Sedative and hypothermic effects of γ-hydroxybutyrate (GHB) in rats alone and in combination with other drugs: Assessment using biotelemetry

Drug and Alcohol Dependence 103 (2009) 137–147

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Sedative and hypothermic effects of ␥-hydroxybutyrate (GHB) in rats alone and in combination with other drugs: Assessment using biotelemetry Petra S. van Nieuwenhuijzen, Iain S. McGregor ∗ School of Psychology, University of Sydney, NSW, 2006, Australia

a r t i c l e

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Article history: Received 6 November 2008 Received in revised form 22 January 2009 Accepted 5 March 2009 Available online 14 May 2009 Keywords: ␥-Hydroxybutyrate (GHB) 3,4-Methylenedioxymethamphetamine (MDMA) Methamphetamine (METH) Telemetry Body temperature Locomotor activity

a b s t r a c t The recreational drug ␥-hydroxybutyrate (GHB) has euphoric effects and can induce sedation and body temperature changes. GHB is frequently combined with other recreational drugs although these interactions are not well characterised. The present study used biotelemetry to provide a fine-grained analysis of the effects of GHB on body temperature and locomotor activity in freely moving rats, and investigated interactions between GHB and 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (METH) and various antagonist drugs. GHB (1000 mg/kg) caused profound sedation for more than 2 h and a complex triphasic effect on body temperature: an initial hypothermia (5–40 min), followed by hyperthermia (40–140 min), followed again by hypothermia (140–360 min). A lower GHB dose (500 mg/kg) also caused sedation but only a hypothermic effect that lasted up to 6 h. The dopamine D1 receptor antagonist SCH 23390 (1 mg/kg), the opioid antagonist naltrexone (1 mg/kg), the benzodiazepine antagonist flumazenil (10 mg/kg), and the 5-HT2A/2C receptor antagonist ritanserin (1 mg/kg) did not prevent the overall sedative or body temperature effects of GHB (1000 mg/kg). However the GABAB antagonist SCH 50911 (50 mg/kg) prevented the hyperthermia induced by GHB (1000 mg/kg). Repeated daily administration of GHB (1000 mg/kg) produced tolerance to the sedative and hyperthermic effects of the drug and cross-tolerance to the sedative effects of the GABAB receptor agonist baclofen (10 mg/kg). A high ambient temperature of 28 ◦ C prevented the hypothermia obtained with GHB (500 mg/kg) at 20 ◦ C, while GHB (500 mg/kg) reduced the hyperthermia and hyperactivity produced by co-administered doses of MDMA (5 mg/kg) or METH (1 mg/kg) at 28 ◦ C. These results further confirm a role for GABAB receptors in the hypothermic and sedative effects of GHB and show an interaction between GHB and MDMA, and GHB and METH, that may be relevant to the experience of recreational users who mix these drugs. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ␥-Hydroxybutyrate (GHB) is a popular drug in many countries and is consumed for its euphoric properties (Sumnall et al., 2008). GHB is also a naturally occurring compound in the mammalian brain where it exists as both a precursor and metabolite of the major inhibitory neurotransmitter GABA. GHB binds to GABAB receptors as a partial agonist and also with high affinity to a G-coupled GHB receptor that is present with high density in the hippocampus, septum and cortex (Lingenhoehl et al., 1999; Maitre, 1997; Wu et al., 2004). The effects of GHB in both humans and rodents range from anxiolytic and reinforcing effects at lower doses to psychomotor impairment, coma and lethality at higher doses (Abanades et al., 2007; Carter et al., 2006; Koek and France, 2008; Schmidt-Mutter et al., 1998; Snead, 1988).

∗ Corresponding author. Tel.: +61 2 9351 3571; fax: +61 2 9351 8023. E-mail address: [email protected] (I.S. McGregor). 0376-8716/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2009.03.004

Studies with laboratory animals have documented dosedependent sedative effects with GHB and a powerful modulation of body temperature. When administered at very low doses (5–10 mg/kg) GHB increases body temperature, while at doses up to 500 mg/kg it causes an opposite hypothermic response in rats (Kaufman et al., 1990). High doses of ␥-butylactone (GBL, a precursor of GHB that is also consumed as a recreational drug) can also increase body temperature in rats (Snead, 1990). The GHB receptor appears to have little or no role in mediating the sedative effects of exogenously administered GHB (Castelli et al., 2004) with most evidence pointing to a key role for GABAB receptors. Thus the hypothermic and sedative effects of GHB are lacking in GABAB receptor knockout mice (Kaupmann et al., 2003; Queva et al., 2003) and are prevented in wild type animals by co-administered GABAB antagonists (Carai et al., 2001), while GABAB receptor agonists such as baclofen produce GHB-like hypothermic and sedative effects (Perry et al., 1998; Queva et al., 2003; Zarrindast and Oveissi, 1988). Further evidence of GABAB receptor involvement in GHB effects comes from cross-tolerance studies. Repeated administration of

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GHB to both mice and rats results in tolerance to its sedative effects (Itzhak and Ali, 2002; Raybon and Boje, 2007; Van Sassenbroeck et al., 2003) and also cross-tolerance to the sedative effects of baclofen (Smith et al., 2006). However, to our knowledge, no previous studies have investigated tolerance and cross-tolerance to the body temperature effects of GHB, and this was a focus in the present study. In addition to its direct effects on GHB and GABAB receptors, GHB produces many other complex neural effects including increased turnover of monoamine neurotransmitters such as dopamine (DA) and serotonin (5-HT) (Gobaille et al., 2002; Maitre, 1997), increased release of neuroactive steroids and neuropeptides (Barbaccia et al., 2002; Pardi and Black, 2006; Van Cauter et al., 2004) and activation of numerous brainstem and limbic nuclei (van Nieuwenhuijzen et al., 2009). The anxiolytic effects of GHB in rats may involve the GABAA -benzodiazepine receptor complex since they are reversed by the benzodiazepine receptor antagonist flumazenil (SchmidtMutter et al., 1998). Similarly some of the behavioural, convulsant and dopaminergic effects of GHB can be reversed by opioid antagonists naloxone and naltrexone in rats (Crosby et al., 1983; Feigenbaum and Howard, 1996; Snead and Bearden, 1980; Vayer et al., 1987). In line with these reports, we examined here whether a range of co-administered dopaminergic, serotonergic, benzodiazepine and GABAB antagonists can affect the sedative and body temperature effects of GHB. GHB is often used in combination with other recreational drugs such as alcohol, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) (Degenhardt and Dunn, 2008). MDMA is a popular and widely used party drug that causes euphoric and empathogenic feelings in users similar to the effects of GHB (Uys and Niesink, 2005). People using MDMA and GHB combinations report that GHB enhances the euphoric effects of MDMA and prevents the ‘comedown’ following the use of MDMA alone (Uys and Niesink, 2005). METH use results in euphoria, heightened attention and increased energy (Winslow et al., 2007). The combined use of GHB and METH has similarly been reported (Degenhardt and Dunn, 2008; Dresen et al., 2007) and one case study suggests that amphetamine reverses the hypotonia seen during GHB overdose (Kohrs et al., 2004). GHB may also be used as a sleep aid to counter the effects of METH (Marshall, 2006). The interactions between GHB and METH and GHB and MDMA are not well documented, often based on self-reports, and were therefore subjected to investigation in the present study. As these drugs are often taken at elevated ambient temperatures in night clubs we also examined how high ambient temperature affects the response to GHB (Snead, 1990). The present experiments used biotelemetry in freely moving rats to provide a more detailed analysis of the dose-dependent effects of GHB on body temperature and locomotor activity and GHB interactions with other drugs. Previous studies with rodents have used traditional methods (e.g. rectal probes) that provide only a limited number of data points over time and involve stressful interventions that can themselves affect body temperature. It was hoped that the use of biotelemetry would provide a more detailed and accurate analysis of GHB effects. 2. Materials and methods

