Anabolic androgenic steroid affects competitive behaviour, behavioural response to ethanol and brain serotonin levels

Behavioural Brain Research 133 (2002) 21 – 29 www.elsevier.com/locate/bbr

Research report

Anabolic androgenic steroid affects competitive behaviour, behavioural response to ethanol and brain serotonin levels Ann-Sophie Lindqvist a,*, Pia Johansson-Steensland b, Fred Nyberg b, Claudia Fahlke a a

b

Department of Psychology, Go¨teborg Uni6ersity, P.O. Box 500, SE-405 30 Go¨teborg, Sweden Department of Pharmaceutical Biosciences, Di6ision of Biological Research on Drug Dependence, Uppsala Uni6ersity, P.O. Box 591, S-751 24 Uppsala, Sweden Received 11 May 2001; received in revised form 17 October 2001; accepted 17 October 2001

Abstract The present study investigated whether anabolic androgenic steroid (AAS) treatment (daily subcutaneous injections during 2 weeks with nandrolone decanoate; 15 mg/kg) affects competitive behaviour, and locomotor activity response to a sedative dose of ethanol (0.5 g ethanol/kg). In addition, levels of brain monoamines were assessed. The results showed that AAS treated animals exhibited enhanced dominant behaviour in the competition test compared to controls. The AAS groups’ locomotor activity was not affected by ethanol in contrast to the controls who showed a sedative locomotor activity. AAS animals had significant lower levels of serotonin in basal forebrain and dorsal striatum compared to controls. These findings further strengthen the fact that AAS affects behaviour, as well as biochemical parameters. Based on previous studies and results from the present study, we hypothesize that AAS abuse may constitute a risk factor for disinhibitory behaviour, partly by affecting the serotonergic system. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Aggression; Anabolic androgenic steroids; Competitive behaviour; Ethanol; Cross-tolerance; Disinhibitory behaviour; Locomotor activity; Nandrolone decanoate; Monoamines; Serotonin

1. Introduction Clinical studies have reported that abuse of anabolic androgenic steroids (AAS) in human, may cause several physical side effects such as cardiovascular problems, liver and gonadal dysfunction, gynecomastia and severe acne (for review see [35,77]). An extensive literature has also documented adverse effects on mental health in AAS abusers. Observed effects are euphoria, irritability, nervous tension, changes in libido, hypomania, mania and psychosis [5,35,73,75,78]. One frequently reported finding is the association between AAS abuse and disinhibitory behaviour [13,20,67,76], a state which includes behaviours such as impulsiveness, aggression, irritability and hostility. AAS abuse has also been connected * Corresponding author. Tel.: +46-31-773-1640; fax: + 46-31-7734628. E-mail address: [email protected] (A.-S. Lindqvist).

with acts of different violent behaviours and crimes [12,16,42,59,64]. It has even been found that psychiatric complications of AAS use, in particular the disinhibitory behaviours impulsiveness and aggression, seem to constitute an increased risk for violent death [70]. Studies on various animal species, including non-human primates, have also shown that AAS affect aggressive behaviours [40,44,50,61]. In recent studies of ours [24,26], we have explored the effects of AAS on defensive aggression. A form of aggression which is also referred to as, for example, hyperreactivity or hyperirritability. This defensive reaction, which is more or less non-testosterone-dependent, occurs in response to a real threat but also to perceived provocation, for example elicited by innocuous stimuli [3]. Thus, we found that defensive behavioural response increased significantly after treatment with the AAS compound nandrolone decanoate [24,26]. This effect was sustained 2

