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Nandrolone decanoate has long-term effects on dominance in a competitive situation in male rats
Physiology & Behavior 84 (2005) 45 – 51
Nandrolone decanoate has long-term effects on dominance in a competitive situation in male rats Ann-Sophie Lindqvist*, Claudia Fahlke Department of Psychology, Go¨ teborg University, P.O. Box 500, SE-405 30 Go¨teborg, Sweden Received 8 June 2004; received in revised form 9 September 2004; accepted 14 October 2004
Abstract The aim of the present study was to examine possible long-term effects of the anabolic androgenic steroid (AAS), nandrolone decanoate (ND), on dominance in a provoking and competitive situation in sexually matured male rats. The experimental group (n=10) received daily injections of ND [15 mg/kg in a volume of 1 ml/kg subcutaneous (s.c.) injection for 14 days]. During the corresponding period, the controls (n=10) were given daily injections of an oil vehicle (1 ml/kg s.c.). All animals were tested in a competitive situation at four occasions after the end of the treatment period (week 5, 8, 11 and 14). Water-deprived pairs of rats, consisting of one ND-treated rat and one control, had to compete for access to water. The results showed that the ND-treated rats approached the water spout significantly more often compared to the controls. During the competition tests, the ND-treated rats spent more time drinking, an effect that was prominent for 11 weeks after the end of the treatment period. The ND-treated rats also displayed more frequently piloerection than the controls. The results indicate that ND has long-term effect on dominance in a provoking and competitive situation. D 2004 Published by Elsevier Inc. Keywords: Anabolic androgenic steroids; Dominance; Intermale social aggression; Long-term effect; Nandrolone decanoate
1. Introduction Studies investigating the acute health effects of anabolic androgenic steroids (AAS) in humans have increased during the last decade. For example, case reports and clinical studies have found a relationship between AAS abuse and behavioural changes, like enhanced impulsiveness, aggression (both verbal and physical) and feelings of irritability and hostility [13,19,37–39,47,51]. It is also common that AAS abuse is associated with acts of violent behaviours and crimes [12,16,30,40,44,49]. Findings from animal studies have, to some extent, supported the literature concerning the acute AAS-induced behavioural changes seen among humans. For example, studies using rodents, as well as primates, have found that AAS stimulate different forms of dominant and aggressive behavioural expressions, relative to non-steroid-
* Corresponding author. Tel.: +46 31 773 1640; fax: +46 31 773 4628. E-mail address: [email protected] (A.-S. Lindqvist). 0031-9384/$ - see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.physbeh.2004.10.012
treated animals [14,23,25,28,29,31,34,36,41,42]. In a recent study of ours [28], it was found that the 19-nor-testosterone AAS compound, nandrolone decanoate (ND), stimulated dominance in a provoking and competitive situation. In the referred study, male rats were daily treated with 15 mg ND/kg for 2 weeks and tested 1 week thereafter. To test establishment of dominant relationships, a pair of water-restricted animals (one ND-treated animal paired with one control) had to compete for access to water. The results showed that the ND-treated animals were more successful in obtaining and maintaining access to the water spout than the controls. Thus, the results indicate that ND has an acute stimulating effect on dominance in a competitive situation. Whether AAS abuse induces long-term behavioural alterations in humans, as well as in animals, is today little investigated. In humans, it is difficult to conduct wellcontrolled studies due to the highly individual variation of AAS abuse patterns (e.g., type of AAS compound, dosage and frequency of administration). In spite of these difficulties, there are some studies that have examined the potential
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long-term behavioural or psychological effects caused by earlier abuse of AAS. Galligani et al. [19] found an enhanced verbal aggression in adult men that had been abstinent from AAS for at least 6 months. Other studies have, however, shown minor or no alterations in aggressive behaviour in past abusers who had been abstinent for 1 year [32,51] or a longer time [46]. As to animal studies, there is, to our knowledge, only one study that has investigated the long-term effects of AAS on aggressive behaviour. In that study, McGinnis et al. [35] showed that the aggressive behaviour was increased when tested 3 weeks after the end of the treatment period, but this difference had disappeared when the rats were retested 12 weeks after the end of treatment. Thus, more knowledge about the possible longterm behavioural effects of AAS is of importance since AAS abuse has become an increased health and societal problem. In the present study, we investigated the possible longterm effect of ND on dominance in a provocative and competitive situation. Animals were daily treated with 15 mg ND per kg for 2 weeks. In humans, the recommended therapeutic dose of ND is 0.4 mg/kg/day (i.m.) [48], but AAS abusers typically administer these steroids in suprapharmacological doses that are 10 to 100 times the therapeutic dose [10,15,18]. The administration schedule used in the present study is approximately 40 times the therapeutic dose and was chosen to mimic the selfadministered heavy human abuse of AAS [11,18,50]. This treatment schedule has earlier been proved to have an acute effect on dominance [28] but also on other behaviours such as reactivity to physical provocations and voluntary ethanol drinking [25] in male rats. To test the possible long-term effect of ND on dominance, animals were exposed for a competitive test situation 5, 8, 11 and 14 weeks after the end of the ND treatment period. A rat is said to act dominantly if its apparent intent is to achieve or maintain high status, i.e., to obtain power, influence or valued prerogatives, over a conspecific [33]. High status also enhances the probability of maintenance of territories which leads to increased access to resources, including food, shelter and mates [20]. Success in a provoking and competitive situation is related to the socalled intermale social aggression [1], which is mainly distinguished by the presence of behaviours such as piloerection and lateral attacks [2,5]. These behaviours are commonly used by the dominant and threatening rat to establish dominance [5].
