Alterations in Anterior Hypothalamic Vasopressin

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ing that 5-HT tone may not play as large a role in the modulation of the adolescent AAS-induced aggressive response as previously thought (Grimes & Melloni ...
Behavioral Neuroscience 2007, Vol. 121, No. 5, 941–948

Copyright 2007 by the American Psychological Association 0735-7044/07/$12.00 DOI: 10.1037/0735-7044.121.5.941

Alterations in Anterior Hypothalamic Vasopressin, but not Serotonin, Correlate With the Temporal Onset of Aggressive Behavior During Adolescent Anabolic–Androgenic Steroid Exposure in Hamsters (Mesocricetus auratus) Jill M. Grimes, Lesley A. Ricci, and Richard H. Melloni Jr. Northeastern University In hamsters (Mesocricetus auratus), anabolic–androgenic steroid (AAS) exposure during adolescence facilitates offensive aggression that is correlated with the enhanced development of the arginine vasopressin (AVP) neural system and reduced development of the serotonin (5-HT) neural system in the anterior hypothalamus (AH). This study examined the temporal onset of these effects by measuring aggression and AH AVP and 5-HT during progressively shorter periods of AAS exposure during adolescent development. The authors tested adolescent hamsters that received AAS for 3, 7, 14, or 28 days for offensive aggression and then examined the hamsters for AVP/5-HT afferent innervation to the AH using immunohistochemistry. While reductions in AH 5-HT afferent innervation were detectable by 7 days of AAS exposure, no concomitant increases in offensive aggression were observed compared to oil-treated littermates. In contrast, by Day 14 of AAS treatment, AH AVP and offensive aggression were significantly higher than oil-treated controls. These data indicate that relatively short-term adolescent AAS exposure alters aggression and AH 5-HT and AVP development, yet only alterations in AH AVP development correlate with temporal onset of the aggressive behavioral phenotype during adolescent AAS exposure. Keywords: anabolic–androgenic steroids, arginine vasopressin, serotonin, anterior hypothalamus, aggression

highly elevated levels of offensive aggression when tested immediately following the exposure period on the first behavioral interaction. The finding that adolescent AAS-treated hamsters demonstrated highly escalated offensive aggression in the absence of prior social interactions and dominance cues suggested that adolescent exposure to AAS stimulated aggression directly, perhaps by affecting the development or activity of neural circuits that regulate this behavior. In hamsters, the anterior hypothalamus (AH) appears to be at the center of a neural network of reciprocal connections between the lateral septum, medial amygdala, and ventrolateral hypothalamus that regulates offensive aggression (Delville, De Vries, & Ferris, 2000). The activity of the entire network is regulated (at least partially) by the activity of centrally released arginine vasopressin (AVP) and serotonin (5-HT) within the AH. In this instance, AH AVP activity has been shown to facilitate offensive aggression that is normally inhibited by AH 5-HT (Ferris et al., 1997; Ferris, Stolberg, & Delville, 1999). The AH appears also to be an important point of convergence for AAS-induced neuroplastic changes in the AVP and 5-HT neural systems that correlate with the expression of the aggressive phenotype. For example, recently we have shown that hamsters stimulated to respond aggressively following the administration of AAS during the majority of adolescent development—postnatal (P) Days 27–56 — display increases in AVP afferent development to the latero-anterior portion of the AH (i.e., the latero-anterior hypothalamic [LAH] nucleus) commensurate with deficits in 5-HT afferent innervation and alterations in 5-HT1A and 1B receptor distri-

In a number of previous studies, we have used developmentally immature Syrian hamsters (Mesocricetus auratus) as an adolescent animal model to examine the link between developmental anabolic–androgenic steroid (AAS) exposure and the behavioral neurobiology of offensive aggression. Behavioral data from these studies indicated that hamsters repeatedly exposed to AAS during relatively short periods of adolescent development (i.e., 14 days; Melloni, Connor, Hang, Harrison, & Ferris, 1997; Melloni & Ferris, 1996) or the majority of the adolescent period (i.e., 28 days; DeLeon, Grimes, & Melloni, 2002; Grimes & Melloni, 2002, 2005, 2006; Grimes, Ricci, & Melloni, 2003, 2006; Harrison, Connor, Nowak, Nash, & Melloni, 2000; Ricci, Grimes, & Melloni, 2007; Ricci, Rasakham, Grimes, & Melloni, 2006) display

Jill M. Grimes, Lesley A. Ricci, and Richard H. Melloni Jr., Behavioral Neuroscience Program, Department of Psychology, Northeastern University. This research was supported by National Institutes of Health (NIH) Grant (R01) DA10547 awarded to Richard H. Melloni Jr., and National Institute on Drug Abuse Predoctoral Fellowship (F31) DA18033 awarded to Jill M. Grimes. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Richard H. Melloni Jr. would like to extend special thanks to K. A. Melloni for her support and encouragement. Correspondence concerning this article should be addressed to Richard H. Melloni Jr., Behavioral Neuroscience Program, Department of Psychology, 125 Nightingale Hall, Northeastern University, 360 Huntington Avenue, Boston, MA 02115. E-mail: [email protected] 941

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butions in this same brain area compared to nonaggressive, vehicle-treated controls, suggesting that increased LAH AVP tone and decreased LAH 5-HT tone modulate the development of the adolescent AAS-induced aggressive phenotype (Grimes & Melloni, 2002, 2005; Harrison et al., 2000; Ricci et al., 2006). This notion was supported by behavioral pharmacology studies employing AVP receptor antagonists and 5-HT receptor agonists (Grimes & Melloni, 2002, 2005; Harrison et al., 2000; Ricci et al., 2006). During withdrawal from repeated adolescent AAS exposure, reductions in LAH AVP afferent innervation correlate with the return to the nonaggressive behavioral phenotype, indicating that at times of increased LAH AVP tone, hamsters responded more aggressively than when levels of LAH AVP were low (Grimes et al., 2006). However, deficits in LAH 5-HT afferent fibers were observed throughout the extended period of withdrawal, suggesting that 5-HT tone may not play as large a role in the modulation of the adolescent AAS-induced aggressive response as previously thought (Grimes & Melloni, 2006). Together, these data indicated that adolescent AAS exposure had short-term, reversible effects on aggression and AH AVP, but not AH 5-HT, correlating alterations in AH AVP with the aggressive behavioral phenotype. These data strengthened the notion that the interactions between AAS and the AH AVP neural system might directly underlie adolescent AASinduced offensive aggression, while at the same time calling into question the role of AH 5-HT and this behavioral response. Given these data, we questioned whether shorter times of exposure to AAS during adolescent development would have similar effects on the development of the LAH AVP and 5-HT neural systems, and whether alterations in the development of one neural system versus the other would correlate with the emergence of the aggressive phenotype during adolescent exposure. It is possible that, either together or alone, these adolescent AAS-induced alterations occur at even earlier times of AAS exposure, stimulating a more rapid development of the aggressive phenotype. To date, however, it is unknown whether brief adolescent AAS exposure predisposes an animal to behave aggressively in response to a threatening stimulus, and whether the behavioral effects of abbreviated time frames of exposure correlate with the altered development of the AH AVP and 5-HT neural systems. We conducted these studies to determine the temporal relationship between the onset of adolescent AAS-induced offensive aggression and the development of the LAH AVP and 5-HT neural systems. First, to determine the time course of adolescent AAS effects on aggression, we measured offensive aggression in adolescent hamsters administered AAS for 3, 7, 14, or 28 days. Then, to determine whether the behavioral effects of adolescent AAS correlated with alterations in LAH AVP or 5-HT, we examined the density of AVP and 5-HT afferent innervation to the AH in these same hamsters 1 day later.

