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Risperidone attenuates and reverses hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) in rats
NeuroToxicology 29 (2008) 1030–1036
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NeuroToxicology
Risperidone attenuates and reverses hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) in rats Katsutoshi Shioda a,c,*, Koichi Nisijima a, Tatsuki Yoshino b, Kyoko Kuboshima a, Tatsunori Iwamura d, Kunio Yui e, Satoshi Kato a a
Department of Psychiatry, Jichi Medical University, Tochigi, Japan Department of Hospital Pharmacy, Jichi Medical University, Tochigi, Japan Tamura city Miyakozi Clinic, Japan d Matsuyama University, Japan e Ashiya University Graduate School, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 November 2007 Accepted 22 July 2008 Available online 5 August 2008
3,4-Methylenedioxymethamphetamine (MDMA, ‘‘ecstasy’’) is a widely used recreational drug. Despite an increase in the number of fatalities related to its use, no definite therapeutic method has been established thus far. In the present study, risperidone’s ability to attenuate MDMA-induced hyperthermia and its mechanism of action were investigated in rats. The pharmacological effect of MDMA was evaluated using microdialysis. In the body temperature experiment, administration of risperidone before and after MDMA administration significantly suppressed MDMA-induced hyperthermia in a dose-dependent fashion. Furthermore, risperidone completely inhibited MDMA-induced hyperthermia at a low ambient temperature. Moreover, pretreatment with ritanserin, ketanserin, or R-96544, all of which are 5-HT2Areceptor antagonists, significantly prevented MDMA-induced hyperthermia. On the other hand, pretreatment with WAY-100635 (a 5-HT1A receptor antagonist), SB 206553 (a 5-HT2B/2C receptor antagonist), or SB 242084 (a 5-HT2C receptor antagonist) did not prevent MDMA-induced hyperthermia. Pretreatment with haloperidol, which blocks the dopamine (DA) receptors D2 and D1, significantly prevented MDMA-induced hyperthermia. However, sulpiride and L-741626, which are D2 receptor blockers, did not prevent MDMA-induced hyperthermia. Pretreatment with SCH 23390 (a D1 receptor antagonist) significantly prevented MDMA-induced hyperthermia. Furthermore, postadministration of ritanserin, haloperidol, and SCH23390 reversed MDMA-induced hyperthermia. These results demonstrate that the mechanism underlying the suppression of MDMA-induced hyperthermia by risperidone is primarily based on the drug’s potent 5-HT2A receptor blocking effect, and to a lesser extent, on its D1 receptor blocking effect. A microdialysis study showed that when MDMA (10 mg/kg) was subcutaneously (s.c.) injected into the rats, the DA and serotonin (5-HT) levels in the anterior hypothalamus of the rats increased approximately 10- and 50-fold, respectively, as compared to their preadministration levels. These increases in the DA and 5-HT levels after MDMA injection were significantly suppressed by pretreatment with risperidone (0.5 mg/kg). This suggested that both the DA and 5-HT systems were involved in the induction of hyperthermia by MDMA. Taken together, the present study’s results indicate that risperidone may be an effective drug for the treatment of MDMA-induced hyperthermia in humans. ß 2008 Elsevier Inc. All rights reserved.
Keywords: MDMA Dopamine Serotonin Microdialysis Risperidone
1. Introduction 3,4-Methylenedioxymethamphetamine (MDMA, ‘‘ecstasy’’) is a widely used recreational drug, and its use has become a major health
* Corresponding author at: Department of Psychiatry, Yakushiji Shimotuke-Shi, Tochigi-Ken 329-0498, Japan. Tel.: +81 285 58 7364; fax: +81 285 44 6198. E-mail address: [email protected] (K. Shioda). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.07.005
issue (Gill et al., 2002; Schifano et al., 2003). The adverse effects of the use of MDMA include incidences of tachycardia, diaphoresis, tremor, hypertension, arrhythmia, and rhabdomyolysis (Hall and Henry, 2006). However, the worst outcome of MDMA ingestion is hyperthermia and the associated serotonin (5-HT) syndrome (Mueller and Korey, 1998; Parrott, 2001). There have been an increasing number of reports on MDMA-induced toxicity accompanied by symptoms of the 5-HT syndrome (Hall and Henry, 2006; Parrott, 2002; Silins et al., 2007). In addition, reports of the
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occurrence of 5-HT syndrome when MDMA is taken together with antidepressants have been increasing; this is because of the increasing use of antidepressants (SSRI, MAO inhibitor, SNRI, etc.; Gillman, 2005; Vuori et al., 2003). In patients with the 5-HT syndrome, hyperthermia can sometimes be fatal (Dowsett, 1996; O’Connor, 1994). In such cases, the chances of survival are very weak if the peak core temperature exceeds 42 8C (Dar and McBrien, 1996). The basic treatment for MDMA-induced hyperthermia includes body temperature reduction and fluid replacement; however, no definite therapeutic method has been established thus far. We have previously reported that prior administration of risperidone prevented hyperthermia and death in an animal model with serotonin syndrome induced by simultaneous administration of 5-hydroxy-L-tryptophan (a 5-HT precursor) and clorgyline (a MAO inhibitor) (Nisijima et al., 2000). We therefore hypothesized that risperidone could prevent MDMA-induced hyperthermia. It is reported that ambient temperature influences MDMA-induced hyperthermia in animals (Gordon et al., 1991; Green et al., 2005) and humans (Freedman et al., 2005). We attempted to demonstrate that a low ambient temperature increases risperidone’s ability to inhibit MDMA-induced hyperthermia. Risperidone is an atypical antipsychotic drug that weakly blocks the activity of the dopamine (DA) D1 receptor and potentially blocks the activities of the D2 and 5-HT2A receptors (Leysen et al., 1988); it also antagonizes the activities of other 5-HT receptor subtypes. In the present study, risperidone’s ability to suppress MDMA-induced hyperthermia was evaluated by performing an experiment using an animal model. It has been suggested that MDMA causes the release of 5HT as well as other neurotransmitters, including DA (Kankaanpaa et al., 1998; White et al., 1996). Body temperature is regulated by 5-HT and DA in the anterior hypothalamus (Yamawaki et al., 1983). We attempted to identify specific 5-HT and DA receptors associated with the induction of hyperthermia by MDMA by administering various 5-HT and DA receptor antagonists prior to MDMA exposure. The rats were injected with MDMA, and microdialysis was used to measure changes in the 5-HT and DA concentrations in the anterior hypothalamus, which plays an important role in the regulation of body temperature. 