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Reduced sensitivity to MDMA-induced facilitation of social behaviour in MDMA pre-exposed rats
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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 1013 – 1021 www.elsevier.com/locate/pnpbp
Reduced sensitivity to MDMA-induced facilitation of social behaviour in MDMA pre-exposed rats Murray R. Thompson a , Paul D. Callaghan a , Glenn E. Hunt b , Iain S. McGregor a,⁎ a
b
School of Psychology, University of Sydney, A18, Sydney, NSW 2006, Australia Department of Psychological Medicine, University of Sydney, Concord Hospital, Sydney, NSW 2139, Australia Received 5 August 2007; received in revised form 24 January 2008; accepted 24 January 2008 Available online 4 February 2008
Abstract The acute effects of the party drug 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) in humans include feelings of love, closeness towards other people and an increased acceptance of others views and feelings. Some evidence suggests that regular MDMA users develop a subsensitivity to the positive effects of the drug and escalate their intake of the drug over time as a result. The current study investigated whether brief exposure to relatively high doses of MDMA in rats produces a subsequent attenuation in the ability of MDMA to enhance social interaction. Male Wistar rats were exposed to either MDMA (4 × 5 mg/kg over 4 h) or vehicle on two consecutive days. Twelve weeks later, MDMA preexposed rats displayed a significantly shorter period of time spent in social interaction than controls when tested in the drug-free state. MDMA pre-exposed rats also showed a blunted prosocial response to MDMA (2.5 mg/kg) relative to controls. This difference was overcome by increasing the MDMA dose to 5 mg/kg. The 5-HT1A agonist 8-OH-DPAT (250 µg/kg but not 125 µg/kg) increased social interaction and this effect did not differ in MDMA and vehicle pre-exposed rats. HPLC analysis showed a small but significant depletion of prefrontal 5-HT and 5-HIAA in MDMA pre-exposed rats. Prefrontal 5-HIAA concentrations were also reduced in the subset of vehicle and MDMA pre-exposed rats that received additional testing with MDMA. These results indicate that treatment with MDMA not only causes lasting reductions in social interaction in rats but causes an attenuation of the prosocial effects of subsequent MDMA administration. The lack of a differential response to 8-OH-DPAT agrees with other findings that the 5-HT1A receptor system remains functionally intact following MDMA pre-exposure and suggests that other neuroadaptations may underlie the lasting social deficits caused by MDMA. © 2008 Elsevier Inc. All rights reserved. Keywords: 5-HT1A; 8-OH-DPAT; Anxiety; Behaviour; MDMA; Social
1. Introduction The ring substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) possesses a unique psychoactive profile with individuals reporting increased emotional experience, extroversion, an openness and willingness to converse with others, euphoria and a sense of peacefulness when under the influence of
Abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; 8-OHDPAT, (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid.; HPLC, high-performance liquid chromatography. ⁎ Corresponding author. Tel.: +61 2 9351 3571; fax: +61 2 9351 8023. E-mail address: [email protected] (I.S. McGregor). 0278-5846/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.01.014
the drug (Baylen and Rosenberg, 2006; Dumont and Verkes, 2006; Vollenweider et al., 1998). The distinctive euphorogenic and prosocial effects of Ecstasy have led to MDMA becoming one of the most popular illicit drugs currently used by young people (Australia Institute of Health and Welfare, 2005; Degenhardt et al., 2004; Soellner, 2005), and a broadening of MDMA use beyond the “club drug” scene of the 1990s has been observed in recent years (Allott and Redman, 2006; Levy et al., 2005; Solowij et al., 1992; Topp et al., 1999; White et al., 2006). Concerns have been raised regarding the long-term residual effects of MDMA use. Ecstasy use in humans has been linked to residual increases in anxiety, depression and memory impairment (Kalechstein et al., 2007; Morgan, 2000; Parrott, 2001; Parrott and Marsden, 2006), and impaired social and emotional
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judgement (Reay et al., 2006). Mirroring human data, rats previously treated with MDMA display long-term residual impairments in memory (McGregor et al., 2003b; Morley et al., 2001; Piper and Meyer, 2004), and an increase in anxiety and depressive-like symptoms (Clemens et al., 2004; Gurtman et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004; Walker et al., 2007). Rats also show reduced social interaction with novel conspecifics for at least 3 months following MDMA exposure, and this effect can be observed even using treatment regimens that do not cause significant lasting alterations in brain serotonin (5-HT) levels (Bull et al., 2003, 2004; Fone et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004). Anecdotal evidence suggests that the positive mood-altering effects of MDMA are reduced following repeated dosing (Parrott, 2005; Solowij et al., 1992) and this may in part result in greater consumption of Ecstasy per session by experienced compared to novice users (Huxster et al., 2006; McCambridge et al., 2005). Reduced serotonergic function after MDMA exposure is evident from attenuated 5-HT release in vivo, decreased firing rates of 5-HT neurons, and diminished neuroendocrine, thermoregulatory and locomotor responses to serotonergic challenges (Baumann et al., 2007; Gartside et al., 1996; Mechan et al., 2001; Series et al., 1994; Shankaran and Gudelsky, 1999). Attenuation of MDMA-induced responses has been shown clinically by reductions in l-tryptophan, dfenfluramine and m-chlorophenylpiperazine induced increases in plasma prolactin, cortisol and growth hormone among cohorts of regular MDMA users (Gerra et al., 2000, 1998; McCann et al., 1994; Price et al., 1989; Verkes et al., 2001). In contrast to its adverse residual effects on social behaviour, MDMA produces an acute facilitation of social interaction in rats that appears to be mediated in part by 5-HT1A and oxytocin receptors (Morley et al., 2005; Thompson et al., 2007). However, is not clear whether acute MDMA-induced social facilitation is attenuated by prior MDMA exposure. Indeed, Ando et al. (2006) recently reported that the prosocial effects of MDMA may become amplified with prior MDMA exposure. However this study used Dark Agouti rats, a strain with significantly higher baseline anxiety and differing acute and long-term behavioural responses to MDMA compared to Wistar or Sprague–Dawley rats (Green and McGregor, 2002). Ando et al. (2006) also tested social interaction following an extremely high (15 mg/kg) single dose of MDMA, a dose that may have conceivably masked subtle tolerance-related phenomena due to a ceiling effect. It was thus of interest to determine whether MDMA pre-exposed Wistar rats would display altered sensitivity to MDMA's acute prosocial effects when lower doses of MDMA were tested. The social response to the 5-HT1A agonist 8-OH-DPAT was also tested in the present study to determine if an attenuation of 5-HT1A agonist-induced social interaction was apparent in MDMA pre-exposed rats. The 5-HT1A system has been implicated in mediating the prosocial effects of MDMA with the 5HT1A antagonist WAY 100,635 blocking the acute prosocial effects of MDMA most likely as a result of an interaction with the neuropeptide oxytocin (Morley et al., 2005; Thompson et al., 2007). If long-term social deficits following MDMA
reflect downregulation of 5-HT1A receptors then it might be expected that MDMA pre-exposed rats would be sub-sensitive to the prosocial effects of 8-OH-DPAT. 2. Methods 2.1. Animals A total of 48 inbred male albino Wistar rats (University of Sydney, Australia) were used in the experiments, aged between 90 and 100 days old and weighing an average of 421 g at the start of testing. The rats were housed in groups of 6 per cage in a temperature-controlled environment (average temperature 22 °C). A 12 h reversed light–dark cycle was in operation (lights off at 8:30 a.m.) and all testing took place in the dark cycle. Food and water were freely available ad libitum. All the experiments were approved by the University of Sydney Animal Ethics Committee. 2.2. Drugs (±)-3,4-methylenedioxymethamphetamine (MDMA) was obtained from Australian Government Analytical Laboratories (Pymble, NSW). MDMA was dissolved in 0.9% saline at a concentration of either 2.5 mg/ml or 5 mg/ml and injected intraperitoneally at a volume of 1 ml/kg. To produce 5-HT depletion in MDMA pre-exposed rats a 4 × 5 mg dose on two consecutive days was used, as in previous studies from our laboratory (Gurtman et al., 2002; McGregor et al., 2003b; Thompson et al., 2004). To examine acute prosocial effects, 2.5 mg/kg and 5 mg/kg doses of MDMA were used, again according to previous studies from our laboratory (Cornish et al., 2003; Morley et al., 2005; Morley and McGregor, 2000). Control rats received equivalent injections of 0.9% saline. The 5-HT1A agonist (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) was purchased from Sigma– Aldrich. 8-OH-DPAT was dissolved in saline at a dose of either 125 µg/kg or 250 µg/kg and injected intraperitoneally at a volume of 1 ml/kg. The 250 µg/kg dose has been shown to increase social interaction in rats in previous studies (Picazo et al., 1995). 2.3. Apparatus and procedures 2.3.1. Acute MDMA administration and effects on body temperature Initial administration of MDMA to produce MDMA preexposed rats for later testing involved procedures reported previously by our group (Thompson et al., 2004). Rats received a 5 mg/kg i.p. injection of MDMA (n = 24) or vehicle (n = 24) every hour for 4 h on 2 consecutive days, giving a cumulative dose of 40 mg/kg. During MDMA or vehicle administration, individual rats were placed in standard operant chambers (30 × 50 × 25.5 cm) with three aluminium walls, one Perspex wall and a metal grid floor. The chambers were enclosed in wooden sound attenuation boxes. Room temperature was maintained at an ambient temperature of 28 °C by a reversecycle air conditioner. High temperatures may exacerbate the 5-