Experimental Procedures Subjects In Syrian hamsters (Mesocricetus auratus), the adolescent period of development can be identified as the time between P25 and P60. Weaning generally occurs P23–P25, with the onset of puberty (as determined by the onset of gonadal maturation) beginning around 1 week later (Miller, Whitsett, Vandenbergh, & Colby,

1977). Testosterone levels start to rise at around P30, reaching near peak levels by P45, and finally peaking P50 –55 (Melloni, Connor, Nash, & Harrison, 1997; Miller et al., 1977). During this developmental time, hamsters wean from their dams, leave the home nest, establish new solitary nest sites, and learn to defend their territory and participate in social dominance hierarchies (Schoenfeld & Leonard, 1985; Whitsett, 1975). For the experimental treatment paradigm, intact preadolescent male hamsters (P21) were obtained from Charles River Laboratories (Wilmington, MA), individually housed in Plexiglas cages, and maintained at ambient room temperature on a reverse light– dark cycle of 14:10-hr light– dark cycle; lights on at 1900. Food and water were provided ad libitum. For aggression testing, stimulus (intruder) males of equal size and weight to the experimental subjects were obtained from Charles River 1 week prior to the behavioral test, group housed with 5 hamsters/cage in large polycarbonate cages, and maintained as above to acclimate to the hamster facility. All intruders were prescreened for avoidance (i.e., disengage and evade) and submission (i.e., tail-up freeze, flee, and fly-away) 1 day prior to the aggression test to control for behavioral differences between stimulus subjects, as previously described (Ferris et al., 1997; Melloni, Connor, Hang, et al., 1997; Ricci, Grimes, & Melloni, 2004; Ricci, Knyshevski, & Melloni, 2005). Intruders that failed to engage residents or displayed submissive postures (⬍5%) were excluded from use in the behavioral assay. All methods and procedures described below were preapproved by the Northeastern University Institutional Animal Care and Use Committee.

Experimental Treatment On P28, hamsters (n ⫽ 76) were weighed and randomly assigned into four groups corresponding to the time at which they would be tested for offensive aggression and killed for immunohistochemistry. Each group was divided into two drug treatment groups: those administered a high-dose mixture of AAS suspended in sesame oil, or those administered sesame oil alone (vehicle control). Hamsters (n ⫽ 6 –10 subjects/drug treatment/group) received daily subcutaneous injections (0.1– 0.2 ml) of an AAS mixture consisting of 2 mg/kg testosterone cypionate, 2 mg/kg nortestosterone, and 1 mg/kg dihydroxytestosterone undecylate (Steraloids Inc., Newport, RI); or injections of sesame oil vehicle for 3, 7, 14, or 28 consecutive days, as previously described (DeLeon, Grimes, & Melloni, 2002; Grimes & Melloni, 2002, 2005; Grimes et al., 2003; Harrison et al., 2000). This daily treatment of AAS was designed to mimic a chronic “heavy use” regimen (Pope & Katz, 1988, 1994). The day after the last injection, the hamsters (n ⫽ 8 –10/drug treatment/group) were tested for offensive aggression (as detailed below) and killed 24 hr later for immunohistochemistry. Specifically, Group 1 (3 days) hamsters were tested for offensive aggression 1 day after a 3-day exposure to AAS and then killed 24 hr later, while hamsters in Groups 2 (7 days), 3 (14 days), and 4 (28 days) were tested and killed following 7, 14, and 28 days of AAS exposure, respectively. AAS- and oil-treated hamsters in each group were killed via transcardial perfusion, and brains were removed and processed for AVP and 5-HT immunohistochemistry, as detailed below.

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Aggression Testing

Image Analysis

We tested experimental subjects for offensive aggression using the resident–intruder paradigm, a well-characterized and ethologically valid model of offensive aggression in Syrian hamsters (Floody & Pfaff, 1977; Lerwill & Makings, 1971). For this measure, an intruder of similar size and weight was introduced into the home cage of experimental subjects and the resident was scored for offensive attack behavior (i.e., total number of lateral, rump, and upright offensive attacks). Briefly, an attack was scored each time the resident hamster chased the intruder and then lunged toward or confined the intruder by upright and sideways threat; each threat was generally followed by a direct attempt to bite the intruder’s flank or rump. Each aggression test lasted 10 min and was scored by an observer unaware of the hamsters’ experimental treatment. No intruder was used for more than one behavioral test. All tests were performed during the first 4 hr of the dark phase under dim red illumination and videotaped for behavioral verification of the findings.

