The effects of an anabolic androgenic steroid and low serotonin on social and non-social behaviors in male rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

The effects of an anabolic androgenic steroid and low serotonin on social and non-social behaviors in male rats Kenneth H. Kubala a , Marilyn Y. McGinnis b,d,⁎, George M. Anderson c , Augustus R. Lumia d a

Texas State University, Department of Psychology, San Marcos, TX 78666, USA Department of Biology, University of Texas at San Antonio, San Antonio, 6900N. Loop 1604 West, San Antonio, TX 78249, USA c Child Study Center, Yale University School of Medicine, New Haven, CT, USA d Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

The behavioral and neurochemical impact of low serotonin (5-HT) was examined in

Accepted 18 July 2008

gonadally intact male rats exposed to an anabolic androgenic steroid (AAS) during puberty.

Available online 28 July 2008

Low 5-HT was induced beginning on postnatal day 26 using parachlorophylalanine (PCPA). Injections of the AAS, testosterone (TP), began on day 40. The rats were tested in both non-

Keywords:

social (locomotor activity and nose poke for food) and social (low-threat and high-threat)

Testosterone

contexts. PCPA and TP + PCPA significantly decreased locomotor activity. PCPA alone

Serotonin

significantly increased nose poke latency compared to controls. Freezing in the PCPA

Aggression

group was significantly elevated compared to TP and TP + PCPA groups, but not compared to

Adolescent

controls. AAS did not affect non-social behaviors. Thus, low serotonin may increase freezing

Dominance

in a non-social context. Following provocation, PCPA and TP + PCPA significantly increased

Locomotor activity

aggression toward smaller non-threatening opponents, suggesting that males with low 5-

PCPA

HT are more aggressive in a low-threat context when provoked. In the resident-pair intruder test, TP significantly increased aggression whereas PCPA did not, suggesting that in a highthreat context, aggression is primarily mediated by AAS. TP + PCPA males were also significantly more aggressive in the high-threat context suggesting that exposure to AAS may override freezing behavior induced by low serotonin. Both PCPA and TP + PCPA significantly and substantially depleted 5-HT and 5-HIAA in all brain regions examined. AAS significantly decreased 5-HIAA levels in the hypothalamus and increased 5-HT levels in the frontal cortex. Following withdrawal from TP + PCPA, most behavioral and neurochemical measures returned to control levels. These data suggest that low serotonin may be a contributing factor in the increased aggression displayed by adolescents who abuse AAS. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

It has been estimated that over one million Americans have abused anabolic androgenic steroids (AAS) (Sturmi and Diorio, 1998). Anabolic androgenic steroid use is no longer limited to

professional or elite athletes, as indicated by the increase in adolescent AAS use (Denham, 2006). This increase in AAS use has been linked to the perceived enhancement of athletic performance and physical appearance (Holland-Hall, 2007; Yesalis and Bahrke, 2002). According to the 2003 Youth Risk

⁎ Corresponding author. Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. E-mail address: [email protected] (M.Y. McGinnis). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.07.065