2. Method 2.1. Animals Twenty sexually mature male Wistar rats, purchased from Mfllegard Breeding Laboratories (Denmark), served as subjects. They were 80–90 days of age and weighing 330–390 g at the beginning of the experiment. Animals were housed in an air-conditioned colony room at a
temperature of 23 8C and a humidity of 50–60% and with reversed light/dark cycle (lights-off 10:00 a.m.–10:00 p.m.). The animals were housed in groups of four per cage (593820 cm; Makrolon 4) and had free access to water and R70 food pellets (Labfor, Lactamin, Vadstena, Sweden) throughout the experiment, if not otherwise stated. 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. The experimental group (n=10) received daily (10:00–11:00 a.m.) subcutaneous (s.c.) injections of the anabolic androgenic steroid nandrolone decanoate (ND; Deca-Durabol, Organon, Oss, Netherlands) of 15 mg/kg in a volume of 1 ml/kg for 14 days. During the corresponding period, the controls (n=10) were given daily injections of oil vehicle (1 ml/kg s.c; arachidis oleum; Apoteket, Sweden). 2.3. Testing procedures All animals were subjected to a provoking and competitive situation at four occasions after the end of the 2week ND/vehicle treatment period. The first test was performed 5 weeks after the treatment period, and the subsequent tests were executed 8, 11 and 14 weeks after the end of the ND treatment period (i.e., 3 weeks between each test occasion). Two days before each test occasion, all animals were subjected to water restrictions in their home cage, except for 1 h (11:00–12:00 a.m.), daily when they had unlimited access to water (test modified from Ref. [1]). On the third day, one ND-treated rat was randomly paired with one control. The dyad was placed in a clear plastic cage (422615 cm; Makrolon 3) which, for both animals, was a neutral environment. The cage was equipped with a water spout that had a suspended cone at the point where the water spout entered the cage, which gave only one rat the opportunity to drink at a time. Immediately after the dyad entered the cage, the following variables were scored by two observers over a 4-min test period: which of the two animals first started to drink water from the spout, the total time (s) each rat spent drinking, number of lateral attacks (a lateral move toward the counterpart) and occurrence of piloerection for each rat. The length of the competition period (i.e., 4 min) was chosen based on previous results, showing that an individual rat on a 23-h water deprivation schedule would drink continuously for the first 4 min of access to a water spout [1,28]. After the 4-min competition period, all competing pairs were separated, and each individual was returned to its original home cage (ND-treated rats and controls were naturally housed separately), where they had ad libitum access to water until the next test occasion occurred. On every test occasion, the ND-treated animals were paired with a new control unfamiliar to them to avoid dominant–submissive pair constellation.
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To assess each rat’s individual drinking time in a noncompetitive environment (i.e., baseline), all animals were kept on water restriction for an additional 23 h after the first competition test (week 5). The individual drinking time during a 4-min period was registered. To observe whether controls would respond differently to the competitive situation when competing against a non-steroid-treated animal, all controls were subjected to an additional competition test 1 week after the last competition test between ND-treated rats and controls (week 15). One control was randomly paired with an unfamiliar control (i.e., not from the same original home cage; n=5 pairs) and thereafter exposed to the competition test procedure. Body weights for all animals were recorded before and immediately after the 2-week ND/vehicle treatments period and on each of the four competitive test occasions. All tests were performed during the dark phase of the light–dark cycle.
Table 1 One nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days) and one control (competing pairs: n=10) had to compete for access to a restricted water spout on four occasions (i.e., 5, 8, 11 and 14 weeks after the end of the treatment period)
2.4. Statistics
to drink significantly more often than the control group: week 8: P=0.04, week 11: P=0.02 and week 14: P=0.02. As seen in Fig. 1 (left panels), the ND-treated animals and controls showed no difference in individual time spent drinking water during the baseline. During the competitive test (i.e., when access to the water spout was restricted to only one animal at a time, thus forcing the animals to compete for access to the spout), the ND-treated animals tended to drink from the water spout for significantly longer time than the control group week 5 ( P=0.05, t=8.0; Fig. 1, middle panels). The difference between the groups was significant in week 8 ( Pb0.05, t=7.5) and week 11 ( Pb0.05, t=8.0). There was no difference in time spent drinking between the two groups on the last test occasion (week 14). When analysing each test occasion separately, without regard to individual baseline figures, ND-treated animals maintained access to the water spout for significantly longer time than controls on each test occasion (week 5: Pb0.01, t=1.0; week 8: Pb0.05, t=0.0; week 11: Pb0.05, t=5.0; week 14: Pb0.05, t=7.5). Presence of piloerection was more frequently observed in the ND-treated animals on all competitive test occasions, although there was only a statistical difference week 8 ( P=0.04) and week 11 ( P=0.05) compared to the control group (Table 1). Regarding lateral attacks, this behaviour occurred more seldom: out of the ND-treated rats, three to four animals showed this behaviour constantly during all four competitive tests, whereas only one control showed this behaviour at week 8 (data not shown). As seen in Fig. 2, the ND-treated animals weighed significantly more than the control group at the start of the 2week treatment period ( Pb0.01, U=13.5) but significantly less at the end of the treatment period ( Pb0.001, U=1.0). The ND-treated animals remained lower in their body weight at each of the four test occasions compared to controls (week 5: Pb0.001, U=0.0; week 8: Pb0.01, U=19.0; week 11: Pb0.01, U=17.5; week 14: Pb0.01, U=15.5).