2. Materials and methods 2.1. Methods All experimental procedures involving animals were approved by the Animal Investigation Committee of our institution and were in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. 2.2. Animals, drug administration, and experimental protocol Male Wistar rats (Clea Japan Inc., Tokyo, Japan) weighing 200– 250 g were used in this study. The rats were housed in cages at 23 1 8C under a 12-h light/dark cycle and were provided free access to food and water. Risperidone (5-HT1A, Ki = 490 nM; 5-HT2A, Ki = 0.6 nM; 5-HT2B, pKi = 7.67; 5-HT2C, pKi = 8.31; D1, Ki = 75 nM; D2, Ki = 3 nM) (Bymaster et al., 1996; Wood et al., 2006), ketanserin (5-HT2A, Ki = 0.38 nM) (Harvey et al., 1999), ritanserin (5-HT2A, Ki = 0.24 nM) (Harvey et al., 1999), WAY-100635 (5-HT1A, Ki = 0.24 nM) (Johansson et al., 1997), SCH 23390 (D1, Ki = 0.76 nM) (Kanba et al., 1994) SB 206553 (5-HT2B, pKi = 8.89; 5-HT2C, pKi = 7.92) (Kennett et al., 1996), SB 242084 (5-HT2C, pKi = 9.0) (Kennett et al., 1997), and L-741626 (D2, pKi = 8.4) (Millan et al., 2000) were purchased from Sigma–Aldrich Co. (Saint Louis, USA). R-96544 (D1, Ki = 1.6 nM; Tocris data) was purchased from Tocris Cookson Ltd. (Bristol, UK). Haloperidol (D1, Ki = 25 nM; D2, Ki = 1 nM) (Bymaster et al., 1996) and sulpiride (D2, Ki = 34 nM) (Kanba
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et al., 1994) were obtained in injection ampoules from Dainippon Sumitomo Pharmaceutical Co., Osaka, Japan, and Astellas Pharma Inc., Tokyo, Japan, respectively. MDMA was synthesized by our collaborating investigator, T. Iwamura—a specialist in synthetic chemistry. Risperidone was dissolved in HCl, and the pH was adjusted in the range of 6–7 by using NaOH. Ritanserin was dissolved in 99.7% acetic acid, and the pH was adjusted in the range of 6–7 by using NaOH. L741626 was dissolved in 50% dimethyl sulfoxide (DMSO) in 0.9% saline. The other drugs were dissolved in 0.9% saline. A volume of 2 mL/kg of all the drugs was injected intraperitoneally into the rats. On the day of the experiment, the rats were placed in individual cages in a room maintained at 24 8C or 20 8C ambient temperature. The rectal temperature of the rats was measured several times, and when the temperature was observed to be stable for approximately 2 h, saline, risperidone (0.5 mg/kg), ketanserin (5 mg/kg), ritanserin (3 mg/kg), WAY-00635 (1 mg/kg), haloperidol (0.5 mg/kg), sulpiride (50 mg/kg), SCH 23390 (0.5 mg/kg), SB 206553 (2.5 mg/kg), SB 242084 (3 mg/kg), L-741626 (0.5 mg/kg), and R-96544 (1 mg/kg) were intraperitoneally injected into the rats. After 15 min, MDMA (10 mg/kg) was injected subcutaneously (s.c.). Thereafter, the rectal temperature was measured every 30 min (preadministration experiment). In the postadministration experiment, MDMA (10 mg/kg) was s.c. injected, and 30 min later, risperidone (0.25 and 0.5 mg/kg), haloperidol (0.25 and 0.5 mg/kg), SCH 23390 (0.25 and 0.5 mg/kg), and ritanserin (1.5 and 3.0 mg/kg) were injected intraperitoneally at 24 8C ambient temperature. In the experiment performed to investigate the effect of a drug on rectal temperature, saline was s.c. injected, and 30 min later, risperidone (0.5 mg/kg), haloperidol (0.5 mg/kg), SCH 23390 (0.5 mg/kg), ritanserin (3 mg/kg), and saline were injected intraperitoneally. In the experiment performed under low ambient temperature conditions, risperidone (0.5 mg/kg) and saline were intraperitoneally injected, and 30 min later, MDMA (10 mg/kg) was injected s.c. at 20 8C ambient temperature. The drug doses were selected as follows. Since prior administration of risperidone (0.5 mg/kg) prevented death in the animal model with the 5-HT syndrome in our previous study (Nisijima et al., 2000), a 0.5 mg/kg dose of risperidone was used. The doses of ketanserin (i.e., 5 mg/kg) and ritanserin (i.e., 3 mg/kg) were selected on the basis of our previous study results (Nisijima et al., 2001). The 1 mg/kg dose of R-96544 was selected on the basis of a report by Ogawa et al. (2002). The 2.5 mg/kg and 3 mg/kg doses of SB 206553 and SB 242084, respectively, were determined on the basis of the reports by Uphouse et al. (2003) and Wolff and Leander (2000), respectively. The 1 mg/kg dose of WAY-100635 was selected on the basis of our previous study results (Nisijima et al., 2001). Since haloperidol blocks the D2 receptor, similar to risperidone (Schotte et al., 1996), a 0.5 mg/kg dose of haloperidol was selected. The 0.5 and 50 mg/kg doses of SCH 23390 and sulpiride, respectively, were selected on the basis of Bronstein’s report (1995). A 0.5 mg/kg dose of L-741626 was determined on the basis of Blessing’s report (2005). 2.3. Measurement of rectal temperature Rectal temperature of the rats was measured every 30 min. The rats were gently restrained while their body temperature was measured. A thermocouple probe connected to a digital thermometer (Shibaura Electronics Co., Tokyo, Japan) was inserted 6 cm into the rectum, and a steady temperature readout was obtained within 10 s of probe insertion. 2.4. Microdialysis study The DA and 5-HT levels in the rat anterior hypothalamus were measured using a previously described microdialysis method