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HT-depleting effects of MDMA in rats (Malberg and Seiden, 1998) and also increase the rewarding and prosocial effects of MDMA (Cornish et al., 2003). The body temperature of all rats was recorded each hour with a Braun Thermoscan Instant Thermometer (IRT 1020) at the time of each injection. Following the 4 h drug administration period, all rats were housed individually in the colony room overnight and replaced back in their home cages the following morning. This procedure prevents the possible lethal effects of ‘aggregation toxicity’ sometimes seen with group housing following high-dose stimulant treatment (Green et al., 1995). 2.3.2. Long-term effects of MDMA pre-exposure on social behaviour Twelve weeks following drug administration, all rats were tested in the social interaction test in a drug-free state with a novel conspecific that had received the same drug pre-exposure. The social interaction test was run on two consecutive days with each rat experiencing a different partner on each of the 2 days. Results over these two days were pooled, providing a final data set with 24 unique pairs of rats per drug condition. The social interaction arena consisted of a black, Perspex box (52 × 52 × 40 cm) dimly lit with red light (40 W). Rats were placed in the test arena for 10 min. A miniature video camera was placed vertically above the social interaction arena that sent a signal into a VCR in a neighbouring room where behaviours were scored live by a trained observer blind to treatment group. Scoring of the social interaction test was based upon the work of File and Seth (2003) but with the total social interaction score subdivided into three categories. These categories were similar to those described by Morley et al. (2005) and consisted of Adjacent lying: duration of side by side contact or ‘huddling’ including climbing under/over conspecific; Anogenital sniffing: duration of sniffing the conspecific's anogenital area; Investigation: duration of sniffing the conspecific apart from anogenital sniffing. A “total social interaction” score was also obtained by summing the scores of these individual behaviours for each pair of rats. The non-social activity of Rearing: standing on hind legs was also scored as previous studies have shown that acute MDMA potently inhibits this behaviour (e.g. Morley et al. (2005)). The testing arena was wiped down with 50% ethanol between every session. 2.3.3. Acute social effects of MDMA on MDMA pre-exposed rats Three days following the last of the baseline social interaction sessions, a subset of 12 MDMA pre-exposed and 12 vehicle preexposed rats was randomly chosen to investigate the acute effects of MDMA or 8-OH-DPAT on social interaction. These 12 MDMA pre-exposed and 12 vehicle pre-exposed animals underwent a further 5 social interaction tests with all 24 of these rats receiving an identical set of tests with low MDMA and 8-OH-DPAT doses. On the first day of testing these vehicle and MDMA-preexposed rats received a dose of 2.5 mg/kg MDMA and were placed in holding cages (40 × 25 × 15 cm opaque box lined with bedding) in the testing room. The room was heated to 28 °C as this temperature serves to facilitate MDMA-induced social interaction
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without affecting baseline social behaviour (Cornish et al., 2003). Twenty minutes following the MDMA injection rats were tested in a 10 min social interaction test with a novel conspecific of identical pre-treatment group (vehicle pre-exposed or MDMA-preexposed). Testing occurred over 2 consecutive days with a different partner used on each day to give a final sample size of 12 unique MDMA pre-exposed and 12 unique vehicle pre-exposed pairs. The same subset of vehicle and MDMA pre-exposed rats was then tested 48 h later on the social interaction test, under the same conditions, but with a 5 mg/kg dose of MDMA and a further novel partner. Testing for this phase of the experiment occurred on a single day to produce a final sample size of 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs. A second challenge day with the 5 mg/kg dose of MDMA was not undertaken as no trend towards differences between vehicle and MDMA pre-exposed rats were observed on the first day of testing. 2.3.4. Acute social effects of 8-OH-DPAT upon MDMA preexposed rats Two days later the vehicle and MDMA pre-exposed rats were tested, using the same procedures, with a 125 µg/kg dose of the 5-HT1A agonist 8-OH-DPAT. This resulted in a final sample size of 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs. One day later, the same subsets of vehicle and MDMA pre-exposed rats were tested with a 250 µg/kg dose of 8-OHDPAT. As with MDMA, the drug was given 20 min prior to the start of the 10 min social interaction test. Again, testing only occurred in 1 day, resulting in 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs for each of the 8-OH-DPAT tests. 2.3.5. HPLC analysis Five days after the final social interaction test with 8-OHDPAT, all rats were rapidly decapitated and the prefrontal cortex was dissected over dry ice and then stored at −80 °C. Subsequent analysis of noradrenaline, dopamine, 5-HIAA (5-hydroxyindoleacetic acid) and 5-HT (5-hydroxytryptamine) was undertaken using high-performance liquid chromatography (HPLC) with electrochemical detection employing standard techniques as described by Clemens et al. (2005). Two samples were excluded from HPLC analysis as wet tissue weights were notably lower than the other prefrontal cortex tissue samples, suggesting an inaccurate dissection. 2.4. Statistical analysis For initial MDMA exposure a repeated-measures Analysis of Variance (ANOVA) was used to compare body temperature in vehicle and MDMA-treated rats across the 4 h of testing on each of the 2 days of treatment. Differences between groups for each hour of testing were subsequently analysed using post hoc contrasts. A one way ANOVA was used to compare the two treatment groups (vehicle and MDMA pre-exposed) on the baseline, acute MDMA and acute 8-OH-DPAT social interaction tests conduced 12–14 weeks after drug pre-exposure. In addition, the acute effects of MDMA and 8-OH-DPAT on social interaction were evaluated by comparing social interaction in vehicle pre-exposed rats on the baseline test with that
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Fig. 1. Mean body temperature in vehicle and MDMA exposed rats on day 1 (left) and day 2 (right) of drug treatment. Body temperature was measured prior to drug administration (0 h) and at 1, 2, 3, and 4 h following the drug. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA and vehicle-exposed groups.
seen in the same rats with acute MDMA and 8-OH-DPAT. This was done with ANOVA followed by post hoc contrasts. For HPLC analysis a 2-way ANOVA was used to analyse the effects of MDMA pre-exposure and subsequent drug challenge with MDMA/8-OH-DPAT on various monoamine levels. The threshold for statistical significance was set at P b 0.05.
Fig. 2. Results from the social interaction test (conducted drug-free) 12 weeks following vehicle or MDMA exposure. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA and vehicle-exposed groups.
3. Results 3.2. Long-term residual effects of MDMA on social interaction 3.1. Acute effects of MDMA on body temperature The effects of vehicle or MDMA administration on body temperature can be seen in Fig. 1. On day 1, a repeated-measures ANOVA revealed a significant temperature difference between vehicle and MDMA-treated rats (F(1,46) = 186.596, P b 0.0001), a significant change in temperature for both treatment groups over the 4 hour test period (F(4,184) = 33.09, P b 0.0001) and a significant treatment by time interaction effect (F(4,184) = 29.30, P b 0.001). Subsequent post hoc analysis revealed no group differences immediately before drug administration. MDMA treatment significantly increased body temperature relative to vehicles on hour 1 (F(1,46) = 26.66, P b 0.001), hour 2 (F(1,46) = 137.44, P b 0.001), hour 3 (F(1,46) = 43.31, P b 0.001) and hour 4 (F(1,46) = 198.77, P b 0.001). The second day of MDMA administration showed a similar general pattern to that seen on day 1 with a significant overall effect of treatment (F(1,46) = 81.10, P b 0.0001), a significant effect of time (F(4,184) = 27.50, P b 0.0001), and a significant treatment by time interaction (F(4,184) = 24.33, P b 0.0001). Unlike day 1, there was no significant body temperature difference between MDMA and vehicle treated rats 1 h after the first injection (F b 1). However, MDMA produced significantly higher body temperatures compared to vehicle treatment at hour 2 (F(1,46) = 81.56, P b 0.001), hour 3 (F(1,46) = 38.15, P b 0.001) and hour 4 (F(1,46) = 307.03, P b 0.001). Although the body temperature of some rats exceeded 40 °C towards the end of the day 1 and 2 test sessions, no lethal effects of MDMA were observed in any of the rats.
