5-Hydroxytryptamine correlates of isolation-induced aggression in mice

European Journal o f Pharmacology 28 ( 1974 ) 326- 337 © North-Holland Publishing Company

5-HYDROXYTRYPTAMINE AGGRESSION IN MICE

CORRELATES

OF ISOLATION-INDUCED

Gordon K. HODGE and Larry L. BUTCHER Department of Psychology, University o f California, Los Angeles, California 90024, U.S.A.

Received 1 April 1974, accepted 3 July 1974 G.K. HODGE and L.L. BUTCHER, 5-Hydroxytryptamine correlates o f isolation-induced aggression in mice, European J. Pharmacol. 28 (1974) 326-337. Drug regimens designed to enhance or impair serotonergic functioning were found to differentially affect isolation-induced fighting in mice. Male albino mice were isolated for at least 4 weeks. Number of fights, attack latencies, and average fight durations were recorded for 15 rain every other day. On intervening days, the locomotor activity of each mouse was measured in stabilimeters. After the baseline for aggression and activity stabilized, drug procedures were instituted. While motility remained unaffected, mice injected with D,L-5-hydroxytryptophan in combination with a peripheral decarboxylase inhibitor (seryltrihydroxybenzylhydrazine)engaged in fewer fights of shorter average duration which were preceded by longer attack latencies. Biochemical analyses indicated that although serotonin levels were increased, catecholamine levels were reduced. A putative inhibitor of tryptophan hydroxylase, p-chloro-N-methylamphetamine (PCMA), was found to increase fighting frequency; attack latencies, average fight durations, and motility measurements did not differ significantly from non-injection performance. At time periods in which fighting was increased, levels of brain serotonin were reduced while catecholamine levels remained unaltered. Although PCMA did not affect motility at time intervals when fighting was increased, locomotor activity was increased for the first 8 hr after administration. Notwithstanding contributions by other putative neurotransmitters, these results suggest that serotonergic mechanisms are involved in the control of isolationinduced aggression in mice.

Catecholamines

Aggression

5-Hydroxytryptamine

1. Introduction Potentiation of serotonergic function by administration o f 5-hydroxytryptophan (5-HTP) or lysergic acid diethylamide, a putative 5-hydroxytryptamine (5-HT) receptor stimulator (And~n et al., 1968), reduces aggression in isolated mice (Yen et al. 1959; Uyeno and Benson, 1965; Welch and Welch, 1969) and muricidal rats (Kulkarni, 1968; Sheard, 1969; Di Chiara et al., 1971; Rewerski et al., 1971). Conversely, decrements in 5-HT function have been correlated with increases in aggression. Koe and Weissman (1966) observed that rats injected with p-chlorophenylalanine (PCPA), an inhibitor o f 5-HT biosynthesis, displayed increased irritability and aggressiveness when handled. Garattini et al. (1969)

Locomotor activity

p-Chlor o-N-methylamphetamine

have reported that isolated male mice which became aggressive had lowered turnover rates of brain 5-HT whereas neither decreased 5-HT turnover nor aggressiveness was detectable in male rats, female mice, or a strain o f mice which did not become aggressive after isolation. Sheard (1969) found that muricidal rats increased mouse killing after PCPA administration correlated with reductions of brain 5-HT and 5-hydroxyindoleacetic acid. Removal of the olfactory bulbs has been shown to lower 5-HT concentrations in the amygdala and to induce muricidal behavior in some natural non-killer rats (Karli et al., 1969). Although the correlation between the decrease in central 5-HT levels and the occurrence o f muricide was not compelling, non-killer animals, both naturally occurring and deolfactorized, would kill mice after

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression being injected with PCPA (Karli et al., 1969; Di Chiara et al., 1971). Recently, Grant et al. (1972) have described increases in muricidal aggression in rats following lesions in the brainstem raphe, a neural region containing a high concentration of 5-HT neuron somata. In a previous paper (Butcher and Dietrich, 1973), we examined shock-elicited aggression in mice following drug regimens preferentially preventing reserpineinduced depletion of either catecholamines or 5-HT. Fighting was increased when central 5-HT, but not catecholamine, levels were reduced. In the present report, we further examine the role of 5-HT in mediating aggressive behavior by investigating the effects on isolation-induced aggression in mice of 5-HTP and p-chloro-N-methylamphetamine (PCMA). The latter compound, by presumably inhibiting tryptophan hydroxylase (Sanders-Bush et al., 1972), produces a long-duration reduction of brain 5-HT (Miller et al., 1970), and, to our knowledge, has not previously been employed in aggression studies. Our investigation of 5-HTP differs from the work of previous investigators in that we have attempted to control for possible extracerebral effects of the 5-HT precursor by pretreating the animals with a peripheral decarboxylase inhibitor (cf. Butcher et al., 1972). Furthermore, the locomotor activity of the mice has been assessed under all drug conditions to determine possible nonspecific motor effects of the pharmacological agents tested (cf. Butcher and Dietrich, 1973). Correlative biochemical data are also presented.