We determined the area covered by AVP- and 5-HT– immunoreactive (ir) fibers within the AH using the Bioquant Nova 5.0 computer-assisted microscopic image analysis software package as previously described (DeLeon, Grimes, Connor, & Melloni, 2002; Grimes & Melloni, 2002, 2005; Grimes et al., 2003). The specific AH aggression area examined comprised the LAH brain region—that is, the brain area in the AH just ventro-lateral to the nucleus circularis and dorso-medial to the medial supraoptic nucleus (see Figure 2). This distinct subregion of the AH proper has been linked to the AVP/5-HT neural control of aggressive behavior in a number of studies (Ferris et al., 1997, 1999), including those investigating the link between the AH AVP and 5-HT neural systems and adolescent AAS-induced aggression (Grimes & Melloni, 2002, 2005, 2006; Grimes et al., 2006; Harrison et al., 2000). Slides from each animal were coded by an experimenter unaware of the experimental conditions. We used Bioquant Nova 5.0 image analysis software running on a Pentium III CSI Open PC computer (R&M Biometrics, Nashville, TN) to identify the AH at the level of the nucleus circularis at low power (4⫻) using a Nikon E600 microscope. At this magnification, a standard computer-generated box was drawn to fit within the particular region of interest. Then, under the same magnification, images were applied a threshold at a standard RGB-scale level empirically determined by observers blinded to treatment conditions to allow detection of stained AVPand 5-HT–ir fibers with moderate to high intensity, while suppressing lightly stained elements. This threshold value was then applied across subjects to control for changes in background staining and differences in foreground staining intensity between hamsters. The illumination was kept constant for all measurements. We identified LAH AVP- and 5-HT–ir fibers in each field using a mouse-driven cursor and then quantified automatically by the Bioquant software. One to two independent measurements of AVP- and 5-HT–ir elements were taken from consecutive sections of each hamster per treatment group, depending on (a) identification of the exact position of the nucleus circularis within the region of interest, and (b) the size of the nucleus in the rostral– caudal plane. Then, the density of AVP- and 5-HT–ir fibers were determined for the entire AH (area density) and used for statistical analysis.

Immunohistochemistry One day following the behavioral test for aggression, AAS and sesame-oil-treated hamsters were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine and the brains fixed by transcardial perfusion with 4% paraformaldehyde. Brains were then cryoprotected by incubating in 30% sucrose in phosphate buffered saline (0.001M KH2PO4, 0.01M Na2HPO4, 0.137M NaCl, 0.003M KCl, pH 7.4) overnight at 4 °C. We cut a consecutive series of 35-␮m coronal sections on a sliding microtome, collected as free-floating sections in 1 ⫻ Phosphate Buffered Saline and labeled for AVP by single-label immunohistochemistry using a modification of an existing protocol (Ricci et al., 2004). For AVP immunohistochemistry, free-floating sections were pretreated with 4.5% H2O2 (30% stock solution) followed by preincubation in 10% normal goat serum (NGS) and 1% bovine serum albumin with 0.6% Triton X-100. Sections were incubated in primary antiserum (1:10,000) for AVP anti-rabbit (DiaSorin) with 10% NGS, 1% bovine serum albumin, and 0.6% Triton X-100 for 24 hr at 33 °C. After primary incubation, sections were incubated in secondary goat anti-rabbit followed by tertiary antisera (Vectastain ABC Elite Kit—rabbit, Vector Labs, Burlingame, CA) for 90 min at room temperature and then labeled with diaminobenzidine (Vector Labs, Burlingame, CA). For 5-HT immunohistochemistry, free-floating sections were pretreated with 3% H2O2 (30% stock solution) followed by preincubation in 20% NGS with 0.6% Triton X-100. Sections were incubated in primary antiserum (1:1,000) for 5-HT anti-rabbit (Protos Biotech, New York, NY) with 20% NGS and 0.6% Triton X-100 for 24 hr at 37 °C. After primary incubation, sections were incubated in secondary goat anti-rabbit followed by tertiary antisera (Vectastain ABC Elite Kit—rabbit, Vector Labs, Burlingame, CA) for 60 min each at room temperature and then labeled with diaminobenzidine (Vector Labs, Burlingame, CA). All sections were mounted on gelatin-coated slides, allowed to air dry, and dehydrated through a series of ethanol and xylene solutions. We then coverslipped the slides using Cytoseal-60 mounting medium (VWR Scientific, West Chester, PA).

Statistics Results from the aggression tests and immunohistochemical staining from AAS-treated hamsters were converted to a percentage of baseline responding compared to the relevant control condition. For these studies, sesame oil treatment was the control condition in each case. We compared all behavioral and neurobiological data using two-way ANOVA, followed by Dunn’s Bonferroni-corrected post hoc (two-tailed) tests when applicable. We analyzed percentage change in lateral attack and AH AVP using linear correlation analysis. The alpha level for all experiments was set at .05.

Results Offensive Aggression AAS administration throughout the developmental period of adolescence produced a time-dependent increase in offensive at-

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tack, such that the longer subjects were exposed to AAS the more attack behaviors they displayed relative to sesame-oil-treated controls, F(1, 3) ⫽ 9.2, p ⬍ .0001. When results were analyzed more closely, there were main effects of both treatment (i.e., AAS exposure) and age (i.e., length of administration). During adolescent development, overall differences in offensive attack, F(1, 62) ⫽ 29.6, p ⬍ .0001, were observed in AAS-treated hamsters when compared with vehicle controls (see Figure 1). Specifically, AAS-treated hamsters directed a higher number of offensive attacks at intruders following 14 ( p ⬍ .05) and 28 ( p ⬍ .01) days of exposure to AAS compared to sesame-oil-treated control subjects (dashed line in Figure 1). The level of aggression observed in hamsters treated with AAS during the majority of adolescent development (i.e., P28 –56, 28 days of exposure) was nearly identical to that described in our previous studies (DeLeon, Grimes, & Melloni, 2002; Grimes & Melloni, 2002, 2005, 2006; Grimes et al., 2003, 2006; Harrison et al., 2000; Ricci, Grimes, & Melloni, 2007; Ricci et al., 2006). In addition, within-AAS-group comparisons indicated that hamsters exposed to AAS for longer periods of time showed significant differences, F(3, 62) ⫽ 9.2, p ⬍ .0001. Specifically, at 14 and 28 days of exposure hamsters displayed significant increases in the number of offensive attacks toward intruders when compared to hamsters exposed for only 3 or 7 days. Hamsters exposed to AAS for 14 days attacked more than those receiving only 3 days of exposure ( p ⬍ .05); whereas at 28 days of exposure, AAS hamsters showed significantly higher attack numbers compared to those receiving 3 ( p ⬍ .01), 7 ( p ⬍ .01), or 14 ( p ⬍ .05) days of AAS, respectively.