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month after the end of the treatment period [26]. Moreover, this behavioural response was further increased when AAS was combined with the stimulant amphetamine [24]. One aim of the present study was to further investigate the effect of AAS on aggressive behaviour, by using a competitive situation where two highly motivated animals compete for the same goal object [2]. This kind of competitive situation has been shown to evoke intermale social aggression, a testosterone-dependent behaviour, where males fight each other in order to establish dominance relationships [3,33]. Whether abuse of AAS may constitute a risk factor for abuse of other drugs in humans, or vice versa, is still unknown. This question is of importance since several survey studies, among teenagers and adults not connected to sport, have reported a concurrent AAS abuse together with other drugs [18,31,32,34,53,79]. We have recently tested the hypothesis that AAS may reinforce the behavioural response to other drugs of abuse, by using an animal model [24,26]. In one of these studies, rats were tested for voluntary ethanol intake after the end of a 2-week treatment period with nandrolone decanoate. It was found that the AAS treated animals drank significantly more alcohol, compared to controls [26]. The mechanism through which AAS stimulates ethanol intake is still unclear, but one possible explanation is that the observed enhanced intake may be due to a cross-tolerance effect [68] between AAS and ethanol, i.e. increased tolerance to ethanol due to pre-treatment of AAS. Thus, a second aim of the present study was to examine how AAS treated animals behaviourally respond to ethanol, by using a dose of ethanol that normally suppresses locomotor activity in untreated rats. The neurobiochemical mechanisms behind the observed behavioural changes, seen in human AAS abusers, are poorly understood. However, several recent animal experimental studies have found that AAS affect various systems in the brain [22,25,27,29,30,36, 47,51,57,63,66,71] that probably can be related to different AAS-induced behaviours. For example, we have recently found a relationship between brain opioid peptides and enhanced aggression [24,26], increased voluntary ethanol intake [26] and lowered fear reactions in threatening situations [26]. One system that is of interest is the neurotransmitter 5-hydroxytryptamine (5HT). Dysfunction in the 5-HT system has been associated with disinhibitory behaviour in humans [72], non-human primates [23], and rodents [52]. Moreover, it is suggested that the serotonergic, as well as the dopaminergic, systems are involved in the rewarding effects of various drugs of abuse [38]. In the present study, we therefore, examined the monoaminergic activity in different brain tissues of the AAS-treated animals.

2. Method

2.1. Animals Twenty-six male Wistar rats purchased from Mo¨ llegard Breeding Laboratories (Denmark) served as subjects. They were 80–90 days of age and weighing 250–300 g at the beginning of the experiment. Animals were housed in an air-conditioned colony room (lightsoff 10:00–22:00 h) at a temperature of 23 °C and a humidity of 50–60%. The rats had free access to water and R70 food pellets (Labfor, Lactamin, Vadstena, Sweden) throughout the experiment. The animals were housed in groups of four per cage (clear plastic cages; 53× 31× 25 cm) throughout the experiment, if not otherwise stated. All behavioural tests were conducted during the dark phase of the light–dark cycle. The experiment was approved by the local ethical committee of the Swedish National Board for Laboratory Animals.

2.2. Steroid administration Animals were randomly divided into two groups. One group (n= 14) received daily subcutaneous (s.c.) injections of nandrolone decanoate (Deca-Durabol®, Organon, Oss, Netherlands) of 15 mg/kg for 14 days. This dose affects behaviours [24,26] and neurochemistry [22,25,27,30,36] in the rat. The other group of animals (n = 12) was given daily injections of oil (arachidis oleum) for 14 days, and served as controls. Arachidis oleum was chosen due to it being the oil-component in Deca-Durabol®.

2.3. Beha6ioural measurements 2.3.1. Competiti6e beha6iours One week after the last injections of nandrolone decanoate, 12 animals were randomly, and individually paired with one of the oil-treated control subjects and housed together in clear plastic cages (42× 25×14 cm). The animals had 3 days to acclimatise to their new environment and its cage mate, meanwhile the animals were subjected to water restrictions except for 1 h (11.00–12.00 h) daily when they had unlimited access to water (modified from [2]). On the 4th day each pair of rats had to compete for the waterspout access. The spout had a suspended cone at the point where the waterspout entered the cage, which gave only one rat the opportunity to drink at a given time. Which animal started drinking, the time the rats spent drinking and aggressive behaviour, such as numbers of pushes, lunge attacks, paw strikes and piloerections, were scored over a 4-min period by two observers. The 4 min competition period was chosen based on earlier results from Albert et al. [2], showing

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that an individual rat on a 23-h water deprivation schedule would drink continuously for the first 4 min of access to a waterspout. After the 4 min competition period, the animals had an additional hour of free access to water but without the suspended cone. Following the competition period, animals were placed in individual cages and kept on water deprivation for an additional day in order to assess each rat’s 4-min drinking baseline.

