Serotonin Type-1A Receptors Modulate Adolescent, Cocaine-Induced Offensive Aggression in Hamsters

Physiology & Behavior 85 (2005) 167 – 176

Serotonin type-1A receptors modulate adolescent, cocaine-induced offensive aggression in hamsters

Irina Knyshevski, Lesley A. Ricci, Thomas E. McCann, Richard H. Melloni Jr.*

Behavioral Neuroscience Program, Department of Psychology, 125 Nightingale Hall, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States

Received 14 November 2004; received in revised form 1 February 2005; accepted 29 March 2005

Abstract

Hamsters repeatedly exposed to cocaine throughout adolescence display highly escalated offensive aggression compared to saline-treated littermates. Recently, we have shown that serotonin neural signaling and development play an important role in adolescent cocaine-induced offensive aggression. This study examined whether the adolescent cocaine-induced aggressive response was modulated by serotonin type 1A (5HT1A) receptors. To test this, adolescent male Syrian hamsters were administered cocaine hydrochloride (0.5 mg/kg, i.p.) throughout adolescent development (P27–57) and then tested for offensive aggression after the administration of the 5HT1A receptor agonist R(+)-8-OH-DPAT(0.1, 0.3, 0.6, 1.0, 1.25 mg/kg, i.p.). R(+)-8-OH-DPAT dose-dependently reduced cocaine-induced offensive aggression, with a significant reduction observed at 0.3 mg/kg for most of the offensive responses measured. Animals treated with higher doses of R(+)-8-OH-DPAT (0.6–1.25 mg/kg) prior to testing showed significant reductions in all measures of offensive aggression and social interest towards intruders (i.e., contact time), indicating more general behavioral inhibition. Adolescent cocaine-treated animals did not differ in body weight from controls, suggesting that the increased aggression was not due to increased body mass. These data support a role for 5HT1A signaling in adolescent cocaine-induced aggression. D 2005 Published by Elsevier Inc.

Keywords: Adolescence; Serotonin; Serotonin 1A receptor; 8-OH-DPAT; Aggression

1. Introduction studies revealed that aggressive, adolescent cocaine-treated hamsters had significant deficits in 5HT afferent innervation The developmental mechanisms and pharmacologic sub- to many areas of the brain implicated in aggression control strates underlying the facilitation of aggressive behavior are when compared to saline-treated littermates, implicating an important focus of the study of the neurobiology of marked serotonergic hypofunctioning in cocaine-treated aggression. Previously, we have used sub-adult Syrian animals [14]. Together, these data supported the notion that hamsters (Mesocricetus auratus) as an animal model to altered 5HT development and signaling between 5HT and examine the consequences of adolescent cocaine exposure on specific 5HT receptors might underlie adolescent cocaine- the behavioral neurobiology of offensive aggression induced aggression. [14,24,43,44]. Behavioral data from these studies showed The inhibitory nature of the 5HT neural system on aggres- that hamsters repeatedly exposed to low doses of cocaine (0.5 sion has been predominately attributed to the action of 5HT at mg/kg/day) during adolescent development displayed highly specific G protein-coupled 5HT receptors, namely the 5HT escalated levels of offensive aggression that is modulated by type 1 (i.e., 5HT1A and 5HT1B) and 5HT type 2 receptors serotonin (5HT) neural signaling, i.e., a neurochemical signal [5,12,36,39,45,46]. For instance, rats treated with eltoprazine, shown previously to inhibit aggression in humans and a mixed 5HT1A/B-receptor agonist, show a dose-dependent animals [40], including hamsters [16–18]. Neuroanatomical decrease in offensive aggression [50]. The 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (i.e., 8-OH- * Corresponding author. Tel.: +1 617 373 3043; fax: +1 617 373 8714. DPAT) effectively decreases aggression in a number of species E-mail address: [email protected] (R.H. Melloni). and models of aggression, including isolation-induced-

0031-9384/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.physbeh.2005.03.020 168 I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176