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(Shioda et al., 2004). In brief, a straight-type cellulose dialysis tubing (length, 1.0 mm; internal diameter, 0.16 mm; molecular weight cutoff, 50,000) was slowly and gently inserted into the right anterior hypothalamus (AP, 1.1. mm; ML, 0.9 mm; DV, 9.2 mm), and fixed in position with acrylic dental cement and three skull screws. The coordinates were selected in relation to the bregma according to the anatomy atlas of Paxinos and Watson (1986). Two days after the surgery, the probe was connected to a microinfusion pump, and the freely moving rats were perfused with Ringer’s solution (147 mM NaCl, 4 mM KCl, and 1.9 mM CaCl2) at a flow rate of 2 mL/min. The perfusate from the right anterior hypothalamic region was collected every 30 min, injected into a high-performance liquid chromatography (HPLC) unit by using an automatic injector (EICOM AS-100; EICOM, Kyoto, Japan), and the DA and 5-HT concentrations were immediately measured. A reverse-phase column (Eicompack CA-5ODS, 150 mm 2.1 mm; EICOM, Kyoto, Japan) was used for separation. The graphite electrode was set at +0.38 V (relative to an Ag/AgCl reference electrode). The mobile phase for the measurements comprised 0.1 M phosphate buffer (pH 6.0), 18% methanol, 80 mg/L sodium 1octanesulfonate, and 50 mg/L ethylenediaminetetraacetic acid. After a 3-h stabilization period, two consecutive dialysate samples were collected to measure the baseline DA and 5-HT levels, following which MDMA (10 mg/kg) was s.c. injected. The DA and 5HT levels were then measured every 30 min. The average DA and 5-HT concentrations before the MDMA injection (two measurements performed every 30 min) were used as the baseline values (=100%). The data are expressed as percentages of the respective baseline values. The average data are shown as means standard error of means (SEMs). 2.5. Histology After the microdialysis experiments, the brains were removed and frozen at 80 8C. In every case, the position of the dialysis probe was macroscopically verified on 150-mm thick serial coronal slices. The data were excluded in the case wherein a blood-filled region was found around a probe site. The data of only those experiments wherein the probe was found to be correctly located were included. 2.6. Statistical analysis Rectal temperature changes from the baseline were calculated at 30 min in each group and statistically analyzed using analysis of variance (ANOVA) with repeated measures, followed by Fisher’s projected least significant difference (PLSD) test or the Bonferroni/ Dunn test. The average data are represented as means SEMs. 3. Results 3.1. Effect of risperidone on MDMA-induced hyperthermia at normal ambient temperature (24 8C) When the rats were s.c. injected with 10 mg/kg MDMA, their rectal temperature increased rapidly; 60 min after MDMA administration, it exceeded 41 8C and then decreased gradually. In contrast, in the group preinjected with risperidone (0.5 mg/kg), the increases in the rectal temperature at 30, 60, 90, and 120 min after MDMA administration were significantly suppressed as compared to that in the saline control group. Further, when risperidone (0.25 and 0.5 mg/kg) was intraperitoneally injected 30 min after MDMA administration, the increase in the rectal temperature from 60 min to 240 min after administration was significantly suppressed as compared to that in the control group (Fig. 1). Administration of
Fig. 1. Effect of risperidone on MDMA-induced hyperthermia (A) Saline and risperidone (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. (B) MDMA (10 mg/kg) was s.c. injected, and 30 min later, risperidone (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of risperidone (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline).
risperidone (0.5 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.4 8C (data not shown). 3.2. Effect of risperidone on MDMA-induced hyperthermia under low ambient temperature (20 8C) conditions When the rats were s.c. injected with 10 mg/kg MDMA, their rectal temperature initially decreased at 30 min after MDMA administration and then gradually increased up to approximately 38.5 8C under low ambient temperature (20 8C) conditions. These temperature conditions significantly attenuated MDMA-induced hyperthermia as compared with that seen at normal ambient temperature (24 8C) (Fig. 2). When risperidone was administered 30 min after 10 mg/kg MDMA was injected s.c., it completely inhibited MDMA-induced hyperthermia under low ambient temperature (20 8C) conditions. 3.3. Extracellular levels of DA and 5-HT in the hypothalamus after MDMA administration and the effect of risperidone pretreatment on these levels Three rats were excluded because a blood-filled region was found around a probe or the probe was incorrectly located. After the subcutaneous injection of 10 mg/kg MDMA, the DA and 5-HT levels in the hypothalamus increased approximately 10-
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and 50-fold, respectively. In contrast to the findings of the control group, risperidone pretreatment significantly suppressed the increases in both the DA and 5-HT levels (Fig. 3). 3.4. Effect of different types of 5-HT receptor antagonists on MDMAinduced hyperthermia
Fig. 2. Effect of risperidone on MDMA-induced hyperthermia under low ambient temperature conditions (20 8C) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, saline and risperidone (0.5 mg/kg) were injected intraperitoneally at normal and low ambient temperatures (24 and 20 8C, respectively). The statistical differences among the groups are indicated as follows: 60 min, 90 min (p < 0.0001) and 120 min (p < 0.01) (saline + 10 mg/kg MDMA group, 20 8C vs. 0.5 mg/kg risperidone + 10 mg/kg MDMA group, 20 8C); 30 min (p < 0.0001), 60 min (p < 0.001), and 90 min (p < 0.05) (0.5 mg/kg risperidone + 10 mg/kg MDMA group, 24 8C vs. 0.5 mg/kg risperidone + 10 mg/kg MDMA group, 20 8C).