Table 1 Overview of experiments. Experiment

Cohort (N)

Drugs and doses (mg/kg)

1. 2.

GHB dose response GHB and SCH 50911

A (7) B (8)

3.

GHB, SCH 23390 and ritanserin

B (8)

4.

GHB, flumazenil and naltrexone

C (8)

5. 6a. 6b.

Baclofen alone Chronic GHB Cross-tolerance to baclofen Elevated temperature (28 ◦ C) GHB and MDMA combined (28 ◦ C) GHB and METH combined (28 ◦ C)

C (8) C (8) C (8) D (8)

GHB (0, 250, 500, 1000) SCH 50911 (50), GHB (1000) SCH 23390 (1), ritanserin (1), GHB (1000) Flumazenil (10), naltrexone (1), GHB (1000) Baclofen (10) GHB (1000) × 10 Baclofen (10) after 10 × GHB (1000) GHB (500)

D (8)

GHB (500), MDMA (5)

D (8)

GHB (500), METH (1)

7. 8. 9.

Cohort (A, B, C or D) refers to the group of animals used in each experiment. Unless indicated all experiments were carried out at an ambient temperature of 20 ◦ C.

elevated ambient temperature was used for testing (see below). All drug testing took place in this room while rats were singly housed. Throughout the experiment rats had access to food and tap water ad libitum. All experimental procedures were conducted in accordance with the Australian Code of Practice for Care and Use of Animals for Scientific Purposes. Ethical approval for experiments was obtained from Sydney University Animal Ethics Committee. In order to minimise the number of animals used, we used a within-subjects design in which most animals were used in more than one experiment (see Table 1). 2.2. Radiotelemetry Radiotelemetry implants (TA10TA-F20, Data Sciences International, St Paul, MN, USA) were used to measure locomotor activity and body temperature in freely moving rats. Signals from the implanted transmitters were detected by a total of 8 RPC1 receivers (Data Sciences International, St Paul, MN, USA) in a multiplexed system. Each implant was calibrated for temperature to conform to the manufacturer’s configuration settings. 2.3. Surgery Rats were anaesthetised with an i.p. injection of 0.75 mg/kg ketamine and 0.5 mg/kg xylazine. After testing of withdrawal reflexes to ensure adequate depth of anaesthesia, a 1 cm midline abdominal incision was made and a radiotelemetry transmitter was placed in the peritoneal cavity according to manufacturer’s protocol (Data Sciences International, St Paul, MN, USA). The individual transmitter serial number was noted for each rat prior to implantation and entered into the computer software. The peritoneal cavity was closed with normal sutures. Rats were allowed a minimum of 7 days to recover from surgery before testing commenced. After surgery rats were continuously housed in the testing room. 2.4. Procedure In each experiment outlined below, core body temperature and locomotor activity were recorded at 1 min intervals from 1 h before to 6 h after drug treatment (total recording time = 7 h). All testing took place in the dark cycle between 10:00 h and 16:00 h. The telemetry signals were sampled during a 10 sec period for each min of testing. RPC1 receivers were placed under each cage and were connected to a computer through a Dataquest PCI card (Data Sciences International, St Paul, MN, USA). Body temperature and locomotor activity data were acquired, processed and analysed using the software package Dataquest ART2.2 (Data Sciences International, St Paul, MN, USA).

2.1. Animals and housing The experiments involved a total of 31 male albino Wistar rats (Animal Resource Centre, Perth, Australia) weighing 300 ± 5 g at the start of the study. Rats were initially housed in a temperature controlled (20 ± 1 ◦ C) colony room on a 12 h reversed light cycle in large plastic tubs of 8, with wood shavings as bedding. After surgery (see below) rats were singly housed in plastic tubs (36 cm × 21 cm × 17.5 cm) to allow recording of individual animals using the biotelemetry system. These plastic tubs were located on a metal rack in a small room separate to the main animal colony. This room was maintained under identical light and temperature conditions to the colony room except at times when an

2.4.1. GHB dose response effects (Experiment 1) To initially assess the dose-dependent effects of GHB on body temperature and locomotor activity we administered 0, 250, 500 and 1000 mg/kg GHB to a total of 7 experimentally naïve rats. All rats received vehicle and each test dose of GHB in counterbalanced order. To ensure a complete washout of GHB, drug treatment days were 48 h apart (Lettieri and Fung, 1979). 2.4.2. SCH 50911 interaction with GHB (Experiment 2) A total of 8 experimentally naïve rats were used in Experiment 2. They received each of the following treatments in counterbalanced order (1) SCH 50911 (50 mg/kg)