[6,46,47,55], alcohol-heightened- [36] and testosterone- dose (0.5 mg/kg) cocaine hydrochloride throughout adoles- dependent [10,51] aggression in mice and offensive aggression cent development (P27–P57) as previously described in rats [13] and hamsters [27]. Perhaps adolescent cocaine [14,24,34,43,44]. As a baseline behavioral control, a exposure stimulates aggressive behavior in hamsters by re- separate set of hamsters (n =10) was treated with saline ducing 5HT signaling through 5HT1A receptors. Indeed, co- throughout adolescence and tested for aggression (see caine treatment has been shown to decrease 5HT1A receptor below) in parallel with the above animals. All animals were activity and expression in brain [3,31,41]. Given this hypo- weighed every other day throughout the treatment regimen to thesis, it should be possible to attenuate the adolescent cocaine- control for weight gain and provide weight comparisons induced aggressive response by enhancing 5HT1A receptor between treatment groups. On the day following the last signaling. To test this hypothesis, animals were treated with injection (P58), cocaine-treated animals were weighed and low-dose cocaine throughout adolescence and then tested for separated randomly into five R(+)-8-OH-DPAT treatment offensive aggression after acute injections of either saline or groups and one vehicle control group (n =5–6 per group). the selective 5HT1A receptor agonist R(+)-8-OH-DPAT. Animals were administered one of five doses of R(+)-8-OH- DPAT (0.1, 0.3, 0.6, 1.0 or 1.25 mg/kg, i.p.) or an equal volume of saline vehicle alone. Doses of R(+)-8-OH-DPAT 2. Methods were chosen that have previously been reported to be effective (0.1–1.25 mg/kg) at attenuating aggressive 2.1. Animals responses across a variety of species utilizing a number of animal models [6,10,13,27,36,46,47,51,55]. All injections Prepubertal male Syrian hamsters (P21) were obtained were performed on unanesthetized animals and took no from Charles River Laboratories (Wilmington, MA), indi- longer than 10 s. After injection, animals were returned to vidually housed in polycarbonate cages and maintained at their home cage. Thirty minutes after injection, animals were ambient room temperature (22–24 -C with 55% relative subjected to aggression testing using the resident–intruder humidity) on a reverse light–dark cycle of (14L:10D, lights paradigm as described below and the experimental animal off at 07:00) as previously described [14,43,44]. Food and (resident) and was scored for general measures of offensive water were provided ad libitium. For aggression testing, aggression (i.e., number of attacks and bites, latency to attack stimulus (intruder) males of equal size and weight to the and bite towards intruders) as previously described experimental animals were obtained from Charles River 1 [14,24,43,44]. In addition, residents were measured for week prior to the behavioral test, group housed at five animals social interest toward intruders (i.e., contact time between per cage in large polycarbonate cages and maintained as resident and intruder) to control for nonspecific effects of above to acclimate to the animal facility. All intruders were 5HT1A receptor agents on behavior. prescreened for low aggression and submission 1 day prior to In a second experiment, adolescent (P27) Syrian hamsters the aggression test to control for behavioral differences (n =42) were treated with low-dose (0.5 mg/kg) cocaine as in between stimulus animals, as previously described [43,44]. Experiment 1. On the day following the last injection (P58), Briefly, stimulus animals were paired in a clean cage free of animals were weighed, separated randomly into three R(+)-8- bedding for a 2-min duration and each animal was scored for OH-DPAT treatment groups and one vehicle control group measures of low aggression (i.e., disengage and evade) and (n =9–13 per group). Animals were administered one of three submission (i.e., tail-up freeze, flee and fly-away). The few doses of R(+)-8-OH-DPAT (0.1, 0.3, 0.6 mg/kg, i.p.) or saline stimulus animals (2–3%) that displayed low aggression or vehicle alone. All injections were performed on unanesthe- submissive behaviors during the prescreening were excluded tized animals and took no longer than 10 s. After injection, from the behavioral assay. All studies using live animals were animals were returned to their home cage. Thirty minutes after approval by The Animal Care and Use Committee at injection, animals were subjected to aggression testing using Northeastern University and all methods used were consistent the resident–intruder paradigm as described below and the with guidelines provided by the National Institutes of Health experimental animal (resident) was scored for more specific for the scientific treatment of animals. and targeted aggressive responses including upright offensive postures, lateral attacks, flank and rump bites, and flank marks 2.2. Drugs as described [23,43,44] to provide a more detailed account of the effect of R(+)-8-OH-DPAT on the aggressive response Cocaine hydrochloride and R(+)-8-OH-DPAT were each resulting from adolescent cocaine exposure. purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in 0.9% normal saline. 2.4. Behavioral testing