Baseline social interaction results 12 week after vehicle or MDMA pre-treatment are presented in Fig. 2. ANOVA revealed that MDMA pre-exposed rats showed significantly reduced levels of total social interaction when compared to vehicle preexposed rats (F(1,46) = 8.95, P b 0.01). Separate analysis of the three subtypes of social behaviour constituting the total
Fig. 3. MDMA-induced behaviours (left panel: 2.5 mg/kg, right panel: 5 mg/kg) in the social interaction test in vehicle and MDMA pre-exposed rats. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA pre-exposed and vehicle pre-exposed groups.
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3.4. Acute prosocial effects of MDMA in MDMA pre-exposed rats The effects of a 2.5 mg/kg dose of MDMA on social interaction in vehicle and MDMA pre-exposed rats are presented in Fig. 3. MDMA-pre-exposed rats given MDMA (2.5 mg/kg) showed significantly less total social interaction (F(1,21) = 7.030, P b 0.05) and adjacent lying (F(1,21)= 5.756, P b 0.05) compared to vehicle pre-exposed rats. No significant group differences were observed for the behavioural subtypes of general investigation (F b 1), anogenital sniffing (F(1,21) = 2.586, P N 0.05) or rearing (F b 1). Fig. 3 illustrates the effects of an acute 5 mg/kg dose of MDMA on social interaction in the MDMA and vehicle preexposed groups. No significant differences or trends towards differences were evident when comparing these groups on social behaviour following this dose (Fs b 1.6). Fig. 4. 8-OH-DPAT-induced behaviours (left panel: 125 µg/kg, right panel: 250 µg/kg) in the social interaction test between vehicle and MDMA preexposed rats. Data represent mean ± SEM.
social interaction score revealed that MDMA pre-exposed rats showed significantly lower levels of investigation than controls (F(1,46) = 7.86, P b 0.01) but did not differ in anogenital sniffing (F b 1), adjacent lying (F(1,46) = 2.82, P N 0.05) or the nonsocial variable of rearing (F b 1.2). 3.3. Acute prosocial effects of MDMA and 8-OH-DPAT in vehicle pre-exposed rats Analysis of social interaction in the vehicle pre-exposed control group across the 4 different acute drug tests conducted with MDMA and 8-OH-DPAT (compare Figs. 2, 3 and 4) revealed that there were significant overall drug effects in this group in levels of total social interaction (F(4,49) = 30.659, P b 0.001). Subsequent post hoc tests revealed that administration of MDMA (2.5 or 5 mg/kg) and 8-OH-DPAT (250 but not 125 µg/kg) significantly increased levels of total social interaction in these control rats relative to the levels seen in these rats in the baseline social interaction test.
3.5. Acute prosocial effects of 8-OH-DPAT in MDMA preexposed rats The effects of 8-OH-DPAT in MDMA pre-exposed and vehicle pre-exposed groups are presented in Fig. 4. No significant group differences were seen in social behaviour following either the 125 or 250 µg/kg doses of 8-OH-DPAT (Fs b 1). 3.6. Neurochemical analysis Results for the HPLC analysis of the prefrontal cortex are presented in Table 1. Rats that had been pre-exposed to MDMA 12 weeks previously exhibited significantly lower levels of 5HIAA (F(1,42) = 5.786, P b 0.05) and 5-HT (F(1,42) = 5.635, P b 0.05) relative to vehicle pre-exposed controls. Exposure to MDMA and 8-OH-DPAT during acute social interaction tests was associated with a significant reduction in 5-HIAA levels (F(1,42) = 18.444, P b 0.001), but not 5-HT, relative to rats that did not take part in that phase of the experiment. Noradrenaline and dopamine levels were unaffected by all treatment regimens used and no significant interactions between drug preexposure and subsequent exposure were obtained for any of the biogenic amines investigated.
Table 1 Results of HPLC analysis in the prefrontal cortex Neurotransmitter
5-HT 5-HIAA Noradrenaline Dopamine
Vehicle/control
Vehicle/MDMA
MDMA/control
MDMA/MDMA
n = 11
n = 12
n = 11
n = 12
612.6 (27.5) 200.8 (8.7) 1335.3 (92.2) 994.6 (205.6)
596.9 (32.0) 159.1 (9.1) 1417.1 (123.5) 1020.7 (167.8)
555.8 (17.3) 174.6 (9.0) 1486.9 (140.5) 1018.4 (76.2)
533.3 (21.3) 145.9 (5.7) 1257.6 (49.4) 750.2 (71.5)
Significance
a a,b
Rats received either vehicle or MDMA exposure. Twelve weeks later a subset of 12 vehicle pre-exposed (vehicle/MDMA) and 12 MDMA pre-exposed (MDMA/ MDMA) rats received two additional doses of MDMA (2.5 and 5 mg/kg on two separate days) as well as two doses of 8-OH-DPAT (125 and 250 µg/kg on two separate days) during social interaction testing. Data represent Mean ± (SEM). Units of measurement are ng/g wet tissue. a P b 0.05 when comparing vehicle groups and MDMA pre-exposed groups. b P b 0.05 when comparing rats challenged later with MDMA and 8-OH-DPAT (groups vehicle/MDMA and MDMA/MDMA) relative to those who were not challenged (groups vehicle/control and MDMA/control).
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4. Discussion The present study demonstrates that MDMA pre-treatment, at doses that decrease prefrontal cortex 5-HT levels, leads to a long-term attenuation of the capacity of low dose MDMA to produce subsequent increases in social interaction. This provides evidence, in a rodent model, for the long-term reductions in the prosocial effects of MDMA that are reported by regular human users of MDMA. As previously demonstrated, the acute administration of 5 mg/ kg MDMA each hour for 4 consecutive hours produced hyperthermia in Wistar rats tested at a 28 °C ambient temperature (Morley et al., 2001; Thompson et al., 2004). Interestingly, the pattern of hyperthermia differed across the two consecutive days of dosing, with a longer latency to an MDMA-induced hyperthermia response on day 2 compared to day 1 (Fig. 1). This effect replicates the earlier finding of Thompson et al. (2004) and indicates a reduced sensitivity to the hyperthermic effects of MDMA, possibly as a result of short term 5-HT depletion and/or a loss of tryptophan hydroxylase activity following the first day of MDMA administration (Baumann et al., 2007). The long-term residual social interaction deficits seen here following prior MDMA treatment replicate previous findings (Bull et al., 2003, 2004; Clemens et al., 2005, 2004; Fone et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004) and the present study further illustrates the lasting nature of this effect, with social interaction deficits evident 12 weeks after brief MDMA exposure (Gurtman et al., 2002; Thompson et al., 2004). This study is the first to characterise lasting reductions in specific subtypes of social interaction following MDMA pre-exposure, with a significant decline in the “general investigation” subtype. To determine whether prior MDMA exposure resulted in attenuation of drug-induced prosocial behaviours, a subset of rats was challenged with two doses of MDMA as well as two doses of the 5-HT1A agonist 8-OH-DPAT. Rats that had been pre-exposed to MDMA 12 weeks previously showed an attenuation of prosocial behaviours 20 min following a low dose (2.5 mg/kg) of MDMA. It is notable that vehicle preexposed rats administered the 2.5 mg/kg dose of MDMA showed much higher levels of total social interaction here than was reported in an earlier study by Morley and McGregor (2000) with this same dose. This may be due to the elevated room temperatures (28 °C) used during social interaction testing in the present study, with high temperatures known to facilitate MDMA's prosocial effects (Cornish et al., 2003). Analysis of social behaviour subtypes revealed that acute administration of MDMA at a dose of 2.5 mg/kg increased adjacent lying to a greater extent in vehicle pre-exposed than in MDMA pre-exposed rats. Adjacent lying is a key acute behavioural effect seen with MDMA in Wistar rats and is mediated through oxytocinergic and 5-HT1A receptor systems (Morley et al., 2005; Thompson et al., 2007). It is interesting to note that the long-term residual MDMA induced social deficits observed when rats were tested in a drug-free state (general investigation) involve a different social behaviour subtype than that deficient under MDMA challenge (adjacent lying). This suggests that MDMA exposure can influence a variety of social
behaviours, with some deficits (i.e. reduced adjacent lying) only evident following further drug challenges. Consistent with this, MDMA pre-exposure has been previously shown to attenuate the effects of drug-induced behaviours, such as the 5-HT2A/2C agonist DOI in the elevated plus maze, while MDMA preexposure by itself had little effect on the amount of time spent on the open arms (Bull et al., 2004). In contrast to the results observed with 2.5 mg/kg dose of MDMA, the prosocial effects of a higher 5 mg/kg dose of MDMA were not significantly affected by MDMA pre-exposure. This indicates that blunting of MDMA's acute effects as a result of preexposure can be overcome with increased MDMA dose. This may provide a rationale for the dose escalation that has been reported in experienced compared to novice MDMA users (Huxster et al., 2006; McCambridge et al., 2005). The 5-HT1A receptor at least partly mediates adjacent lying behaviours following acute MDMA treatment (Morley et al., 2005), and this was further confirmed in the present study with an MDMA-like increase in adjacent lying obtained with the 5HT1A receptor agonist 8-OH-DPAT. Interestingly however, rats pre-exposed to MDMA did not appear to be deficient in their prosocial response to 8-OH-DPAT. Here it must be noted that there was a large “all or nothing” increase in the prosocial effects of 8-OH-PDAT between the 125 and 250 µg/kg doses used in the present study such that possible group differences at intermediate doses cannot be ruled out. Nonetheless, the current results lead to the tentative conclusion that the 5-HT1A system's role in the production of affiliative prosocial behaviours remains intact following MDMA pre-exposure. There have been contrasting reports concerning the long-term effects of MDMA administration on 5-HT1A receptor expression, with binding studies showing either no effect (McGregor et al., 2003a; Piper et al., 2006) or subtle effects upon receptor concentration in specific brain regions (Aguirre et al., 1998, 1997). Functional assays of the 5-HT1A receptor following MDMA pre-treatment have also produced mixed results. 8-OHDPAT-induced forepaw treading and serotonin syndrome are attenuated by MDMA pre-treatment (Granoff and Ashby, 2001; Piper et al., 2006), while 8-OH-DPAT-induced hypothermic responses were unaltered (Granoff and Ashby, 2001; McNamara et al., 1995; Mechan et al., 2001; Piper et al., 2006). Thus although the present results suggest little effect of MDMA pre-exposure on 8-OH-DPAT-induced social behaviours, effects on other nonsocial behaviours cannot be ruled out. Rearing behaviour, which is greatly reduced by acute MDMA or 8-OH-DPAT treatment (Thompson et al., 2007), was attenuated here by MDMA to a similar extent in vehicle and MDMA pre-exposed rats. Previous studies have indicated that MDMA-induced attenuation of rearing behaviours in the social interaction test is unaffected by co-administration of a range of different 5-HT receptor antagonists (Morley et al., 2005) or the oxytocin antagonist tocinoic acid (Thompson et al., 2007). Other studies suggest that inhibitory effects of acute 8-OHDPAT on rearing may involve mesencephalic dopaminergic mechanisms (e.g. Hillegaart et al., 2000) and this may explain why MDMA pre-exposure did not modify the sensitivity of this particular behaviour to acute pharmacological challenge.