2.3.2. Locomotor acti6ity and ethanol injection One week after the competition test, all animals were tested for locomotor activity responses to ethanol. Seven AAS treated animals and six oil-treated controls were randomly chosen to receive intraperitoneal (i.p.) injections of a 0.5 g ethanol/kg, dissolved in 0.9% saline solution. This dose was chosen on the basis of preliminary observations indicating that an untreated rat given this dose of ethanol would respond with a significant decreased locomotor activity (unpublished data by C. Fahlke). The remaining animals (AAS: n = 7 and control: n= 6) were given corresponding volume of saline solution. The subjects were given the injections 15 min before the test and their locomotor activity was registered, during 1 h, in test chambers made of plexiglas boxes (70× 70×35 cm high; Kungsbacka Ma¨ t och Reglerteknik AB). Each test box was surrounded by two series of invisible infrared photocell beams to measure spontaneous motor behaviour. The two series of infrared beams were situated 14 and 4 cm from the box floor. The photocells were situated 9 cm apart. The lower grid of infrared beams registered locomotion. Counts occurred when the rat moved horizontally, showing predominately ambulatory behaviour. Rearing behaviour, controlled by the high level series of infrared beams, was registered every time the rat raised itself onto its haunches. Peripheral locomotion was observed by measuring the time the animal was spending in the corners. This behaviour was registered by both sets of beams. The chambers were cleaned with hot water between the tests. 2.4. Biological measurements At the end of the experiment, including an alcohol washout period of 2 weeks, the animals were decapitated in a separate room. The systemic effect of the nandrolone decanoate was investigated by weighing the wet thymus gland. It is well known that steroids bind to glucocorticoid receptors in thymus and by a negative feedback mechanism induce thymus atrophy [6]. In addition, we also weighed the adrenal glands since the size of the thymus is reciprocal to the circulating corticosterone levels [1]. The brains were rapidly dissected out and placed on a chilled petri dish. The following tissue parts of the

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brain were taken for analysis: basal forebrain (medial frontal cortex, nucleus accumbens, olfactory tubercle, septum), dorsal striatum (caudate-putamen), hippocampus, amygdala and hemispheres (remaining cortical tissues). The tissues were weighed and kept at −80 °C until analysed for dopamine (DA), norepinephrine (NE), serotonin (5-HT), homovanillic acid (HVA), 3,4dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) with high-pressure liquid chromatography with electrochemical detection (HPLC-ED). The frozen tissues were homogenized in 0.1 M perchloric acid containing Na2-EDTA (2 mg/ml), glutathione (0.5 mg/ml) and D,L-alpha-methyl-DOPA (100 ng/ml), using Branson Sonifier 250. The samples were centrifuged for 10 min (10 000*G, 4 °C) and the supernatants were taken for analysis. The HPLC-ED system consisted of a Gynkotek P580 pump, a CMA 200 autosampler and a stainless steel column (4.6×150 mm) packed with Nucleosil RP18 5u (Jones Chromatography). Separation occurred in a mobile phase (pH 2.74) made up of K2HPO4 (0.012 M) and citric acid (0.04 M), containing Na-octyl-sulphate (63 mg/l) Na2-EDTA (20 mg/l) and methanol (8%), the flow rate being 0.8 ml/min. HPLC-ED employed Antec Decade electrochemical detector including VT-03Hy-Ref. Currents were monitored using a Chromelion PC1 software.

2.5. Statistics Between-groups comparisons of behaviour and biological measurements were employed by the Mann– Whitney U-test (StatView, Abacus). Competitioninduced changes in time spent drinking water were assessed by computing the difference between the figures for competition test and baseline. These difference scores were used in the statistical calculations. The results are presented as median9median absolute deviation (MAD; i.e. the median of the set of differences between each data point and the median of the data). Two-tailed levels of significance were used.

3. Results

3.1. Competiti6e beha6iour During the competition test, the AAS-treated animals had a significantly lower body weight compared to the control animals (2799 14 and 303917 g, respectively; U=22, PB 0.01). As seen in Fig. 1 (left panels), the two groups showed no difference in time spent drinking during baseline. When access to water was restricted to only one animal at a time, ten AAS animals of the 12

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competing pairs were the ones that first approached the waterspout. The AAS-treated animals also maintained access to the water spout more than two times as long as the control animals (U =24, P B 0.01; Fig. 1, right panels). Regarding the competitive behaviours, there were no differences in numbers of pushes between the groups (AAS: 11.59 4.0 and controls: 10.59 5.5). No signs of aggressive rage attacks such as lunge attacks and paw strikes were observed, but some AAS animals showed piloerection during the drinking time. This behaviour was never observed in the control animals.