2. Materials and methods

2.1. Experimental animals and isolation procedures It is well established that certain strains of mice, if isolated from weaning, will display aggressive responses when adults (see Valzelli, 1969). We used male mice weanlings of the Swiss-Webster SIM/WS strain (Simonsen Laboratories; Gilroy, California; U.S.A.). Except when they were placed in the behavioral testing apparatus, the mice were individually housed in stainless steel cages (17 × 26 × 12 cm) under conditions of constant illumination, temperature (22°C), and relative humidity (50%) for the duration of the experimental series. Food and water were ad

327

libitum. After a 4 week period of isolation, the mice, weighing 3 0 - 4 0 g, were either (1) randomly assigned to fighting pairs which remained intact throughout the behavioral studies or (2) injected with 5-HTP, PCMA, or the respective vehicles and sacrificed for biochemical analyses of brain dopamine (DA), noradrenaline (NA), and 5-HT.

2. 2. Definition and measurement of aggression When two isolated mice are placed together in a small chamber, aggressive behavior typically ensues characterized by (1) certain precursor behaviors such as tail rattling, chasing, and threatening postures (Scott, 1970) which precede (2) actual physical contact (e.g., biting, striking with the forepaws, scratching or fighting). Fig. 1 illustrates typical fighting behavior in non-injected isolated mice. In our experiments, fighting was measured in a round plastic bowl having a diameter of 22 cm and a depth of 10 cm (fig. 2). The bowl was divided into two halves by a removable aluminum plate (fig. 2); a layer of wood shavings covered the bottom of the bowl (not illustrated in fig. 2). At the start of the testing session, one mouse of an aggressive pair was placed in the bowl on one side of the plate and the other mouse was put on the other side (fig. 2). A clear plexiglass cover was then placed on top of the bowl. After removing the aluminum dividing plate through a slit in the cover, fighting between the 2 mice was recorded for 15 min. The next aggression testing session did not occur until 48 hr later to minimize fatigue factors which may have decreased aggressive behavior if the animals were tested each day (Scott, 1946). The basic datum recorded was actual physical contact or fighting. Tail rattling, threatening postures, and other precursor behaviors leading up to, but not including, physical contact were not quantified. A fight was scored whenever one mouse was observed to bite or vigorously strike the other at least once, and was considered terminated when physical contact ceased. To minimize variability, a single observer scored fighting for all experiments reported here. Three variables were used in characterizing the aggression data for each 15 min testing period: (1)number of fights, (2) attack latency, the time interval between raising the dividing plate in the testing chamber

328

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression ing the total duration of fighting by the number of fights. 2.3. Pharmacological agents The drugs used, their form, and their source of supply are as follows: D,L-5-HTP monohydrate (Calbiochem, Inc.; L a Jolla, California; U.S.A.), N 1(D,L-seryl)-N2 (2,3,4-trihydroxybenzyl)hydrazine (Ro 4-4602/1; courtesy of Dr. W.E. Scott; Hoffman-La Roche, Inc.; Nutley, New Jersey; U.S.A.), and PCMA" HC1 (Ro 4-6861 / 1; courtesy of Dr. W.E. Scott; Hoffman-La Roche, Inc.; Nutley New Jersey; U.S.A.). Doses are expressed in terms of the above forms of the pharmacological agents. All drugs were given i.p. in a volume of 0.9% saline equivalent to 1 ml per 100 g body weight. Solutions of 5-HTP were prepared individually for each animal immediately before injection. This drug was dissolved in 0.3 ml saline with gentle warming and injected in doses of 100, 200, and 400 mg/kg. To minimize potentially toxic peripheral effects of the 5-HT precursor, 25 mg/kg Ro 4-4602 was always injected 30 min prior to 5-HTP (Selden and Martin, 1970; Butcher et al., 1972). In lower doses (i.e., 2 5 - 5 0 mg/kg), Ro 4-4602 preferentially blocks extracerebral decarboxylase activity (Seiden and Martin, 1970). PCMA was administered in doses of 4, 10, and 20 mg/kg. 2.4. Behavioral-pharmacological procedures

Fig. 1. Typical fighting behavior of non-injected isolated mice. and the occurrence of the first fight, and (3) average fight duration, a derived measure calculated by divid-