length of fibers from these neurons into the LAH (see Figure 2A). AAS administration during adolescence produced a timedependent increase in AVP-ir such that the longer the hamsters were exposed to AAS, the higher the density of AVP-ir in the LAH relative to sesame-oil-treated controls, F(1, 3) ⫽ 9.2, p ⬍ .0001. When results were analyzed more closely, main effects of both treatment (i.e., AAS exposure) and age (i.e., length of administration) were observed on the density of AVP-ir in the LAH. During adolescent AAS exposure, overall differences in AVP-ir staining, F(1, 36) ⫽ 27.01, p ⬍ .0001, were observed in the LAH of AAS-treated hamsters when compared with vehicle controls (see Figure 2, black bars in graph). Specifically, AAS-treated hamsters displayed a significant increase in the density of AVP-ir fibers in the LAH at 14 days of exposure to AAS ( p ⬍ .01) and continuing through 28 ( p ⬍ .01) days of AAS treatment compared to sesameoil-treated controls (dashed line in Figure 2). The difference in the density of AVP-ir fibers in the LAH observed of these subjects was nearly identical to that described in our previous studies employing a treatment regimen that encompassed nearly the entire adolescent period (Grimes et al, 2006; Harrison et al., 2000). In addition, within-AAS-group comparisons indicated a main effect of exposure time on AVP-ir density in the LAH, F(3, 36) ⫽ 10.2, p ⬍ .0001. Specifically, hamsters exposed to AAS for 14 days displayed significant increases in the mean area covered by AVP-ir fibers in the LAH when compared to hamsters exposed to AAS for 3 ( p ⬍ .01) or 7 ( p ⬍ .01) days. Similarly, at 28 days of exposure, AVP-ir in the LAH was significantly higher than that observed in hamsters receiving 3 ( p ⬍ .01) or 7 ( p ⬍ .01) days of AAS, respectively.

LAH AVP Immunohistochemistry A high density of AVP-ir was found in the LAH of both AASand vehicle-treated hamsters. A dense staining pattern was distributed in the neuronal somata region located in the nucleus circularis and medial supraoptic nucleus and appeared consistent along the

Figure 1. Comparison of offensive attacks during the course of adolescent AAS treatment. Comparisons to vehicle-treated controls: * p ⬍ .05, ** p ⬍ .01. Within-group comparisons of adolescent AAS-treated hamsters at 3 and 7 days versus 14 days of exposure and 3, 7, and 14 days versus 28 days of exposure, respectively: # p ⬍ .05, # # p ⬍ .01, Mann–Whitney U tests (two-tailed). Data are expressed as percentage of vehicle responding, as indicated by the dashed line at 100% on graph. Error bars represent the standard error of the mean. AAS ⫽ anabolic–androgenic steroids. Hyphen indicates no statistical data.

LAH 5-HT Immunohistochemistry In previous studies, we and others have used immunohistochemical staining of 5-HT fibers and varicosities as a sensitive marker of the development of 5-HT afferent projections into the LAH (DeLeon, Grimes, Connor, & Melloni, 2002; Delville, Melloni, & Ferris, 1998; Grimes & Melloni, 2002; Taravosh-Lahn, Bastida, & Delville, 2005, 2006). As described previously (Grimes & Melloni, 2002, 2006), a high density of 5-HT-ir staining (i.e., fibers and varicosities) was found in the LAH of vehicle-treated hamsters (see Figure 2B). AAS administration throughout the developmental period of adolescence produced a decrease in 5-HT-ir such that hamsters exposed to AAS for longer than 3 days showed less 5-HT-ir density in the LAH, relative to sesame-oil-treated controls F(1, 3) ⫽ 9.2, p ⬍ .0001. When results were analyzed more closely, main effects of both treatment (i.e., AAS exposure) and age (i.e., length of administration) were observed on the density of 5-HT-ir. During adolescent AAS exposure, overall differences in 5-HT-ir staining, F(1, 38) ⫽ 17.8, p ⬍ .0001, were observed in the LAH of AAS-treated subjects when compared with vehicle controls (see Figure 2, white bars in graph). Specifically, AAS-treated subjects displayed a significant decrease in the density of 5-HT-ir varicosities and fibers in the LAH by 7 days of exposure to AAS ( p ⬍ .01) and continuing through 14 ( p ⬍ 0.01) and 28 ( p ⬍ .01) days of AAS treatment compared to sesame oil control hamsters (dashed line in Figure 2). The difference in the density of 5-HT-ir fibers in the LAH observed of these hamsters was nearly identical to that described in our previous studies employing a treatment regimen that encompassed nearly the entire adolescent period

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Figure 2. Darkfield photomicrograph showing immunoreactive labeling for AVP (A) and 5-HT (B) in the latero-anterior hypothalamic brain region (seen as white neuronal somata and afferent fiber streams for AVP and varicosities and puncta for 5-HT). Graph depicts the comparison of the density of AVP (black bars) and 5-HT (white bars) afferent fibers in the anterior hypothalamus during the course of adolescent AAS treatment. Comparisons to vehicle-treated controls: ** p ⬍ .01. Within-group comparisons of adolescent AAS-treated subjects at 3 and 7 days versus 14 and 28 days of exposure for AVP (black bars) and 3 days versus 7, 14, and 28 days of exposure for 5-HT (white bars), respectively: # # p ⬍ .01, Student’s t test, two-tailed. Data are expressed as percentage of vehicle responding, as indicated by the 100% dashed lines in graph. Error bars represent the standard error of the mean. AVP ⫽ arginine vasopressin; AAS ⫽ anabolic– androgenic steroids; AH ⫽ anterior hypothalamus; LAH ⫽ latero-anterior hypothalamus; NC ⫽ nucleus circularis; OC ⫽ optic chiasm.

(Grimes & Melloni, 2002, 2006). In addition, within AAS group comparisons indicated a main effect of age, F(3, 38) ⫽ 8.7, p ⬍ .001. Specifically, hamsters exposed to AAS for 7 ( p ⬍ .01), 14 ( p ⬍ .01), and 28 ( p ⬍ .01) days displayed significant decreases in the number of 5-HT-ir fibers and varicosities when compared to hamsters exposed to AAS for only 3 days of adolescent development.

Correlational Analysis: Offensive Aggression and LAH AVP/5-HT Immunohistochemistry When percentage change data for offensive attacks and density of LAH AVP-ir were collapsed across drug exposure time, a direct linear correlation (r ⫽ .758, p ⬍ .01) was observed between LAH AVP and attack behavior, with increases in AVP afferent innervation to the LAH correlating with higher levels of offensive attack. No correlation between 5-HT afferent innervation to the LAH and offensive aggression was observed (r ⫽ .131, p ⬎ .05).