3.2. Locomotor acti6ity and ethanol injection AAS and control animals, which received a saline-injection, showed similar locomotor activity responses (Fig. 2, left panels). There were no differences in locomotor activity between the ethanol-treated AAS group compared to AAS animals receiving saline. Nor were there any differences in activity levels when comparing ethanol-treated AAS animals with ethanol-treated controls (Fig. 2, right panels). However, control rats receiving ethanol showed a significant reduced locomotor activity compared to the saline-injected control animals (U =5, P B 0.05). As seen in Table 1, all animals, regardless of group or treatments, showed the same activity of rearing behaviour and peripheral locomotion.

3.3. Biological measurements There were no significant differences in any biological variables between the two subgroups of AAS-

Fig. 2. Median 9 MAD locomotor activity (cumulative counts for 60 min) in animals pre-treated with the (AAS) nandrolone decanoate (daily s.c. injections of 15 mg/kg for 14 days) or with arachidis oleum controls. At the time for the locomotor activity test animals received an injection of 0.5 g ethanol/kg or saline, 15 min before the test. AAS-ethanol: n =7; AAS-saline: n =7; control-ethanol: n = 6; control-saline: n = 6. Control-saline vs. control-ethanol: *PB 0.05 (Mann– Whitney U-test).

treated animals (i.e. exposed for ethanol or saline-injections during the locomotor activity test), or between the two matching control subgroups. Thus, the subgroups were therefore, combined to their original cohorts (AAS: n= 14; controls: n= 12) for the statistical analysis of the biological variables. There were no differences in body weight between the AAS and control animals at the start of the experiment (Table 2), whereas the AAS-treated group did not gain as much weight as the controls during the 2-week treatment period (U= 38, PB 0.05). This group difference in body weight had disappeared at the time of decapitation (Table 2). The systemic effect of nandrolone decanoate treatment was measured by weighing the thymus gland. A significant reduction in thymus weight was observed in the AAS animals (28.059 2.86 mg/100 g body weight), compared to controls (69.12911.11 mg/100 g body Table 1 Median9 MAD rearing behaviour (counts) and peripheral locomotor activity (counts) in animals pre-treated with the (AAS) nandrolone decanoate (daily s.c. injections of 15 mg/kg for 14 days), or with arachidis oleum (controls)

Fig. 1. Time spent drinking (s) during baseline (left panels) and competition test (right panels) when water drinking was restricted to one animal. The competitive pairs were one rat pre-treated with the (AAS) nandrolone decanoate (daily s.c. injections of 15 mg/kg for 14 days) and one arachidis oleum-treated control. Total pairs were n= 12.Values are expressed as median 9 MAD. **P\ 0.01 (Mann – Whitney U-test).

AAS-saline (n = 7) Control-saline (n =6) AAS-ethanol (n = 7) Control-ethanol (n = 6)

Rearing

Peripheral locomotion

215 9 60 325 9 72 236 9 45 223 9 15

49.6 9 10.2 54.2 9 4.8 43.1 94.8 62.8 911.5

At the time for the locomotor activity test (60 min) animals received an injection of 0.5 g ethanol/kg, or saline, 15 min before the test. The differences between the groups are not statistically significant (Mann– Whitney U-test)

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Table 2 Median 9MAD body weight (g) in rats

AAS Control

Start of experiment

End of the 2-week treatment period

Time of decapitation

236 915 240 911

266 98* 281 913

284 915 309 944

Treated with the (AAS; n= 14) nandrolone decanoate (daily s.c. injections of 15 mg/kg for 14 days) or with arachidis oleum (controls; n= 12) at start of experiment, end of the 2-week treatment period and at time of decapitation. * PB0.05 (Mann–Whitney U-test).

weight; U =18, PB0.001). There was also a statistically significant difference in adrenal weights, expressed as two adrenals mg/kg body weight, between the AAS animals (18.199 2 mg), and the controls (13.1192.45 mg; U =28; PB 0.01). Biochemical analyses showed that AAS rats, compared to control rats, displayed significant lower levels of 5-HT in basal forebrain (U =42, P B 0.05), and dorsal striatum (U =45, P B 0.05; Table 3). There was a trend toward a lowered level of 5-HT in hippocampus and amygdala, although not statistical significant (P= 0.06 for both brain areas). The concentrations of NA, DA, DOPAC and HVA were not significantly altered in any of the brain regions, except for that the AAS group had lower levels of 5-HIAA in dorsal striatum, compared to controls (U =40, P B0.05; Table 3). The ratios of 5-HIAA/5-HT or DOPAC +HVA/DA in the AAS treated animals were not altered, compared to the controls (data not shown).