Before drug procedures were instituted, a noninjection control baseline for aggression and locomotor activity was established for each mouse pair and for each mouse in the pairs, respectively. The fighting behavior was assessed every other day for a total of 7 days prior to the first drug administration session. On alternate days, we quantified the locomotor activity of the mice by placing them individually in stabilimeters (Davis and Ellison, 1964). Activity counts were recorded for 15 min. Data collected over the first 2 week period constituted the initial non-injection baseline for aggression and motility. Thereafter, following a given drug session, another pharmacological agent was not administered until both fighting and activity measures again returned to levels comparable to those observed under non-injection conditions. After the initial 2 week period of baseline mea-

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

329

Fig. 2. Apparatus used to observe fighting behavior. A = removable aluminum plate. B = plastic bowl.

surements, the mice were randomly assigned to 1 of 3 groups. The 2 experimental groups received either 5-HTP or PCMA. The third group was comprised of control animals which received either no injections, saline, or 25 mg/kg Ro 44602 administered 30 min prior to saline. Both members of the individual aggressive pairs were injected. Control mice were run on the same days that the experimental animals received either 5-HTP or PCMA. In the 5-HTP study, activity and aggression data were collected on the same mice, and every mouse in this group received, in random order, each of the 3 doses of 5-HTP. Aggression and motility measurements were made 60 min after injections. At least 48 hr intervened between successive drug administrations. From our own observations and previous research (Butcher et al., 1972), it is known that the 48 hr inter-injection interval far exceeds the duration of action of the serotonin precursor. Because of the relatively long duration of action of PCMA (Miller et al., 1970), the mice in this drug group were injected only once. Behavioral observations commenced 24 hr after PCMA administration and continued at 24 hr intervals thereafter, alternating between aggression and motility testing sessions, until the baseline again returned to control levels.

Some animals were tested for aggression first whereas the remaining mice had motility measurements first. The 24 hr interval was chosen as the initial time interval studied because Miller et al. (1970) have shown that the short term actions of PCMA (i.e., 0 . 5 - 4 hr post injection) include normal or slightly elevated brain levels of NA and 5-HT, due perhaps to the monoamine oxidase inhibiting property of the drug. Finally, Jouvet (1967) has found that p-chloroamphetamine, by presumably impairing serotonergic functioning in a manner comparable to that of PCMA, produces abnormalities in the sleep of cats. Aggressive behavior in rats has also been reported to accompany PCPA-induced alterations in sleep (Mouret et al., 1968). Similarly, preliminary observations in this laboratory have indicated that increases in aggressiveness followed PCMA administration. Since disrupted patterns of sleep, possibly resulting from lowered levels of 5-HT, might account for increased irritability and aggressiveness (Morgane and Stem, 1972), it was necessary to consider the possible sleepdisrupting actions of PCMA. To estimate this possible effect of PCMA, we made gross observations of the animals' behavior and continuously monitored their locomotor activity for 72 hr.

330

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

Biochemical analyses were performed on unfought, isolated mice to determine levels of whole brain monoamines after some of the drug conditions. Following injections at time periods corresponding to latencies which had preceded behavioral observations, the mice were killed by decapitation, their brains rapidly removed, weighed, and homogenized in cold (0°C) 0.4 N perchloric acid. Levels of DA and NA were assayed according to the method of Shellenberger and Gordon (1971), and 5-HT was determined in the alumina supernatants by the ninhydrin procedure of Snyder et al. (1965).

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3.1. Baseline recovery following repeated injections Over the duration of the experiments, neither aggression nor locomotor activity was appreciably affected either by the repeated injection procedure or by the length of time the experiments encompassed (fig. 3). The effects of repeated injections were assessed between groups with the Wald-Wolfowitz runs test (Siegel, 1956). Each of the 22 non-injection sessions of the drug-injected group (pooled 5-HTP and PCMA animals) were compared with corresponding sessions of the control group which received either no injections, saline, or Ro 4-4602 in combination with saline. There were no significant (p > 0.05) differences between the 2 groups on any of the 4 measures. Temporal effects accruing across sessions were evaluated on the means within each group by a runs up and down test (Bradley, 1968). The relative flatness of the curves depicting number of fights, latency, and activity reflects the nonsignificance (p > 0.05) obtained. Although tests on average fight durations of both groups were significant ( p < 0.05), the drop in durations occurring across days 1 and 2 accounted for the effect, since tests on the last 20 days were nonsignificant (p > 0.05).

3. 2. Effects of 5-HTP 3. 2.1. Aggression and locomotor activity As shown in fig. 4, 5-HTP in combination with Ro

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Fig. 3. Non-injection baseline for aggression and locomotor activity (all points represent non-injection means ± S.E.M.). Animals receiving either no injections, saline injections or Ro 4-4602 in combination with saline injections are indicated by open triangles (for measures of aggression, n = 13 pairs; for locomotor activity, n = 28 mice). Drug-injected animals receiving either Ro 4-4602 + 5-HTP or PCMA injections at

various intervals (not indicated), between which at least 2 non-injection baseline sessions (sessions 8-22; see text, section 2.4.) were interposed, are indicated by closed circles (for measures of aggression, n = 32 pairs; for locomotor activity, n = 85 mice).