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Behavior Surveillance System, administered by the Centers for Disease Control and Prevention, 6.8% of adolescent males and 5.3% of adolescent females experiment with anabolic steroids at some point in their lifetime. Adolescents may be the most susceptible to the deleterious effects of AAS (Holland-Hall, 2007; Yesalis and Bahrke, 2002) such as indiscriminate, unprovoked aggression (Pope and Katz, 1988), and impulsivity (Elliot and Goldberg, 1996). Because rising hormone levels associated with adolescence have been shown to influence brain development (Romeo, 2003; Sisk and Foster, 2004; Spear, 2000), AAS abuse during this hormonally sensitive period may permanently alter normal adult social behavior patterns. In humans AAS increases aggression in both adult and adolescent users (Yesalis and Bahrke, 1995; Burnett and Kleiman, 1994; Choi and Pope, 1994; Galligani et al., 1996; Pope and Katz, 1990; Pope et al., 2000). The AAS testosterone also potentiates aggression in both adolescent and adult male rats, mice, and hamsters (see Clark and Henderson, 2003; McGinnis, 2004 for reviews). Two factors which influence AASinduced aggression in adolescent male rodents are the animals' ability to make appropriate social discriminations, and their response to physical provocation. With regard to social discrimination, male rats receiving testosterone (TP) can discriminate between castrated and non-castrated opponents as well as between ovariectomized and intact females (Breuer et al., 2001, Cunningham and McGinnis, 2006). Thus, AAS-induced aggression does not appear to be indiscriminate, but is predicated, in part, on the threat potential of the opponent animal. The second factor, physical provocation, elicits aggression in males exposed to AAS (McGinnis et al., 2002a,b; Farrell and McGinnis, 2004). These results suggest that the AAS, testosterone (TP), lowers the threshold to respond aggressively to physical (actual) provocation or to a perceived threat (anticipated provocation) (McGinnis et al., 2002a,b). Low serotonin has been associated with increased aggression and dominance in both humans and animals (Matte and Tornow, 1978; Miczek et al., 2002; Sewell et al., 1982). Studies investigating a possible link between adolescent AAS exposure and low 5-HT have shown that AAS lowers serotonin in some brain regions, but not others. For example, AAS lowers hypothalamic and forebrain serotonin immunoreactivity in adolescent male hamsters (Grimes and Melloni, 2006). In rats, Bonson et al. (1994) reported a decrease in serotonin in hippocampus, but not striatum or frontal cortex, whereas Keleta et al. (2007) found that AAS decreased serotonin levels in the striatum, but not in hypothalamus or frontal cortex. These data suggest a possible role for serotonin in the modulation of AAS-induced aggression. However, differences between the behavioral effects of low serotonin and the effects of high AAS levels have been reported. For example, social behaviors such as sexual motivation were enhanced only by AAS, and the highest levels of aggression were seen in males with both high AAS and low serotonin (Keleta et al., 2007). In contrast, non-social behaviors, such as locomotor activity and irritability, were affected by low serotonin, but not by AAS exposure (Keleta et al., 2007). The aims of this experiment were threefold. First, to provide a less dramatic, but more clinically relevant level of serotonin depletion, we employed a very low PCPA (para-

chlorophylalanine) dose (50 mg/kg). Keleta et al. (2007) used a relatively low dose of PCPA (100 mg/kg) to induce low serotonin. However, in spite of this low chronic PCPA dose, it was still sufficient to induce a dramatic depletion in brain serotonin. Second, we examined the behavior of AAS and PCPA-treated males in two social and non-social conditions. Keleta et al. (2007) found that both low serotonin and AAS increased intermale aggression, but only low serotonin affected locomotor activity. With this in mind, we developed additional tests to distinguish between the effects of AAS and the effects of low serotonin. We employed two non-social tests, the open field test used by Keleta et al. (2007) and a nose poke test for a food reward to assess differences between low serotonin and AAS in the response to a novel environment (Spoont, 1992). To further differentiate the impact of low serotonin and AAS on the elicitation of aggression, we employed two distinct tests for aggression: a low-threat test and a high-threat test. For the low-threat test, we used a smaller, immature, male opponent to simulate a social interaction which typically does not elicit aggression in rats (Thor and Flannelly, 1976). In the high-threat test the elicitation of aggression was achieved by placing the experimental male into the home cage of a long-term paired male and female (resident-pair intruder test), creating a situation which elicits high levels of aggression (Flannelly and Lore, 1977). Thus, these two distinct tests for aggression allow for further understanding of the role of both the social environment (resident- –pair vs home cage) and the stimulus qualities of the opponents (non-threatening prepubertal male vs mature resident male) in the expression of aggression. Third, we examined the effects of withdrawal from both PCPA and AAS on both behavior and serotonin to determine the longterm effects of adolescent exposure to low serotonin and AAS. Our hypothesis was that low serotonin would induce a nonspecific reactivity in both non-social and social contexts, whereas the influence of AAS exposure would be expressed primarily within a social context.

2.

Results

2.1.

Non-social behavior tests

2.1.1.