Discussion To determine the time course of adolescent AAS effects on aggression, we measured offensive aggression in adolescent ham-

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sters administered AAS for 3, 7, 14, or 28 days. Replicating previous results from our laboratory (DeLeon, Grimes, & Melloni, 2002; Grimes & Melloni, 2002, 2005, 2006; Grimes et al., 2003, 2006; Harrison et al., 2000; Melloni, Connor, Hang, et al., 1997; Melloni & Ferris, 1996; Ricci et al., 2006, 2007), hamsters treated with AAS during the majority of the adolescent period (i.e., 28 days) or for a shorter period of adolescent development (i.e., 14 days) were significantly more aggressive than vehicle-treated controls. However, hamsters examined after progressively shorter treatment periods (i.e., 7 and 3 days of exposure) showed no such differences in aggressive responding compared to vehicle-treated littermates. Thus, a shorter period of exposure to AAS during adolescence (i.e., 2 weeks) was sufficient to significantly increase the aggressive behavior of experimental subjects. These data are significant, indicating that temporal onset of AAS-induced aggression can occur after relatively brief exposure times if the drug is administered during adolescent development. Most studies to date, including those from this laboratory, have examined the aggression-eliciting effects of adolescent AAS exposure after extended periods of administration—that is, during the majority of the adolescent period (i.e., 4 weeks; DeLeon, Grimes, and Melloni, 2002; Grimes & Melloni, 2002, 2005, 2006; Grimes et al., 2003, 2006; Harrison et al., 2000; Ricci et al., 2006, 2007). In each of these studies, AAS-treated hamsters displayed an intense and mature aggressive phenotype when tested during the late-pubertal stage of development using the standard resident–intruder paradigm. Similarly, pubertal male Long–Evans rats administered testosterone propionate, nandrolone decanoate, or stanozolol individually or together as a mixture showed increased aggressive responding after 4⫹ weeks of exposure, dependent on the type of AAS administered (Cunningham & McGinnis, 2006; Farrell & McGinnis, 2003, 2004; Wesson & McGinnis, 2006). In these studies, a significant increase in the resident–intruder and neutral arena aggression was observed only in testosterone-treated rats; however, in each case, increases in aggressive responding were only observed following tail pinch stimulus, indicating the requirement for physical provocation for display of the aggressive phenotype in rats (Wesson & McGinnis, 2006). That notwithstanding, results from both these lines of research indicate that longer exposure times to AAS during adolescent development (i.e., ⱖ4 weeks) have dramatic aggression-eliciting effects; however, it is clear that the specific effect greatly depends upon the type of AAS used, the species of the animal, the dosing and treatment regimen, and the behavioral testing paradigm employed. These data, along with the findings from the present study indicating that shorter periods of exposure to AAS during adolescent development enhance aggressive responding (i.e., 2 weeks), are significant in that they indicate that even short-term use of these substances by adolescent youths may pose a significant mental health risk. Next, to determine the temporal relationship between the onset of adolescent AAS-induced offensive aggression and the developmental alterations in the AH AVP and 5-HT neural systems, we examined aggression and the development of AVP and 5-HT afferent fibers to the LAH following 3, 7, 14, and 28 days of adolescent AAS exposure. Replicating previous results from our laboratory (Grimes et al., 2006; Grimes & Melloni, 2002, 2006; Harrison et al., 2000), hamsters treated with AAS during the majority of the adolescent period (i.e., 4 weeks) displayed significant increases in LAH AVP afferent fiber density and decreases in

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LAH 5-HT afferent innervation compared to controls. Hamsters examined after shorter treatment periods displayed alterations in both LAH AVP and 5-HT afferent development; however, these differences emerged at different times during the treatment period. Specifically, subjects examined for LAH AVP after very short treatment periods (i.e., 3 and 7 days of exposure) showed no differences in afferent innervation compared to vehicle-treated controls. In fact, it was not until 14 days of exposure that hamsters treated with AAS displayed significant increases in LAH AVP afferent density. When aggression levels and extent of LAH AVP afferent innervation were examined for each time point, a strong, positive correlation between LAH AVP fiber density and levels of offensive aggression was observed, indicating that at times of increased LAH AVP tone, subjects respond more aggressively than when levels of LAH AVP are low. This correlation between aggression and AH AVP across a shorter term of exposure further strengthens the notion that the interactions between AAS and the LAH AVP neural system directly underlie adolescent AASinduced alterations in offensive aggression. Conversely, upon examination of the AH 5-HT system, it was found that hamsters treated with AAS showed significant deficits in LAH 5-HT following only 7 days of exposure. These results show that dramatic reductions in LAH 5-HT afferent innervation/development occur well before any observable changes in aggressive behavior during the course of adolescent AAS exposure. Together these data suggest that changes in the development of the 5-HT neural system alone in the LAH may not explain the temporal onset of the aggressive phenotype that occurs during adolescent AAS exposure. These data are novel and significant in that no studies to date have linked changes in the development of defined neural systems with the temporal onset of the aggressive behavioral phenotype during adolescent AAS exposure. Further, these behavioral and neurobiologic data are in direct accord with those observed following AAS withdrawal, that is, alterations in AH AVP, but not 5-HT, correlate with the aggressive phenotype, providing a second line of direct evidence for a causal role of AH AVP, but perhaps not 5-HT, in adolescent AAS-induced aggression. The finding here that the development of the LAH AVP neural system correlates with the emergence of the aggressive phenotype is not surprising, given studies examining the relationship between developmental AVP and aggression. For instance, the temporal onset of flank marking behavior (i.e., an AVP-mediated stereotypic behavior that is part of the ethogram of offensive aggression) in hamsters correlates with two to threefold increases in AVP levels in the AH, suggesting that the development of the AH AVP neural system contributes to the temporal onset of flank marking behavior in hamsters (Ferris et al., 1996). In prairie voles, exposure to AVP early in the postnatal developmental period correlates with increased aggression in adult subjects, indicating that early increases in AVP activity can have lasting effects on aggressive behavior (Stribley & Carter, 1999). Thus, marked increases in AVP activity during development are associated with processes that predispose subjects to behave in an aggressive fashion. Together with findings presented in this report, these data provide compelling evidence that developmental alterations in AVP activity can have dramatic impact on the behavioral phenotype presented by animal species. Conversely, the finding that low levels of 5-HT do not correlate with the emergence of the aggressive phenotype is surprising, given the vast literature indicating sup-