4-4602 produced a dose dependent decrease in the number of fights and a corresponding increase in attack latency. While there were fewer fights at a dose of 200 mg/kg 5-HTP, the average fight durations were comparable to pre-injection levels.-Average fight durations for doses of 400 mg/kg 5-HTP, however, were much shorter. Motility was not altered by the 5-HTP and Ro 4-4602 combination, nor did the animals appear sedated or lethargic. Although the relative dominant-subordinate standing of each mouse in a pair remained unaffected by 5-HTP, there were drug-related differences in the expression of this relationship. Specifically, the dominants less frequently directed aggressive displays to-

G.K. Hodge, L.L. Butcher, 5.Hydroxytryptamine and aggression

331

Table 1 Levels of dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in whole mouse brain after injection of Ro 4-4602 and D,L-5-hydroxytryptophan (5-HTP). Monoamine in brain (ug/g, mean ± S.E.M.)*,**

Drug regimen

DA

NA

5-HT

A. Non-injected

1.31 -+0.141 (4)

0.51 -+0.055 (4)

0.85 ± 0.143 (4)

B. Ro 4-4602, 25 mg/kg + 0.9% saline

1.09 ± 0.064 (4)

0.45 + 0.025 (4)

0.84 ± 0.094 (4)

0.62 + 0.041 (4)

0.29 ± 0.010 (4)

3.50 ± 0.336 (4)

C. Ro 4-4602,

25 mg/kg + 5-HTP, 400 mg/kg

* Numbers of brains upon which analyses were performed are shown in parentheses. ** For each monoamine, a one-way analysis of variance revealed that significant differences existed among the various treatment conditions (DA: F = 14.59, dr= 2,9, p < 0.01; NA: F = 10.04, d f = 2,9, p < 0.01; 5-HT: F = 49.61, dr= 2,9, p < 0.01). A posteriori analyses comparing each treatment condition with every other were performed according to the Newman-Keuls procedure (Winer, 1971). For DA, comparisons A-C and B-C were significant at p < 0.01. For NA, comparisons A-C and B-C were significant at p < 0.01 and p < 0.05, respectively. For 5-HT, comparisons A-C and B-C were significant at p < 0.01. For all monoamines, the A-B comparisons were not significant (p > 0.05). ward the subordinates, and any aggressiveness which was displayed usually did not lead to fighting. For example, whereas tail rattling by the aggressive dominants would likely precede fighting under non-injec40

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3.2.2. Biochemistry The 400 mg/kg dose of 5-HTP which produced the greatest reduction in fighting increased central whole brain levels of 5-HT to 3 - 4 times that of control values (table 1). Reductions in catecholamine levels Fig. 4. Effect of Ro 4-4602 + 5-HTP on aggression and locomotor activity 1 hr after injections. For measures of aggression and activity, each bar represents the mean and the S.E.M. for 7-8 mouse pairs and 10-14 mice, respectively. The t-test for correlated means (two-tailed) was used in comparing non-injected behavior (open bars) with druginjected behavior (closed bars) of the same mice observed during the following session. *p < 0.05. VEH = vehicle-injected (25 mg/kg Ro 4-4602 + 0.9% saline).

332

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also occurred, perhaps as a consequence of the endogenous catecholamines being displaced by the newly formed 5-HT (Butcher et al., 1972).

3.3. Effects of PCMA 3. 3. I. Aggression and locomotor activity The number of fights was increased 24 and 48 hr after injection of 20 mg/kg PCMA (figs. 5 and 6). Several of the dominant animals inflicted more injurious wounds on the subordinates than was normal. Although average fight durations did not differ from pre-injection values, the amount of time fighting was significantly increased 48 hr following administration of the 20 mg/kg PCMA dose (mean non-injected duration of 18.8 sec vs. mean PCMA-injected duration of 35.8sec; t = 2.90, df = 5, p < 0.05). With few exceptions (see below) the subordinates of the 24 and 48 hr groups did not become more aggressive; rather, the increases in fighting were usually attributable to

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the normally aggressive dominants becoming even more so. In addition to some hyper-reactivity (increased vocalizations, exaggerated movements, etc.), tail rattling was observed in 4 subordinates (one from the 24 hr and 3 from the 48 hr groups). Tail rattling has been found indicative of either impending 'fight' or 'flight' in mice (Scott and Fredericson, 1951). But 'under the conditions of the experiments reported here, tail rattling was more commonly associated with the imminent occurrence of fighting customarily instigated by the tail rattling animal. In any case, tail rattling by the subordinates was conspicuously incongruous with their usual submissive behavior. Moreover, 2 subordinate mice from the 48 hr group actually did engage in fighting. For the 24 and 48 hr periods (figs. 5 and 6), neither attack latencies nor locomotor activity was affected by any dose of PCMA, and at 72 hr post-injection, no observed behaviors were different from the non-injection baseline sessions. Over the course of the first 8 hr following injections, the animals that received 10 or 20 mg/kg