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Reduction in MDMA-stimulated social behaviour and reduced baseline social behaviour after prior MDMA exposure may therefore involve a number of non-5-HT1A receptor mechanisms. This might include lasting alterations in the density of other serotonergic receptors and/or the 5-HT transporter in key regions underlying social behaviour such as the amygdala and hypothalamus (see McGregor et al., 2003a). Prior MDMA ‘neurodegenerative’ treatment attenuates the neurochemical responses to subsequent acute MDMA administration, including reductions in evoked 5-HT release in vivo (Series et al., 1994; Shankaran and Gudelsky, 1999), and this may also be a key mechanism in the pre-exposure effect reported here. However non-serotonergic mechanisms may also be involved. We have recently shown that MDMA stimulates the release of the neuropeptide oxytocin in rats and that the oxytocin receptor antagonist tocinoic acid can attenuate the acute increases in social interaction produced by MDMA (Thompson et al., 2007). It is therefore possible that decreased sensitivity to the social effects of MDMA as a result of prior MDMA exposure may reflect some long-term blunting of oxytocinergic circuitry by MDMA and this is a possibility that warrants further investigation. While MDMA pre-exposure caused lasting reductions in 5-HT and 5-HIAA concentrations in the present study, the additional subsequent testing with MDMA and 8-OH-DPAT conducted 12 weeks later did not further alter prefrontal cortex 5-HT levels. This most likely reflects the acute MDMA doses used being below the threshold required to deplete 5-HT (Green et al., 2003). However, prefrontal cortex 5-HIAA concentrations were significantly reduced in rats receiving these additional tests with MDMA and 8-OH-DPAT, irrespective of pre-exposure status. There was a relatively short period of time (9 days) in these rats between the last acute test with MDMA and brain dissection, such that MDMA-induced inhibition of tryptophan hydroxylase may have still been apparent (O'Shea et al., 2006; Stone et al., 1987). In addition, the effects of MDMA may have been potentiated due to the high ambient room temperatures used during social interaction testing. High temperatures have been shown to accentuate MDMA-induced reductions in 5-HT/5-HIAA forebrain content and inactivation of tryptophan hydroxylase (Che et al., 1995; Malberg and Seiden, 1998). Acute 8-OH-DPAT treatment does not significantly alter forebrain biogenic amine concentrations, and thus would be unlikely to be involved in the effect on 5-HIAA (Kennett et al., 1987; Larsson et al., 1990). The results in the present study are in contrast to those of Ando et al. (2006) who reported an augmented adjacent lying effect to acute MDMA (15 mg/kg) in Dark Agouti rats that had been previously exposed to a 15 mg/kg dose of MDMA. In a further contrast between these two studies, Ando et al. (2006) found no residual deficits in social interaction behaviours, although an increase in aggressive behaviours was reported in MDMA pre-exposed rats. Clearly, the high dose (15 mg/kg) used by Ando et al. (2006) may have not allowed subtle MDMA pre-exposure effects to be observed, given that pre-exposure effects were only seen in the present study with 2.5 mg/kg and not 5 mg/kg of MDMA. However, this does not explain the enhanced acute prosocial effects of MDMA seen in the Ando
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et al. (2006) study in MDMA pre-exposed rats and this may represent a fundamental strain difference. The present study also observed no aggressive episodes in any rats during the social interaction test, further emphasising the behavioural differences between these two rat strains (Green and McGregor, 2002). Clearly a direct behavioural and neurochemical comparison of the social response of Albino Wistar and Dark Agouti rat strains to MDMA would be a useful exercise. 5. Conclusions Overall, the present study extends the previous findings of subsensitivity to MDMA following prior MDMA exposure to include results from the social interaction test. These results mirror those reports of tolerance to the prosocial effects of MDMA in human users and may help to explain the dose escalation sometimes seen in experienced MDMA users. Acknowledgements Support was contributed by the National Health and Medical Research Council of Australia. References Aguirre N, Frechilla D, Garcia-Osta A, Lasheras B, Del Rio J. Differential regulation by methylenedioxymethamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex, and brainstem: the role of corticosteroids. J Neurochem 1997;68:1099–105. Aguirre N, Ballaz S, Lasheras B, Del Rio J. MDMA (‘Ecstasy’) enhances 5-HT1A receptor density and 8-OH-DPAT-induced hypothermia: blockade by drugs preventing 5-hydroxytryptamine depletion. Eur J Pharmacol 1998;346:181–8. Allott K, Redman J. Patterns of use and harm reduction practices of ecstasy users in Australia. Drug Alcohol Depend 2006;82:168–76. Ando RD, Benko A, Ferrington L, Kirilly E, Kelly PA, Bagdy G. Partial lesion of the serotonergic system by a single dose of MDMA results in behavioural disinhibition and enhances acute MDMA-induced social behaviour on the social interaction test. Neuropharmacology 2006;50:884–96. Australia Institute of Health and Welfare. 2004 National Drug Strategy Household Survey: First Results. AIHW; 2005. cat. no.PHE 57. Baumann MH, Wang X, Rothman RB. 3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings. Psychopharmacology (Berl) 2007;189:407–24. Baylen CA, Rosenberg H. A review of the acute subjective effects of MDMA/ ecstasy. Addiction 2006;101:933–47. Bull EJ, Hutson PH, Fone KC. Reduced social interaction following 3,4methylenedioxymethamphetamine is not associated with enhanced 5-HT 2C receptor responsivity. Neuropharmacology 2003;44:439–48. Bull EJ, Hutson PH, Fone KC. Decreased social behaviour following 3,4methylenedioxymethamphetamine (MDMA) is accompanied by changes in 5-HT2A receptor responsivity. Neuropharmacology 2004;46:202–10. Che S, Johnson M, Hanson GR, Gibb JW. Body temperature effect on methylenedioxymethamphetamine-induced acute decrease in tryptophan hydroxylase activity. Eur J Pharmacol 1995;293:447–53. Clemens KJ, Van Nieuwenhuyzen PS, Li KM, Cornish JL, Hunt GE, McGregor IS. MDMA (“ecstasy”), methamphetamine and their combination: long-term changes in social interaction and neurochemistry in the rat. Psychopharmacology (Berl) 2004;173:318–25. Clemens KJ, Cornish JL, Li KM, Hunt GE, McGregor IS. MDMA (‘Ecstasy’) and methamphetamine combined: order of administration influences hyperthermic and long-term adverse effects in female rats. Neuropharmacology 2005;49: 195–207.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 1013 – 1021 www.elsevier.com/locate/pnpbp
Reduced sensitivity to MDMA-induced facilitation of social behaviour in MDMA pre-exposed rats Murray R. Thompson a , Paul D. Callaghan a , Glenn E. Hunt b , Iain S. McGregor a,⁎ a
b
School of Psychology, University of Sydney, A18, Sydney, NSW 2006, Australia Department of Psychological Medicine, University of Sydney, Concord Hospital, Sydney, NSW 2139, Australia Received 5 August 2007; received in revised form 24 January 2008; accepted 24 January 2008 Available online 4 February 2008
Abstract The acute effects of the party drug 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) in humans include feelings of love, closeness towards other people and an increased acceptance of others views and feelings. Some evidence suggests that regular MDMA users develop a subsensitivity to the positive effects of the drug and escalate their intake of the drug over time as a result. The current study investigated whether brief exposure to relatively high doses of MDMA in rats produces a subsequent attenuation in the ability of MDMA to enhance social interaction. Male Wistar rats were exposed to either MDMA (4 × 5 mg/kg over 4 h) or vehicle on two consecutive days. Twelve weeks later, MDMA preexposed rats displayed a significantly shorter period of time spent in social interaction than controls when tested in the drug-free state. MDMA pre-exposed rats also showed a blunted prosocial response to MDMA (2.5 mg/kg) relative to controls. This difference was overcome by increasing the MDMA dose to 5 mg/kg. The 5-HT1A agonist 8-OH-DPAT (250 µg/kg but not 125 µg/kg) increased social interaction and this effect did not differ in MDMA and vehicle pre-exposed rats. HPLC analysis showed a small but significant depletion of prefrontal 5-HT and 5-HIAA in MDMA pre-exposed rats. Prefrontal 5-HIAA concentrations were also reduced in the subset of vehicle and MDMA pre-exposed rats that received additional testing with MDMA. These results indicate that treatment with MDMA not only causes lasting reductions in social interaction in rats but causes an attenuation of the prosocial effects of subsequent MDMA administration. The lack of a differential response to 8-OH-DPAT agrees with other findings that the 5-HT1A receptor system remains functionally intact following MDMA pre-exposure and suggests that other neuroadaptations may underlie the lasting social deficits caused by MDMA. © 2008 Elsevier Inc. All rights reserved. Keywords: 5-HT1A; 8-OH-DPAT; Anxiety; Behaviour; MDMA; Social