Fig. 3. Extracellular levels of (A) 5-HT and (B) DA in the hypothalamus after MDMA administration and the effect of risperidone pretreatment on these levels Saline and risperidone (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. The average DA and 5-HT concentrations before the MDMA injection (2 measurements performed every 30 min) were used as the baseline values (=100%). The DA and 5-HT values are represented as mean % of control SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline), and ****p < 0.0001 (vs. saline).
Pretreatment with ketanserin (5 mg/kg) and ritanserin (3 mg/ kg), which exert antagonistic effects on the 5-HT2A receptor, significantly prevented MDMA-induced hyperthermia. Pretreatment with R-96544 (1 mg/kg), a 5-HT2A receptor antagonist, also significantly prevented MDMA-induced hyperthermia (Fig. 4A). Post-treatment with ritanserin (3 mg/kg) significantly reversed MDMA-induced hyperthermia. A low dose of ritanserin (1.5 mg/kg) also significantly reversed MDMA-induced hyperthermia. Ritanserin was assumed to reverse MDMAinduced hyperthermia in a dose-dependent manner (Fig. 4B). Administration of ritanserin (3 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.4 8C (data not shown). However, pretreatment with SB 242084 (2.5 mg/kg), a 5-HT2C receptor antagonist, and SB 206553 (3.0 mg/kg), a 5-HT2B/2C receptor antagonist, did not prevent MDMA-induced hyperthermia (Fig. 5). Pretreatment with WAY-100635 (1 mg/kg), a 5-HT1A
Fig. 4. Effect of 5-HT2A antagonists on MDMA-induced hyperthermia (A) Saline, ritanserin (3 mg/kg), ketanserin (5 mg/kg), and R-96544 (1 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline). (B) MDMA (10 mg/kg) was injected s.c., and 30 min later, ritanserin (1.5 and 3 mg/kg) was intraperitoneally injected. For clarity, the results of the administration of ritanserin (3 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline).
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Fig. 5. Effects of the 5-HT2C antagonist SB 242084 and the 5-HT2B/2C antagonist SB 206553 on MDMA-induced hyperthermia Saline, SB242084 (2.5 mg/kg), and SB206553 (3 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs.
receptor antagonist, also did not suppress MDMA-induced hyperthermia (Fig. 6). 3.5. Effect of different types of DA receptor antagonists on MDMAinduced hyperthermia
Fig. 7. Effects of the pretreatments with haloperidol and SCH 23390 on MDMAinduced hyperthermia Saline, haloperidol (0.5 mg/kg), and SCH 23390 (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline).
significantly reversed MDMA-induced hyperthermia. Administration of SCH 23390 (0.5 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.8 8C (data not shown).
Pretreatment with haloperidol (0.5 mg/kg), which exerts potential antagonistic effects on the D2 and D1 receptors, significantly prevented MDMA-induced hyperthermia. Pretreatment with SCH 23390 (0.5 mg/kg), a selective D1 receptor antagonist, also significantly prevented MDMA-induced hyperthermia (Fig. 7). Post-treatment with haloperidol (0.5 mg/kg) significantly reversed MDMA-induced hyperthermia. Administration of haloperidol (0.5 mg/kg) alone exhibited no effect on the rectal temperature of the rats (data not shown). A low dose of haloperidol (0.25 mg/kg) also significantly reversed MDMA-induced hyperthermia (Fig. 8A). Haloperidol was assumed to reverse MDMA-induced hyperthermia in a dose-dependent manner. Post-treatment with SCH 23390 (0.5 mg/kg) significantly reversed MDMA-induced hyperthermia. (Fig. 8B). A low dose of SCH 23390 (0.25 mg/kg) also
Fig. 6. Effect of the 5-HT1A antagonist WAY-100635 on MDMA-induced hyperthermia Saline and WAY-100635 (1.0 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs.
Fig. 8. Effects of the post-treatments with haloperidol and SCH 23390 on MDMAinduced hyperthermia (A) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, haloperidol (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of haloperidol (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline). (B) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, SCH 23390 (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of SCH 23390 (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline).
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Fig. 9. Effect of D2 receptor antagonists on MDMA-induced hyperthermia saline, sulpiride (50 mg/kg), and L-741626 (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was s.c. injected. Values are represented as means SEMs.