Since the distributions of the behavioural data were skewed, nonparametric methods were employed for the statistical analyses [45]. Between-group comparisons (NDtreated animals versus controls and controls versus controls) of number of lateral attacks and time spent drinking during the competition tests were analysed with the Wilcoxon matched-pairs signed rank test. The use of a within-group test was chosen since the result from a competing pair is dependent on each specific pair constellation. Statistical analyses of time spent drinking during the competition tests (week 5, 8, 11 and 14) were based upon individual differences between each of the four competition tests versus drinking time during the individual baseline test. These differences were used in the statistical calculations for between-group comparisons with the Wilcoxon matchedpairs signed rank test. Mann–Whitney U test was employed when analysing individual time spent drinking during the baseline test. Group comparisons of nominal data (which animal started to drink and occurrence of piloerection) were analysed with the Binominal test [45]. Between-group comparisons for body weight on each test occasion were based upon individual differences between the present and former body weight value. These differences were used in the statistical calculation and analysed by help of Mann– Whitney U test. Data are presented as medianFmedian absolute deviation (MAD). Two-tailed levels of significance were used.
3. Results As seen in Table 1, three ND-treated animals and seven controls first started to drink from the water spout at the first test occasion (week 5; ns). At the three subsequent test occasions, the ND-treated animals were the ones who started
Number of weeks after the end of the 2-week treatment period 5 weeks
8 weeks
11 weeks
14 weeks
Start drinking ND 3 Control 7
8** 2
9*** 1
9*** 1
Piloerection ND 10 control 3
8** 0
5* 0
3 0
Shown are number of animals that first started drinking and showed piloerection. ND vs. control. * P=0.05. ** P=0.04. *** P=0.02 (Binominal tests).
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Fig. 1. MedianFMAD values for drinking time (s) in a noncompetitive test situation (i.e., baseline; left panels). Drinking time values are also shown for the subsequent competition tests (i.e., week 5, 8, 11 and 14; middle panels). Time spent drinking at each test occasions was registered over a 4-min period. During the competition tests, access to the water spout was only available for one animal at a time. One nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days) and one control had to compete for the access to the spout. Total pairs were n=10. ND vs. control: (*)P=0.05; *Pb0.05 (Wilcoxon signed rank test). Right panels show the drinking time for controls when they were randomly paired with each other (n=5 pairs).
One week after the last competition test, all controls were randomly paired with each other (i.e., controls versus controls) and thereafter tested once more in the competitive test situation. The median time (s) each group spent drinking from the water spout was 83F23 and 68F9 (ns, see Fig. 1; right panels) and medianFMAD value for both groups together was 71F19. No signs of lateral attacks or piloerection were observed in any of the controls during this competition test.
Fig. 2. MedianFMAD values for body weight (g) in rats treated with nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days; n=10) or with oil (n=10) at the start of the experiment, end of the 2-week treatment period and thereafter 5, 8, 11 and 14 weeks after the end of the treatment. Total pairs were n=10. ND vs. control: **P=0.01; ***Pb0.001 (Mann–Whitney U test).
4. Discussion In a previous study of ours [28], we found that ND had an acute stimulating effect on establishing dominance in a provoking and competitive situation. The aim of the present study was to examine whether the same ND treatment schedule (15 mg/kg/day for 2 weeks) renders long-term effect on dominance. The main finding indicates that the ND-induced dominance remained for more than 2 months after the cessation of the ND treatment period. More specifically, the ND-treated rats obtained and maintained access to the water spout for longer period of time compared to the controls. This effect was significant 11 weeks after the end of the ND treatment period. It was also evident that the ND-treated rats more frequently showed piloerection and to some extent lateral attacks. Together with our earlier results [28], we can thus conclude that ND treatment has acute, as well as long-term effects, on dominance in a provoking and competitive situation. Lateral attacks were only seen in three to four ND-treated rats, and the same rats showed this behaviour on all four competitive tests, whereas only one control showed this behaviour at one occasion (week 8). This finding is in accordance with Breuer et al. [9], who found no difference in attack/fight behaviour, which includes lateral attacks, between ND-treated rats and controls when tested in a neutral environment. Moreover, it has been found that ND has less effect on attack/fights in comparison to threat (which includes piloerection) [35] in a neutral environment. Studies have shown that nontreated gonadal intact male rats, but also testosterone-propionate-treated rats, exhibit more aggression toward an opponent in their home environment than in a neutral environment [4,9]. Thus, it is possible that the reason for the low frequency of lateral attacks in the present study may be due to the fact that animals were tested in a neutral environment.