1. Introduction The ring substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) possesses a unique psychoactive profile with individuals reporting increased emotional experience, extroversion, an openness and willingness to converse with others, euphoria and a sense of peacefulness when under the influence of
Abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; 8-OHDPAT, (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid.; HPLC, high-performance liquid chromatography. ⁎ Corresponding author. Tel.: +61 2 9351 3571; fax: +61 2 9351 8023. E-mail address: [email protected] (I.S. McGregor). 0278-5846/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.01.014
the drug (Baylen and Rosenberg, 2006; Dumont and Verkes, 2006; Vollenweider et al., 1998). The distinctive euphorogenic and prosocial effects of Ecstasy have led to MDMA becoming one of the most popular illicit drugs currently used by young people (Australia Institute of Health and Welfare, 2005; Degenhardt et al., 2004; Soellner, 2005), and a broadening of MDMA use beyond the “club drug” scene of the 1990s has been observed in recent years (Allott and Redman, 2006; Levy et al., 2005; Solowij et al., 1992; Topp et al., 1999; White et al., 2006). Concerns have been raised regarding the long-term residual effects of MDMA use. Ecstasy use in humans has been linked to residual increases in anxiety, depression and memory impairment (Kalechstein et al., 2007; Morgan, 2000; Parrott, 2001; Parrott and Marsden, 2006), and impaired social and emotional
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judgement (Reay et al., 2006). Mirroring human data, rats previously treated with MDMA display long-term residual impairments in memory (McGregor et al., 2003b; Morley et al., 2001; Piper and Meyer, 2004), and an increase in anxiety and depressive-like symptoms (Clemens et al., 2004; Gurtman et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004; Walker et al., 2007). Rats also show reduced social interaction with novel conspecifics for at least 3 months following MDMA exposure, and this effect can be observed even using treatment regimens that do not cause significant lasting alterations in brain serotonin (5-HT) levels (Bull et al., 2003, 2004; Fone et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004). Anecdotal evidence suggests that the positive mood-altering effects of MDMA are reduced following repeated dosing (Parrott, 2005; Solowij et al., 1992) and this may in part result in greater consumption of Ecstasy per session by experienced compared to novice users (Huxster et al., 2006; McCambridge et al., 2005). Reduced serotonergic function after MDMA exposure is evident from attenuated 5-HT release in vivo, decreased firing rates of 5-HT neurons, and diminished neuroendocrine, thermoregulatory and locomotor responses to serotonergic challenges (Baumann et al., 2007; Gartside et al., 1996; Mechan et al., 2001; Series et al., 1994; Shankaran and Gudelsky, 1999). Attenuation of MDMA-induced responses has been shown clinically by reductions in l-tryptophan, dfenfluramine and m-chlorophenylpiperazine induced increases in plasma prolactin, cortisol and growth hormone among cohorts of regular MDMA users (Gerra et al., 2000, 1998; McCann et al., 1994; Price et al., 1989; Verkes et al., 2001). In contrast to its adverse residual effects on social behaviour, MDMA produces an acute facilitation of social interaction in rats that appears to be mediated in part by 5-HT1A and oxytocin receptors (Morley et al., 2005; Thompson et al., 2007). However, is not clear whether acute MDMA-induced social facilitation is attenuated by prior MDMA exposure. Indeed, Ando et al. (2006) recently reported that the prosocial effects of MDMA may become amplified with prior MDMA exposure. However this study used Dark Agouti rats, a strain with significantly higher baseline anxiety and differing acute and long-term behavioural responses to MDMA compared to Wistar or Sprague–Dawley rats (Green and McGregor, 2002). Ando et al. (2006) also tested social interaction following an extremely high (15 mg/kg) single dose of MDMA, a dose that may have conceivably masked subtle tolerance-related phenomena due to a ceiling effect. It was thus of interest to determine whether MDMA pre-exposed Wistar rats would display altered sensitivity to MDMA's acute prosocial effects when lower doses of MDMA were tested. The social response to the 5-HT1A agonist 8-OH-DPAT was also tested in the present study to determine if an attenuation of 5-HT1A agonist-induced social interaction was apparent in MDMA pre-exposed rats. The 5-HT1A system has been implicated in mediating the prosocial effects of MDMA with the 5HT1A antagonist WAY 100,635 blocking the acute prosocial effects of MDMA most likely as a result of an interaction with the neuropeptide oxytocin (Morley et al., 2005; Thompson et al., 2007). If long-term social deficits following MDMA
reflect downregulation of 5-HT1A receptors then it might be expected that MDMA pre-exposed rats would be sub-sensitive to the prosocial effects of 8-OH-DPAT. 2. Methods 2.1. Animals A total of 48 inbred male albino Wistar rats (University of Sydney, Australia) were used in the experiments, aged between 90 and 100 days old and weighing an average of 421 g at the start of testing. The rats were housed in groups of 6 per cage in a temperature-controlled environment (average temperature 22 °C). A 12 h reversed light–dark cycle was in operation (lights off at 8:30 a.m.) and all testing took place in the dark cycle. Food and water were freely available ad libitum. All the experiments were approved by the University of Sydney Animal Ethics Committee. 2.2. Drugs (±)-3,4-methylenedioxymethamphetamine (MDMA) was obtained from Australian Government Analytical Laboratories (Pymble, NSW). MDMA was dissolved in 0.9% saline at a concentration of either 2.5 mg/ml or 5 mg/ml and injected intraperitoneally at a volume of 1 ml/kg. To produce 5-HT depletion in MDMA pre-exposed rats a 4 × 5 mg dose on two consecutive days was used, as in previous studies from our laboratory (Gurtman et al., 2002; McGregor et al., 2003b; Thompson et al., 2004). To examine acute prosocial effects, 2.5 mg/kg and 5 mg/kg doses of MDMA were used, again according to previous studies from our laboratory (Cornish et al., 2003; Morley et al., 2005; Morley and McGregor, 2000). Control rats received equivalent injections of 0.9% saline. The 5-HT1A agonist (±)-8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) was purchased from Sigma– Aldrich. 8-OH-DPAT was dissolved in saline at a dose of either 125 µg/kg or 250 µg/kg and injected intraperitoneally at a volume of 1 ml/kg. The 250 µg/kg dose has been shown to increase social interaction in rats in previous studies (Picazo et al., 1995). 2.3. Apparatus and procedures 2.3.1. Acute MDMA administration and effects on body temperature Initial administration of MDMA to produce MDMA preexposed rats for later testing involved procedures reported previously by our group (Thompson et al., 2004). Rats received a 5 mg/kg i.p. injection of MDMA (n = 24) or vehicle (n = 24) every hour for 4 h on 2 consecutive days, giving a cumulative dose of 40 mg/kg. During MDMA or vehicle administration, individual rats were placed in standard operant chambers (30 × 50 × 25.5 cm) with three aluminium walls, one Perspex wall and a metal grid floor. The chambers were enclosed in wooden sound attenuation boxes. Room temperature was maintained at an ambient temperature of 28 °C by a reversecycle air conditioner. High temperatures may exacerbate the 5-