P.S. van Nieuwenhuijzen, I.S. McGregor / Drug and Alcohol Dependence 103 (2009) 137–147 and GHB (1000 mg/kg), (2) vehicle and GHB (1000 mg/kg) and (3) vehicle and vehicle. The first injection was administered 30 min before the second (Barbaccia, 2004) and each test was 48 h apart. 2.4.3. SCH 23390 and ritanserin interaction with GHB (Experiment 3) One week after Experiment 2, the same group of rats used in Experiment 2 received in counterbalanced order (1) SCH 23390 (1 mg/kg) and GHB (1000 mg/kg), (2) ritanserin (1 mg/kg) and GHB (1000 mg/kg), (3) vehicle and GHB (1000 mg/kg) and (4) vehicle and vehicle. The first injection (of antagonist) was administered 30 min before GHB (Catalina et al., 2002; Verty et al., 2004). Each test was 48 h apart. 2.4.4. Flumazenil and naltrexone interaction with GHB (Experiment 4) A total of 8 experimentally naïve rats were used in Experiment 4. The rats received in a counterbalanced order, (1) flumazenil (10 mg/kg) and GHB (1000 mg/kg), (2) naltrexone (1 mg/kg) and GHB (1000 mg/kg), (3) vehicle and GHB (1000 mg/kg) or (4) vehicle and vehicle. The first injection (of antagonist) was always administered 30 min before the second (of GHB) (Gallate et al., 2004; SchmidtMutter et al., 1998) and each test was separated by 48 h. 2.4.5. Baclofen given alone (Experiment 5) The same group of rats (n = 8) previously used in Experiment 4 received either (±)-baclofen (10 mg/kg) or vehicle in a counterbalanced order over two treatment days separated by 24 h. Since the half-life of baclofen is 4–5 h it was expected that baclofen levels would have dropped to minimal level after 24 h (Smith et al., 1999). The dose of baclofen was chosen based on our previous work demonstrating that this dose parallels the effects of 500 mg/kg GHB (van Nieuwenhuijzen et al., 2009). 2.4.6. Tolerance to repeated GHB (Experiment 6a) and cross-tolerance with baclofen (Experiment 6b) Commencing 7 days after Experiment 5, the rats (n = 8) previously used in Experiments 4 and 5 received either GHB (1000 mg/kg) (n = 4) or vehicle (n = 4) for 10 consecutive days. On Day 11, all rats received a challenge dose of 10 mg/kg (±)baclofen and body temperature and locomotor activity were assessed. 2.4.7. GHB effects in elevated ambient temperature (28 ◦ C) (Experiment 7) A total of 8 experimentally naïve rats received either GHB (500 mg/kg) or saline over two days. For the 7 h of data recording (from 1 h before to 6 h after drug administration) the ambient temperature was 28 ◦ C to simulate the hot and sweaty environment where drugs are often taken (McGregor et al., 2003). After this the room was returned to a temperature of 20 ◦ C. Room temperature was elevated using a reverse cycle air conditioning unit (Electrolux, Sydney, Australia), attached to a temperature regulator set to 28 ◦ C resulting in a constant elevated ambient temperature. 2.4.8. MDMA interaction with GHB in elevated ambient temperature (28 ◦ C) (Experiment 8) The same rats used in Experiment 7 (n = 8) were also used in Experiment 8, which commenced 7 days later. In a counterbalanced order rats received one of the following 3 treatments: (1) MDMA (5 mg/kg) and vehicle, (2) MDMA (5 mg/kg) and GHB (500 mg/kg), or (3) vehicle and vehicle. The second injection was administered immediately after the first. Each treatment was separated by 48 h to ensure a complete washout. During the 7 h of data recording (from 1 h before to 6 h after drug administration) the ambient temperature was 28 ◦ C, after which the room was returned to a temperature of 20 ◦ C. 2.4.9. METH interaction with GHB in elevated ambient temperature (28 ◦ C) (Experiment 9) The same rats used in Experiments 7 and 8 (n = 8) were used in Experiment 9, which commenced 4 days later. In a counterbalanced order rats received each of 3 treatments: (1) METH (1 mg/kg) and vehicle, (2) METH (1 mg/kg) and GHB (500 mg/kg) and (3) vehicle and vehicle. The second injection was administered immediately after the first. Each treatment was separated by 48 h to ensure drug washout. During the 7 h of data recording the ambient temperature was 28 ◦ C, and thereafter 20 ◦ C. 2.5. Drugs GHB, (±)-baclofen, naltrexone hydrochloride, flumazenil, ritanserin and R(±)-SCH 23390 hydrochloride were purchased from Sigma (Sydney, Australia). SCH 50911 was purchased from Tocris (Sydney, Australia). 3,4-Methylenedioxymethampetamine and methamphetamine, in the form of racemic hydrochloride salts, were purchased from the Australian Government Analytical Laboratories (Pymble, NSW). All doses refer to the salt. GHB was dissolved in distilled water and administered intraperitoneally (i.p.) at a volume of 2 ml/kg. MDMA, METH, SCH 23390 and SCH 50911 were dissolved in physiological saline and administered i.p. at a volume of 1 ml/kg. Naltrexone and flumazenil were dissolved in a solution of Tween 80 (5%) and ethanol (5%), suspended in physiological saline (90%), and administered i.p. at a volume of

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1 ml/kg. (±)-Baclofen was initially dissolved in 1 ml of 0.1 mM NaOH to a concentration of 100 mg/ml, then further diluted in saline to a final concentration of 10 mg/ml. Ritanserin was dissolved in 50% DMSO and administered i.p. at a volume of 1 ml/kg. All solutions were made freshly on the day of the experiment. Table 1 gives an overview and summary of the drugs and doses used across the experiments. 2.6. Statistical analysis Planned contrasts (within-subjects repeated measures ANOVA) were used to compare body temperature across specific treatments. Body temperature data were averaged across 30 min bins over the 6 h of testing for the purpose of this analysis. For each experiment, the minimum and maximum temperatures reached were also compared across treatments using planned contrasts. Planned contrasts were also used to analyse locomotor activity. Locomotor activity data (in arbitrary units) were summed into 10 min bins for the 6 h of testing, starting from the time of injection. All statistical analysis was conducted using SPSS 16 for Macintosh and the minimum level of statistical significance was set at p < 0.05.

3. Results The F values for analysis of body temperature and locomotor activity effects across the different experiments on are shown in Tables 2 and 3 respectively. Table 4 shows the average minimum and maximum temperature reached after each drug treatment. 3.1. Effect of GHB on body temperature and locomotor activity Fig. 1 shows the effect of various doses of GHB on body temperature (Fig. 1a–c) over time and total locomotor activity (Fig. 1d). The 500 mg/kg dose of GHB significantly decreased body temperature Table 2 F values and significance for planned contrasts: body temperature data. Experiment

Treatment

Treatment × Time (min)