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These results are in line with the present study showing that all 10 ND-treated animals displayed piloerection on the first test occasion (week 5), whereas only three controls showed this behaviour. On the same test occasion, seven controls out of ten first approached and started to drink from the water spout. It is possible that the controls’ behaviour (first approaching the spout to drink, i.e., obtaining control over a valuable resource) provoked the ND-treated rats to show piloerection to obtain access to the water spout. At the subsequent tests (week 8, 11 and 14), no presence of piloerection was observed among the controls, and the number of ND-treated rats showing piloerection declined over these test occasions. It should be noted that the decreased presence of piloerection over the subsequent tests could not be due to a habituation of an already established dominant–submissive pair constellation since the NDtreated rats always were paired with an unfamiliar control at each test occasion. Thus, the dominance of the NDtreated rats, manifested by their obtaining and maintaining access to the water spout during the subsequent tests, may have been achieved by other nonobserved cues than piloerection. For example, it is known that ultrasonic vocalization and odours are of importance to establish dominance [5]. On the two last test occasions (week 11 and 14), the majority of the controls never made any effort to even approach the water spout, instead, they stayed on the side in the cage opposite to where the water spout and the NDtreated rats were located. According to Blanchard et al. [6], subordinate rats almost never fight in the presence of a dominant conspecific. Noteworthy, when controls were randomly paired with another control (week 15), both control groups spent more time drinking compared to when they competed against an ND-treated rat. Thus, the NDtreated rats’ success in obtaining and maintaining access to water may therefore not be due to a predisposed submissive behavioural profile of the control group. Instead, the findings further strengthen the hypothesis that ND stimulates establishment of dominant relationships. All AAS compounds are synthetic derivates of testosterone, but they vary, however, in their androgenic and anabolic profile. In a comparison between several AAS compounds as to their affinity to the androgen receptor in the brain and the prostate of the rat, it has been shown that ND is as potent as endogenous testosterone, while other AAS compounds, such as the 17-alkyl derivates (e.g., stanozolol), are less potent [43]. Although ND levels were not measured in the present study, it is likely that the used testosterone analogue, ND, is the cause to the increased dominance seen in the ND-treated rats. For example, Albert et al. [1] have shown that animals with testosterone implants achieve success in the specific type of competition situation used in the present study, and the aggressive behaviours that these animals displayed were mainly piloerection and lateral attacks. In addition, it has been found that castration results in a decreased intermale social aggression and loss of
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dominance in a competitive situation [3]. Moreover, testosterone-treated male rats also improve their success rate in competition tasks, such as food reward [7] and copulation with a sexually receptive female [31]. McGinnis et al. [35] have investigated the long-term effect of AAS on aggression. In that study, rats were exposed to a resident–intruder situation 3 and 12 weeks after having been treated with either testosterone, ND or stanozolol (5 mg/kg, 5 days per week for 12 weeks). After 3 weeks, only rats treated with testosterone propionate showed enhanced aggressive behaviours, an effect that was eliminated 12 weeks after the end of the treatment. It may not be relevant to compare our study with that of McGinnis et al. [35] since the dose and treatment schedule of ND was quite different from the present study. Interestingly, however, the authors suggest a potential reversibility of AAS-induced aggression following withdrawal from the steroid treatment. This possibility is important since one of the most common acute psychiatric side-effect of AAS abuse in humans is increased aggression [49]. Thus, it cannot be excluded that the ND-induced dominance observed in the present study eventually returns to a more bnormalQ expression of behaviour. On the other hand, there is a possibility that experiences of success in a competitive situation influence the forthcoming behavioural responses when retesting the animals, even if ND is no longer present in the system. One way to avoid any possible influences of habituation would be to test a new batch of animals on each test occasion. The ND-treated animals had lower body weight compared to their competing control rivals. The results from the present study showed that the body weight of the NDtreated animals decreased up to 8 weeks after the end of the ND-treatment. Thereafter, they had a more or less similar body weight gain development as the controls, although their body weight was nearly 100 g less. The results of lower body weight of the ND-treated rats is in agreement with previous studies of ours [25,28] and others [24,26]. The lowered body weight gain of the ND-treated rats may be due to reduced food consumption. Lindblom et al. [27] have shown that, when using the same treatment regime as in the present study and when the same amount of food was given to controls as the comparable ND-treated animals consumed the previous day, there was no difference in body weight gain between the ND-treated rats and controls. They also found that ND treatment reduced the mRNA levels of the h-endorphin precursor POMC in hypothalamus [27]. Since POMC is the prohormone for a-melanocyte stimulating hormone that is known to participate in the central control of body weight homeostasis [17], the authors suggest that some of the metabolic consequences of ND may be the result of alteration in the melanocortin system. Hence, the reduced body weight observed in the ND-treated rats was probably the result of lowered food intake, although the physiological mechanism behind the decreased food intake is not investigated in this study. Nevertheless, although the ND-treated rats were smaller, they were more
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successful in terms of time of possessing the water bottle. These results of smaller but more successful ND-treated rats are in agreement with other studies [22,28]. Taken together, the results from the present study indicate that ND treatment has relatively long-term effects on dominance and success in a provoking and competitive situation. Further studies in our laboratory aim to investigate whether the ND-induced long-term dominance is reversible following ND treatment. Another question still to be answered is what possible neural system may mediate these behaviours. Especially the serotonergic system may be of interest since previous studies have shown that AAS may modulate aggressive behaviours through this system [7,8,21,28]. In an earlier study of ours [28], we found that the same treatment regime, as used in the present study, resulted in decreased serotonin concentrations in the basal forebrain and the dorsal striatum in the rat. Thus, a cautious interpretation of the present data may be that the observed increased dominance is an effect resulting from a dysfunction in the serotonergic neurotransmitter system induced by the ND treatment.
Acknowledgements The skillful technical assistance of Mrs. Birgit Linder and the statistical advice of late Associate Professor Ernest H3rd are gratefully acknowledged. This work was financially supported by grants from the Swedish National Centre for Research in Sports (No 144/02), L7ngmanska Kulturfonden, Wilhelm och Martina Lundgrens Vetenskapsfond 1, Sigurd och Elsa Goljes Minne, The Royal Society of Arts and Sciences in Gfteborg, Swedish Medical Research Council (13447-03C) and Swedish Alcohol Monopoly Foundation for Alcohol Research (00/4:3).