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

PCMA were more active than either the 4 mg/kg PCMA or the saline-injected groups (fig. 7). From 1 to 3 hr after injections, the fur of the 20 mg/kg group was wet from salivation and lacrimation. While the 4 mg/kg PCMA and the saline groups were also active throughout the first several hr their behavior was generally less agitated and more frequently interspersed with immotive periods wherein the mice were often observed eating or curled up with opened or closed eyes. Additional periods of greater motility, occurring at 17, 29, and 52 hr, further distinguished the 20 mg/kg PCMA animals from the mice given

333

saline, 4, or 10 mg/kg PCMA (fig. 7). While these results are not interpreted as supportive evidence for PCMA-induced sleep deprivation, they are nevertheless completely compatible with studies which have demonstrated anomalies in sleep following impairment of serotonergic functioning (Jouvet, 1967; Mouret et al., 1968). t t 2 . Biochemistry The increased number of fights following 20 mg/kg PCMA administration was accompanied by a decrease in whole brain levels of 5-HT at 24 and

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HOURS Fig. 7. Effect o f PCMA injections on locomotor activity during a subsequent 72 hr period (injections occurred at 0 hr; each point represents the mean for 6 - 1 2 mice). For the following time intervals after injections, one-way analyses of variance indicated that significant differences existed between the treatment conditions: 1 hr: F = 6.38, d f = 3,22, p < 0.01; 2 hr: F = 4.99, d f = 3,20, p < 0.01 ; 3 hr: F = 8.17, d f = 3,20, p < 0.01; 4 hr: F = 7.47, d f = 3,22, p < 0.01 ; 5 hr: F = 14.45, d f = 3,29, p < 0.01 ; 6 hr: F = 5.51, d f = 3,31, p < 0.01; 7 hr: F = 3.39, d r = 3,31,p < 0.05; 8 hr: F = 3.47, d f = 3,31,p < 0.05; 17 hr: F = 3.17, d r = 3,29, p < 0.05; 52 hr: F = 4.87, d f = 3,21, p < 0.05. A posteriori analyses comparing each treatment condition with the saline-injected control were performed according to Dunnett's procedure (Winer, 1971). 1 hr after injections, comparisons A - C and A - D were significant at p < 0.05 and p < 0.01, respectively. 2 hr after injections, comparisons A - C and A - D were significant at p < 0.025. 3 hr after injections, comparisons A - C and A - D were significant at p < 0.01 and p < 0.025, respectively. 4 hr after injections, comparisons A - C and A - D were significant at p < 0.01.5 hr after injections, comparisons A - C and A - D were significant at p < 0.01 and p < 0.005, respectively. 6, 7, 8, and 52 hr after injections, the A - D c o m p a r i s o n s were significant at p < 0.01, p < 0.05, p < 0.05, and p < 0.005, respectively. For the preceding 10 time intervals, no other comparisons were significant (p > 0.05).

334

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

Table 2 Levels of dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in whole mouse brain after injection of p-chloro-N-methylamphetamine (PCMA) at different times following administration. Drug regimen

Monoamine in brain (#g/g, mean +-S.E.M.)*,** DA

NA

5-HT

A. 0.9% saline 24-72 hr (pooled)

1.13 +-0.114 (6)

0.50-+ 0.040 (6)

0.81 +- 0.074 (7)

B. PCMA, 20 mg/kg 24 hr

1.03 -+0.075 (8)

0.50 -+ 0°025 (8)

0.60 -+ 0.051 (8)

C~ PCMA, 20 mg/kg 48 hr

1.08 +- 0.085 (7)

0.47 -+ 0.017 (7)

0.56 +- 0.056 (7)

D. PCMA, 20mg/kg 72 hr

0.83 -+ 0.069 (6)

0.39 -+ 0.024 (6)

0.65 -+ 0.031 (6)

E. PCMA, 20 mg/kg 3 months

1.35 -+ 0.146 (4)

0.58 -+ 0.041 (4)

0.89 +- 0.101 (4)

* Numbers of brains upon which analyses were performed are shown in parentheses. ** For each monoanime, a one-way analysis of variance revealed that significant differences existed among the different treatment conditions (DA: F = 3.15, df = 4,26, p < 0.05; NA: F = 4.64, df = 4,26, p < 0.01; 5-HT: F = 4.61, dr= 4,27, p < 0.01). A posteriori analyses comparing each treatment condition with the saline-injected control were performed according to Dunnett's procedure (Winer, 1971). For DA, no comparisons with control were significant (19 > 0.05). For NA, comparison A - D was significant at p < 0.025; comparisons A-B, A-C, and A - E were not significant (p > 0.05). For 5-HT, comparison A-B was significant at p < 0.05; comparison A-C was significant at p < 0.025; comparisons A - D and A - E were not significant (p > 0.05).