Open field test

In the open field test the number of center and vertical crosses were combined to yield a measure of locomotor activity for each animal. Both the PCPA alone group and the TP + PCPA group exhibited significantly less locomotor activity (p < .05) than controls, as shown in Fig. 1. The locomotor activity of the TP alone group did not differ significantly from controls. Following withdrawal from the combination of TP + PCPA locomotor activity returned to control levels.

2.1.2.

Nose poke test

Nose poke frequency, duration and latency as well as the duration of freezing were recorded. The PCPA group exhibited a significantly (p < .05) longer latency to nose poke relative to controls as shown in Fig. 2. The duration of freezing was significantly higher in the PCPA group compared to TP alone

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Fig. 1 – Mean (±sem) number of total crosses for each group. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), control (n = 5), withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to controls.

and TP + PCPA groups but was not significantly different from controls (Fig. 3). No significant differences in nose poke frequency or duration were found between any of the treatment groups (data not shown). Following withdrawal from TP + PCPA all of the nose poke measures were comparable to controls.

2.2.

Social behavior tests

2.2.1.

Intermale aggression with smaller males

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Fig. 3 – Mean (±sem) duration of freezing during the nose poke test for each group. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), control (n = 5), withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to the groups shown by the lines.

PCPA and controls and was not significantly different from either group. Provocation following PCPA alone or TP + PCPA treatment elicited significantly (p < .05) more dominance mounts than

In the absence of provocation, males in the TP + PCPA group exhibited significantly (p < .05) more dominance mounts than controls (Fig. 4A). Dominance mounts were elevated in the PCPA alone group, but this did not significantly differ from controls. Dominance mounts of males receiving TP alone were not significantly different from controls. The composite aggression scores, shown in Fig. 4B, did not differ significantly from controls. Following withdrawal from TP + PCPA, dominance mounts returned to control levels. The composite aggression score was midway between TP +

Fig. 2 – Mean (±sem) latency to nose poke for each group. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), control (n = 5), withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to controls.

Fig. 4 – Mean (±sem) number of dominance mounts (panel A) and composite aggression score (panel B) in the absence of provocation. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), control (n = 5), withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to controls.

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controls, as shown in Fig. 5A. TP alone increased aggression, but this did not reach statistical significance (p < 0.08). Following withdrawal from TP + PCPA, the number of dominance mounts fell significantly (p < 0. 01) and was comparable to controls. The composite aggression scores for both the PCPA alone and the TP + PCPA males were significantly (p < .05) higher than controls, as shown in Fig. 5B. The TP group exhibited increased aggression, though this was not significantly different from controls. Following withdrawal from the combination of TP + PCPA the composite aggression score fell to a point intermediate between TP + PCPA and control levels and was not significantly different from either group.

2.2.2.

Resident-pair intruder test

In the resident-pair intruder test both the TP and the TP+ PCPA animals displayed significantly (p < .05) higher composite aggression scores compared to controls, as shown in Fig. 6. There was no significant difference from the control group in

Fig. 6 – Mean (±sem) composite aggression score for each group in the resident-pair intruder test. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), Cont (n = 5), Withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to controls.

the composite aggression score for the group receiving PCPA alone. After withdrawal from the combination of TP+ PCPA composite aggression score did not fall to control levels, but

Table 1 – Brain serotonin (5-HT) and 5-HIAA levels (ng/g) as percent of control

Fig. 5 – Mean (± sem) number of dominance mounts (panel A) and composite aggression score (panel B) following physical provocation. PCPA (n = 6), TP (n = 6), TP + PCPA (n = 12), control (n = 5), withdrawal (n = 5). *Indicates a significant (p < .05) difference relative to controls. #Indicates a significant (p < .01) difference relative to groups shown by the lines.