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Nandrolone decanoate has long-term effects on dominance in a competitive situation in male rats Ann-Sophie Lindqvist*, Claudia Fahlke Department of Psychology, Go¨ teborg University, P.O. Box 500, SE-405 30 Go¨teborg, Sweden Received 8 June 2004; received in revised form 9 September 2004; accepted 14 October 2004
Abstract The aim of the present study was to examine possible long-term effects of the anabolic androgenic steroid (AAS), nandrolone decanoate (ND), on dominance in a provoking and competitive situation in sexually matured male rats. The experimental group (n=10) received daily injections of ND [15 mg/kg in a volume of 1 ml/kg subcutaneous (s.c.) injection for 14 days]. During the corresponding period, the controls (n=10) were given daily injections of an oil vehicle (1 ml/kg s.c.). All animals were tested in a competitive situation at four occasions after the end of the treatment period (week 5, 8, 11 and 14). Water-deprived pairs of rats, consisting of one ND-treated rat and one control, had to compete for access to water. The results showed that the ND-treated rats approached the water spout significantly more often compared to the controls. During the competition tests, the ND-treated rats spent more time drinking, an effect that was prominent for 11 weeks after the end of the treatment period. The ND-treated rats also displayed more frequently piloerection than the controls. The results indicate that ND has long-term effect on dominance in a provoking and competitive situation. D 2004 Published by Elsevier Inc. Keywords: Anabolic androgenic steroids; Dominance; Intermale social aggression; Long-term effect; Nandrolone decanoate
1. Introduction Studies investigating the acute health effects of anabolic androgenic steroids (AAS) in humans have increased during the last decade. For example, case reports and clinical studies have found a relationship between AAS abuse and behavioural changes, like enhanced impulsiveness, aggression (both verbal and physical) and feelings of irritability and hostility [13,19,37–39,47,51]. It is also common that AAS abuse is associated with acts of violent behaviours and crimes [12,16,30,40,44,49]. Findings from animal studies have, to some extent, supported the literature concerning the acute AAS-induced behavioural changes seen among humans. For example, studies using rodents, as well as primates, have found that AAS stimulate different forms of dominant and aggressive behavioural expressions, relative to non-steroid-
* Corresponding author. Tel.: +46 31 773 1640; fax: +46 31 773 4628. E-mail address: [email protected] (A.-S. Lindqvist). 0031-9384/$ - see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.physbeh.2004.10.012
treated animals [14,23,25,28,29,31,34,36,41,42]. In a recent study of ours [28], it was found that the 19-nor-testosterone AAS compound, nandrolone decanoate (ND), stimulated dominance in a provoking and competitive situation. In the referred study, male rats were daily treated with 15 mg ND/kg for 2 weeks and tested 1 week thereafter. To test establishment of dominant relationships, a pair of water-restricted animals (one ND-treated animal paired with one control) had to compete for access to water. The results showed that the ND-treated animals were more successful in obtaining and maintaining access to the water spout than the controls. Thus, the results indicate that ND has an acute stimulating effect on dominance in a competitive situation. Whether AAS abuse induces long-term behavioural alterations in humans, as well as in animals, is today little investigated. In humans, it is difficult to conduct wellcontrolled studies due to the highly individual variation of AAS abuse patterns (e.g., type of AAS compound, dosage and frequency of administration). In spite of these difficulties, there are some studies that have examined the potential
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long-term behavioural or psychological effects caused by earlier abuse of AAS. Galligani et al. [19] found an enhanced verbal aggression in adult men that had been abstinent from AAS for at least 6 months. Other studies have, however, shown minor or no alterations in aggressive behaviour in past abusers who had been abstinent for 1 year [32,51] or a longer time [46]. As to animal studies, there is, to our knowledge, only one study that has investigated the long-term effects of AAS on aggressive behaviour. In that study, McGinnis et al. [35] showed that the aggressive behaviour was increased when tested 3 weeks after the end of the treatment period, but this difference had disappeared when the rats were retested 12 weeks after the end of treatment. Thus, more knowledge about the possible longterm behavioural effects of AAS is of importance since AAS abuse has become an increased health and societal problem. In the present study, we investigated the possible longterm effect of ND on dominance in a provocative and competitive situation. Animals were daily treated with 15 mg ND per kg for 2 weeks. In humans, the recommended therapeutic dose of ND is 0.4 mg/kg/day (i.m.) [48], but AAS abusers typically administer these steroids in suprapharmacological doses that are 10 to 100 times the therapeutic dose [10,15,18]. The administration schedule used in the present study is approximately 40 times the therapeutic dose and was chosen to mimic the selfadministered heavy human abuse of AAS [11,18,50]. This treatment schedule has earlier been proved to have an acute effect on dominance [28] but also on other behaviours such as reactivity to physical provocations and voluntary ethanol drinking [25] in male rats. To test the possible long-term effect of ND on dominance, animals were exposed for a competitive test situation 5, 8, 11 and 14 weeks after the end of the ND treatment period. A rat is said to act dominantly if its apparent intent is to achieve or maintain high status, i.e., to obtain power, influence or valued prerogatives, over a conspecific [33]. High status also enhances the probability of maintenance of territories which leads to increased access to resources, including food, shelter and mates [20]. Success in a provoking and competitive situation is related to the socalled intermale social aggression [1], which is mainly distinguished by the presence of behaviours such as piloerection and lateral attacks [2,5]. These behaviours are commonly used by the dominant and threatening rat to establish dominance [5].