48 hr (table 2). Brain catecholamine levels remained unchanged until 72 hr after injections when NA levels were reduced. Serotonin levels, however, were then no longer declining and, if anything, were beginning to rise. While the lowered levels of 5-HT were associated with increased fighting (cf. figs. 5 and 6 with table 2), there were no similar behavioral consequences correlated with lowered NA levels since fighting had returned to normal by 72 hr.

4. Discussion The results o f the present study implicate serotonergic mechanisms as important mediators o f aggressive behavior in isolated mice. By presumably facilitating 5-hydroxytryptaminergic systems, but not excluding effects on catecholaminergic mechanisms, 5-HTP administration inhibited aggressive behavior. Conversely, decreasing the efficacy o f serotonergic

mechanisms by injecting PCMA produced an increase in fighting and aggressive displays. It has been suggested that the mitigating effects which certain drugs have on aggression may be due to the general, nonspecific sedative effects of these compounds (Yen et al., 1959; Kr~iak and Steinberg, 1969). But in the context of the present experiments, 'sedation' is an inadequate descriptive term because while fighting was depressed other forms of behavior remained unaltered. Spontaneous locomotor activity was not impaired following doses of 5-HTP which were efficacious in reducing fighting. Nor did the mice, dominants or subordinates, appear sedated, lethargic, or suffering from muscular or m o t o r impairment. The dominant animals retained and even intensified non-hostile activities such as selfgrooming and digging in the wood shavings at the b o t t o m of the testing chamber. Subordinate mice also continued to manifest behaviors commensurate with those of their non-injected state (e.g., vocalizations and maintenance o f submissive postures).

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

Although 5-HTP would appear to have inhibitory effects on aggressive behavior, our biochemical results failed to provide compelling evidence that the decreased fighting was predominantly due to increased potentiation of serotonergic systems. While 5-HTP produced 4-fold increases in whole brain 5-HT levels, catecholamine levels were simultaneously reduced. Gunne (1969), in summarizing several studies, has found that when sham rage was evoked in cats by supracollicular transsection or by electrical stimulation of the amygdala or lateral hypothalamus, there were concurrent decreases in central NA. Gunne (1969) proposed that these results indicated an increased utilization of NA during the aggressive episodes. While sham rage in cats may bear little semblance to isolation-induced aggression in mice, it is possible that the decreased fighting we observed in mice given 5-HTP may have been the result of either (1) reduced NA levels (and/or DA levels as indicated by our results; see table 1) or (2) a combination of reduced levels of NA (and/or DA) and increased levels of 5-HT. In support of this second contention are the recent findings of Ellison and Bresler (1974) who have reported increased shock-elicited aggression in rats following simultaneous reductions of serotonin and catecholamine levels after administration of 6-hydroxydopamine and/or PCPA; reduced catecholamine levels per se were ineffective in increasing the incidence of fighting. Conversely, it has also been reported that aggression can be induced in mice (Lycke et al., 1969) and in rats (Benkert et al., 1973) by increasing catecholamine levels after L-dihydroxyphenylalanine injections while concurrently decreasing 5-HT levels with PCPA. The preceding results, as well as others (Scheel-Kriiger and Randrup, 1968; Welch and Welch, 1969; Eichelman et al., 1972), suggests that a balance in amine levels and/or turnover may be responsible for the mediation of some forms of aggressive behavior. The present results with PCMA, although in complete agreement with data from our earlier study (Butcher and Dietrich, 1973), are in contrast to experiments using PCPA in which aggression in mice was either unchanged (Lycke et al., 1969) or actually reduced (Welch and Welch, 1969; Benkert et al., 1973). Although these earlier PCPA experiments did not include appropriate biochemical analyses for the