5-HT

5-HIAA

Frontal cortex PCPA TP TP + PCPA Withdrawal

22.4** 173.0** 18.5** 106.6

20.3** 90.1 12.8** 95.1

Hypothalamus PCPA TP TP + PCPA Withdrawal

44.9** 184.5 27.5** 111.0

24.2** 60.8** 19.4** 101.3

Hippocampus PCPA TP TP + PCPA Withdrawal

23.1** 112.5 18.0** 83.6

12.9** 78.6 9.2** 76.4**

Striatum PCPA TP TP + PCPA Withdrawal

21.3** 98.4 11.8** 81.6

4.9** 85.9 2.6** 80.4

Brainstem PCPA TP TP + PCPA Withdrawal

33.1** 110.2 37.5** 105.1

34.9** 142.5 33.9** 87.4

Values are expressed as percent of gonadally intact vehicle-injected controls. n = 6 for control, PCPA, TP and TP + PCPA groups and n = 5 for the withdrawal group. *Indicates a significant (p < 0.05) difference relative to controls. **Indicates a significant (p < 0.01) difference relative to controls.

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remained elevated, though not significantly different from either TP+ PCPA or control groups.

2.3.

Neurochemical analysis

HPLC revealed that 5-HT concentrations in the cortex, striatum, hypothalamus, brainstem and hippocampus were significantly (p < .01) depleted in both the PCPA alone and the TP + PCPA groups compared to controls, as shown in Table 1. PCPA alone reduced 5-HT 55.1%–78.7% compared to controls. TP + PCPA reduced 5-HT concentrations 62.5%–88.2% compared to controls. In the frontal cortex 5-HT concentrations in the TP alone group were significantly (p < .01) higher than controls. The TP group did not significantly differ from the control group in any other brain region. Ten days after withdrawal from the combination of TP + PCPA 5-HT concentrations were comparable to controls in all regions examined. Regional 5-HIAA concentrations for the different treatment regimens are presented in Table 1. In general, these changes were similar to those observed with regard to 5-HT. However, the increase in 5-HT seen in frontal cortex with TP treatment was not seen for 5-HIAA. In addition there was a significant reduction in 5-HIAA in hypothalamus in the TP-treated males. After 10 days of withdrawal from TP + PCPA the levels of 5HIAA were still significantly reduced in the hippocampus.

3.

Discussion

We wished to compare directly the individual and combined effects of lowered central 5-HT functioning and anabolic androgenic steroid exposure on non-social and social behaviors in peripubertal male rats. Two non-social behaviors were examined in this study. The first, locomotor activity, was tested in a novel open field environment. Previous studies using PCPA doses ranging from 150–1000 mg/kg reported a significant reduction locomotor activity (Dringenberg et al., 1995; Matte and Tornow, 1978). Chronic administration of a relative low PCPA dose (100 mg/kg) also significantly decreased locomotor activity (Keleta et al., 2007). In the current study we found that a very low PCPA dose (50 mg/kg) was also effective in decreasing locomotor activity. It is possible that even lower PCPA doses would be needed to demonstrate a level of serotonin depletion that would no longer suppress locomotor activity. In contrast to the effects of PCPA, chronic exposure to high doses of testosterone did not affect locomotor activity. This is consistent with previous reports (Keleta et al., 2007; Salvador et al., 1999). The second non-social behavior test employed a nose poke for a food reward. We found that PCPA-treated males displayed a significantly longer latency to nose poke for a food reward. They also spent more time freezing than controls, though this did not achieve statistical significance. The increased freezing by PCPA-treated males occurred even in the presence of a highly palatable reinforcer, suggesting that low serotonin may heighten the perceived threat of a novel environment (Spoont, 1992). Interestingly, both the TP and TP + PCPA males exhibited significantly shorter freezing durations than did the PCPA alone males. Thus, males with low serotonin respond to a novel environment by freezing,