2. Method 2.1. Animals Twenty sexually mature male Wistar rats, purchased from Mfllegard Breeding Laboratories (Denmark), served as subjects. They were 80–90 days of age and weighing 330–390 g at the beginning of the experiment. Animals were housed in an air-conditioned colony room at a
temperature of 23 8C and a humidity of 50–60% and with reversed light/dark cycle (lights-off 10:00 a.m.–10:00 p.m.). The animals were housed in groups of four per cage (593820 cm; Makrolon 4) and had free access to water and R70 food pellets (Labfor, Lactamin, Vadstena, Sweden) throughout the experiment, if not otherwise stated. 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. The experimental group (n=10) received daily (10:00–11:00 a.m.) subcutaneous (s.c.) injections of the anabolic androgenic steroid nandrolone decanoate (ND; Deca-Durabol, Organon, Oss, Netherlands) of 15 mg/kg in a volume of 1 ml/kg for 14 days. During the corresponding period, the controls (n=10) were given daily injections of oil vehicle (1 ml/kg s.c; arachidis oleum; Apoteket, Sweden). 2.3. Testing procedures All animals were subjected to a provoking and competitive situation at four occasions after the end of the 2week ND/vehicle treatment period. The first test was performed 5 weeks after the treatment period, and the subsequent tests were executed 8, 11 and 14 weeks after the end of the ND treatment period (i.e., 3 weeks between each test occasion). Two days before each test occasion, all animals were subjected to water restrictions in their home cage, except for 1 h (11:00–12:00 a.m.), daily when they had unlimited access to water (test modified from Ref. [1]). On the third day, one ND-treated rat was randomly paired with one control. The dyad was placed in a clear plastic cage (422615 cm; Makrolon 3) which, for both animals, was a neutral environment. The cage was equipped with a water spout that had a suspended cone at the point where the water spout entered the cage, which gave only one rat the opportunity to drink at a time. Immediately after the dyad entered the cage, the following variables were scored by two observers over a 4-min test period: which of the two animals first started to drink water from the spout, the total time (s) each rat spent drinking, number of lateral attacks (a lateral move toward the counterpart) and occurrence of piloerection for each rat. The length of the competition period (i.e., 4 min) was chosen based on previous results, showing that an individual rat on a 23-h water deprivation schedule would drink continuously for the first 4 min of access to a water spout [1,28]. After the 4-min competition period, all competing pairs were separated, and each individual was returned to its original home cage (ND-treated rats and controls were naturally housed separately), where they had ad libitum access to water until the next test occasion occurred. On every test occasion, the ND-treated animals were paired with a new control unfamiliar to them to avoid dominant–submissive pair constellation.
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To assess each rat’s individual drinking time in a noncompetitive environment (i.e., baseline), all animals were kept on water restriction for an additional 23 h after the first competition test (week 5). The individual drinking time during a 4-min period was registered. To observe whether controls would respond differently to the competitive situation when competing against a non-steroid-treated animal, all controls were subjected to an additional competition test 1 week after the last competition test between ND-treated rats and controls (week 15). One control was randomly paired with an unfamiliar control (i.e., not from the same original home cage; n=5 pairs) and thereafter exposed to the competition test procedure. Body weights for all animals were recorded before and immediately after the 2-week ND/vehicle treatments period and on each of the four competitive test occasions. All tests were performed during the dark phase of the light–dark cycle.
Table 1 One nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days) and one control (competing pairs: n=10) had to compete for access to a restricted water spout on four occasions (i.e., 5, 8, 11 and 14 weeks after the end of the treatment period)
2.4. Statistics
to drink significantly more often than the control group: week 8: P=0.04, week 11: P=0.02 and week 14: P=0.02. As seen in Fig. 1 (left panels), the ND-treated animals and controls showed no difference in individual time spent drinking water during the baseline. During the competitive test (i.e., when access to the water spout was restricted to only one animal at a time, thus forcing the animals to compete for access to the spout), the ND-treated animals tended to drink from the water spout for significantly longer time than the control group week 5 ( P=0.05, t=8.0; Fig. 1, middle panels). The difference between the groups was significant in week 8 ( Pb0.05, t=7.5) and week 11 ( Pb0.05, t=8.0). There was no difference in time spent drinking between the two groups on the last test occasion (week 14). When analysing each test occasion separately, without regard to individual baseline figures, ND-treated animals maintained access to the water spout for significantly longer time than controls on each test occasion (week 5: Pb0.01, t=1.0; week 8: Pb0.05, t=0.0; week 11: Pb0.05, t=5.0; week 14: Pb0.05, t=7.5). Presence of piloerection was more frequently observed in the ND-treated animals on all competitive test occasions, although there was only a statistical difference week 8 ( P=0.04) and week 11 ( P=0.05) compared to the control group (Table 1). Regarding lateral attacks, this behaviour occurred more seldom: out of the ND-treated rats, three to four animals showed this behaviour constantly during all four competitive tests, whereas only one control showed this behaviour at week 8 (data not shown). As seen in Fig. 2, the ND-treated animals weighed significantly more than the control group at the start of the 2week treatment period ( Pb0.01, U=13.5) but significantly less at the end of the treatment period ( Pb0.001, U=1.0). The ND-treated animals remained lower in their body weight at each of the four test occasions compared to controls (week 5: Pb0.001, U=0.0; week 8: Pb0.01, U=19.0; week 11: Pb0.01, U=17.5; week 14: Pb0.01, U=15.5).