4. Discussion The present results show that the nandrolone decanoate treated animals were more dominant in the competitive situation. They started to drink, and were more successful in maintaining access to the waterspout, even though the AAS animals had lower body weight compared to their oil treated cage mates. These results are in line with other animal experimental studies, using various types of testosterone treatments [3]. Interestingly, it has been documented, in humans, that winners of physical competitive matches have higher testosterone levels than losers [19,21,49]. Even winners of non-physical face-to-face competition (tournament chess play) showed higher testosterone levels than losers [48]. There was no difference in the amount of pushes between the groups in the competitive situation. However, it should be mentioned that our observation was that the capacity of the AAS animals to take possession of the waterspout, depended more in making their way to the spout and persistence in staying there. In some cases AAS animals even showed piloerections, a behaviour that was never seen in the controls. However,

other social aggressive behaviours, such as lunge attack and paw strike, were never observed between the competitive pairs. Although, several studies have found enhanced social aggression in AAS treated animals [40,44,50,61], there are also contradictory findings, reporting no or minor effects of AAS on social aggression [14,46]. One possible explanation for the absence of social aggression in this study may be that the animals were housed with their competitor for a period prior to the competition test. Thus, it is possible that the competitive pair already had established their relationship before the test situation. Similarly, Albert et al. [2] found that animals with testosterone implant housed together with animals without testosterone implant for a short period prior to the competition test, had more success in the competitive situation than the controls, but the testosterone implanted animals did not display more aggression than their cage mates. Presumably, enhanced aggression might have been displayed if the AAS-treated animal had been housed with a female prior to the competition test since this is well known to trigger intermale social aggression [41]. Taken together, the social dominant behaviour seen in the competition test is probably related to the androgenic properties of nandrolone decanoate and although we observed low level of social aggressive behaviours, it should be noted that the same dose and duration of treatment with nandrolone decanoate as in the present study, enhances other forms of aggression, such as defensive behavioural responses to innocuous stimulus [24,26]. Another finding of the present study was that the AAS animals’ locomotor activity, after a high dose of alcohol, was unaffected compared to saline treated AAS animals. In contrast to the AAS treated subjects, and as expected, the ethanol-treated controls showed a significantly decreased locomotor activity when compared to the saline treated controls. Long-term treatment or intake of one drug is known to cause cross-tolerance to a second drug [68]. Whether abuse of AAS per se, develops tolerance or cross-tolerance to other drugs, is still unknown. However, it has been reported that male bodybuilders, actively taking AAS, have a delayed response in detecting the effects of ethanol [43]. Similarly, it has been shown that AAS users have fewer episodes of euphoria, and dysphoria,

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after cocaine intake [43]. In addition, survey studies have reported a concurrent abuse of AAS with other drugs of abuse, among individuals not connected to sports [18,31,32,34,53,79], and AAS users are more likely than non-users to abuse other drugs [18,53]. In fact, it has recently been observed that AAS abuse may led to cocaine misuse [55] and opioid dependence [4,74]. It should also be noted that in a recent study of ours [26] it was found that AAS treated animals drank more alcohol in a free-choice situation compared to controls. Thus case reports, survey studies and our experimental studies support the hypothesis, recently proposed by Arvary and Pope [4], that AAS may serve as a gateway for abuse, or even dependence and cross-tolerance to, other drugs. We did not find any difference in locomotor activity between saline-treated AAS animals and controls. Previous studies on this topic have not been consistent. Several studies have found a decrease in locomotor activity [10,11,26], and others have reported no effect of AAS on locomotor activity [7,14,28,46,54,62]. The divergent results may depend upon different methodological issues, e.g. dose of AAS, duration of treatment, type of steroid, but also animal species and strains used. Further studies, preferably using similar type of methodology, are needed in order to clarify the specific effect of AAS on locomotor activity. In line with recent studies [25,26], the nandrolone decanoate treatment caused thymus atrophy, indicating that the treatment had a systemic effect [6]. AAS