25

whereas the behavior of males with high testosterone is not suppressed by novelty. When exposed to both low serotonin and high testosterone, the TP overrode the effect of low serotonin. Thus, AAS may actually counteract the effects of PCPA on freezing behavior in this non-social context. These results suggest that the behavior of males with low serotonin and those with high testosterone may be modulated by different neural mechanisms. In order to differentiate the effects of high testosterone and low serotonin in a social context, we employed two tests of aggression: a low-threat test and a high-threat test. In the lowthreat test, males were tested for aggression against smaller, non-threatening, gonadally intact males both with and without physical provocation. In the high-threat test, males were tested for aggression in the home cage of a resident-pair (male and female). With regard to the effects of AAS on aggression, Keleta et al., (2007) found that TP increased aggression toward gonadally intact males of similar age and body weight. However, AAS-treated rats typically show lower levels of aggression toward non-threatening opponents such as castrated males (Breuer et al., 2001, McGinnis, 2004). In the current study we found that AAS males were not aggressive toward smaller opponents in the absence of provocation. Although AAS males were more aggressive toward smaller opponents after provocation, this was not significantly different from controls. These data are consistent with the notion that the potential threat of the opponent may be a relevant factor in predicting the likelihood that AAS males will exhibit aggression (McGinnis, 2004). Studies in both humans and animals have suggested a link between serotonin and aggression (Matte and Tornow, 1978; Miczek et al., 1975; Miczek et al., 2002; Sewell et al., 1982). Keleta et al., (2007) found that PCPA increased aggression toward males of similar age and body weight. However, it was not known whether PCPA-treated males would be aggressive toward non-threatening opponents. An interesting finding in the current study is that while AAS-treated males were not aggressive toward smaller opponents, PCPA-treated males (PCPA alone and TP + PCPA) showed increased aggression in the absence of provocation, which was significant in the TP + PCPA group for dominance mounts. Moreover, when provoked, only the PCPA and TP + PCPA-treated rats, not the TP males, displayed a significant increase in aggression toward the smaller males. This is consistent with the hypothesis of Spoont (1992) who proposed that low brain serotonin heightens sensitivity to social provocation or threat. Thus, the “species-specific defense reaction” (Bolles, 1970) of the PCPAtreated rat was to react by attacking rather than freeze. In contrast, the AAS males displayed aggression only after provocation. They were thus able to discriminate the nature of the potential threat. Previously, we proposed that AAS may lower the threshold to act aggressively toward a potentially threatening stimulus (Breuer et al., 2001; Cunningham and McGinnis, 2006; McGinnis et al., 2002a,b; Farrell and McGinnis, 2004). The residentpair intruder test was designed to provide a high-threat social context. In the resident-pair intruder test experimental animals were placed in the home cage of a long-term pair consisting of an adult male and a sexually receptive female.

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This paradigm has been found to be very effective in eliciting attacks by the resident male (Thor and Flannelly, 1976). In contrast to the low-threat aggression paradigm where the experimental male was the resident, in this paradigm, the experimental male was the intruder. We found that AAS alone, or combination with PCPA, significantly increased aggression toward the resident male in this high-threat context. Notably, the PCPA animals did not exhibit heightened aggression in this test. A central hypothesis was that low serotonin would induce non-specific reactivity in both non-social and social situations, whereas AAS exposure would induce an aggressive response primarily within a social context. In fact, low 5-HT had a major impact on non-social behaviors, while AAS did not affect either locomotor activity or nose poke. In a social context, AAS males were aggressive toward males in the highthreat resident-pair intruder context. Yet, these same AAStreated males did not display high levels of aggression in the low-threat context using smaller males. In contrast, PCPA males were aggressive in the low-threat condition, but not in the high-threat condition. These data suggest that while both AAS and serotonin induce aggression, the aggression is mediated by different factors. We propose that low serotonin is involved in modulating the ‘species-specific defense reaction’, and that whether the animal fights or freezes is determined by the social or environmental context. Recent studies suggest that 5-HT and its metabolites are affected by exposure to AAS during adolescence (Grimes and Melloni, 2006; Lindqvist et al., 2002; Keleta et al., 2007). In the present study, we found that TP did not significantly decrease 5-HT in brain. However, TP did significantly increase 5-HT levels in the frontal cortex, which is consistent with our previous findings (Keleta et al., 2007). TP also significantly decreased 5-HIAA in the hypothalamus, an area not only associated with aggression (Grimes and Melloni, 2006; Delville et al., 2000; Taravosh-Lahn et al., 2006), but also an area with high levels of androgen receptors (McGinnis et al., 1981). In spite of the AAS-induced changes in 5-HT and 5-HIAA, AAS exposure in comparison to PCPA had a modest effect on brain serotonin. This would suggest that the behavioral consequences of AAS exposure may not be due to solely lowering brain serotonin. In order to more closely approximate reductions in 5-HT functioning reported in humans (eg. reductions associated with aggression and methylenedioxymethamphetamine (MDMA, “ecstasy” use and aggression)) (McCann et al., 1999, Leckman et al., 1990) we employed a much lower (50 mg/kg) PCPA dose than that used in previous animal studies (Dringenberg et al., 1995; Matte and Tornow, 1978; Miczek et al., 2002; Sewell et al., 1982). In a previous study from this laboratory (Keleta et al., 2007) we reported that chronic administration of a relatively low (100 mg/kg) dose of PCPA induced a severe 5-HT depletion. Although in the present study we expected that half the Keleta et al. (2007) PCPA dose (50 mg/kg) would lead to a moderate depletion of 5-HT, we found that it was not substantially different from the higher, 100 mg/kg dose (55.1% to 88.2% vs 71% to 94%). Notably, this level of 5-HT depletion, which is much lower than in most previous studies, was sufficient to affect both non-social as well as social behaviors.