Since the distributions of the behavioural data were skewed, nonparametric methods were employed for the statistical analyses [45]. Between-group comparisons (NDtreated animals versus controls and controls versus controls) of number of lateral attacks and time spent drinking during the competition tests were analysed with the Wilcoxon matched-pairs signed rank test. The use of a within-group test was chosen since the result from a competing pair is dependent on each specific pair constellation. Statistical analyses of time spent drinking during the competition tests (week 5, 8, 11 and 14) were based upon individual differences between each of the four competition tests versus drinking time during the individual baseline test. These differences were used in the statistical calculations for between-group comparisons with the Wilcoxon matchedpairs signed rank test. Mann–Whitney U test was employed when analysing individual time spent drinking during the baseline test. Group comparisons of nominal data (which animal started to drink and occurrence of piloerection) were analysed with the Binominal test [45]. Between-group comparisons for body weight on each test occasion were based upon individual differences between the present and former body weight value. These differences were used in the statistical calculation and analysed by help of Mann– Whitney U test. Data are presented as medianFmedian absolute deviation (MAD). Two-tailed levels of significance were used.
3. Results As seen in Table 1, three ND-treated animals and seven controls first started to drink from the water spout at the first test occasion (week 5; ns). At the three subsequent test occasions, the ND-treated animals were the ones who started
Number of weeks after the end of the 2-week treatment period 5 weeks
8 weeks
11 weeks
14 weeks
Start drinking ND 3 Control 7
8** 2
9*** 1
9*** 1
Piloerection ND 10 control 3
8** 0
5* 0
3 0
Shown are number of animals that first started drinking and showed piloerection. ND vs. control. * P=0.05. ** P=0.04. *** P=0.02 (Binominal tests).
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Fig. 1. MedianFMAD values for drinking time (s) in a noncompetitive test situation (i.e., baseline; left panels). Drinking time values are also shown for the subsequent competition tests (i.e., week 5, 8, 11 and 14; middle panels). Time spent drinking at each test occasions was registered over a 4-min period. During the competition tests, access to the water spout was only available for one animal at a time. One nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days) and one control had to compete for the access to the spout. Total pairs were n=10. ND vs. control: (*)P=0.05; *Pb0.05 (Wilcoxon signed rank test). Right panels show the drinking time for controls when they were randomly paired with each other (n=5 pairs).
One week after the last competition test, all controls were randomly paired with each other (i.e., controls versus controls) and thereafter tested once more in the competitive test situation. The median time (s) each group spent drinking from the water spout was 83F23 and 68F9 (ns, see Fig. 1; right panels) and medianFMAD value for both groups together was 71F19. No signs of lateral attacks or piloerection were observed in any of the controls during this competition test.
Fig. 2. MedianFMAD values for body weight (g) in rats treated with nandrolone decanoate (ND; daily s.c. injections of 15 mg/kg for 14 days; n=10) or with oil (n=10) at the start of the experiment, end of the 2-week treatment period and thereafter 5, 8, 11 and 14 weeks after the end of the treatment. Total pairs were n=10. ND vs. control: **P=0.01; ***Pb0.001 (Mann–Whitney U test).
4. Discussion In a previous study of ours [28], we found that ND had an acute stimulating effect on establishing dominance in a provoking and competitive situation. The aim of the present study was to examine whether the same ND treatment schedule (15 mg/kg/day for 2 weeks) renders long-term effect on dominance. The main finding indicates that the ND-induced dominance remained for more than 2 months after the cessation of the ND treatment period. More specifically, the ND-treated rats obtained and maintained access to the water spout for longer period of time compared to the controls. This effect was significant 11 weeks after the end of the ND treatment period. It was also evident that the ND-treated rats more frequently showed piloerection and to some extent lateral attacks. Together with our earlier results [28], we can thus conclude that ND treatment has acute, as well as long-term effects, on dominance in a provoking and competitive situation. Lateral attacks were only seen in three to four ND-treated rats, and the same rats showed this behaviour on all four competitive tests, whereas only one control showed this behaviour at one occasion (week 8). This finding is in accordance with Breuer et al. [9], who found no difference in attack/fight behaviour, which includes lateral attacks, between ND-treated rats and controls when tested in a neutral environment. Moreover, it has been found that ND has less effect on attack/fights in comparison to threat (which includes piloerection) [35] in a neutral environment. Studies have shown that nontreated gonadal intact male rats, but also testosterone-propionate-treated rats, exhibit more aggression toward an opponent in their home environment than in a neutral environment [4,9]. Thus, it is possible that the reason for the low frequency of lateral attacks in the present study may be due to the fact that animals were tested in a neutral environment.