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effect of the drug on monoamines, PCPA is known to reduce NA as well as 5-HT levels (Miller et al., 1970). PCPA has been found, in fact, to lower both central serotonin and catecholamine levels in rats while having disparate effects on shock-elicited aggression (cf. Conner et al., 1970 with Ellison and Bresler, 1974). Since the actions of PCPA on monoamine levels are contingent upon the time periods following administration (Miller et al., 1970), the different time course effects of the drug on the dynamics of monoaminergic interactions might explain the correspondingly different effects PCPA has been reported to have on aggression. PCMA, however, at certain doses and latencies, appears to have a more specific action on central 5~HT containing neurons than does PCPA (Miller et al., 1970), and may therefore be more useful than PCPA in delineating the respective components of aggressive behavior subserved by either serotonin or the catecholamines. Although serotonergic mechanisms seem implicated as mediators of certain components of aggression, 5-HT functioning is probably also involved in the maintenance of other related behaviors. From 1 to 8 and 17 hr following PCMA treatment, we observed marked increases in excitability and locomotor activity. To some extent these elevations in activity and the disruptions in sleep and other behavioral patterns following PCPA or PCMA treatment may be partially attributable to alterations in NA levels which can occur up to 8 days or 24 hr after PCPA or PCMA injections, respectively (Miller et al., 1970). But NA involvement would seemingly not account for either the increased activity seen at 29 and 52 hr after PCMA injections or the hyper-reactivity observed in some mice at 24 and 48 hr after administrations since NA levels were then essentially normal. Although we observed normal locomotor activity during 15 rain intervals measured at 24, 48, and 72 hr after PCMA injections, the increased activity at 29 and 52 hr suggests that increases in aggression were_not the only changes in behavior correlated with lowered levels of 5-HT. One possibility is that the constellation of behaviors characterized by increased excitability, irritability, reactivity, sleep disturbances, and aggression are all inextricable manifestations of varying degrees of serotonergic functioning or dysfunctioning. Nonetheless, and while not precluding the importance of other transmitters, the existence of 5-hydroxytrypt-

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G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression

aminergic mechanisms underlying aggressive behavior seem very likely, although the nature o f the interplay arising b e t w e e n 5-HT and o t h e r systems in the exact f o r m u l a t i o n o f this behavior remains equivocable.

Acknowledgements For performing the biochemical analyses, the authors wish to thank Dr. Edward Geller and his staff at the Brentwood V.A. Hospital; Los Angeles, California. For their assislance in recording some of the activity measurements, we thank Paul C. Mclntire and Bill Weisman. This research was supported in part by grant NS-10928 from the National Institute of Neurological Diseases and Stroke.

References And6n, N.-E., H. Corrodi, K. Fuxe and T. H6kfelt, 1968, Evidence for a 5-hydroxytryptamine receptor stimulation by lysergic acid diethylamide, Brit. J. Pharmacol. 34, 1. Benkert, O., A. Renz and N. Matussek, 1973, Dopamine, noradreline and 5-hydroxytryptamine in relation to motor activity, fighting and mounting behavior. II. L-DOPA and D,L-threodihydroxyphenylserine in combination with Ro 4-4602 apd parachlorophenylalanine, Neuropharmacology 12, 187. Bradley, J.V.,: 1968, Distribution-free Statistical Tests (Prentice-HaU, Inc., Englewood Cliffs, N.J.). Butcher, L.L and A.P. Dietrich, 1973, Effects of shock-elicited aggression in mice of preferentially protecting brain monoamines against the depleting action of reserpine, Naunyn-Schmiedeb. Arch. Pharmacol. 277, 61. Butcher, L.L., J. Engel and K. Fuxe, 1972, Behavioral, biochemical, and histochemical analyses of the central effects of monoamine precursors after peripheral decarboxylase inhibition, Brain Res. 41,387. Conner, R.L., J.M. Stolk, J.D. Barchas, W.C. Dement and S. Levine, 1970, The effect of parachlorophenylalanine (PCPA) on shock-induced fighting behavior in rats, Physiol. Behav. 5, 1221. Davis, J.F. and G.D. Ellison, 1964, A general purpose activity recorder with variable sensitivity, J. Exptl. Anal. Behav. 7, 117. Di Chiara, G., R, Camba and P.F. Spano, 1971, Evidence for inhibition by brain serotonin of mouse killing behavior in rats, Nature 233,272. Eichelman, B.S., N.B. Thoa and K.Y. Ng, 1972, Facilitated aggression in the rat following 6-hydroxydopamine administration, Physiol. Behav. 8, 1. Eilison, G.D. : and D.E. Bresler, 1974, Tests of emotional behavior in rats following depletion of norepinephrine, of serotonin, or of both, Psychopharmacologia (Berlin) 34, 275.