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HT-depleting effects of MDMA in rats (Malberg and Seiden, 1998) and also increase the rewarding and prosocial effects of MDMA (Cornish et al., 2003). The body temperature of all rats was recorded each hour with a Braun Thermoscan Instant Thermometer (IRT 1020) at the time of each injection. Following the 4 h drug administration period, all rats were housed individually in the colony room overnight and replaced back in their home cages the following morning. This procedure prevents the possible lethal effects of ‘aggregation toxicity’ sometimes seen with group housing following high-dose stimulant treatment (Green et al., 1995). 2.3.2. Long-term effects of MDMA pre-exposure on social behaviour Twelve weeks following drug administration, all rats were tested in the social interaction test in a drug-free state with a novel conspecific that had received the same drug pre-exposure. The social interaction test was run on two consecutive days with each rat experiencing a different partner on each of the 2 days. Results over these two days were pooled, providing a final data set with 24 unique pairs of rats per drug condition. The social interaction arena consisted of a black, Perspex box (52 × 52 × 40 cm) dimly lit with red light (40 W). Rats were placed in the test arena for 10 min. A miniature video camera was placed vertically above the social interaction arena that sent a signal into a VCR in a neighbouring room where behaviours were scored live by a trained observer blind to treatment group. Scoring of the social interaction test was based upon the work of File and Seth (2003) but with the total social interaction score subdivided into three categories. These categories were similar to those described by Morley et al. (2005) and consisted of Adjacent lying: duration of side by side contact or ‘huddling’ including climbing under/over conspecific; Anogenital sniffing: duration of sniffing the conspecific's anogenital area; Investigation: duration of sniffing the conspecific apart from anogenital sniffing. A “total social interaction” score was also obtained by summing the scores of these individual behaviours for each pair of rats. The non-social activity of Rearing: standing on hind legs was also scored as previous studies have shown that acute MDMA potently inhibits this behaviour (e.g. Morley et al. (2005)). The testing arena was wiped down with 50% ethanol between every session. 2.3.3. Acute social effects of MDMA on MDMA pre-exposed rats Three days following the last of the baseline social interaction sessions, a subset of 12 MDMA pre-exposed and 12 vehicle preexposed rats was randomly chosen to investigate the acute effects of MDMA or 8-OH-DPAT on social interaction. These 12 MDMA pre-exposed and 12 vehicle pre-exposed animals underwent a further 5 social interaction tests with all 24 of these rats receiving an identical set of tests with low MDMA and 8-OH-DPAT doses. On the first day of testing these vehicle and MDMA-preexposed rats received a dose of 2.5 mg/kg MDMA and were placed in holding cages (40 × 25 × 15 cm opaque box lined with bedding) in the testing room. The room was heated to 28 °C as this temperature serves to facilitate MDMA-induced social interaction
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without affecting baseline social behaviour (Cornish et al., 2003). Twenty minutes following the MDMA injection rats were tested in a 10 min social interaction test with a novel conspecific of identical pre-treatment group (vehicle pre-exposed or MDMA-preexposed). Testing occurred over 2 consecutive days with a different partner used on each day to give a final sample size of 12 unique MDMA pre-exposed and 12 unique vehicle pre-exposed pairs. The same subset of vehicle and MDMA pre-exposed rats was then tested 48 h later on the social interaction test, under the same conditions, but with a 5 mg/kg dose of MDMA and a further novel partner. Testing for this phase of the experiment occurred on a single day to produce a final sample size of 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs. A second challenge day with the 5 mg/kg dose of MDMA was not undertaken as no trend towards differences between vehicle and MDMA pre-exposed rats were observed on the first day of testing. 2.3.4. Acute social effects of 8-OH-DPAT upon MDMA preexposed rats Two days later the vehicle and MDMA pre-exposed rats were tested, using the same procedures, with a 125 µg/kg dose of the 5-HT1A agonist 8-OH-DPAT. This resulted in a final sample size of 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs. One day later, the same subsets of vehicle and MDMA pre-exposed rats were tested with a 250 µg/kg dose of 8-OHDPAT. As with MDMA, the drug was given 20 min prior to the start of the 10 min social interaction test. Again, testing only occurred in 1 day, resulting in 6 MDMA pre-exposed and 6 vehicle pre-exposed pairs for each of the 8-OH-DPAT tests. 2.3.5. HPLC analysis Five days after the final social interaction test with 8-OHDPAT, all rats were rapidly decapitated and the prefrontal cortex was dissected over dry ice and then stored at −80 °C. Subsequent analysis of noradrenaline, dopamine, 5-HIAA (5-hydroxyindoleacetic acid) and 5-HT (5-hydroxytryptamine) was undertaken using high-performance liquid chromatography (HPLC) with electrochemical detection employing standard techniques as described by Clemens et al. (2005). Two samples were excluded from HPLC analysis as wet tissue weights were notably lower than the other prefrontal cortex tissue samples, suggesting an inaccurate dissection. 2.4. Statistical analysis For initial MDMA exposure a repeated-measures Analysis of Variance (ANOVA) was used to compare body temperature in vehicle and MDMA-treated rats across the 4 h of testing on each of the 2 days of treatment. Differences between groups for each hour of testing were subsequently analysed using post hoc contrasts. A one way ANOVA was used to compare the two treatment groups (vehicle and MDMA pre-exposed) on the baseline, acute MDMA and acute 8-OH-DPAT social interaction tests conduced 12–14 weeks after drug pre-exposure. In addition, the acute effects of MDMA and 8-OH-DPAT on social interaction were evaluated by comparing social interaction in vehicle pre-exposed rats on the baseline test with that
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Fig. 1. Mean body temperature in vehicle and MDMA exposed rats on day 1 (left) and day 2 (right) of drug treatment. Body temperature was measured prior to drug administration (0 h) and at 1, 2, 3, and 4 h following the drug. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA and vehicle-exposed groups.
seen in the same rats with acute MDMA and 8-OH-DPAT. This was done with ANOVA followed by post hoc contrasts. For HPLC analysis a 2-way ANOVA was used to analyse the effects of MDMA pre-exposure and subsequent drug challenge with MDMA/8-OH-DPAT on various monoamine levels. The threshold for statistical significance was set at P b 0.05.
Fig. 2. Results from the social interaction test (conducted drug-free) 12 weeks following vehicle or MDMA exposure. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA and vehicle-exposed groups.
3. Results 3.2. Long-term residual effects of MDMA on social interaction 3.1. Acute effects of MDMA on body temperature The effects of vehicle or MDMA administration on body temperature can be seen in Fig. 1. On day 1, a repeated-measures ANOVA revealed a significant temperature difference between vehicle and MDMA-treated rats (F(1,46) = 186.596, P b 0.0001), a significant change in temperature for both treatment groups over the 4 hour test period (F(4,184) = 33.09, P b 0.0001) and a significant treatment by time interaction effect (F(4,184) = 29.30, P b 0.001). Subsequent post hoc analysis revealed no group differences immediately before drug administration. MDMA treatment significantly increased body temperature relative to vehicles on hour 1 (F(1,46) = 26.66, P b 0.001), hour 2 (F(1,46) = 137.44, P b 0.001), hour 3 (F(1,46) = 43.31, P b 0.001) and hour 4 (F(1,46) = 198.77, P b 0.001). The second day of MDMA administration showed a similar general pattern to that seen on day 1 with a significant overall effect of treatment (F(1,46) = 81.10, P b 0.0001), a significant effect of time (F(4,184) = 27.50, P b 0.0001), and a significant treatment by time interaction (F(4,184) = 24.33, P b 0.0001). Unlike day 1, there was no significant body temperature difference between MDMA and vehicle treated rats 1 h after the first injection (F b 1). However, MDMA produced significantly higher body temperatures compared to vehicle treatment at hour 2 (F(1,46) = 81.56, P b 0.001), hour 3 (F(1,46) = 38.15, P b 0.001) and hour 4 (F(1,46) = 307.03, P b 0.001). Although the body temperature of some rats exceeded 40 °C towards the end of the day 1 and 2 test sessions, no lethal effects of MDMA were observed in any of the rats.