With regard to the combined effects of TP + PCPA, the nonsocial behaviors, locomotor activity and nose poke, returned to control levels follow withdrawal. In the social behavior tests, in both low-threat and high-threat conditions, the withdrawal of TP + PCPA resulted in levels of behavior intermediate between TP + PCPA and control levels. In spite of a clear reduction in aggression following TP + PCPA withdrawal, males still displayed moderately elevated composite aggression scores with and without provocation against smaller opponents. A similar intermediate level of aggression occurred in the resident-pair test. Because the parameters of aggression displayed under these two levels of threat were only partially decreased, they were not significantly different from either the TP + PCPA or the control group. This suggests that 10 days after withdrawal from TP and PCPA, aggression had not fully returned to control levels. These results present at least two possibilities. First, the higher aggression level compared to controls may be the result of repeated testing however, data previously published from our laboratory, using a similar repeated aggression test paradigm, suggest that this is unlikely. In these earlier studies, the control animals did not show any increase in aggression over time (Feinberg et al., 1997; McGinnis et al., 2002a,b; Farrell and McGinnis, 2004). It is also possible that withdrawal from TP + PCPA results in a only a partial normalization of social behavior in spite of the restoration of brain 5-HT and 5-HIAA (with the exception of 5HIAA in the hippocampus). It is not clear whether early low 5HT in brain may sensitizes the animal to the impact of AAS and that re-exposure to novel or threatening conditions may potentate aggression in comparison to males with normal 5HT levels in puberty. Further studies using a longer withdrawal period and further testing may answer this question. The combination of high AAS and low serotonin resulted in two interesting effects. First, only the males receiving TP + PCPA were aggressive towards smaller males in the absence of provocation. This is consistent with our previous report using opponent males of a similar age and body weight (Keleta et al., 2007). One possible explanation is that the combination of high AAS and low serotonin alters the animal's ability to make appropriate discriminations in a social context (Spoont, 1992), although this could also be related to serotonin-induced alterations in autonomic function. Second, TP significantly reduced the impact of low serotonin on freezing duration. In fact, as can be seen, TP alone and TP + PCPA freezing durations were virtually identical. This suggests that exposure to AAS may override the freezing behavior induced by low serotonin and potentially increase the display of aggression. This view is supported by the results in the high-threat test, where PCPA males were not aggressive, but when TP was present, they were as aggressive as males receiving TP alone. These results, while speculative, suggest that adolescents with low serotonin disorders who abuse anabolic androgenic steroids might be at greater risk for engaging in increased aggressive behavior.

4.

Experimental procedures

Gonadally intact male Long–Evans rats received from Charles River Laboratories (Wilmington, MA) were employed in this study. The males were twenty-six days old upon arrival.