1. GHB dose response VEH vs. 250 GHB VEH vs. 500 GHB VEH vs. 1000 GHB

F(1,6) = 2.80 F(1,6) = 21.13** F(1,6) = 0.28

F(11,66) = 0.31 F(11,66) = 0.92 F(11,66) = 8.85***

2. SCH 50911 (50 mg/kg) and GHB (1000 mg/kg) GHB vs. VEH F(1,7) = 2.74 GHB vs. SCH 50911 + GHB F(1,7) = 35.82**

F(11,77) = 11.70*** F(11,77) = 40.47***

3. SCH 23390 (1 mg/kg), ritanserin (1 mg/kg) and GHB (1000 mg/kg) GHB vs. VEH F(1,7) = 38.37*** F(11,77) = 6.49*** F(11,77) = 10.58*** GHB vs. SCH 23390 + GHB F(1,7) = 0.01 F(11,77) = 12.31*** GHB vs. ritanserin + GHB F(1,7) = 0.09 4. Flumazenil (10 mg/kg), naltrexone (1 mg/kg) and GHB (1000 mg/kg) GHB vs. VEH F(1,7) = 1.91 F(11,77) = 8.04*** F(11,77) = 1.53 GHB vs. FLU + GHB F(1,7) = 0.14 F(11,77) = 2.59** GHB vs. NX + GHB F(1,7) = 0.77 5. Baclofen (10 mg/kg) VEH vs. baclofen

F(1,7) = 6.76*

F(11,77) = 4.61***

6a. Tolerance to GHB (1000 mg/kg) Day 1 vs. Day 10

F(1,3) = 3.70

F(11,33) = 1.17

6b. Baclofen (10 mg/kg) after 10 daily administration of GHB (1000 mg/kg) F(11,66) = 1.24 VEH vs. GHB pre-treatment F(1,6) = 2.47 7. Elevated temperature (28 ◦ C) VEH vs. 500 GHB

F(1,7) = 0.51

F(11,77) = 0.74

◦

8. GHB (500 mg/kg) and MDMA (5 mg/kg) (28 C) VEH vs. MDMA F(1,7) = 13.45** VEH vs. MDMA/GHB F(1,7) = 4.91 MDMA vs. MDMA/GHB F(1,7) = 2.25

F(11,77) = 2.78** F(11,77) = 1.49 F(11,77) = 0.29

9. GHB (500 mg/kg) and METH (1 mg/kg) (28 ◦ C) VEH vs. METH F(1,7) = 55.70*** VEH vs. METH/GHB F(1,7) = 2.10 METH vs. METH/GHB F(1,7) = 20.01**

F(11,77) = 4.08*** F(11,77) = 0.78 F(11,77) = 12.51***

Unless indicated all experiments were carried out at 20 ◦ C. Asterisks indicate significant changes in body temperature: *p < 0.05, **p < 0.01 and ***p < 0.001.

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Table 3 F values and significance for planned contrasts: locomotor activity results. Experiment 1. GHB dose response VEH vs. 250 GHB VEH vs. 500 GHB VEH vs. 1000 GHB

Treatment F(1,6) = 0.47 F(1,6) = 9.15* F(1,6) = 39.79***

2. SCH 50911 (50 mg/kg) and GHB (1000 mg/kg) GHB vs. VEH F(1.7) = 5.96* GHB vs. SCH 50911 + GHB F(1.7) = 0.21

Treatment × time (min) F(36,216) = 0.78 F(36,216) = 0.96 F(36,216) = 0.89 F(36,252) = 1.53* F(36,252) = 1.74**

3. SCH 23390 (1 mg/kg), ritanserin (1 mg/kg) and GHB (1000 mg/kg) F(36,252) = 3.02*** GHB vs. VEH F(1,7) = 18.27** F(36,252) = 2.63*** GHB vs. SCH 23390 + GHB F(1,7) = 4.62 GHB vs. ritanserin + GHB F(1,7) = 2.7 F(36,252) = 1.25 4. Flumazenil (10 mg/kg), naltrexone (1 mg/kg) and GHB (1000 mg/kg) F(36,252) = 3.98*** GHB vs. VEH F(1,7) = 24.52** F(36,252) = 1.11 GHB vs. FLU + GHB F(1,7) = 3.89 F(36,25) = 1.80** GHB vs. NX + GHB F(1,7) = 0.13 5. Baclofen (10 mg/kg) VEH vs. baclofen

F(1,7) = 17.90**

6a. Tolerance to GHB (1000 mg/kg) Day 1 vs. Day 10 F(1,3) = 9.20*

F(36,252) = 6.28*** F(36,108) = 0.82

6b. Baclofen (10 mg/kg) after 10 daily administration of GHB (1000 mg/kg) F(36,252) = 1.53* VEH v GHB pre-treatment F(1,7) = 0.75 7. Elevated temperature (28 ◦ C) VEH vs. 500 GHB

F(1,7) = 0.85

F(36,252) = 1.05

◦

8. GHB (500 mg/kg) and MDMA (5 mg/kg) (28 C) VEH vs. MDMA F(1,7) = 13.61** VEH vs. MDMA/GHB F(1,7) = 0.01 MDMA vs. MDMA/GHB F(1,7) = 5.85*

F(36,252) = 3.41*** F(36,252) = 1.38 F(36,252) = 2.47***

9. GHB (500 mg/kg) and METH (1 mg/kg) (28 ◦ C) VEH vs. METH F(1,7) = 77.11*** VEH vs. METH/GHB F(1,7) = 4.54 METH vs. METH/GHB F(1,7) = 27.42**

F(36,252) = 9.03*** F(36,252) = 1.70* F(36,252) = 9.40***

Table 4 Minimum and maximum temperature reached in the 6 h after drug or vehicle administration. Experiment

Minimum, ◦ C (SEM)

Maximum, ◦ C (SEM)

1. GHB dose response VEH 250 GHB 500 GHB 1000 GHB

37.0 (0.09) 36.7 (0.15) 36.3 (0.11)** 36.5 (0.15)

38.2 (0.06) 38.2 (0.09) 38.0 (0.06)* 38.9 (0.19)**

2. SCH 50911 (50 mg/kg) and GHB (1000 mg/kg) VEH 37.1 (0.07)### GHB 36.1 (0.12) SCH 50911 + GHB 35.6 (0.11)###

38.2 (0.10)## 39.1 (0.16) 38.0 (0.04)###

3. SCH 23390 (1 mg/kg), ritanserin (1 mg/kg) and GHB (1000 mg/kg) 38.4 (0.07) VEH 37.1 (0.07)### GHB 36.1 (0.12) 38.5 (0.08) SCH 23390 + GHB 36.3 (0.15) 38.3 (0.07) 38.5 (0.15) Ritanserin + GHB 35.5 (0.14)## 4. Flumazenil (10 mg/kg), naltrexone (1 mg/kg) and GHB (1000 mg/kg) 38.2 (0.16) VEH 36.6 (0.15)# GHB 36.1 (0.09) 38.4 (0.11) FLU + GHB 36.1 (0.09) 38.5 (0.10) NX + GHB 36.1 (0.09) 38.4 (0.19) 5. Baclofen (10 mg/kg) VEH Baclofen