2.3. Experimental treatment Experimental animals were tested for offensive aggression using the resident–intruder paradigm, a well-charac- In the first experiment, adolescent (P27) Syrian hamsters terized and ethologically valid model of offensive aggression (n =33) received daily intraperitoneal (i.p.) injections of low- in Syrian hamsters [20,30]. Briefly, an intruder of similar size I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176 169 and weight was introduced into the home cage of the significantly decreased the number of attacks (0.1 mg/kg experimental animal (resident) and the resident was scored [Z =2.40, p <0.05], 0.3 mg/kg [Z =2.72, p <0.01]) and bites for either (1) general measures of offensive aggression (i.e., (0.3 mg/kg [Z =1.92, p =0.05]) of cocaine treated animals number of attacks and bites, latency to attack and bite towards intruders when compared with saline-treated con- towards intruders) or (2) more specific and targeted trols. As shown in Fig. 1A, hamsters treated with low-dose aggressive responses including upright offensive postures, cocaine throughout adolescence showed high levels of lateral attacks, flank and rump bites, and flank marks. An offensive attack when administered saline prior to the attack was scored each time the resident animal would aggression test. Indeed, the intensity of the aggressive pursue and then either: (1) lunge toward and/or (2) confine encounter (i.e., number of attacks and bites during the test the intruder by upright and sideways threat; each generally period) was extremely high (greater than 45 attacks and 20 followed by a direct attempt to bite the intruder’s dorsal rump bites) in nearly all (5 out of 6) of the saline-treated animals and/or flank target area(s). The latency to attack and bite was tested. Conversely, adolescent cocaine-treated animals defined as the period of time between the beginning of the administered R(+)-8-OH-DPAT prior to the aggression test behavioral test and the first attack and bite of the residents displayed significantly less offensive aggression compared toward an intruder. In the case of no attacks and/or bites, to controls. In fact, half of the animals in each low-dose (0.1 latencies to attack and bite were assigned the maximum and 0.3 mg/kg of R(+)-8-OH-DPAT) treatment group (n =3 latency (i.e., 600 s). In addition, residents were measured for each) scored greater than a 50% reduction in the number of social interest toward intruders (i.e., contact time between attacks and bites during the behavioral test. Animals treated resident and intruder) to control for nonspecific effects of with moderate (0.6 mg/kg) and high (1.0 and 1.25 mg/kg) 5HT1A receptor agents on behavior. Contact time was doses of R(+)-8-OH-DPAT showed a complete elimination defined as the period of time during which the resident of attack and bite behavior compared to saline-treated control deliberately initiated contact with the intruder either through animals (attacks and bites; 0.6–1.25 mg/kg [Z =2.92, olfactory investigation (i.e., sniffing) or aggression. Each p <0.01 each comparison]). All R(+)-8-OH-DPAT-treated aggression test lasted for 10 min and was videotaped and animals in these three R(+)-8-OH-DPAT treatment groups coded manually by two observers unaware of the hamsters’ failed to attack intruders during the test period. Since attack experimental treatment. Differences in scores for all behav- and bites were eliminated in animals treated with moderate to iors measured were less than 5% between the two observers. high R(+)-8-OH-DPAT doses, between treatment group No intruder was used for more than one behavioral test and comparisons did not present a dose-dependent pattern all animals were tested during the first 4 h of the dark cycle between R(+)-8-OH-DPAT and aggression. under dim-red illumination to control for circadian influen- In addition, 5HT1A receptor activation with R(+)-8-OH- ces on behavioral responding. DPAT produced an overall effect on the initiation of offensive attack ( F(5,32) =4.28, p =0.05) and bite behavior 2.5. Statistical analysis ( F(5,32) =17.64, p <0.001), with an effective dose beginning at 0.6 mg/kg. Specifically, cocaine-treated hamsters admin- Results from the aggression tests were compared istered moderate to high (0.6–1.25 mg/kg) doses of R(+)-8- between saline- and R(+)-8-OH-DPAT treatment groups. OH-DPAT displayed a significantly slower time to attack For the R(+)-8-OH-DPAT dose–response, saline was the (0.6 mg/kg [t =2.79], 1.0 mg/kg [t =2.18], 1.25 mg/kg control condition. Nonparametric data (number of general [t =3.11], p <0.01 each comparison) and bite (0.6–1.25 mg/ and targeted bites and attacks) were compared by Mann– kg [t =6.27], p <0.01 each comparison) intruders than Whitney U-tests (two-tailed) and Kruskal–Wallis ANOVAs. saline-treated animals (Fig. 1B). Indeed, the initiation of Parametric data (latency to first bite and attack, contact time the aggressive response was rapid in all cocaine-treated and body weight) were compared by ANOVA and Fischer’s hamsters administered saline and those treated with low PLSD post-hoc (two-tailed). For all studies, significance doses (0.1–0.3 mg/kg) of R(+)-8-OH-DPAT, with nearly all was defined as p <0.05. (i.e., 5 out of 6) animals attacking within the first minute and biting within 5 min of the start of the behavioral test. By comparison, over 90% of the animals administered moder- 3. Results ate to high doses (0.6–1.25 mg/kg) of R(+)-8-OH-DPAT prior to the aggression test did not score a single attack or 3.1. Effects on general measures of adolescent cocaine- bite on intruders. By virtue of the fact that the majority of induced offensive aggression R(+)-8-OH-DPAT-treated animals (12 out of 18) failed to attack, attack latencies for all R(+)-8-OH-DPAT treatment 5HT1A receptor activation with R(+)-8-OH-DPAT pro- groups were approximately 4–5 min later, toward the end of duced an overall effect on offensive attack (v2 =24.44, the test period. 2 p <0.001) and bite behavior (v =20.18, p <0.01) with Finally, 5HT1A receptor activation with R(+)-8-OH- lowest effective doses of 0.1 mg/kg and 0.3 mg/kg, DPAT also produced an overall effect on contact time (i.e., respectively. At these doses, R(+)-8-OH-DPAT treatment a measure of social interest in intruders) ( F(5,32) =7.08, 170 I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176