Pretreatment with sulpiride (50 mg/kg) and L-741626 (0.5 mg/ kg), which are selective D2 receptor antagonists, did not prevent MDMA-induced hyperthermia (Fig. 9). 4. Discussion No established pharmacological treatment for MDMA-induced hyperthermia is available thus far. A muscle relaxant, dantrolene, has been used in some cases. Although treatment with dantrolene has been recommended (Kunitz et al., 2003; Mallick and Bodenham, 1997; Singarajan and Lavies, 1992; Singarajan and Lavies, 1992), its effectiveness remains controversial (Hall and Henry, 2006; Rusyniak and Sprague, 2005). In previous animal experiments, an atypical antipsychotic drug, clozapine (Blessing et al., 2003); a gamma-aminobutyric acid type B receptor (GABAB) agonist, baclofen (Bexis et al., 2004); and an a1 and b1,2,3 adrenergic receptor antagonist, carvedilol (Sprague et al., 2005) have also been reported to be effective against MDMA-induced hyperthermia. On the basis of our previous findings (Nisijima et al., 2000), we hypothesized that risperidone would be effective against MDMA-induced hyperthermia. Further, we demonstrated that risperidone prevents as well as reverses MDMA-induced hyperthermia in an animal model. Risperidone is an atypical antipsychotic drug with a potent 5HT2A receptor blocking effect. Since it blocks other 5-HT receptor subtypes also (Schmidt et al., 2001), we attempted to identify the 5HT receptor subtype that plays the most important role in inhibiting MDMA-hyperthermia. MDMA-induced hyperthermia in rats was found to be significantly prevented by other 5-HT2A receptor antagonists, including ketanserin, ritanserin, and R-96544. Furthermore, ritanserin reversed MDMA-induced hyperthermia. However, SB 206553 (a 5-HT2B/2C receptor antagonist), SB 242084 (a 5-HT2C receptor antagonist), and WAY-100635 (a 5HT1A receptor antagonist) did not prevent MDMA-induced hyperthermia. These results suggest that the inhibitory effect of risperidone on MDMA-induced hyperthermia is mainly due to its potent 5-HT2A receptor antagonism. The 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induces hyperthermia in animals (Mazzola-Pomietto et al., 1995). Moreover, Herin et al. (2005) reported that the selective 5-HT2A receptor antagonist M 100907 prevents MDMA-induced hyperthermia in rats. The findings of these two studies are consistent with those of the present study. DA, as well as 5-HT, is known to be involved in the central control of thermoregulation (Yamada et al., 1988; Yamawaki et al., 1983). In the present study, we investigated the effects of several DA
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antagonists on MDMA-induced hyperthermia. Haloperidol and the D1 receptor antagonist SCH 23390 significantly attenuated and reversed MDMA-induced hyperthermia, but the D2 receptor antagonists – sulpiride and L-741626 – did not attenuate MDMAinduced hyperthermia at all. Although haloperidol is a typical antipsychotic drug with a potent D2 receptor blocking effect, it also has a relatively strong D1 receptor blocking effect (Schmidt et al., 2001). In fact, its inhibitory effect on MDMA-induced hyperthermia may be due to its D1 receptor blocking effect. These results suggest that the induction of hyperthermia by MDMA may be related to the activities of both the 5-HT2A and D1 receptors. Risperidone exerts a weak antagonistic effect on the D1 receptor (Ki = 75 nM) as compared to haloperidol (Ki = 25 nM) (Bymaster et al., 1996). This suggests that MDMA-induced hyperthermia may not only be inhibited by the D1 receptor blocking effect of risperidone but also by its potent 5-HT2A receptor blocking effect. The acute toxicity of MDMA is considered to be due to the release of a neurotransmitter by the 5-HT and other neurons, including the dopaminergic neurons (Parrott, 2002). In fact, in vivo microdialysis experiments demonstrate that MDMA increases the extracellular levels of 5-HT and DA in several regions of the rat brain; however, the effects of MDMA on 5-HT are greater than on DA (Gough et al., 2002; Mechan et al., 2002). In the present study, we found that the 5-HT and DA levels in the anterior hypothalamus of the rat increased 50- and 10-fold, respectively, after the subcutaneous administration of 10 mg/kg MDMA. The rate of increase in the DA and 5-HT levels appeared to be markedly higher in the present study than in previous studies. The changes in the 5HT and DA levels in the anterior hypothalamus have not been measured previously. However, in the present study, the increase in the 5-HT levels was observed to be greater than that in the DA levels, which corroborates the previously reported findings. As compared to the increases in the 5-HT and DA levels in the control group, those in the group pretreated with risperidone were significantly suppressed. Ago et al. (2007) reported that ritanserin suppressed methamphetamine-induced 5-HT release in the prefrontal cortex. They assumed that activation of the 5-TH2A receptor might lead to an enhancement in the methamphetamine-induced 5HT release. However, in the present study, the drug investigated by us (i.e., MDMA) and the region in which the DA and 5-HT levels were measured (i.e., the anterior hypothalamus) differed from those considered in the study conducted by Ago et al. The MDMA-induced increase in the 5-HT level was suppressed by pretreatment with risperidone may be due to the potent antagonistic effect of risperidone on the 5-HT2A receptor. However, the exact underlying mechanism remains unclear. Nash (1990) and Schmidt et al. (1992) reported that 5-HT2A receptor antagonists, including ketanserin and MDL 100907, reduce MDMA-induced DA release in the striatum. Activation of 5-HT2A receptors has been reported to increase DA synthesis in the brain (Huang and Nichols, 1993). These previous findings are consistent with our results; the MDMA-induced increase in the DA levels was attenuated by risperidone pretreatment. Our experiments demonstrated that the MDMA-induced increase in body temperature at a low ambient temperature (20 8C) was less than that at normal ambient temperature (24 8C). Our study results confirm those of the previous studies (Gordon et al., 1991; Green et al., 2005), and our result is reasonable because acute MDMA injection has been shown to inhibit the normal heatloss mechanism in rats (Mechan et al., 2002). Moreover, risperidone significantly reversed MDMA-induced hyperthermia in rats at normal ambient temperature (24 8C) and completely inhibited it at a low ambient temperature (20 8C). These results indicate that body cooling together with risperidone administration may be more effective against MDMA-induced hyperthermia in humans.