36.7 (0.14) 35.6 (0.22)**

6a. Tolerance to GHB (1000 mg/kg) Day 1 35.9 (0.42) Day 10 36.4 (0.17)

38.1 (0.15) 37.9 (0.13) 38.7 (0.10) 38.5 (0.08)ˆ

6b. Baclofen (10 mg/kg) after 10 daily administration of GHB (1000 mg/kg) VEH pre-treatment 36.0 (0.02) 37.9 (0.11) GHB pre-treatment 36.6 (0.27) 38.1 (0.15) 7. Elevated temperature (28 ◦ C) VEH 36.7 (0.12) 500 GHB 36.6 (0.05)

37.6 (0.12) 37.6 (0.05)

Unless indicated all experiments were carried out at 20 ◦ C. Asterisks indicate significant changes in locomotor activity: *p < 0.05, **p < 0.01, ***p < 0.001.

8. GHB (500 mg/kg) and MDMA (5 mg/kg) (28 ◦ C) VEH 36.6 (0.07) MDMA 37.2 (0.10)*** MDMA/GHB 36.8 (0.07)

38.0 (0.04) 39.1 (0.23)** 38.5 (0.26)

relative to vehicle treatment (p < 0.01) (Fig. 1b). The 1000 mg/kg dose of GHB induced a triphasic effect on body temperature: an initial hypothermia followed by hyperthermia then a hypothermic response, before returning to baseline, resulting in a significant treatment × time interaction (p < 0.001) (Fig. 1c). The maximum body temperature reached after 1000 mg/kg GHB, 38.9 ◦ C, was significantly higher than after vehicle treatment (p < 0.001) (Table 4). The minimum body temperature reached after 500 mg/kg GHB was 36.3 ◦ C, significantly lower than with vehicle treatment (p < 0.01) (Fig. 1b). The lowest, 250 mg/kg dose of GHB, did not alter body temperature relative to vehicle treatment. Locomotor activity (Fig. 1d) was dramatically reduced after 1000 mg/kg GHB (p < 0.001); 500 mg/kg GHB produced a smaller decrease (p < 0.01) and 250 mg/kg GHB had no significant effect on locomotor activity.

9. GHB (500 mg/kg) and METH (1 mg/kg) (28 ◦ C) VEH 36.6 (0.09) METH 37.0 (0.10)* METH/GHB 36.6 (0.17)

37.8 (0.13) 38.3 (0.13)*** 38.1 (0.10)*

3.2. SCH 50911 interaction with GHB SCH 50911 (50 mg/kg) prevented the hyperthermia seen with 1000 mg/kg GHB (p < 0.01) (Fig. 2a). Treatment with SCH 50911 + GHB produced a minimum body temperature of 35.6 ◦ C, which was significantly lower than that in the GHB alone group (36.1 ◦ C) (p < 0.001). Conversely the peak body temperature in the GHB alone group was 39.1 ◦ C, which was significantly higher than the maximum temperature after SCH 50911 + GHB (38.0 ◦ C) (p < 0.001). GHB decreased locomotor activity compared to vehicle (p < 0.05). Pre-treatment with SCH 50911 only had relatively subtle (treatment × time) effects on the sedation caused by GHB with a faster recovery from sedation (Fig. 2b).

Unless indicated all experiments were carried out at 20 ◦ C Asterisks indicates significant difference in body temperature compared to vehicle treatment: *p < 0.05, **p < 0.01 and ***p < 0.001. Hash indicates significant difference in body temperature compared to GHB treatment: # p < 0.05 and ### p < 0.001. ˆp < 0.05 compared to Day 1.

3.3. SCH 23390 and ritanserin interaction with GHB Pre-treatment with ritanserin resulted in a larger decrease in body temperature over time than with GHB alone (p < 0.001) (Fig. 3a). The minimum temperature reached after ritanserin + GHB was 35.5 ◦ C, significantly lower than that reached after GHB alone (36.1 ◦ C) (p < 0.01) (Fig. 3a). Pre-treatment with SCH 23390 did not affect the body temperature effects of GHB (1000 mg/kg). Total locomotor activity was not affected by pre-treatment with either SCH 23390 or ritanserin (Fig. 3b). 3.4. Flumazenil and naltrexone interaction with GHB There was no significant effect of flumazenil (10 mg/kg) or naltrexone (1 mg/kg) on the body temperature (Fig. 3c) or overall locomotor activity (Fig. 3d) changes induced by GHB (1000 mg/kg). 3.5. Baclofen (±)-Baclofen (10 mg/kg) decreased body temperature relative to vehicle treatment (p < 0.05) (Fig. 4a) and resulted in a mean min-

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Fig. 1. Body temperature (mean + SEM) over time in rats treated with (a) 250 mg/kg, (b) 500 mg/kg, and (c) 1000 mg/kg of GHB, relative to vehicle treatment. (d) Total locomotor activity + SEM for the 6 h after drug administration, for each treatment condition. *p < 0.05 and ***p < 0.001 for GHB dose vs. vehicle. n = 7 for each graph. Light vertical lines on x-axis at time = 0 indicates time of vehicle or drug administration.

Fig. 2. (a) Body temperature (mean + SEM) and (b) locomotor activity over time in rats treated with vehicle + vehicle, vehicle + 1000 mg/kg GHB or 50 mg/kg SCH 50911 (SCH5) + 1000 mg/kg GHB. Insert represents total locomotor activity + SEM over the 6 h after drug administration. *p < 0.05 vs. GHB. n = 8 for each graph. Light vertical lines on x-axis at time = −30 and time = 0 indicates times of vehicle, or drug administration.