Baseline social interaction results 12 week after vehicle or MDMA pre-treatment are presented in Fig. 2. ANOVA revealed that MDMA pre-exposed rats showed significantly reduced levels of total social interaction when compared to vehicle preexposed rats (F(1,46) = 8.95, P b 0.01). Separate analysis of the three subtypes of social behaviour constituting the total
Fig. 3. MDMA-induced behaviours (left panel: 2.5 mg/kg, right panel: 5 mg/kg) in the social interaction test in vehicle and MDMA pre-exposed rats. Data represent mean ± SEM. ⁎P b 0.05, comparing MDMA pre-exposed and vehicle pre-exposed groups.
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3.4. Acute prosocial effects of MDMA in MDMA pre-exposed rats The effects of a 2.5 mg/kg dose of MDMA on social interaction in vehicle and MDMA pre-exposed rats are presented in Fig. 3. MDMA-pre-exposed rats given MDMA (2.5 mg/kg) showed significantly less total social interaction (F(1,21) = 7.030, P b 0.05) and adjacent lying (F(1,21)= 5.756, P b 0.05) compared to vehicle pre-exposed rats. No significant group differences were observed for the behavioural subtypes of general investigation (F b 1), anogenital sniffing (F(1,21) = 2.586, P N 0.05) or rearing (F b 1). Fig. 3 illustrates the effects of an acute 5 mg/kg dose of MDMA on social interaction in the MDMA and vehicle preexposed groups. No significant differences or trends towards differences were evident when comparing these groups on social behaviour following this dose (Fs b 1.6). Fig. 4. 8-OH-DPAT-induced behaviours (left panel: 125 µg/kg, right panel: 250 µg/kg) in the social interaction test between vehicle and MDMA preexposed rats. Data represent mean ± SEM.
social interaction score revealed that MDMA pre-exposed rats showed significantly lower levels of investigation than controls (F(1,46) = 7.86, P b 0.01) but did not differ in anogenital sniffing (F b 1), adjacent lying (F(1,46) = 2.82, P N 0.05) or the nonsocial variable of rearing (F b 1.2). 3.3. Acute prosocial effects of MDMA and 8-OH-DPAT in vehicle pre-exposed rats Analysis of social interaction in the vehicle pre-exposed control group across the 4 different acute drug tests conducted with MDMA and 8-OH-DPAT (compare Figs. 2, 3 and 4) revealed that there were significant overall drug effects in this group in levels of total social interaction (F(4,49) = 30.659, P b 0.001). Subsequent post hoc tests revealed that administration of MDMA (2.5 or 5 mg/kg) and 8-OH-DPAT (250 but not 125 µg/kg) significantly increased levels of total social interaction in these control rats relative to the levels seen in these rats in the baseline social interaction test.
3.5. Acute prosocial effects of 8-OH-DPAT in MDMA preexposed rats The effects of 8-OH-DPAT in MDMA pre-exposed and vehicle pre-exposed groups are presented in Fig. 4. No significant group differences were seen in social behaviour following either the 125 or 250 µg/kg doses of 8-OH-DPAT (Fs b 1). 3.6. Neurochemical analysis Results for the HPLC analysis of the prefrontal cortex are presented in Table 1. Rats that had been pre-exposed to MDMA 12 weeks previously exhibited significantly lower levels of 5HIAA (F(1,42) = 5.786, P b 0.05) and 5-HT (F(1,42) = 5.635, P b 0.05) relative to vehicle pre-exposed controls. Exposure to MDMA and 8-OH-DPAT during acute social interaction tests was associated with a significant reduction in 5-HIAA levels (F(1,42) = 18.444, P b 0.001), but not 5-HT, relative to rats that did not take part in that phase of the experiment. Noradrenaline and dopamine levels were unaffected by all treatment regimens used and no significant interactions between drug preexposure and subsequent exposure were obtained for any of the biogenic amines investigated.
Table 1 Results of HPLC analysis in the prefrontal cortex Neurotransmitter
5-HT 5-HIAA Noradrenaline Dopamine
Vehicle/control
Vehicle/MDMA
MDMA/control
MDMA/MDMA
n = 11
n = 12
n = 11
n = 12
612.6 (27.5) 200.8 (8.7) 1335.3 (92.2) 994.6 (205.6)
596.9 (32.0) 159.1 (9.1) 1417.1 (123.5) 1020.7 (167.8)
555.8 (17.3) 174.6 (9.0) 1486.9 (140.5) 1018.4 (76.2)
533.3 (21.3) 145.9 (5.7) 1257.6 (49.4) 750.2 (71.5)
Significance
a a,b
Rats received either vehicle or MDMA exposure. Twelve weeks later a subset of 12 vehicle pre-exposed (vehicle/MDMA) and 12 MDMA pre-exposed (MDMA/ MDMA) rats received two additional doses of MDMA (2.5 and 5 mg/kg on two separate days) as well as two doses of 8-OH-DPAT (125 and 250 µg/kg on two separate days) during social interaction testing. Data represent Mean ± (SEM). Units of measurement are ng/g wet tissue. a P b 0.05 when comparing vehicle groups and MDMA pre-exposed groups. b P b 0.05 when comparing rats challenged later with MDMA and 8-OH-DPAT (groups vehicle/MDMA and MDMA/MDMA) relative to those who were not challenged (groups vehicle/control and MDMA/control).
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4. Discussion The present study demonstrates that MDMA pre-treatment, at doses that decrease prefrontal cortex 5-HT levels, leads to a long-term attenuation of the capacity of low dose MDMA to produce subsequent increases in social interaction. This provides evidence, in a rodent model, for the long-term reductions in the prosocial effects of MDMA that are reported by regular human users of MDMA. As previously demonstrated, the acute administration of 5 mg/ kg MDMA each hour for 4 consecutive hours produced hyperthermia in Wistar rats tested at a 28 °C ambient temperature (Morley et al., 2001; Thompson et al., 2004). Interestingly, the pattern of hyperthermia differed across the two consecutive days of dosing, with a longer latency to an MDMA-induced hyperthermia response on day 2 compared to day 1 (Fig. 1). This effect replicates the earlier finding of Thompson et al. (2004) and indicates a reduced sensitivity to the hyperthermic effects of MDMA, possibly as a result of short term 5-HT depletion and/or a loss of tryptophan hydroxylase activity following the first day of MDMA administration (Baumann et al., 2007). The long-term residual social interaction deficits seen here following prior MDMA treatment replicate previous findings (Bull et al., 2003, 2004; Clemens et al., 2005, 2004; Fone et al., 2002; McGregor et al., 2003a,b; Morley et al., 2001, 2004) and the present study further illustrates the lasting nature of this effect, with social interaction deficits evident 12 weeks after brief MDMA exposure (Gurtman et al., 2002; Thompson et al., 2004). This study is the first to characterise lasting reductions in specific subtypes of social interaction following MDMA pre-exposure, with a significant decline in the “general investigation” subtype. To determine whether prior MDMA exposure resulted in attenuation of drug-induced prosocial behaviours, a subset of rats was challenged with two doses of MDMA as well as two doses of the 5-HT1A agonist 8-OH-DPAT. Rats that had been pre-exposed to MDMA 12 weeks previously showed an attenuation of prosocial behaviours 20 min following a low dose (2.5 mg/kg) of MDMA. It is notable that vehicle preexposed rats administered the 2.5 mg/kg dose of MDMA showed much higher levels of total social interaction here than was reported in an earlier study by Morley and McGregor (2000) with this same dose. This may be due to the elevated room temperatures (28 °C) used during social interaction testing in the present study, with high temperatures known to facilitate MDMA's prosocial effects (Cornish et al., 2003). Analysis of social behaviour subtypes revealed that acute administration of MDMA at a dose of 2.5 mg/kg increased adjacent lying to a greater extent in vehicle pre-exposed than in MDMA pre-exposed rats. Adjacent lying is a key acute behavioural effect seen with MDMA in Wistar rats and is mediated through oxytocinergic and 5-HT1A receptor systems (Morley et al., 2005; Thompson et al., 2007). It is interesting to note that the long-term residual MDMA induced social deficits observed when rats were tested in a drug-free state (general investigation) involve a different social behaviour subtype than that deficient under MDMA challenge (adjacent lying). This suggests that MDMA exposure can influence a variety of social
behaviours, with some deficits (i.e. reduced adjacent lying) only evident following further drug challenges. Consistent with this, MDMA pre-exposure has been previously shown to attenuate the effects of drug-induced behaviours, such as the 5-HT2A/2C agonist DOI in the elevated plus maze, while MDMA preexposure by itself had little effect on the amount of time spent on the open arms (Bull et al., 2004). In contrast to the results observed with 2.5 mg/kg dose of MDMA, the prosocial effects of a higher 5 mg/kg dose of MDMA were not significantly affected by MDMA pre-exposure. This indicates that blunting of MDMA's acute effects as a result of preexposure can be overcome with increased MDMA dose. This may provide a rationale for the dose escalation that has been reported in experienced compared to novice MDMA users (Huxster et al., 2006; McCambridge et al., 2005). The 5-HT1A receptor at least partly mediates adjacent lying behaviours following acute MDMA treatment (Morley et al., 2005), and this was further confirmed in the present study with an MDMA-like increase in adjacent lying obtained with the 5HT1A receptor agonist 8-OH-DPAT. Interestingly however, rats pre-exposed to MDMA did not appear to be deficient in their prosocial response to 8-OH-DPAT. Here it must be noted that there was a large “all or nothing” increase in the prosocial effects of 8-OH-PDAT between the 125 and 250 µg/kg doses used in the present study such that possible group differences at intermediate doses cannot be ruled out. Nonetheless, the current results lead to the tentative conclusion that the 5-HT1A system's role in the production of affiliative prosocial behaviours remains intact following MDMA pre-exposure. There have been contrasting reports concerning the long-term effects of MDMA administration on 5-HT1A receptor expression, with binding studies showing either no effect (McGregor et al., 2003a; Piper et al., 2006) or subtle effects upon receptor concentration in specific brain regions (Aguirre et al., 1998, 1997). Functional assays of the 5-HT1A receptor following MDMA pre-treatment have also produced mixed results. 8-OHDPAT-induced forepaw treading and serotonin syndrome are attenuated by MDMA pre-treatment (Granoff and Ashby, 2001; Piper et al., 2006), while 8-OH-DPAT-induced hypothermic responses were unaltered (Granoff and Ashby, 2001; McNamara et al., 1995; Mechan et al., 2001; Piper et al., 2006). Thus although the present results suggest little effect of MDMA pre-exposure on 8-OH-DPAT-induced social behaviours, effects on other nonsocial behaviours cannot be ruled out. Rearing behaviour, which is greatly reduced by acute MDMA or 8-OH-DPAT treatment (Thompson et al., 2007), was attenuated here by MDMA to a similar extent in vehicle and MDMA pre-exposed rats. Previous studies have indicated that MDMA-induced attenuation of rearing behaviours in the social interaction test is unaffected by co-administration of a range of different 5-HT receptor antagonists (Morley et al., 2005) or the oxytocin antagonist tocinoic acid (Thompson et al., 2007). Other studies suggest that inhibitory effects of acute 8-OHDPAT on rearing may involve mesencephalic dopaminergic mechanisms (e.g. Hillegaart et al., 2000) and this may explain why MDMA pre-exposure did not modify the sensitivity of this particular behaviour to acute pharmacological challenge.