A Attacks Bites 90 30 44-111

5-41 60 0-29 * 20 4-66

* 30 ** 7-53 10 0-23 ** ** ** Number (Median,Range)

Number (Median,Range) ** ** ** 0-17 0-7 0-1 0-1 0-2 0-4 0 0 Saline 0.1 0.3 0.6 1 1.25 Saline 0.1 0.3 0.6 1 1.25 Dose 8-OHDPAT (mg/kg) Dose 8-OHDPAT (mg/kg)

B Attack Latency Bite Latency 800 800

** ** ** ** ** 600 ** 600

400 400

200 200 Time (Seconds+SEM) Time (Seconds+SEM)

0 0 Saline 0.1 0.3 0.6 1 1.25 Saline 0.1 0.3 0.6 1 1.25 Dose 8-OHDPAT (mg/kg) Dose 8-OHDPAT (mg/kg) C 600 Contact Time

400

** ** 200 ** Time (Seconds+SEM)

0 Saline 0.1 0.3 0.6 1 1.25 Dose 8-OHDPAT (mg/kg)

Fig. 1. R(+)-8-OH-DPAT treatment decreases general measures of offensive aggression in adolescent cocaine-treated hamsters. (A) Aggression intensity (i.e., number of attacks and bites) in saline- and R(+)-8-OH-DPAT-treated residents. (B) Aggression initiation (i.e., mean latency to first attack and bite) in saline- and 8-OH-DPAT-treated residents. (C) Contact time in saline- and R(+)-8-OH-DPAT-treated residents. Dashed lines in (A–C) represent the baseline behavioral response of non-aggressive, saline (i.e., non-cocaine)-treated residents. Bars denote S.E.M. *p <0.05, **p <0.01. p <0.001), with an effective dose beginning at 0.6 mg/kg than did those administered low (0.1–0.3 mg/kg) doses of (Fig. 1C). Indeed, all animals treated with moderate to high agonist. For instance, compared to the lowest dose of R(+)- doses (0.6–1.25 mg/kg) of R(+)-8-OH-DPAT showed 8-OH-DPAT tested (i.e., 0.1 mg/kg), moderate (0.6 mg/kg) marked reductions in contact time compared to saline- and high (1.0 and 1.25 mg/kg) doses of agonist significantly treated controls (0.6 mg/kg [t =3.56], 1.0 mg/kg [t =3.21], reduce contact time (0.6 mg/kg [t =3.42, p <0.01], 1.0 mg/ 1.25 mg/kg [t =4.18], p <0.01 each comparison). Con- kg [t =3.07, p <0.05], 1.25 mg/kg [t =4.04, p <0.01]). versely, the low-dose R(+)-8-OH-DPAT treatment groups Similar results were obtained comparing the higher of the (0.1–0.3 mg/kg) showed no significant difference in the two low doses of R(+)-8-OH-DPAT (i.e., 0.3 mg/kg) to total duration of physical contact with intruders compared to moderate (0.6 mg/kg) and high (1.0 and 1.25 mg/kg) dose saline treated animals. Between treatment group compar- treatment groups (0.6 mg/kg [t =2.81, p <0.01], 1.0 mg/kg isons indicated that hamsters treated with moderate (0.6 mg/ [t =2.46, p <0.05], 1.25 mg/kg [t =3.43, p <0.01]). Animals kg) and high (1.0–1.25 mg/kg) doses of R(+)-8-OH-DPAT in the moderate to high-dose R(+)-8-OH-DPAT treatment also spent significantly less time interacting with intruders groups showed reduced locomotive behavior (data not I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176 171 shown) and spent far more time (greater than 7–8 min) dose tested (0.1 mg/kg), R(+)-8-OH-DPAT significantly engaging