pressive effects of 5-HT on aggression in humans (Brown et al., 1982; Coccaro & Kavoussi, 1997; Kruesi et al., 1990; Linnoila et al., 1983) and in many animal models and species (Higley et al., 1992; Kyes, Botchin, Kaplan, Manuck, & Mann, 1995; Sijbesma et al., 1991; Vergnes, Depaulis, Boehrer, & Kempf, 1988), including hamsters (Delville, Mansour, & Ferris, 1996; Ferris et al., 1997, 1999). Each of these studies, however, considers 5-HT activity in adult individuals with an appropriately matured 5-HT neural system. Conversely, in adolescent hamsters, the 5-HT neural system is still developing, and significant increases in 5-HT-containing afferent fibers are observed in several brain sites implicated in aggression control (e.g., the AH) across pubertal development and into adulthood (Taravosh-Lahn et al., 2005, 2006). Perhaps the capacity of 5-HT to modulate aggression is different at times when the 5-HT is plastic (in development) versus static (adulthood). Indeed, in adolescent hamsters where the 5-HT neural system is plastic, low doses of fluoxetine (i.e., a selective 5-HT reuptake inhibitor that increases extracellular 5-HT levels) actually enhance aggressive responding, while higher levels reduce them (DeLeon, Grimes, Connor, & Melloni, 2002; Grimes & Melloni, 2002; Taravosh-Lahn et al., 2006), indicating that large increases in available extracellular 5-HT are required to suppress aggression. Similarly, socially subjugated adolescent hamsters display subtle but significant increases in 5-HT afferent development to the AH correlated with context-dependent aggressive responding that is thought to represent the responsiveness of the 5-HT neural system to threat (i.e., the higher the threat, the higher the response of the 5-HT system and the lower the aggressive phenotype; Delville et al., 1998). Along similar lines, early life social defeat stress is correlated with an increase in the number of 5-HT neurons expressing fos (i.e., a sensitive marker of neuronal activity) in rats, indicating an increase in the activity of 5-HT neurons in subjugated animals (Gardner, Trhrivikraman, Lightman, Plotsky, & Lowry, 2005). In adult hamsters, low doses of fluoxetine have no effect on aggression, while high doses reduce aggressive responding (Ferris et al., 1997; Taravosh-Lahn et al., 2006), and aggressive responding is associated with a reduced 5-HT neural signaling (Delville et al., 1996; Ferris et al., 1997, 1999; Higley et al., 1992; Kyes et al., 1995; Sijbesma et al., 1991; Vergnes et al., 1988). It is possible, then, that 5-HT’s role in the regulation of the aggressive phenotype may be strongly developmentally regulated and dependent upon the developmental or neural plastic state of the 5-HT neural system itself, including that of selective 5-HT receptor populations. For instance, 5-HT1A receptors have been shown to modulate aggressive behavior in many animal species and models of aggression (Bell & Hobson, 1994; Bonson, Johnson, Fiorella, Rabin, & Winter, 1994; Bonson & Winter, 1992; Cologer-Clifford, Simon, Richter, Smoluk, & Lu, 1999; de Boer, Lesourd, Mocaer, & Koolhaas, 1999; Miczek, Hussain, & Faccidomo, 1998; Sanchez, Arnt, Hyttel, & Moltzen, 1993; Sanchez & Hyttel, 1994; Simon, Cologer-Clifford, Lu, McKenna, & Hu, 1998; White, Kucharik, & Moyer, 1991), including hamsters (Joppa, Rowe, & Meisel, 1997; Knyshevski, Ricci, McCann, & Melloni, 2005). In hamsters, adolescence is associated with increased 5-HT1A receptor expression in the same neural sites that also show increased 5-HT during adolescent development (Taravosh-Lahn et al., 2006). Since adolescent, AAS-induced aggression is modulated by 5-HT1A activity and expression (Ricci et al., 2006), the examination of the role of this receptor subtype in the temporal onset of

AGGRESSION IN STEROID-EXPOSED ADOLESCENT HAMSTERS

adolescent AAS-induced offensive aggression may provide further insight into the role of 5-HT activity and functioning in the development and generation of the aggressive response. It is also possible that 5-HT’s role in the regulation of aggression may be highly dependent upon the state of activity/development of other neural systems that interact with 5-HT (e.g., the LAH AVP neural system—see above). Indeed, together with data from AAS withdrawal studies indicating a correlation between aggression and AH AVP (Grimes et al., 2006), but not 5-HT (Grimes & Melloni, 2006), these data strengthen the notion that the interactions between AAS and the AH AVP neural system might directly underlie adolescent AAS-induced offensive aggression, and hint that AH 5-HT may play a more permissive role in this behavioral response. In summary, the studies presented in this article provide the first examination of the temporal onset of the effects of AAS exposure on offensive aggression, and on the LAH AVP and 5-HT neural systems during progressively longer periods of AAS exposure during adolescent development. These findings show that offensive aggression and LAH AVP afferent development were significantly higher in AAS-treated hamsters than controls after 14 days of drug exposure and that reductions in LAH 5-HT afferent development occur 1 full week prior to the observed changes in aggressive responding. These data suggest that adolescent AAS exposure has short-term effects on AH AVP and 5-HT development, yet only alterations in LAH AVP development correlate with the temporal onset of the aggressive behavioral phenotype during AAS exposure, providing a second line of evidence for a causal role of LAH AVP activity and function in adolescent AASinduced offensive aggression.