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These results are in line with the present study showing that all 10 ND-treated animals displayed piloerection on the first test occasion (week 5), whereas only three controls showed this behaviour. On the same test occasion, seven controls out of ten first approached and started to drink from the water spout. It is possible that the controls’ behaviour (first approaching the spout to drink, i.e., obtaining control over a valuable resource) provoked the ND-treated rats to show piloerection to obtain access to the water spout. At the subsequent tests (week 8, 11 and 14), no presence of piloerection was observed among the controls, and the number of ND-treated rats showing piloerection declined over these test occasions. It should be noted that the decreased presence of piloerection over the subsequent tests could not be due to a habituation of an already established dominant–submissive pair constellation since the NDtreated rats always were paired with an unfamiliar control at each test occasion. Thus, the dominance of the NDtreated rats, manifested by their obtaining and maintaining access to the water spout during the subsequent tests, may have been achieved by other nonobserved cues than piloerection. For example, it is known that ultrasonic vocalization and odours are of importance to establish dominance [5]. On the two last test occasions (week 11 and 14), the majority of the controls never made any effort to even approach the water spout, instead, they stayed on the side in the cage opposite to where the water spout and the NDtreated rats were located. According to Blanchard et al. [6], subordinate rats almost never fight in the presence of a dominant conspecific. Noteworthy, when controls were randomly paired with another control (week 15), both control groups spent more time drinking compared to when they competed against an ND-treated rat. Thus, the NDtreated rats’ success in obtaining and maintaining access to water may therefore not be due to a predisposed submissive behavioural profile of the control group. Instead, the findings further strengthen the hypothesis that ND stimulates establishment of dominant relationships. All AAS compounds are synthetic derivates of testosterone, but they vary, however, in their androgenic and anabolic profile. In a comparison between several AAS compounds as to their affinity to the androgen receptor in the brain and the prostate of the rat, it has been shown that ND is as potent as endogenous testosterone, while other AAS compounds, such as the 17-alkyl derivates (e.g., stanozolol), are less potent [43]. Although ND levels were not measured in the present study, it is likely that the used testosterone analogue, ND, is the cause to the increased dominance seen in the ND-treated rats. For example, Albert et al. [1] have shown that animals with testosterone implants achieve success in the specific type of competition situation used in the present study, and the aggressive behaviours that these animals displayed were mainly piloerection and lateral attacks. In addition, it has been found that castration results in a decreased intermale social aggression and loss of
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dominance in a competitive situation [3]. Moreover, testosterone-treated male rats also improve their success rate in competition tasks, such as food reward [7] and copulation with a sexually receptive female [31]. McGinnis et al. [35] have investigated the long-term effect of AAS on aggression. In that study, rats were exposed to a resident–intruder situation 3 and 12 weeks after having been treated with either testosterone, ND or stanozolol (5 mg/kg, 5 days per week for 12 weeks). After 3 weeks, only rats treated with testosterone propionate showed enhanced aggressive behaviours, an effect that was eliminated 12 weeks after the end of the treatment. It may not be relevant to compare our study with that of McGinnis et al. [35] since the dose and treatment schedule of ND was quite different from the present study. Interestingly, however, the authors suggest a potential reversibility of AAS-induced aggression following withdrawal from the steroid treatment. This possibility is important since one of the most common acute psychiatric side-effect of AAS abuse in humans is increased aggression [49]. Thus, it cannot be excluded that the ND-induced dominance observed in the present study eventually returns to a more bnormalQ expression of behaviour. On the other hand, there is a possibility that experiences of success in a competitive situation influence the forthcoming behavioural responses when retesting the animals, even if ND is no longer present in the system. One way to avoid any possible influences of habituation would be to test a new batch of animals on each test occasion. The ND-treated animals had lower body weight compared to their competing control rivals. The results from the present study showed that the body weight of the NDtreated animals decreased up to 8 weeks after the end of the ND-treatment. Thereafter, they had a more or less similar body weight gain development as the controls, although their body weight was nearly 100 g less. The results of lower body weight of the ND-treated rats is in agreement with previous studies of ours [25,28] and others [24,26]. The lowered body weight gain of the ND-treated rats may be due to reduced food consumption. Lindblom et al. [27] have shown that, when using the same treatment regime as in the present study and when the same amount of food was given to controls as the comparable ND-treated animals consumed the previous day, there was no difference in body weight gain between the ND-treated rats and controls. They also found that ND treatment reduced the mRNA levels of the h-endorphin precursor POMC in hypothalamus [27]. Since POMC is the prohormone for a-melanocyte stimulating hormone that is known to participate in the central control of body weight homeostasis [17], the authors suggest that some of the metabolic consequences of ND may be the result of alteration in the melanocortin system. Hence, the reduced body weight observed in the ND-treated rats was probably the result of lowered food intake, although the physiological mechanism behind the decreased food intake is not investigated in this study. Nevertheless, although the ND-treated rats were smaller, they were more
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successful in terms of time of possessing the water bottle. These results of smaller but more successful ND-treated rats are in agreement with other studies [22,28]. Taken together, the results from the present study indicate that ND treatment has relatively long-term effects on dominance and success in a provoking and competitive situation. Further studies in our laboratory aim to investigate whether the ND-induced long-term dominance is reversible following ND treatment. Another question still to be answered is what possible neural system may mediate these behaviours. Especially the serotonergic system may be of interest since previous studies have shown that AAS may modulate aggressive behaviours through this system [7,8,21,28]. In an earlier study of ours [28], we found that the same treatment regime, as used in the present study, resulted in decreased serotonin concentrations in the basal forebrain and the dorsal striatum in the rat. Thus, a cautious interpretation of the present data may be that the observed increased dominance is an effect resulting from a dysfunction in the serotonergic neurotransmitter system induced by the ND treatment.
Acknowledgements The skillful technical assistance of Mrs. Birgit Linder and the statistical advice of late Associate Professor Ernest H3rd are gratefully acknowledged. This work was financially supported by grants from the Swedish National Centre for Research in Sports (No 144/02), L7ngmanska Kulturfonden, Wilhelm och Martina Lundgrens Vetenskapsfond 1, Sigurd och Elsa Goljes Minne, The Royal Society of Arts and Sciences in Gfteborg, Swedish Medical Research Council (13447-03C) and Swedish Alcohol Monopoly Foundation for Alcohol Research (00/4:3).
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