in non-social behaviors (e.g., grooming and reduced the number of lateral attacks (Z =2.45, p <0.05) digging) during the test period. compared to saline-treated animals, as did the higher doses tested (lateral attacks; 0.3 mg/kg [Z =3.68, p <0.001] and 3.2. Effects on select measures of adolescent cocaine- 0.6 mg/kg [Z =3.13, p <0.001]) (Fig. 2). Similarly, at a low induced offensive aggression dosage of 0.3 mg/kg, R(+)-8-OH-DPAT significantly reduced the number of flank bites (Z =2.69, p <0.01) When examined more precisely, 5HT1A receptor activa- compared to saline-treated animals, as did the higher dose tion with low doses (0.1–0.3 mg/kg) of R(+)-8-OH-DPAT (0.6 mg/kg [Z =3.13, p <0.01]) (Fig. 2A). Conversely, at produced an overall effect on several specific and targeted both low effective doses (0.1 and 0.3 mg/kg), R(+)-8-OH- offensive responses, including lateral attack (v2 =15.49, DPAT did not produce any significant differences in upright p <0.001) and flank bites (v2 =7.75, p <0.05). At the lowest offensive attacks (0.1 mg/kg [Z =1.39, p =0.17], 0.3 mg/kg

A Lateral Attacks Flank Bites 90 30

34-98 60 20 * 5-37 2-68 0-29

30 10 *** 2-57 ** Number (Median,Range) *** Number (Median,Range) 0-23 ** 0-12 0-1 0 0 Saline 0.1 0.3 0.6 Saline 0.1 0.3 0.6 Dose 8-OHDPAT (mg/kg) Dose 8-OHDPAT (mg/kg)

B Attack Latency Bite Latency 800 800 *** 600 600 **

400 400

200 200 Time (Seconds+SEM) Time (Seconds+SEM)

0 0 Saline 0.1 0.3 0.6 Saline 0.1 0.3 0.6 Dose 8-OHDPAT (mg/kg) Dose 8-OHDPAT (mg/kg) C 600 Contact Time

400

*** 200 Time (Seconds+SEM)

0 Saline 0.1 0.3 0.6 Dose 8-OHDPAT (mg/kg)

Fig. 2. Anti-aggressive effects of low-to-moderate doses of R(+)-8-OH-DPAT on the offensive response pattern of adolescent cocaine-treated hamsters. (A) Low-dose agonist treatment (0.1–0.3 mg/kg) selectively decreases specific and targeted measures of aggression intensity (i.e., number of lateral attacks and flank bites) in aggressive adolescent cocaine-treated residents, while (B) aggression initiation (i.e., mean latency to first attack and bite) are only affected at doses, which reduce social interest (i.e., contact time) as in (C). Bars denote S.E.M. *p <0.05, **p <0.01, ***p <0.001. 172 I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176

Cocaine Saline 150 offensive aggression [24] that could be blocked by enhancing 5HT signaling [14]. Animals that respond aggressively following adolescent cocaine exposure showed significant 120 decreases in 5HT afferent innervation to many areas of the hamster brain important for aggression control [14]. 90 Together, these data suggested that adolescent cocaine exposure stimulated aggression by suppressing the activity 60 and development of the 5HT neural system implicated in the

Mean Weight (Grams+SEM) inhibition of offensive aggression. Such alterations in 5HT