References Bell, R., & Hobson, H. (1994). 5-HT1A receptor influences on rodent social and agonistic behavior: A review and empirical study. Neuroscience & Biobehavioral Reviews, 18, 325–338. Bonson, K. R., Johnson, R. G., Fiorella, D., Rabin, R. A., & Winter, J. C. (1994). Serotonergic control of androgen-induced dominance. Pharmacology Biochemistry and Behavior, 49, 313–322. Bonson, K. R., & Winter, J. C. (1992). Reversal of testosterone-induced dominance by the serotonergic agonist quipazine. Pharmacology Biochemistry and Behavior, 42, 809 – 813. Brown, G. L., Ebert, M. H., Goyer, P. F., Jimerson, D. C., Klein, W. J., Bunney, W. E., et al. (1982). Aggression, suicide, and serotonin: Relationships to CSF amine metabolites. American Journal of Psychiatry, 139, 741–746. Coccaro, E. F., & Kavoussi, R. J. (1997). Fluoxetine and impulsive aggressive behavior in personality-disordered subjects. Archives of General Psychiatry, 54, 1081–1088. Cologer-Clifford, A., Simon, N. G., Richter, M. L., Smoluk, S. A., & Lu, S. (1999). Androgens and estrogens modulate 5-HT1A and 5-HT1B agonist effects on aggression. Physiology & Behavior, 65, 823– 828. Cunningham, R. L., & McGinnis, M. Y. (2006). Physical provocation of pubertal anabolic androgenic steroid exposed male rats elicits aggression towards females. Hormones and Behavior, 50, 410 – 416. de Boer, S. F., Lesourd, M., Mocaer, E., & Koolhaas, J. M. (1999). Selective antiaggressive effects of alnespirone in resident–intruder test are mediated via 5-hydroxytryptamine1A receptors: A comparative pharmacological study with 8-hydroxy-2-dipropylaminotetralin, ipsapirone, buspirone, eltoprazine, and WAY-100635. Journal of Pharmacology and Experimental Therapeutics, 288, 1125–1133. DeLeon, K. R., Grimes, J. M., Connor, D. F., & Melloni, R. H., Jr. (2002). Adolescent cocaine exposure and offensive aggression: Involvement of

947

serotonin neural signaling and innervation in male Syrian hamsters. Behavioural Brain Research, 133, 211–220. DeLeon, K. R., Grimes, J. M., & Melloni, R. H., Jr. (2002). Repeated anabolic-androgenic steroid treatment during adolescence increases vasopressin V(1A) receptor binding in Syrian hamsters: Correlation with offensive aggression. Hormones and Behavior, 42, 182–191. Delville, Y., De Vries, G. J., & Ferris, C. F. (2000). Neural connections of the anterior hypothalamus and agonistic behavior in golden hamsters. Brain, Behavior and Evolution, 55, 53–76. Delville, Y., Mansour, K. M., & Ferris, C. F. (1996). Serotonin blocks vasopressin-facilitated offensive aggression: Interactions within the ventrolateral hypothalamus of golden hamsters. Physiology & Behavior, 59, 813– 816. Delville, Y., Melloni, R. H., Jr., & Ferris, C. F. (1998). Behavioral and neurobiological consequences of social subjugation during puberty in golden hamsters. Journal of Neuroscience, 18, 2667–2672. Farrell, S. F., & McGinnis, M. Y. (2003). Effects of pubertal anabolic– androgenic steroid (AAS) administration on reproductive and aggressive behaviors in male rats. Behavioral Neuroscience, 117, 904 –911. Farrell, S. F., & McGinnis, M. Y. (2004). Long-term effects of pubertal anabolic–androgenic steroid exposure on reproductive and aggressive behaviors in male rats. Hormones and Behavior, 46, 193–203. Ferris, C. F., Delville, Y., Brewer, J. A., Mansour, K., Yules, B., & Melloni, R. H., Jr. (1996). Vasopressin and developmental onset of flank marking behavior in golden hamsters. Journal of Neurobiology, 30, 192–204. Ferris, C. F., Melloni, R. H., Jr., Koppel, G., Perry, K. W., Fuller, R. W., & Delville, Y. (1997). Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters. Journal of Neuroscience, 17, 4331– 4340. Ferris, C. F., Stolberg, T., & Delville, Y. (1999). Serotonin regulation of aggressive behavior in male golden hamsters (Mesocricetus auratus). Behavioral Neuroscience, 113, 804 – 815. Floody, O. R., & Pfaff, D. W. (1977). Aggressive behavior in female hamsters: The hormonal basis for fluctuations in female aggressiveness correlated with estrous state. Journal of Comparative and Physiological Psychology, 91, 443– 464. Gardner, K. L., Trhrivikraman, L. V., Lightman, S. L., Plotsky, P. M., & Lowry, C. A. (2005). Early life experience alters behavior during social defeat: Focus on serotonergic systems. Neuroscience, 136, 181–191. Grimes, J. M., & Melloni, R. H., Jr. (2002). Serotonin modulates offensive attack in adolescent anabolic steroid-treated hamsters. Pharmacology Biochemistry and Behavior, 73, 713–721. Grimes, J. M., & Melloni, R. H., Jr. (2005). Serotonin 1B receptor activity and expression modulate the aggression-stimulating effects of adolescent anabolic steroid exposure in hamsters. Behavioral Neuroscience, 119, 1184 –1194. Grimes, J. M., & Melloni, R. H., Jr. (2006). Prolonged alterations in the serotonin neural system following the cessation of adolescent anabolicandrogenic steroid exposure in hamsters (Mesocricetus auratus). Behavioral Neuroscience, 120, 1242–1251. Grimes, J. M., Ricci, L. A., & Melloni, R. H., Jr. (2003). Glutamic acid decarboxylase (GAD65) immunoreactivity in brains of aggressive, adolescent anabolic steroid-treated hamsters. Hormones and Behavior, 44, 271–280. Grimes, J. M., Ricci, L. A., & Melloni, R. H., Jr. (2006). Plasticity in anterior hypothalamic vasopressin correlates with aggression during anabolic/androgenic steroid withdrawal. Behavioral Neuroscience, 120, 115–124. Harrison, R. J., Connor, D. F., Nowak, C., Nash, K., & Melloni, R. H., Jr. (2000). Chronic anabolic–androgenic steroid treatment during adolescence increases anterior hypothalamic vasopressin and aggression in intact hamsters. Psychoneuroendocrinology, 25, 317–338. Higley, J. D., Mehlman, P. T., Taub, D. M., Higley, S. B., Suomi, S. J.,