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In conclusion, risperidone attenuates and reverses the MDMAelicited increase in body temperature of rats. The results of this study suggest that risperidone inhibits MDMA-induced hyperthermia primarily by blocking the activities of the 5-HT2A receptors. In addition, hyperthermia may be inhibited, in part, by the blocking of the D1 receptor by risperidone. Risperidone is used as an atypical antipsychotic drug worldwide. Our results indicate risperidone to be a potentially useful drug for treating MDMA-induced acute hyperthermia in humans. Conflict of interest We carried out all experiments without company support. References Ago Y, Nakamura S, Kajita N, Uda M, Hashimoto H, Baba A, et al. Ritanserin reverses repeated methamphetamine-induced behavioral and neurochemical sensitization in mice. Synapse 2007;61:757–63. Bexis S, Benjamin BD, Ong J, White JM, Irvine RJ. Baclofen prevents MDMA-induced rise in core body temperature in rats. Drug Alcohol Depend 2004;74:89–96. Blessing WW. Clozapine increases cutaneous blood flow and reduced sympathetic cutaneous vasomotor altering response (SCVARs) in rats: comparison with effects of haloperidol. Psychopharmacology 2005;181:518–28. Blessing WW, Seaman B, Pedersen NP, Ootsuka Y. Clozapine reverses hyperthermia and sympathetically mediated cutaneous vasoconstriction induced by 3,4-methylenedioxymethamphetamine (Ecstasy) in rabbits and rats. J Neurosci 2003;23:6385– 91. Bronstein DM, Hong JS. Effects of sulpiride and SCH 23390 on methamphetamineinduced changes in body temperature and lethality. J Pharmacol Exp Ther 1995;274:943–50. Bymaster FP, Calligaro DO, Falcone JF, Marsh RD, Moore NA, Tye NC, et al. Radioreceptor binding profile of the atypical antipsychotic olanzapine. Neuropsychopharmacology 1996;14(2):87–96. Dar KJ, McBrien ME. MDMA induced hyperthermia: report of a fatality and review of current therapy. Intens Care Med 1996;22:995–6. Dowsett RP. Deaths attributed to ‘‘ecstasy’’ overdose. Med J Aust 1996;164:700. Freedman RR, Johanson CE, Tancer ME. Thermoregulatory effects of 3,4-methylenedioxymethamphetamine (MDMA) in humans. Psychopharmacology 2005;183: 248–56. Gill JR, Hayes JA, deSouza IS, Marker E, Stajic M. Ecstasy (MDMA) deaths in New York City: a case series and review of the literature. J Forensic Sci 2002;47:121–6. Gillman PK. Monoamine oxidase inhibitors, opioid analgesics and serotonin toxicity. Br J Anaesth 2005;95:434–41. Gordon CJ, Watkinson WP, O’Callaghan JP, Miller DB. Effects of 3,4-methylenedioxymethamphetamine on autonomic thermoregulatory responses of the rat. Pharmacol Biochem Behav 1991;38:339–44. Gough B, Iman SZ, Blough B, Slikker W, Ali S. Comparative effects of substituted amphetamines (PMA, MDMA, and METH) on monoamines in rat caudate: a microdialysis study. Ann NY Acad Sci 2002;965:410–20. Green AR, O’Shea E, Saadat KS, Elliott JM, Colado MI. Studies on the effect of MDMA (‘ecstasy’) on the body temperature of rats housed at different ambient room temperatures. Br J Pharmacol 2005;146:306–12. Hall AP, Henry JA. Acute toxic effects of ‘Ecstasy’ (MDMA) and related compounds: overview of pathophysiology and clinical management. Br J Anaesth 2006;96: 678–85. Harvey JA, Welsh SE, Hood H, Romano AG. Effect of 5-HT2 receptor antagonists on a cranial nerve reflex in the rabbit: evidence for inverse agonism. Psychopharmacology (Berl) 1999;141:162–8. Herin DV, Liu S, Ullrich T, Rice KC, Cunningham KA. Role of the serotonin 5-HT2A receptor in the hyperlocomotive and hyperthermic effects of (+)-3,4-methylenedioxymethamphetamine. Psychopharmacology 2005;178:505–13. Huang X, Nichols DE. 5-HT2 receptor-mediated potentiation of dopamine synthesis and central serotonergic deficits. Eur J Pharmacol 1993;238:291–6. Johansson L, Sohn D, Thorberg SO, Jackson DM, Kelder D, Larsson LG, et al. The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist NAD-299. J Pharmacol Exp Ther 1997;283:216–25. Kanba S, Suzuki E, Nomura S, Nakaki T, Yagi G, Asai M, et al. Affinity of neuroleptics for D1 receptor of human brain striatum. J Psychiatry Neurosci 1994;19:265–9. Kankaanpaa A, Meririnne E, Lillsunde P, Seppala T. The acute effects of amphetamine derivatives on extracellular serotonin and dopamine levels in rat nucleus accumbens. Pharmacol Biochem Behav 1998;59:1003–9. Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, et al. In vitro and in vivo profile of SB 206553 a potent 5-HT2C/5-HT2B receptor antagonist with anxiolyticlike properties. Br J Pharmacol 1996;117:427–34. Kennett GA, Wood MD, Bright F, Trail B, Riley G, Holland V, et al. SB 242084 a selective and brain penetrant 5-HT2C receptor antagonist. Neuropharmacology 1997;36:609–20. Kunitz O, Ince A, Kuhlen R, Rossaint R. Hyperpyrexia and rhabdomyolysis after ecstasy (MDMA) intoxication]. Anaesthesist 2003;52:511–5.