30 development and function might have drastic consequences 1357911131517192123252729 on the activity and expression of the 5HT receptor pools that Treatment Day subserve the anti-aggressive response of 5HT. Indeed, the Fig. 3. Growth curves during the experimental treatment period. Body inhibitory nature of 5HT on aggression has been predom- weight gained in animals treated with low dose (0.5 mg/kg/day) cocaine inately attributed to the activity of 5HT type 1 (i.e., 5HT1A (squares) or saline (triangles) for 30 consecutive days during adolescence and 5HT1B) and type 2 receptors [5,12,36,39,45,46]. (P27–57). Bars denote S.E.M. Recently, we have localized and mapped the 5HT1A receptor to each aggression area of the hamster brain (data not [Z =1.1, p =0.27]), rump bites (0.1 mg/kg [Z =1.35, shown). Perhaps exposure to cocaine during adolescence p =0.16], 0.3 mg/kg [Z =1.59, p =0.11]), flank marks (0.1 stimulates aggression by altering 5HT signaling through mg/kg [Z =0.46, p =0.64], 0.3 mg/kg [Z =1.13, p =0.25]). 5HT1A receptors in these brain areas. Indeed, cocaine 5HT1A receptor activation with R(+)-8-OH-DPAT pro- treatment has been previously shown to decrease 5HT1A duced an overall effect on the latency to attack ( F(5,35) = receptor activity and expression [3,31,41]. To address this 8.63, p <0.001) and bite ( F(5,35) =9.06, p <0.001), with an question, we performed experimental manipulations effective dose beginning at 0.6 mg/kg. A significant employing the selective 5HT1A agonist R(+)-8-OH-DPAT increase in attack (t =3.17, p <0.01) and bite (t =18.03, to examine the possibility that 5HT1A receptors might play p <0.001) latency was observed at 0.6 mg/kg compared to in important role in adolescent cocaine-induced aggressive saline-treated animals (Fig. 2B). However, as observed in behavior. The behavioral data presented here support our Experiment 1, at this moderate dose of R(+)-8-OH-DPAT, hypothesis that 5HT1A receptors play an inhibitory role in significant reductions in social interest towards intruders adolescent cocaine-induced offensive aggression. For (i.e., contact time with conspecifics) (t =6.69, p <0.001) are instance, the IP administration of saline to cocaine-treated observed that are not seen with lower doses of agonist (0.1 hamsters had no effect on aggressive responding. These mg/kg [t =0.49] and 0.3 mg/kg [t =2.03], p >0.05 each animals showed equally high levels of offensive aggression comparison) (Fig. 2C). analogous to that observed in our previous studies [14,24,43,44]. Nearly all saline-treated animals (14 out of 3.3. Body weight 15) showed a very high intensity of aggression as (defined by the total amount of attacks and bites, lateral attacks and Prior to treatment, at P27, initial body weights were not flank bites) and a quick onset (initiation) of the aggressive significantly different (TS.D.) between cocaine-treated and response (defined by the latency to the first attack and bite). control hamsters (cocaine-treated, 63.15T4.07 g vs. control, In fact, the response pattern and high level of aggression 64.08T4.9 g). Following 30 days of treatment with cocaine, seen in saline-treated animals exceeds that of experienced at P58, each of the treatment groups showed a significant adult ‘‘fighters’’ (i.e., hamsters trained to respond aggres- increase in body weight (cocaine-treated, 116.53T7.75 g vs. sively by repeated exposure to conspecifics) stimulated to control, 122.08T10.46 g) (Fig. 3). However, there was no respond hyper-aggressively by activation of the neural significant difference in total body weight gain observed circuits stimulating the aggressive response [17]. (t =1.51, p >0.1) between cocaine-treated (57.4T3.6 g) and Conversely, the i.p. administration of the selective 5HT1A control (63.2T5.5 g) groups. receptor agonist R(+)-8-OH-DPAT dose-dependently reduced adolescent cocaine-induced aggression. In the first experiment, R(+)-8-OH-DPAT-treated animals showed a 4. Discussion greater than 40% decrease in the number of attacks and a 60% decrease in the number of bites, with an effective doses Serotonin has been observed to play an inhibitory role in beginning at 0.1 mg/kg (attacks) and 0.3 mg/kg (bites), aggression in humans [7,8,28,32] and in many animal respectively. However, the low doses of R(+)-8-OH-DPAT models and species [26,29,49,54], including hamsters [16– (i.e., 0.1–0.3 mg/kg) observed to reduce aggression 18,22]. Recently, we showed that hamsters repeatedly intensity (i.e., the number of attacks and bites during the treated with low doses of cocaine (0.5 mg/kg/day) through- test period) had no affect on the initiation of the aggressive out adolescent development display significantly escalated response (i.e., latency to attack and/or bite). Higher doses of I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176 173