Garattini, S., E. Giacolone and L. Valzelli, 1969, Biochemical changes during isolation-induced aggressiveness in mice, in: Aggressive Behaviour, eds. S. Garattini and E.B. Sigg (John Wiley and Sons, Inc., New York) p. 179. Grant, L.D., G.R. Breese and J.L. Howard, 1972, CNS catecholamine and serotonin inhibition of muricide: Evidence for monoamine interactions, Abstr. Second Ann. Meet. Soc. Neurosci., Houston, p. 130. Gunne, L.-M., 1969, Brain catecholamines in the rage response evoked by intracerebral stimulation and ablation, in: Aggressive Behaviour, eds. S. Garattini and E.B. Sigg (John Wiley and Sons, Inc., New York) p. 238. Jouvet, M., 1967, Regulation neuro-humorale des 6tats de sommeil, in: Biological and Clinical Aspects of the Central Nervous System, ed. C.M. Jacottet (Buchdruckerei Kohlhepp AG, Basel) p. 112. Karli, P., M. Vergnes and Fo Didiergiorges, 1969, Rat-mouse interspecific aggressive behaviour and its manipulation by brain ablation and by brain stimulation, in: Aggressive Behaviour, eds. S. Garattini and E.B. Sigg (John Wiley and Sons, Inc., New York) p. 47. Koe, B. and A. Weissman, 1966, p-Chlorophenylalanine: A specific depletor of brain serotonin, J. Pharmacol. Exptl. Therap. 154, 499. Krgiak, M. and H. Steinberg, 1969, Psychopharmacological aspects of aggression: A review of the literature and some new experiments, J. Psychosom. Res. 13,243. Kulkarni, A.S., 1968, Muricidal block produced by 5-hydroxytryptophan and various drugs, Life Sci. 7, 125. Lycke, E., K. Modigh and B.-E. Roos, 1969, Aggression in mice associated with changes in the monoamine-metabolism of the brain, Experientia 25, 951. Miller, F.P., R.H. Cox, W.R. Snodgrass and R.P. Maickel, 1970, Comparative effects of p-chlorophenylalanine, p-chloroamphetamine and p-chloro-N-methylamphetamine on rat brain norepinephrine, serotonin and 5-hydroxyindole-3-acetic acid, Biochem. Pharmacol. 19, 435. Morgane, P.J. and W.C. Stern, 1972, Relationship of sleep to neuroanatomical circuits, biochemistry, and behaviour, Ann, N.Y. Acad. Sci. 193, 95. Mouret, J., P. Bobillier and M. Jouvet, 1968, Insomnia following parachlorophenylalanine in the rat, European J. Pharmacol. 5, 17. Rewerski, W., W. Kostowski, T. Piechcki and M. Rylski, 1971, The effects of some hallucinogens on aggressiveness of mice and rats, Pharmacology 5, 314. Sanders-Bush, E., J.A. Bushing and F. Sulser, 1972, p-Chloroamphetamine. Inhibition of cerebral tryptophan hydroxylase, Biochem. Pharmacol. 21, 1501. Scheel-Kriiger, J. and A. Randrup, 1968, Aggressive behaviour provoked by pargyline in rats pretreated with diethyldithiocarbamate, J. Pharm. Pharmacol. 20, 948. Scott, J.P., 1946, Incomplete adjustment caused by frustration of untrained fighting mice, J. Comp. Psychol. 39, 379. Scott, J.P., 1970, Agnostic behavior of mice and rats: A

G.K. Hodge, L.L. Butcher, 5-Hydroxytryptamine and aggression review, in: Animal Aggression: Selected Readings, ed. C.tL Southwick (Van Nostrand Reinhold Company, New York) p. 103. Scott, J.P. and E. Fredericson, 1951, The causes of fighting in mice and rats, Physiol. Zool. 24,273. Seiden, L.S. and T.W. Martin, 1970, Potentiation of effects of L-dopa on conditioned avoidance behavior by inhibition of extracerebral dopa decarboxylase, Physiol. Behav. 6,453. Sheard, M., 1969, The effect of p-chlorophenylalanine on Behavior in rats: Relation to brain serotonin and 5-hydroxyindoleacetic acid, Brain Res. 15,524. SheUenberger, M.K. and J.H. Gordon, 1971, A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas, Anal. Biochem. 39, 356. Siegel, S., 1956, Nonparametric Statistics for the Behavioral Sciences (McGraw-Hill Book Company, New York).

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Snyder, S.H., J. Axelrod and M. Zweig, 1965, A sensitive and specific fluorescence assay for tissue serotonin, Biochem. Pharmacol. 14, 831. Uyeno, E.T. and W.M. Benson, 1965, Effects of lysergic acid diethylamide on attack behavior of male albino mice, Psychopharmacologia (Berlin) 7, 20. Valzelli, L., 1969, Aggressive behaviour induced by isolation, in: Aggressive Behaviour, eds. S. Garattini and E.B. Sigg (John Wiley and Sons, Inc., New York) p. 70. Welch, B.L. and A.S. Welch, 1969, Aggression and the biogenic amine neurohumors, in: Aggressive Behaviour, eds. S. Garattini and E.B. Sigg (John Wiley and Sons, Inc., New York) p. 188. Winer, B.J., 1971, Statistical Principles in Experimental Design, 2nd edn. (McGraw-Hill Book Company, New York). Yen, C.Y., R.L. Stanger and N. Millman, 1959, Ataractic suppression of isolation-induced aggressive behavior, Arch. Intern. Pharmacodyn. 123, 179.