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Female rats weighing between 225–250 g were used for the resident-pair intruder test. They were ovariectomized and implanted with one silastic capsule (1.47-mm i.d. × 1.96-mm o. d. × 5-mm length) packed with 50% crystalline estradiol as described before (McGinnis et al., 1981). Intact opponent males weighed between 200 and 250 g and were separately housed upon arrival and used for the aggression tests. All animals were housed in Nalgene cages with cardboard bedding. Food and water were provided ad libitum. The room in which the animals were kept was maintained a 12:12 light–dark cycle with the lights turned off at noon. Cages were cleaned weekly and the bedding was changed in each cage, unless specified otherwise by the procedure for a particular behavior test. Behavior tests were conducted between 1200 and 1700 h and were independently scored by two experimenters. All tests were video taped. All procedures followed guidelines established for the care and use of laboratory animals by the National Institute of Health. The experimental animals were assigned to one of five groups based on the smallest weight deviation possible between each group. The TP group received a combination of the AAS testosterone propionate (TP) plus saline. The PCPA group received PCPA (4-Chloro-DL-phenlalanine methyl ester) plus PEG (polyethyleneglycol mol weight 200). The TP + PCPA group received both TP and PCPA. The withdrawal group also received both TP and PCPA and then went through 15 days of withdrawal prior to sacrifice. The control group received the vehicle substances, PEG and saline. PCPA injections, began on day 26. Each of the animals either received 50 mg/kg of PCPA (Sigma Chemicals, St. Louis, MO) or the same volume of saline. In a previous study employing a 100 mg/kg dose, PCPA severely depleted serotonin levels (Keleta et al., 2006). The current 50 mg/kg dose was used in an attempt to approximate a more physiological serotonin depletion range. The PCPA injections were administered three times a week for the duration of the experiment except for the withdrawal group. On day 40, which corresponds to the first day of puberty for the experimental animals (Korenbrot et al., 1977), TP or PEG were injected subcutaneously (5 mg/kg). This dose of testosterone has been previously shown to produce significantly elevated levels of aggression, and is comparable to doses used by humans (Breuer et al., 2001; McGinnis et al., 2002a,b; Pope et al., 2000; Yates et al., 1992). TP injections were given five days a week for the duration of the experiment for all groups except for the withdrawal group. Table 2 indicates the age and sequence for each of the behavioral tests.

4.1.

Non-social behavior tests

An open field test was employed to examine locomotor activity. The test took place in a square (60 cm× 60 cm× 30 cm) wooden

apparatus which had a floor that was divided into four (30 × 30 cm) quadrants. In the center of the floor, a circle 10 cm in diameter was defined by using black tape. Each rat was randomly placed in one of the four quadrants and observed for 5 min. During this time the number of center and vertical crosses was recorded. The container was cleaned with alcohol after each trial. Behaviors were operationally defined as: vertical crosses — the number of times the experimental animal moved from one quadrant to another with all four paws crossing into the next quadrant, and center crosses — the number of times the animal placed all four paws in the center circle. A nose poke test was performed to assess the animal's response to a food reinforcer in a novel environment. For this test, the animals were exposed to both the odor and taste of Oreo cookies by placing a piece of the cookie in the experimental cage the day before the test. One day prior to the test a piece of cookie was placed in the experimental cage and the animal was allowed to consume a small piece of the cookie. The animals were then food deprived for 24 h prior to the nose poke test. The experimental apparatus was a 16 inch long × 6 inch wide × 12 inch high plexiglas cage with two small holes 3 in. apart. A funnel was attached to one hole with Oreo cookie pieces placed in it. This procedure allowed the animal to smell the Oreo but not consume it. The frequency and latency of the nose pokes as well as the duration of freezing were recorded. Behaviors were operationally defined as: Nose poke — the number of times each animal put its nose in the hole containing the Oreo pieces; Freezing — the amount of time the experimental animal remained in an immobile position; and Nose poke latency — the amount of time to initiate the first nose poke.

4.2.