R(+)-8-OH-DPAT (i.e., 0.6 mg/kg and above) were required aggression initiation (i.e., latency to attack and bite) and to significantly affect the onset of aggressive responding. social interest (i.e., contact time), indicating general non- But, at these moderate-to-high doses, R(+)-8-OH-DPAT also specific behavioral inhibition. The fact that there was no significantly reduced contact time between experimental difference in contact time between saline- and R(+)-8-OH- animals and intruders, indicating a reduction in social DPAT-treated animals at low doses (0.1–0.3 mg/kg) interest towards conspecifics. This suggests that, at higher indicated that animals in both groups were equally doses (i.e., 0.6–1.25 mg/kg), R(+)-8-OH-DPAT may act in a interested in intruders and that the blockade of 5HT action non-specific fashion, generally affecting the overall behav- through the 5HT1A receptor did not attenuate cocaine- ioral state of the animal. Not surprisingly, animals in the induced offensive aggression through a similar nonspecific high-dose R(+)-8-OH-DPAT treatment group also displayed behavioral process. reduced locomotor activity often associated with ‘‘5HT The finding that 5HT1A activation (via R(+)-8-OH- syndrome’’, agreeing with observations from several studies DPAT) produces a dose dependent reduction of adolescent [19,33,39]. These reductions in social interest and locomo- cocaine-induced aggression combined with the explicit tion are somewhat surprising since there is evidence that the reduction of select target behaviors suggests that there moderate-to-high-dose regimen used in this study has been may be a specific regulatory mechanism of action for this observed to selectively reduce aggressive responding in receptor. Indeed, it is interesting that, while the effective mice [6,10,46,55]. anti-aggressive dose of R(+)-8-OH-DPAT (0.3 mg/kg) in Since acute 5HT1A receptor stimulation with low doses these studies is toward the higher dose range of what has (0.1–0.3 mg/kg) of R(+)-8-OH-DPAT dose-dependently previously been observed as an effective dose in other reduced only certain aspects of the adolescent cocaine- models of aggression [6,13,35,47], this dose did not induced aggressive response (i.e., aggression intensity but completely block the expression of all aspects of the not initiation), it was possible that R(+)-8-OH-DPAT was cocaine-induced aggressive response. A plausible explan- acting in a highly discriminating anti-aggressive fashion, ation for the ability of the low doses of R(+)-8-OH-DPAT to having 5HT1A-selective effects on specific and targeted block selective aspects of the adolescent cocaine-induced measures of the aggressive response. To address this issue, aggressive response may lie in the complicated nature of the the anti-aggressive properties of the effective doses (0.1– role of 5HT1A receptors in aggression. Indeed, the inhibitory 0.3 mg/kg) of R(+)-8-OH-DPAT were investigated examin- effect of 5HT1A receptor signaling on offensive aggression ing several more specific and targeted determinants of has been shown to be the result of 5HT action on offensive aggression. Consistent with the findings from the postsynaptic heteroreceptors in forebrain regions implicated first study, R(+)-8-OH-DPAT effectively reduced offensive in aggression stimulation and somatodendritic autoreceptors displays. In particular, the intensity of the aggressive on 5HT neurons in the raphe nuclei. Activation of response (i.e., amount and type of attacking and biting somatodendritic autoreceptors inhibits frequency of firing behavior) was significantly less in R(+)-8-OH-DPAT-treated of 5HT neurons in the raphe, selectively reducing 5HT animals vs. saline-treated counterparts, whereas the initia- release in areas of the brain receiving 5HT afferent tion of the aggressive encounter (i.e., latency to attack and innervation [21]. This mechanism of action has been bite) remained unchanged by low doses of R(+)-8-OH- implicated in the anti-aggressive properties of 5HT1A DPAT. Specifically, along with a greater than 40% and 60% agonists [11,38]. By this mechanism then, the anti-aggres- decrease in the total number of attack and bites, respectively, sive properties of low doses (0.1 mg/kg and 0.3 mg/kg) of R(+)-8-OH-DPAT-treated animals showed a greater than 5HT1A agonists on adolescent cocaine-induced offensive 60% decrease in the number of lateral attacks (at 0.1 mg/kg) aggression would be due to decreased 5HT activity in those and a 90% decrease in flank bites (at 0.3 mg/kg) compared areas of the brain involved in aggression regulation. This to saline-treated controls. No effect of R(+)-8-OH-DPAT mechanism seems unlikely, however, in light of previous was noted on upright offensive attacks, rump bites and/or reports from our lab showing that the highly escalated flank marks. Most interestingly, attack and bite behaviors aggressive phenotype resulting from adolescent cocaine which target either the flank or hind quarter region of the exposure could be reduced by enhancing 5HT activity, and intruder are highly organized and intense, adult forms of that aggressive, adolescent cocaine-treated hamsters had aggressive behavior [15,56]. These data suggest that 5HT1A significant deficits in 5HT afferent innervation to areas of activity may play an important role in modulating (i.e., the brain implicated in aggression control, namely the inhibiting) highly intense, mature aspects of the aggressive anterior hypothalamus (AH), bed nucleus of the stria response in adolescent cocaine-treated animals. Further, the terminalis (BNST), corticomedial (CoMeA) and medial selective nature of the anti-aggressive properties of R(+)-8- (MeA) amygdala, and lateral septum (LS); together impli- OH-DPAT at low doses (0.1–0.3 mg/kg) suggests that the cating decreased 5HT activity in aggressive, cocaine- attenuation of overall aggression observed in the first study exposed animals [14]. was not due to a more general behavioral affect of R(+)-8- Using pharmacodynamic reasoning, the loss of 5HT’s OH-DPAT. In fact, results from the second study agree with trophic influence to the aforementioned brain areas in the first, in that moderate doses of R(+)-8-OH-DPAT reduce aggressive, adolescent cocaine-treated animals may cause 174 I. Knyshevski et al. / Physiology & Behavior 85 (2005) 167–176