948

GRIMES, RICCI, AND MELLONI

Vickers, J. H., et al. (1992). Cerebrospinal fluid monoamine and adrenal correlates of aggression in free-ranging rhesus monkeys. Archives of General Psychiatry, 49, 436 – 441. Joppa, M. A., Rowe, R. K., & Meisel, R. L. (1997). Effects of serotonin 1A or 1B receptor agonists on social aggression in male and female Syrian hamsters. Pharmacology Biochemistry and Behavior, 58, 349 –353. Knyshevski, I., Ricci, L. A., McCann, T. E., & Melloni, R. H., Jr. (2005). Serotonin type-1A receptors modulate adolescent, cocaine-induced offensive aggression in hamsters. Physiology & Behavior, 85, 167–176. Kruesi, M. J., Rapoport, J. L., Hamburger, S., Hibbs, E., Potter, W. Z., Lenane, M., et al. (1990). Cerebrospinal fluid monoamine metabolites, aggression, and impulsivity in disruptive behavior disorders of children and adolescents. Archives of General Psychiatry, 47, 419 – 426. Kyes, R. C., Botchin, M. B., Kaplan, J. R., Manuck, S. B., & Mann, J. J. (1995). Aggression and brain serotonergic responsivity: Response to slides in male macaques. Physiology & Behavior, 57, 205–208. Lerwill, C. J., & Makings, P. (1971). The agonistic behavior of the golden hamster. Animal Behavior, 19, 714 –721. Linnoila, M., Virkkunen, M., Scheinin, M., Nuutila, A., Rimon, R., & Goodwin, F. K. (1983). Low cerebrospinal fluid 5-hydroxyindoleacetic acid concentration differentiates impulsive from nonimpulsive violent behavior. Life Sciences, 33, 2609 –2614. Melloni, R. H., Jr., Connor, D. F., Hang, P. T., Harrison, R. J., & Ferris, C. F. (1997). Anabolic–androgenic steroid exposure during adolescence and aggressive behavior in golden hamsters. Physiology & Behavior, 61, 359 –364. Melloni, R. H., Jr., Connor, D. F., Nash, K., & Harrison, R. J. (1997). Chronic anabolic steroid exposure during adolescence stimulates vasopressin-dependent aggression in hamsters. Journal of Neuroscience Abstracts, 23, 1870. Melloni, R. H., Jr., & Ferris, C. F. (1996). Adolescent anabolic steroid use and aggressive behavior in golden hamsters. Annals of the New York Academy of Sciences, 794, 372–375. Miczek, K. A., Hussain, S., & Faccidomo, S. (1998). Alcohol-heightened aggression in mice: Attenuation by 5-HT1A receptor agonists. Psychopharmacology, 139, 160 –168. Miller, L. L., Whitsett, J. M., Vandenbergh, J. G., & Colby, D. R. (1977). Physical and behavioral aspects of sexual maturation in male golden hamsters. Journal of Comparative and Physiological Psychology, 91, 245–259. Pope, H. G., Jr., & Katz, D. L. (1988). Affective and psychotic symptoms associated with anabolic steroid use. American Journal of Psychiatry, 145, 487– 490. Pope, H. G., Jr., & Katz, D. L. (1994). Psychiatric and medical effects of anabolic–androgenic steroid use: A controlled study of 160 athletes. Archives of General Psychiatry, 51, 375–382. Ricci, L. A., Grimes, J. M., & Melloni, R. H., Jr. (2004). Serotonin type-3 receptors modulate the aggression-stimulating effects of adolescent cocaine exposure. Behavioral Neuroscience, 118, 1097–1110. Ricci, L. A., Grimes, J. M., & Melloni, R. H., Jr. (2007). Lasting changes in neuronal activation patterns in select forebrain regions of aggressive, adolescent anabolic/androgenic steroid-treated hamsters. Behavioural Brain Research, 176, 344 –352.

Ricci, L. A., Knyshevski, I., & Melloni, R. H., Jr. (2005). Serotonin type-3 receptors stimulate offensive aggression in Syrian hamsters. Behavioral Brain Research, 156, 19 –29. Ricci, L. A., Rasakham, S., Grimes, J. M., & Melloni, R. H., Jr. (2006). Serotonin 1A receptor activity and expression modulate adolescent anabolic/androgenic steroid induced aggression in hamsters. Pharmacology Biochemistry and Behavior, 85, 1–11. Sanchez, C., Arnt, J., Hyttel, J., & Moltzen, E. K. (1993). The role of serotonergic mechanisms in inhibition of isolation-induced aggression in male mice. Psychopharmacology, 110, 53–59. Sanchez, C., & Hyttel, J. (1994). Isolation-induced aggression in mice: Effects of 5-hydroxytryptamine uptake inhibitors and involvement of postsynaptic 5-HT1A receptors. European Journal of Pharmacology, 264, 241–247. Schoenfeld, T. A., & Leonard, C. M. (1985). Behavioral development in the Syrian golden hamster. In H. I. Siegel (Ed.), The hamster: Reproduction and behavior (pp. 289 –321). New York: Plenum Press. Sijbesma, H., Schipper, J., de Kloet, E. R., Mos, J., van Aken, H., & Olivier, B. (1991). Postsynaptic 5-HT1 receptors and offensive aggression in rats: A combined behavioural and autoradiographic study with eltoprazine. Pharmacology Biochemistry and Behavior, 38, 447– 458. Simon, N. G., Cologer-Clifford, A., Lu, S. F., McKenna, S. E., & Hu, S. (1998). Testosterone and its metabolites modulate 5-HT1A and 5-HT1B agonist effects on intermale aggression. Neuroscience & Biobehavioral Reviews, 23, 325–336. Stribley, J. M., & Carter, C. S. (1999). Developmental exposure to vasopressin increases aggression in adult prairie voles. Proceedings of the National Academy of Sciences, USA, 96, 12601–12604. Taravosh-Lahn, K., Bastida, C., & Delville, Y. (2005). Serotonin and the development of agonistic behavior in golden hamsters. Program No. 76.245. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2005. Retrieved October 13, 2006, from http:// sfn.scholarone.com/itin2005/index.htm Taravosh-Lahn, K., Bastida, C., & Delville, Y. (2006). Differential responsiveness to fluoxetine during puberty. Behavioral Neuroscience, 120, 1084 –1092. Vergnes, M., Depaulis, A., Boehrer, A., & Kempf, E. (1988). Selective increase of offensive behavior in the rat following intrahypothalamic 5,7-DHT-induced serotonin depletion. Behavioural Brain Research, 29, 85–91. Wesson, D. W., & McGinnis, M. Y. (2006). Stacking anabolic androgenic steroids (AAS) during puberty in rats: A neuroendocrine and behavioral assessment. Pharmacology Biochemistry and Behavior, 83, 410 – 419. White, S. M., Kucharik, R. F., & Moyer, J. A. (1991). Effects of serotonergic agents on isolation-induced aggression. Pharmacology Biochemistry and Behavior, 39, 729 –736. Whitsett, J. M. (1975). The development of aggressive and marking behavior in intact and castrated male hamsters. Hormones and Behavior, 6, 47–57.

Received November 14, 2006 Revision received March 8, 2007 Accepted March 23, 2007 䡲