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Fig. 3. (a) Body temperature (mean + SEM) over time and (b) total locomotor activity + SEM over 6 h after drug administration in rats treated with treated with vehicle + vehicle, vehicle + 1000 mg/kg GHB, 1 mg/kg SCH 23390 (SCH2) + 1000 mg/kg GHB or 1 mg/kg ritanserin (RIT) + 1000 mg/kg GHB. (c) Body temperature (mean + SEM) over time and (d) total locomotor activity + SEM over 6 h after drug administration in rats treated with vehicle + vehicle, vehicle + 1000 mg/kg GHB, 10 mg/kg flumazenil (FLU) + 1000 mg/kg GHB or 1 mg/kg naltrexone (NX) + 1000 mg/kg GHB. **p < 0.01 vs. GHB. n = 8 for each graph. Light vertical lines on x-axis at time = −30 and time = 0 indicates times of vehicle, or drug administration.

imum temperature of 35.6 ◦ C which was lower than with vehicle treatment (p < 0.05). Baclofen also significantly reduced locomotor activity compared with vehicle treatment with almost complete immobility for more than an hour after treatment (p < 0.01) (Fig. 4b). 3.6. Tolerance and cross-tolerance to baclofen effects Administration of GHB (1000 mg/kg) for 10 days did not change the tri-phasic effect of GHB on body temperature. However the

maximum temperature reached on Day 1 (38.7 ◦ C) was significantly higher to that on Day 10 (38.5 ◦ C) (p < 0.05) (Fig. 5a). The sedative effect of GHB on Day 1 was greater than that on Day 10 as shown by greater mean total activity on Day 10 compared to Day 1 (p < 0.05) (Fig. 5b). Rats treated with vehicle for 10 days showed a tendency towards a slightly lower minimum body temperature after a challenge injection of 10 mg/kg baclofen (36.0 ◦ C) than rats receiving equivalent treatment with GHB (36.6 ◦ C) (p = 0.06) (Fig. 5c). Baclofen caused

Fig. 4. (a) Body temperature (mean + SEM) and (b) locomotor activity over time in rats treated with vehicle or 10 mg/kg baclofen (BAC). Insert represents total locomotor activity + SEM over 6 h after drug administration. **p < 0.01 vs. vehicle. n = 8 for each graph. Light vertical lines on x-axis at time = 0 indicates time of vehicle or drug administration.

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Fig. 5. (a) Body temperature (mean + SEM) and (b) locomotor activity over time on first (Day 1) or tenth (Day 10) day of GHB (1000 mg/kg) administration. Insert represents total locomotor activity + SEM over 6 h after drug administration. *p < 0.05 vs. Day 1. (c) Body temperature (mean + SEM) and (d) locomotor activity over time in rats pre-treated for 10 days with vehicle or GHB 1000 mg/kg then challenged with 10 mg/kg baclofen (BAC). Insert represents total locomotor activity + SEM over 6 h after drug administration. **p < 0.01 vs. vehicle pre-treated group. n = 4 for each graph. Light vertical lines on x-axis at time = 0 indicates time of vehicle or drug administration.

a greater suppression of locomotor activity in vehicle pre-treated rats than GHB pre-treated rats (p < 0.01) (Fig. 5d).

4. Discussion 4.1. Dose response effects

3.7. GHB effects in elevated ambient temperature (28 ◦ C) GHB (500 mg/kg) administered at an elevated ambient temperature (28 ◦ C) did not alter body temperature or locomotor activity relative to vehicle treatment (data not shown). 3.8. MDMA interaction with GHB at an elevated ambient temperature (28 ◦ C) MDMA administration increased body temperature (p < 0.01) and the co-administration of GHB attenuated this hyperthermic response (Fig. 6a). MDMA increased body temperature to a maximum of 39.1 ◦ C, significantly higher than vehicle treatment (p < 0.01) (Table 4). Total locomotor activity was increased by MDMA, relative to vehicle treatment (p < 0.01) with coadministered GHB preventing this effect (Fig. 6b). 3.9. METH interaction with GHB at an elevated ambient temperature (28 ◦ C) METH increased body temperature relative to vehicle treatment (p < 0.001) and GHB combined with METH (p < 0.01) (Fig. 6c). METH alone resulted in a higher mean maximum temperature of 38.3 ◦ C relative to vehicle (Fig. 6c) METH increased total locomotor activity (p < 0.001) while coadministered GHB prevented this effect (Fig. 6d).

A key finding in the present study is the triphasic effect of a high 1000 mg/kg dose of GHB on body temperature over a 6-h period following administration. An initial drop in body temperature was followed by a large hyperthermic spike, after which a further long-lasting hypothermia was evident (Fig. 1c). The initial drop in body temperature after 1000 mg/kg GHB was somewhat more pronounced in Experiments 2, 3, 4 and 6 than in Experiment 1, perhaps due to the higher pre-drug baseline temperatures in this first experiment. The lower 500 mg/kg dose of GHB produced only a marked hypothermic component that lasted for up to 6 h. Given that GHB is metabolised rapidly with a half-life of approximately 1.2 h (Lettieri and Fung, 1979), the long hypothermic component seen with the 1000 mg/kg dose may reflect a dose-related effect: with the high dose coming to resemble the effects of the 500 mg/kg at a time when approximately half of the drug dose has been metabolized. Hyperthermia has been reported in rats after 5 and 10 mg/kg GHB (Kaufman et al., 1990) and the present study reports the novel finding of a hyperthermic response after 1000 mg/kg GHB. The hyperthermia seen after administration of low doses of GHB has been attributed to the GHB-receptors whereas the effects at higher doses have been attributed to the GABAB receptor (Kaufman et al., 1990). Snead (1990) showed that administration of a high dose (600 mg/kg) of ␥-butylactone (a GHB precursor and pro-drug) in rats resulted in a hyperthermic response of +2 ◦ C that lasted for an

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Fig. 6. (a) Body temperature (mean + SEM) and (b) locomotor activity over time in rats treated with vehicle, 5 mg/kg MDMA or 5 mg/kg MDMA + 500 mg/kg GHB. Insert represents total locomotor activity + SEM over 6 h after drug administration, **p < 0.01 vs. vehicle. (c) Body temperature (mean + SEM) and (d) mean of locomotor activity over time in rats treated with vehicle, 1 mg/kg METH or 1 mg/kg METH + 500 mg/kg GHB. Insert represents total locomotor activity over 6 h after drug administration, ***p < 0.001 vs. vehicle. n = 8 for each graph. Light vertical lines on x-axis at time = 0 indicates time of vehicle or drug administration.