treated animals also had enlarged adrenal glands. The enlargement may be due to the inverse relationship between thymus and adrenals, where the size of the thymus is reciprocal to the circulating corticosterone levels [1]. Whether AAS have a direct effect on the thymus gland and thereby affecting the adrenals, or vice versa, cannot be concluded from this study. However, our data indicate that long-term treatment of AAS has widespread effects, including alterations on the thymus as well as the adrenal glands. Indeed, it is well known that the hypothalamic-pituitary-adrenal (HPA) axis, including circulatory glucocorticoid level, is more or less sensitive to drugs. In fact, the HPA axis is activated by almost any kind of threat to the homeostasis [56]. For example, chronic ethanol treatment produces substantial hyperstimulation of the HPA axis, as indicated by increased adrenal weight and decreased thymus weight [65]. The present results of the biochemical analysis indicate that of all monoamines examined, nandrolone decanoate treatment mainly affected the 5-HT activity. Thus the 5-HT activity was lowered in the basal forebrain and dorsal striatum, and there was also a trend for reduced 5-HT activity in the hippocampus and amygdala. Of the metabolites, only the level of 5-HIAA in the dorsal striatum was reduced. Neither were there any significant differences in monoamine turnover between the AAS treated animals and the controls. These results are in contrast to the findings from Thiblin and colleagues [69]. They recently reported an increase of

Table 3 Biochemical analysis of monoamines and their metabolites Monoamines

Metabolites

NE

DA

5-HT

Limbic forebrain AAS Control

1121 950 1105 9 53

3093 9 111 3290 9 247

819 9 18* 934 9 23

Dorsal striatum AAS Control

1469 10 1349 10

96699 180 94639 293

Hippocampus AAS Control

577 929 597 918

Amygdala AAS Control Hemispheres AAS Control

DOPAC

HVA

5-HIAA

598 9 27 598 9 34

234 9 14 250 9 16

497 9 12 535 9 24

417 915* 494 9 13

1236 9 37 1261 949

721 9 37 797 9 45

536 9 15* 5909 22

9.17 9 0.46 8.37 9 0.80

362 9 16 403 913

5.52 9 0.36 6.62 9 0.43

2.88 9 0.37 3.25 9 0.48

3769 14 389 9 11

6489 28 597 9 25

6609 151 709 9 111

728 9 22 783 9 20

113 924 107 913

64 9 12 64 9 7

4699 19 5129 15

4349 24 4229 22

539 6 879 33

313 9 10 324 916

25 91 29 96

36 9 13 25 9 3

2139 6 2289 10

Monoamines (norepinephrine [NE], dopamine [DA] and serotonin [5-HT]) and their metabolites (3,4-dihydroxyphenylacetic acid [DOPAC], homovanillic acid [HVA] and 5-hydroxyindoleacetic acid [5-HIAA]), in different brain parts of rats treated with the (AAS; n = 14) nandrolone decanoate (daily s.c. injections of 15 mg/kg for 14 days) or with arachidis oleum (controls; n = 12). The concentrations are given in ng/g wet tissue weight (median 9MAD). * PB0.05 (Mann–Whitney U-test).