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Reduction in MDMA-stimulated social behaviour and reduced baseline social behaviour after prior MDMA exposure may therefore involve a number of non-5-HT1A receptor mechanisms. This might include lasting alterations in the density of other serotonergic receptors and/or the 5-HT transporter in key regions underlying social behaviour such as the amygdala and hypothalamus (see McGregor et al., 2003a). Prior MDMA ‘neurodegenerative’ treatment attenuates the neurochemical responses to subsequent acute MDMA administration, including reductions in evoked 5-HT release in vivo (Series et al., 1994; Shankaran and Gudelsky, 1999), and this may also be a key mechanism in the pre-exposure effect reported here. However non-serotonergic mechanisms may also be involved. We have recently shown that MDMA stimulates the release of the neuropeptide oxytocin in rats and that the oxytocin receptor antagonist tocinoic acid can attenuate the acute increases in social interaction produced by MDMA (Thompson et al., 2007). It is therefore possible that decreased sensitivity to the social effects of MDMA as a result of prior MDMA exposure may reflect some long-term blunting of oxytocinergic circuitry by MDMA and this is a possibility that warrants further investigation. While MDMA pre-exposure caused lasting reductions in 5-HT and 5-HIAA concentrations in the present study, the additional subsequent testing with MDMA and 8-OH-DPAT conducted 12 weeks later did not further alter prefrontal cortex 5-HT levels. This most likely reflects the acute MDMA doses used being below the threshold required to deplete 5-HT (Green et al., 2003). However, prefrontal cortex 5-HIAA concentrations were significantly reduced in rats receiving these additional tests with MDMA and 8-OH-DPAT, irrespective of pre-exposure status. There was a relatively short period of time (9 days) in these rats between the last acute test with MDMA and brain dissection, such that MDMA-induced inhibition of tryptophan hydroxylase may have still been apparent (O'Shea et al., 2006; Stone et al., 1987). In addition, the effects of MDMA may have been potentiated due to the high ambient room temperatures used during social interaction testing. High temperatures have been shown to accentuate MDMA-induced reductions in 5-HT/5-HIAA forebrain content and inactivation of tryptophan hydroxylase (Che et al., 1995; Malberg and Seiden, 1998). Acute 8-OH-DPAT treatment does not significantly alter forebrain biogenic amine concentrations, and thus would be unlikely to be involved in the effect on 5-HIAA (Kennett et al., 1987; Larsson et al., 1990). The results in the present study are in contrast to those of Ando et al. (2006) who reported an augmented adjacent lying effect to acute MDMA (15 mg/kg) in Dark Agouti rats that had been previously exposed to a 15 mg/kg dose of MDMA. In a further contrast between these two studies, Ando et al. (2006) found no residual deficits in social interaction behaviours, although an increase in aggressive behaviours was reported in MDMA pre-exposed rats. Clearly, the high dose (15 mg/kg) used by Ando et al. (2006) may have not allowed subtle MDMA pre-exposure effects to be observed, given that pre-exposure effects were only seen in the present study with 2.5 mg/kg and not 5 mg/kg of MDMA. However, this does not explain the enhanced acute prosocial effects of MDMA seen in the Ando
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et al. (2006) study in MDMA pre-exposed rats and this may represent a fundamental strain difference. The present study also observed no aggressive episodes in any rats during the social interaction test, further emphasising the behavioural differences between these two rat strains (Green and McGregor, 2002). Clearly a direct behavioural and neurochemical comparison of the social response of Albino Wistar and Dark Agouti rat strains to MDMA would be a useful exercise. 5. Conclusions Overall, the present study extends the previous findings of subsensitivity to MDMA following prior MDMA exposure to include results from the social interaction test. These results mirror those reports of tolerance to the prosocial effects of MDMA in human users and may help to explain the dose escalation sometimes seen in experienced MDMA users. Acknowledgements Support was contributed by the National Health and Medical Research Council of Australia. References Aguirre N, Frechilla D, Garcia-Osta A, Lasheras B, Del Rio J. Differential regulation by methylenedioxymethamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex, and brainstem: the role of corticosteroids. J Neurochem 1997;68:1099–105. Aguirre N, Ballaz S, Lasheras B, Del Rio J. MDMA (‘Ecstasy’) enhances 5-HT1A receptor density and 8-OH-DPAT-induced hypothermia: blockade by drugs preventing 5-hydroxytryptamine depletion. Eur J Pharmacol 1998;346:181–8. Allott K, Redman J. Patterns of use and harm reduction practices of ecstasy users in Australia. Drug Alcohol Depend 2006;82:168–76. Ando RD, Benko A, Ferrington L, Kirilly E, Kelly PA, Bagdy G. Partial lesion of the serotonergic system by a single dose of MDMA results in behavioural disinhibition and enhances acute MDMA-induced social behaviour on the social interaction test. Neuropharmacology 2006;50:884–96. Australia Institute of Health and Welfare. 2004 National Drug Strategy Household Survey: First Results. AIHW; 2005. cat. no.PHE 57. Baumann MH, Wang X, Rothman RB. 3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings. Psychopharmacology (Berl) 2007;189:407–24. Baylen CA, Rosenberg H. A review of the acute subjective effects of MDMA/ ecstasy. Addiction 2006;101:933–47. Bull EJ, Hutson PH, Fone KC. Reduced social interaction following 3,4methylenedioxymethamphetamine is not associated with enhanced 5-HT 2C receptor responsivity. Neuropharmacology 2003;44:439–48. Bull EJ, Hutson PH, Fone KC. Decreased social behaviour following 3,4methylenedioxymethamphetamine (MDMA) is accompanied by changes in 5-HT2A receptor responsivity. Neuropharmacology 2004;46:202–10. Che S, Johnson M, Hanson GR, Gibb JW. Body temperature effect on methylenedioxymethamphetamine-induced acute decrease in tryptophan hydroxylase activity. Eur J Pharmacol 1995;293:447–53. Clemens KJ, Van Nieuwenhuyzen PS, Li KM, Cornish JL, Hunt GE, McGregor IS. MDMA (“ecstasy”), methamphetamine and their combination: long-term changes in social interaction and neurochemistry in the rat. Psychopharmacology (Berl) 2004;173:318–25. Clemens KJ, Cornish JL, Li KM, Hunt GE, McGregor IS. MDMA (‘Ecstasy’) and methamphetamine combined: order of administration influences hyperthermic and long-term adverse effects in female rats. Neuropharmacology 2005;49: 195–207.
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