hour and was followed by hypothermia Similarly, Zarrindast and Oveissi (1988) showed that 20 mg/kg baclofen when administered to rats resulted in an initial hypothermia followed by marked hyperthermia that lasted for 3 h. The hyperthermic response seen after a high dose of GHB therefore seems likely to involve GABAB receptors and this was further confirmed in the present study by our finding in Experiment 2, that SCH 50911, a GABAB receptor antagonist, reversed GHB-induced hyperthermia. This is consistent with the absence of a body temperature response to GHB in GABAB receptor knockout mice (Queva et al., 2003). In line with previous reports in rodents (Carai et al., 2001; Queva et al., 2003; Smith et al., 2006) GHB caused dose-dependent sedation, including prolonged periods of complete immobility following the 1000 mg/kg dose. This agrees with the effects in humans, including loss of gross motor skills, sedation, sleep and coma (Abanades et al., 2007; Carter et al., 2006). Informal observations by the experiments suggested that rats appeared to suddenly awake from the 1000 mg/kg dose. This is reminiscent of GHB overdoses in humans, in which after a period of unconsciousness users awake rapidly feeling refreshed and with no residual lasting effects (Chin et al., 1998; Van Sassenbroeck et al., 2007). 4.2. Receptor mechanisms underlying GHB (1000 mg/kg) effects Pre-treatment with SCH 50911 (50 mg/kg), a selective GABAB receptor antagonist prior to GHB resulted in a drop in body temperature instead of the triphasic effect observed with 1000 mg/kg GHB alone. This dose of SCH 50911 also reduced GHB-induced sedation over time. The hypothermia seen after SCH 50911 + GHB was larger than the hypothermia seen after 500 mg/kg GHB, and was similar to the hypothermia seen after 10 mg/kg baclofen, with both

treatments resulting in a minimum temperature of 35.6 ◦ C. Unfortunately, further experiments using higher doses of SCH 50911 were precluded due to the high costs of this compound. Nonetheless the 50 mg/kg dose was partly effective in reversing some of the GHB effects in agreement with previous studies. Thus SCH 50911 (20 mg/kg) prevented the reduction in acetylcholine levels seen after the administration of 200 and 500 mg/kg GHB to male rats (Nava et al., 2001) while 50 mg/kg SCH 50911 exacerbated the withdrawal syndrome seen after the repeated administration of 1,4-butanediol, a GHB precursor, in rats (Carai et al. (2005b). Flumazenil is a benzodiazepine antagonist that attenuates the anxiolytic effects of GHB in rats (Schmidt-Mutter et al., 1998), although it does not prevent the lethal effects of high dose GHB in mice (Carai et al., 2005a). In the present study pre-treatment with flumazenil did not affect GHB-induced changes in body temperature or locomotor activity suggesting little involvement of benzodiazepine receptors in these effects. Naltrexone, an opioid antagonist, also failed to affect GHBinduced changes on body temperature and locomotor activity. Higher doses of naltrexone (4 mg/kg and 10 mg/kg) prevented GBL induced changes in dopamine content, seizures, hypothermia and 3 –5 cyclic-guanosine monophosphate when administered to rats (Crosby et al., 1983; Snead and Bearden, 1980; Vayer et al., 1987 and Feigenbaum and Howard, 1996) reversed the inhibitory effect of GHB (500 mg/kg) on dopamine release by pre-treatment with 0.8 mg/kg naltrexone in rats. In the present study a moderate dose (1 mg/kg) of naltrexone was used, which by itself causes a slight decrease in body temperature (Eikelboom, 1987). It is possible that a higher dose of naltrexone would have reduced the hyperthermia seen after GHB, perhaps via functional antagonism rather than direct inhibition of the effects of GHB (Eikelboom, 1987).

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GHB affects the turnover of dopamine and 5-HT in the brain and can also affect the synthesis of these neurotransmitters (Gobaille et al., 2002; Maitre, 1997), which are also important in the regulation of body temperature (Nisijima et al., 2001). To explore the involvement of these neurotransmitters in the effects of GHB we assessed the effects of co-administering a D1 receptor antagonist, SCH 23390, and a 5HT2A/2C -receptor antagonist, ritanserin with GHB (Janssen, 1985; Velasco and Luchsinger, 1998). Neither drug altered the increase in body temperature or sedative effects of GHB. Ritanserin tended to magnify the initial decrease in body temperature seen after GHB indicating that the initial hypothermia may be potentiated by the inhibition of serotonergic action. Ritanserin is capable of reversing MDMA-induced hyperthermia (Shioda et al., 2008) and hyperthermia associated with the 5-HT syndrome in rats (Nisijima et al., 2001). In mice treated with dehydroepiandosterone, pre-treatment with ritanserin exacerbated the hypothermia seen after dehydroepiandosterone alone (Catalina et al., 2002). 5-HT induced hypothermia in rats was blocked only by very low doses of ritanserin (0.01 mg/kg) and higher doses diminished this effect (Colpaert et al., 1985). From these studies it is clear that ritanserin affects body temperature responses, although the precise mechanism(s) behind the augmentation of the hypothermic response mediated by GHB requires further investigation. SCH 23390 is effective in reversing MDMA-induced hyperthermia in laboratory animals (Benamar et al., 2008; Mechan et al., 2002). However, in the present study this D1 receptor antagonist was without effect indicating that dopamine D1 receptors are most likely not involved in the body temperature effects of GHB. Together these data reinforce the importance of the GABAB receptor in mediating the effects of GHB on locomotor activity and body temperature. Future investigations would benefit from administration of a greater range of dose combinations of antagonists and GHB to further investigate the mechanisms involved in GHB’s pharmacological effects. The present study might have also studied a GHB-receptor antagonist (e.g. NCS-382): however GABAB receptor knockout mice (that still have functional GHB receptors) show an absence of sedative and body temperature effects of GHB (1000 mg/kg) indicating that these effects are mediated by GABAB receptors and not GHB-receptors (Kaupmann et al., 2003). In addition NCS-382 does not reverse GHB’s sedative, ataxic or discriminative stimulus effects (Castelli et al., 2004). 4.3. Baclofen, tolerance to GHB and cross-tolerance with baclofen Administration of baclofen (10 mg/kg) resulted in marked hypothermia and sedation in accordance with previous studies (Perry et al., 1998; Queva et al., 2003; Zarrindast and Oveissi, 1988). The sedation seen after 10 mg/kg baclofen was similar to the sedative effects of 500 mg/kg GHB in agreement with our recent report (van Nieuwenhuijzen et al., 2009). Daily administration of a high dose of GHB (1000 mg/kg) resulted in modest tolerance to the hyperthermic effects of the drug assessed on the 10th day of administration and a considerably shorter duration of sedation on this day. To our knowledge this is the first report of tolerance to body temperature effects of GHB although tolerance to the sedative effects are well-established (Bania et al., 2003; Itzhak and Ali, 2002; Raybon and Boje, 2007). We also report here that repeated exposure to GHB provides some tolerance to the hypothermia and sedation produced by baclofen (10 mg/kg). This cross-tolerance further underlines the centrality of the GABAB receptor in the physiological and sedative effects of GHB and agrees with other reports (e.g. Smith et al., 2006). Indeed, drug discrimination studies confirm that baclofen substitutes well for GHB in GHB-trained rats (Baker et al., 2008; Carter et al., 2003; Colombo et al., 1998).