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5-HIAA/5-HT ratio in different brain parts of AAS treated animals, whereas the 5-HT synthesis rate was unaffected. Even the DA synthesis and DOPAC+ HVA/DA ratio in the striatum was increased. However in that study, compared to the present, animals received one injection of AAS per week for 6 weeks, the dose was much lower, and other AAS compounds than nandrolone decanoate were used. A hypothesis that the authors formed to shear light on their results was that a decreased activity of 5-HT, may reflect a compensatory down regulatory mechanism of a stressed 5-HT system, and that the dose used in their study was too low to trigger this mechanism. Another possibility, explaining the conflicting results may be the time interval between AAS treatment and decapitation. In our study, the animals’ brains were taken out 4 weeks after the last AAS injection, whereas Thiblin et al. [69] decapitated their animals 2 days after the final injection. It is possible that AAS-induced changes in monoaminergic activity differs over time, depending on when brain are taken for analysis in relation to the last AAS injection. Although, the divergent results between Thiblin et al. [69] and the present study, it should be emphasized that both these studies, are to our knowledge, the first studies investigating the effect of AAS on monoamine levels in the rat brain. Studies have rather focused on the relationship between the androgenic part of AAS, i.e. testosterone, and the monoaminergic system, mainly finding a decreased 5-HT activity following testosterone treatment [8,9,45]. Therefore, it is of great importance to further explore the effects of AAS on monoaminergic system, especially with focus on the effect of various AAS compounds, doses, treatment schedules, and time intervals between treatment and decapitation. Results from such studies may have important implications for understanding and clarifying to what extend the monoaminergic system can be related to the observed AAS-induced disinhibitory behaviours, for example increased dominant and defensive behavioural responses [24,26], and decreased fear reaction in threatening situations [26], but also enhanced ethanol intake [26]. The findings of decreased 5-HT activity in the present study, is of interest since a dysfunction of the 5-HT system has been associated with disinhibitory psychopathology. Firstly, there has been an extensive literature, which implicates a dysfunction of 5-HT in disorders involving aggressive and violent behaviours [23,52,72], and secondly, clinical studies have demonstrated a relationship between serotonergic dysfunction and drug abuse, especially excessive alcohol consumption [23,37]. These behaviours (i.e. impulsiveness, aggression, violentness and excessive alcohol intake), and a possible serotonergic dysfunction, have been found to characterize a subgroup of alcoholics, the so-called type II alcoholism [15]. These individuals have an early

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onset of problem drinking, but also co-abuse of other drugs, and history of impulsive and violent behaviour [15,39]. Similar to the type II alcoholics, there are studies indicating an increased disinhibitory psychopathology [5,13,20,67,76] and excessive violent behaviours [12,16,42,59,64] in individuals abusing AAS. Whether AAS abuse induces psychopathological behavioural changes, or if individuals with personality disorders abuse AAS as a part of their disorder is still unclear. However, there are some clinical evidences, suggesting that AAS abuse may be a function of antisocial personality disorder [60,76], whereas results from other studies suggest that AAS abuse rather pave the way for disinhibitory behaviour [17,20,58,67]. As for the suggested serotonergic dysfunction, seen in type II alcoholism, there is today no evidence for a disturbed 5-HT function in AAS abusers. However, the results from the present study indicate that long-term AAS treatment causes a dysfunction in the 5-HT system. Thus, our hypothesis is that AAS abuse may induce type II-like disinhibitory features, partly by affecting the 5-HT system. Acknowledgements The technical assistance of Birgit Linder and AnnMarie Dahlgren is gratefully acknowledged. This work was supported by grants from the Swedish Alcohol Monopoly Foundation for Alcohol Research (00/4:1), the Swedish Medical Research Council (K01-21X13447-02B and Grant No 9459), the Swedish Council for Research in Humanities and the Social Sciences (F0416-1999), Stiftelsen Sigurd och Elsa Goljes Minne, Stiftelsen Lars Hiertas Minne, Wilhelm och Martina Lundgrens Vetenskapsfond, Ra˚ dman och Fru Ernst Collianders Stiftelse fo¨ r va¨ lgo¨ rande a¨ ndama˚ l, Stiftelsen Clas Groschinskys minnesfond, Stiftelsen La¨ ngmanska kulturfonden and Svenska Sa¨ llskapet fo¨ r medicinsk forskning. References [1] Akana SF, Cascio CS, Shinsako J, Dallman MF. Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am J Physiol 1985;249:R527 – 32. [2] Albert DJ, Dyson EM, Walsh ML. Competitive behavior in male rats: aggression and success enhanced by medial hypothalamic lesions as well as by testosterone implants. Physiol Behav 1987;40:695 – 701. [3] Albert DJ, Walsh ML, Jonik RH. Aggression in humans: what is its biological foundation? Neurosci Biobehav Rev 1993;17:405 – 25. [4] Arvary D, Pope HG. Anabolic-androgenic steroids as a gateway to opioid dependence. N Engl J Med 2000;342:1532. [5] Bahrke MS, Yesalis CE III, Wright JE. Psychological and behavioural effects of endogenous testosterone and anabolic-androgenic steroids. An update. Sports Med 1996;22:367 –90.

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