the up-regulation of heteroreceptor pools (including 5HT1A action of the anti-aggressive properties of R(+)-8-OH-DPAT receptors), resulting in postsynaptic systems more sensitive in adolescent cocaine-induced aggression, as well as the to 5HT1A agonists. Since activation of postsynaptic 5HT1A neurodevelopmental effects of adolescent cocaine exposure receptors cause a hyperpolarizing potassium current, reduc- on the 5HT1A and 5HT7 neural systems. ing postsynaptic neuronal activity [1,52], a more plausible In summary, the studies presented in this paper provide explanation for the selective anti-aggressive effects of the first examination of the role of 5HT1A receptors in 5HT1A agonists on adolescent cocaine-induced offensive adolescent cocaine-induced aggression. These findings aggression may be explained by a straightforward dose show that increases in offensive aggression resulting from effect on signaling through highly sensitive postsynaptic adolescent cocaine treatment are modulated by 5HT1A heteroreceptors located on aggression-facilitating neurons in receptor activity, suggesting that 5HT1A receptor activity these brain regions (e.g., arginine vasopressin [AVP] is an important component of the adolescent cocaine- neurons in the AH). Indeed, recently, we have co-localized induced aggressive response. These findings provide a 5HT1A receptors onto AH AVP neurons (unpublished direct link between adolescent cocaine, 5HT1A receptor observations) and the site-specific activation of postsynaptic function and the regulation of adolescent cocaine-induced 5HT1A heteroreceptors in this site (as well as in the CoMeA offensive aggression in hamsters. and medial preoptic area) have been shown to reduce offensive displays [9,11,18]. However, postsynaptic activation also mediates a variety of functional responses Acknowledgments including many of the symptoms of 5HT syndrome [37,48,53]. It is possible then that, in a compromised 5HT The authors would like to thank Jill M. Grimes for neural system (as seen following adolescent cocaine critical reading of the manuscript. R.H.M. would like to exposure) with highly sensitive 5HT1A heteroreceptor pools, extend special thanks to Dr. K.A. Melloni for support and low doses (e.g., 0.1–0.3 mg/kg) of 5HT1A agonists encouragement. This work was supported by NIH research selectively reduce distinct displays of offensive responding grant RO1 DA10547 from NIDA to R.H.M. and a Research (e.g., attack vs. bite behavior), while slightly higher doses Development and Scholarship Fund Award from the Office (greater than or equal to 0.3 mg/kg but less than 0.6 mg/kg) of the Provost at Northeastern University. 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