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4.4. GHB, MDMA and METH interactions at an elevated ambient temperature GHB is regularly used in clubs and at dance parties, which are often hot and humid environments (Barker et al., 2007), and it was therefore of interest to investigate how this might modulate GHB effects. Disruption of normal thermoregulatory processes by various drugs can cause core temperature to be more greatly affected by ambient temperature (Green et al., 2005; Lomax and Daniel, 1990; Rosow et al., 1980; Schlosberg, 1983). In the present study, elevated ambient temperature (28 ◦ C) prevented the hypothermia observed with GHB (500 mg/kg) at 20 ◦ C. This agrees with Lin et al. (1979) who found that an ambient temperature of 30 ◦ C prevented the hypothermia seen at 22 ◦ C with GHB (300 mg/kg), while Snead (1990) showed that ambient temperatures of 32 and 35 ◦ C prevented the hypothermic effect of GBL (200 and 400 mg/kg) at 26 ◦ C. Increased ambient temperature prevents the opportunity for heat loss, thus minimising the effects of normally hypothermic drugs (Ghosh and Poddar, 1993). Interestingly, no differences in locomotor activity were seen between vehicle and GHB treatment when the drug was administered in elevated room temperature. This is most likely due to the decreased activity of control animals in a hot environment (Finger, 1976; Hargreaves et al., 2007) rather than any heat-driven loss of GHB’s sedative effects. MDMA administered at normal or elevated ambient temperature is well known to cause hyperthermia, an effect replicated here (Green et al., 2005; Hargreaves et al., 2007). However, GHB co-administered with MDMA decreased the maximum temperature reached and the duration of MDMA-induced hyperthermia (Experiment 8). Interestingly, this occurred under conditions under which GHB by itself did not affect body temperature (Experiment 7). This effect recalls a report that baclofen (3 mg/kg) prevents MDMAinduced hyperthermia in rats (Bexis et al., 2004). GHB can inhibit dopamine release (Cruz et al., 2004; Hechler et al., 1993) and this may conceivably also play a role in GHB attenuation of MDMA hyperthermia, which involves dopaminergic mechanisms (Mechan et al., 2002). Drugs that attenuate MDMA-induced hyperthermia usually prevent some of the lasting 5-HT depletion produced by MDMA in rats, raising the possibility that GHB may have such protective effects (Green et al., 2004; Morley et al., 2004). Future research might therefore measure 5-HT levels in the brain after the administration of MDMA and MDMA/GHB combinations to test for this possibility. In a previous study, the hyperactivity caused by MDMA in rats was not affected by baclofen (3 mg/kg) (Bexis et al., 2004). However, in the present study MDMA hyperactivity was clearly prevented by the co-administration of GHB. The increase in locomotor activity after MDMA is a result of both serotonergic and dopaminergic receptor systems (Bankson and Cunningham, 2001). Both D1 and D2 receptor antagonists are capable of reversing MDMA induced hyperlocomotion (Bubar et al., 2004). The inhibition of dopamine release by GHB could therefore prevent MDMA-induced hyperlocomotion (Cruz et al., 2004; Hechler et al., 1993). METH (1 mg/kg), like MDMA, also caused marked hyperactivity that lasted for 4 h with core body temperature also increased (Experiment 9). The co-administration of GHB (500 mg/kg) completely prevented METH-induced hyperactivity and hyperthermia. As with MDMA, METH-induced hyperthermia contributes to the mortality and neurotoxicity associated with this drug (Sandoval et al., 2000) and hypothermia can be protective against METH neurotoxicity (Bowyer et al., 1994). METH hyperactivity reflects increased dopamine release and decreased GABAergic influence (Caligiuri and Buitenhuys, 2005) while GHB inhibits dopamine release and promotes GABAergic inhibitory processes (Maitre, 1997). These opposing effects could

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explain the reversal of METH hyperactivity by GHB. Alternatively, the interaction between GHB and MDMA and GHB and METH may reflect a simple summation of stimulatory and inhibitory processes that do not necessarily share a common neurochemical substrate. The present study administered GHB/MDMA and GHB/METH at the same time, so future studies would benefit from administering GHB and MDMA or GHB and METH in different orders and with a delay between injections to investigate whether the order of dosing, or time interval between dosing, changes outcomes. Order of dosing has certainly been found to be an important determinant of the body temperature changes seen with the co-administration of MDMA and METH to rats (Clemens et al., 2005). 4.5. Conclusions The present study reports the first investigation of the effects of GHB on body temperature and locomotor activity in rats using a biotelemetry system, and the first to analyse the effects of coadministered MDMA and METH on GHB effects. The study shows for the first time a complex triphasic effect on body temperature after a high dose of GHB, with a powerful hyperthermic response that is reversed by the GABAB receptor antagonist SCH 50911. This reversal, as well as the cross-tolerance between GHB and baclofen, confirms the importance of the GABAB receptor in the effects of GHB. In addition, GHB strongly attenuated the hyperthermic and hyperactive responses to moderate doses of MDMA and METH. These effects could have implications for party drug users for whom hyperthermia can be one of the major hazards of MDMA and METH use. Other behavioural, cognitive and neurotoxic sequelae of GHB and MDMA/METH combinations would clearly be a worthy topic for further investigation. Role of funding source Research was funded by an NHMRC grant awarded to I.S. McGregor; the NHMRC had no further role in study design; in collection, analysis and interpretation of data; in the writing of the report or the decision to submit the paper for publication. Contributors Authors Petra S. van Nieuwenhuijzen and Iain S. McGregor both contributed to the study design. Petra S. van Nieuwenhuijzen wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement This work was supported by a National Health and Medical Research Council grant to ISM. References Abanades, S., Farre, M., Barral, D., Torrens, M., Closas, N., Langohr, K., Pastor, A., de la Torre, R., 2007. Relative abuse liability of gamma-hydroxybutyric acid, flunitrazepam, and ethanol in club drug users. J. Clin. Psychopharmacol. 27, 625–638. Baker, L.E., Searcy, G.D., Pynnonen, D.M., Poling, A., 2008. Differentiating the discriminative stimulus effects of gamma-hydroxybutyrate and ethanol in a three-choice drug discrimination procedure in rats. Pharmacol. Biochem. Behav. 89, 598–607. Bania, T.C., Ashar, T., Press, G., Carey, P.M., 2003. Gamma-hydroxybutyric acid tolerance and withdrawal in a rat model. Acad. Emerg. Med. 10, 697–704. Bankson, M.G., Cunningham, K.A., 2001. 3,4-Methylenedioxymethamphetamine (MDMA) as a unique model of serotonin receptor function and serotonindopamine interactions. J. Pharmacol. Exp. Ther. 297, 846–852.

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