the open field and water consumption in the rats. Moreover, biochemical analysis showed dramatically reduced brain 5-HT concentrations in
animals treated with p-chlorophenylalanine. It seems, therefore, unlikely that an impairment of central serotonergic mechanisms enhances dominance simply due to stimulation of general activity and for an increase in the motivation to drink. The precise mechanisms underlying the stimulatory effect upon dominance which occurs following both pharmacological and surgical impairment of the 5-HT neurons remain to be identified. The data obtained in the present study should be discussed together with other results showing increased irritability and aggressiveness in animals with destroyed 5-HT neurons [3, 6, 12, 23, 25] and pharmacologically-induced suppression of 5-HT synthesis [19,20]. It must be, however, pointed out that dominance is not simply connected with increased aggressivity. Both terms denote a variety of forms of aggression and domination which occur in different situations. In the extention to the findings mentioned above, the result of this study indicates that the D-S relationship in rats competing for water seems to be also closely associated with the brain 5-HT neurons. Obviously, the mechanism of dominance is not simply related merely to serotonergic neurons and other transmitter systems are involved in the mechanism of this behavioral phenomenon. It is commonly accepted that changes in one transmitter system lead to the alterations in other transmitter systems. For example, lesions of the 5-HT neurons have been reported to increase the activity of noradrenergic neurons and modulate response to drugs acting upon dopaminergic neurotransmission (see 112]). The results of the study have also implication for research on mechanism of depression and should be also discussed in terms of "antidepressive'" efficacy of antiserotonergic drugs and other treatments leading to the reduced brain 5-HT neurotransmission [13,17]. Recently we have found that social dominance produced in rats competing for food may also provide a suitable method for the selection of antidepressive drugs [ 14]. ACKNOWLEDGMENTS This paper was supported by Polish Academy of Sciences, Grant Nr 10.4. The authors are grateful to Ms. Anna Reszka and Ms. Elzbieta Zawadzka for technical assistance.
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