Social behavior tests

Two types of aggression tests were performed. The first was a low-threat test, in which males were tested for aggression in their own home cage against smaller, non-threatening opponents, both with and without physical provocation. The second test was a high-threat test, in which males were tested for aggression in the home cage of a resident-pair (male and female). For the low-threat aggression tests with and without physical provocation the bedding in the experimental animal's home cage was not cleaned for two weeks prior to aggression tests. The experimental animals' weight ranged from 300 to 325 g at time of testing. To provide a nonthreatening stimulus, smaller, immature, gonadally intact males weighing between 200 and 250 g were used as opponents. This procedure was adapted from Thor and Flannelly (1976). The opponent male was placed in experimental animals home cage for 10 min and the frequencies and latencies of the following behaviors were recorded: (1)

Table 2 – Time line for experimental procedures Day:

26

40

62

69

75

77

85

95

100

PCPA injection began

TP injection began

Open field test

Intermale aggression test

Resident-pair aggression test

Nose poke test

Sacrifice stop injection to withdrawal group

Behavior test withdrawal group

Sacrifice withdrawal group

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dominance mount — the number of times the experimental animal attempted to mount the opponent male; (2) threats — an experimental animal threatening an opponent, defined by pilo-erection and threatening stances by the experimental animal; (3) boxing — both the experimental male and opponent standing on their hind legs and striking each other with their front limbs; (4) dominance postures — a posture by the experimental animal in which the opponent lies in a supine position with the experimental animal on top; (5) lateral kicks — kicks occurring from the experimental animal on the opponent male; (6) composite aggression score — the sum of dominance mounts, threats, boxing, dominance postures and lateral kicks. Only those behaviors initiated by the experimental rat were used in the analysis. In the low-threat aggression tests physical provocation was presented as described in previous studies (Farrell and McGinnis, 2004; McGinnis, 2004; McGinnis et al., 2002a,b). In the current study, experimental males received a mild physical provocation with a tail pinch via forceps once every minute during the 10 minute test. Aggression measures were recorded as described above. To assess aggression in a high-threat situation, a residentpair intruder test was conducted (Flannelly and Lore, 1977). For this test an individual pair consisting of a sexually mature male and a mature ovariectomized female implanted subcutaneously with a 5-mm silastic capsule filled with estradiol, were housed together for at least two weeks prior to testing. Three hours before the resident-intruder test the female was injected with 1 mg of progesterone to induce sexual receptivity. Each experimental male was placed into the cage of the resident-pair. The number of aggressive encounters initiated by the experimental males was recorded for 10 min in the same manner as described above. The withdrawal group was tested again 10 days after their last injection of TP + PCPA. The entire battery of both nonsocial (open field and nose poke) and social (intermale aggression and resident-intruder aggression) behavioral tests were performed (see Table 2). At the conclusion of behavior testing the rats were sacrificed using a lethal dose of chloral hydrate. Animals received their final injections of TP and PCPA 1 h before they were sacrificed. Five brain regions (frontal cortex, striatum, hypothalamus, dorsal hippocampus, and brain stem) were dissected out according to the method described by Keleta et al. (2007) and stored at −80 °C. The withdrawal group was sacrificed in the same manner as the first four groups 15 days after their last injection to assess the reversibility of the PCPA effects on brain chemistry. In accordance with a previous study, reverse-phase HPLC (Janusonis et al., 2006, Keleta et al., 2007) was then used to determine the 5-HT and 5-HIAA concentrations in the frontal cortex, striatum, hypothalamus, dorsal hippocampus, and brain stem of the experimental animals. HPLC analysis was carried out after the addition of an internal standard solution containing antioxidants and N-methyl serotonin, homogenization by sonification, and centrifugation. Levels of neurochemicals were determined fluorometrically after automated injection of supernatant and separation on a 25 × 0.46 Altex Ultrasphere reverse C18 column.

Comparisons of the groups by analysis of variance (ANOVA) were performed using the software package, Statview. Fisher's PLSD was used for post hoc analysis. All statistical comparisons were based on a two-tailed test with the significance value set at p < .05 for individual group comparisons.

Acknowledgments The authors would like to acknowledge Mr. Albert Davis for his excellent technical assistance. Supported by NIH grant DA10886 to MYM.

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