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NeuroToxicology
Risperidone attenuates and reverses hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) in rats Katsutoshi Shioda a,c,*, Koichi Nisijima a, Tatsuki Yoshino b, Kyoko Kuboshima a, Tatsunori Iwamura d, Kunio Yui e, Satoshi Kato a a
Department of Psychiatry, Jichi Medical University, Tochigi, Japan Department of Hospital Pharmacy, Jichi Medical University, Tochigi, Japan Tamura city Miyakozi Clinic, Japan d Matsuyama University, Japan e Ashiya University Graduate School, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 November 2007 Accepted 22 July 2008 Available online 5 August 2008
3,4-Methylenedioxymethamphetamine (MDMA, ‘‘ecstasy’’) is a widely used recreational drug. Despite an increase in the number of fatalities related to its use, no definite therapeutic method has been established thus far. In the present study, risperidone’s ability to attenuate MDMA-induced hyperthermia and its mechanism of action were investigated in rats. The pharmacological effect of MDMA was evaluated using microdialysis. In the body temperature experiment, administration of risperidone before and after MDMA administration significantly suppressed MDMA-induced hyperthermia in a dose-dependent fashion. Furthermore, risperidone completely inhibited MDMA-induced hyperthermia at a low ambient temperature. Moreover, pretreatment with ritanserin, ketanserin, or R-96544, all of which are 5-HT2Areceptor antagonists, significantly prevented MDMA-induced hyperthermia. On the other hand, pretreatment with WAY-100635 (a 5-HT1A receptor antagonist), SB 206553 (a 5-HT2B/2C receptor antagonist), or SB 242084 (a 5-HT2C receptor antagonist) did not prevent MDMA-induced hyperthermia. Pretreatment with haloperidol, which blocks the dopamine (DA) receptors D2 and D1, significantly prevented MDMA-induced hyperthermia. However, sulpiride and L-741626, which are D2 receptor blockers, did not prevent MDMA-induced hyperthermia. Pretreatment with SCH 23390 (a D1 receptor antagonist) significantly prevented MDMA-induced hyperthermia. Furthermore, postadministration of ritanserin, haloperidol, and SCH23390 reversed MDMA-induced hyperthermia. These results demonstrate that the mechanism underlying the suppression of MDMA-induced hyperthermia by risperidone is primarily based on the drug’s potent 5-HT2A receptor blocking effect, and to a lesser extent, on its D1 receptor blocking effect. A microdialysis study showed that when MDMA (10 mg/kg) was subcutaneously (s.c.) injected into the rats, the DA and serotonin (5-HT) levels in the anterior hypothalamus of the rats increased approximately 10- and 50-fold, respectively, as compared to their preadministration levels. These increases in the DA and 5-HT levels after MDMA injection were significantly suppressed by pretreatment with risperidone (0.5 mg/kg). This suggested that both the DA and 5-HT systems were involved in the induction of hyperthermia by MDMA. Taken together, the present study’s results indicate that risperidone may be an effective drug for the treatment of MDMA-induced hyperthermia in humans. ß 2008 Elsevier Inc. All rights reserved.
Keywords: MDMA Dopamine Serotonin Microdialysis Risperidone
1. Introduction 3,4-Methylenedioxymethamphetamine (MDMA, ‘‘ecstasy’’) is a widely used recreational drug, and its use has become a major health
* Corresponding author at: Department of Psychiatry, Yakushiji Shimotuke-Shi, Tochigi-Ken 329-0498, Japan. Tel.: +81 285 58 7364; fax: +81 285 44 6198. E-mail address: [email protected] (K. Shioda). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.07.005
issue (Gill et al., 2002; Schifano et al., 2003). The adverse effects of the use of MDMA include incidences of tachycardia, diaphoresis, tremor, hypertension, arrhythmia, and rhabdomyolysis (Hall and Henry, 2006). However, the worst outcome of MDMA ingestion is hyperthermia and the associated serotonin (5-HT) syndrome (Mueller and Korey, 1998; Parrott, 2001). There have been an increasing number of reports on MDMA-induced toxicity accompanied by symptoms of the 5-HT syndrome (Hall and Henry, 2006; Parrott, 2002; Silins et al., 2007). In addition, reports of the
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occurrence of 5-HT syndrome when MDMA is taken together with antidepressants have been increasing; this is because of the increasing use of antidepressants (SSRI, MAO inhibitor, SNRI, etc.; Gillman, 2005; Vuori et al., 2003). In patients with the 5-HT syndrome, hyperthermia can sometimes be fatal (Dowsett, 1996; O’Connor, 1994). In such cases, the chances of survival are very weak if the peak core temperature exceeds 42 8C (Dar and McBrien, 1996). The basic treatment for MDMA-induced hyperthermia includes body temperature reduction and fluid replacement; however, no definite therapeutic method has been established thus far. We have previously reported that prior administration of risperidone prevented hyperthermia and death in an animal model with serotonin syndrome induced by simultaneous administration of 5-hydroxy-L-tryptophan (a 5-HT precursor) and clorgyline (a MAO inhibitor) (Nisijima et al., 2000). We therefore hypothesized that risperidone could prevent MDMA-induced hyperthermia. It is reported that ambient temperature influences MDMA-induced hyperthermia in animals (Gordon et al., 1991; Green et al., 2005) and humans (Freedman et al., 2005). We attempted to demonstrate that a low ambient temperature increases risperidone’s ability to inhibit MDMA-induced hyperthermia. Risperidone is an atypical antipsychotic drug that weakly blocks the activity of the dopamine (DA) D1 receptor and potentially blocks the activities of the D2 and 5-HT2A receptors (Leysen et al., 1988); it also antagonizes the activities of other 5-HT receptor subtypes. In the present study, risperidone’s ability to suppress MDMA-induced hyperthermia was evaluated by performing an experiment using an animal model. It has been suggested that MDMA causes the release of 5HT as well as other neurotransmitters, including DA (Kankaanpaa et al., 1998; White et al., 1996). Body temperature is regulated by 5-HT and DA in the anterior hypothalamus (Yamawaki et al., 1983). We attempted to identify specific 5-HT and DA receptors associated with the induction of hyperthermia by MDMA by administering various 5-HT and DA receptor antagonists prior to MDMA exposure. The rats were injected with MDMA, and microdialysis was used to measure changes in the 5-HT and DA concentrations in the anterior hypothalamus, which plays an important role in the regulation of body temperature. 2. Materials and methods 2.1. Methods All experimental procedures involving animals were approved by the Animal Investigation Committee of our institution and were in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. 2.2. Animals, drug administration, and experimental protocol Male Wistar rats (Clea Japan Inc., Tokyo, Japan) weighing 200– 250 g were used in this study. The rats were housed in cages at 23 1 8C under a 12-h light/dark cycle and were provided free access to food and water. Risperidone (5-HT1A, Ki = 490 nM; 5-HT2A, Ki = 0.6 nM; 5-HT2B, pKi = 7.67; 5-HT2C, pKi = 8.31; D1, Ki = 75 nM; D2, Ki = 3 nM) (Bymaster et al., 1996; Wood et al., 2006), ketanserin (5-HT2A, Ki = 0.38 nM) (Harvey et al., 1999), ritanserin (5-HT2A, Ki = 0.24 nM) (Harvey et al., 1999), WAY-100635 (5-HT1A, Ki = 0.24 nM) (Johansson et al., 1997), SCH 23390 (D1, Ki = 0.76 nM) (Kanba et al., 1994) SB 206553 (5-HT2B, pKi = 8.89; 5-HT2C, pKi = 7.92) (Kennett et al., 1996), SB 242084 (5-HT2C, pKi = 9.0) (Kennett et al., 1997), and L-741626 (D2, pKi = 8.4) (Millan et al., 2000) were purchased from Sigma–Aldrich Co. (Saint Louis, USA). R-96544 (D1, Ki = 1.6 nM; Tocris data) was purchased from Tocris Cookson Ltd. (Bristol, UK). Haloperidol (D1, Ki = 25 nM; D2, Ki = 1 nM) (Bymaster et al., 1996) and sulpiride (D2, Ki = 34 nM) (Kanba
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et al., 1994) were obtained in injection ampoules from Dainippon Sumitomo Pharmaceutical Co., Osaka, Japan, and Astellas Pharma Inc., Tokyo, Japan, respectively. MDMA was synthesized by our collaborating investigator, T. Iwamura—a specialist in synthetic chemistry. Risperidone was dissolved in HCl, and the pH was adjusted in the range of 6–7 by using NaOH. Ritanserin was dissolved in 99.7% acetic acid, and the pH was adjusted in the range of 6–7 by using NaOH. L741626 was dissolved in 50% dimethyl sulfoxide (DMSO) in 0.9% saline. The other drugs were dissolved in 0.9% saline. A volume of 2 mL/kg of all the drugs was injected intraperitoneally into the rats. On the day of the experiment, the rats were placed in individual cages in a room maintained at 24 8C or 20 8C ambient temperature. The rectal temperature of the rats was measured several times, and when the temperature was observed to be stable for approximately 2 h, saline, risperidone (0.5 mg/kg), ketanserin (5 mg/kg), ritanserin (3 mg/kg), WAY-00635 (1 mg/kg), haloperidol (0.5 mg/kg), sulpiride (50 mg/kg), SCH 23390 (0.5 mg/kg), SB 206553 (2.5 mg/kg), SB 242084 (3 mg/kg), L-741626 (0.5 mg/kg), and R-96544 (1 mg/kg) were intraperitoneally injected into the rats. After 15 min, MDMA (10 mg/kg) was injected subcutaneously (s.c.). Thereafter, the rectal temperature was measured every 30 min (preadministration experiment). In the postadministration experiment, MDMA (10 mg/kg) was s.c. injected, and 30 min later, risperidone (0.25 and 0.5 mg/kg), haloperidol (0.25 and 0.5 mg/kg), SCH 23390 (0.25 and 0.5 mg/kg), and ritanserin (1.5 and 3.0 mg/kg) were injected intraperitoneally at 24 8C ambient temperature. In the experiment performed to investigate the effect of a drug on rectal temperature, saline was s.c. injected, and 30 min later, risperidone (0.5 mg/kg), haloperidol (0.5 mg/kg), SCH 23390 (0.5 mg/kg), ritanserin (3 mg/kg), and saline were injected intraperitoneally. In the experiment performed under low ambient temperature conditions, risperidone (0.5 mg/kg) and saline were intraperitoneally injected, and 30 min later, MDMA (10 mg/kg) was injected s.c. at 20 8C ambient temperature. The drug doses were selected as follows. Since prior administration of risperidone (0.5 mg/kg) prevented death in the animal model with the 5-HT syndrome in our previous study (Nisijima et al., 2000), a 0.5 mg/kg dose of risperidone was used. The doses of ketanserin (i.e., 5 mg/kg) and ritanserin (i.e., 3 mg/kg) were selected on the basis of our previous study results (Nisijima et al., 2001). The 1 mg/kg dose of R-96544 was selected on the basis of a report by Ogawa et al. (2002). The 2.5 mg/kg and 3 mg/kg doses of SB 206553 and SB 242084, respectively, were determined on the basis of the reports by Uphouse et al. (2003) and Wolff and Leander (2000), respectively. The 1 mg/kg dose of WAY-100635 was selected on the basis of our previous study results (Nisijima et al., 2001). Since haloperidol blocks the D2 receptor, similar to risperidone (Schotte et al., 1996), a 0.5 mg/kg dose of haloperidol was selected. The 0.5 and 50 mg/kg doses of SCH 23390 and sulpiride, respectively, were selected on the basis of Bronstein’s report (1995). A 0.5 mg/kg dose of L-741626 was determined on the basis of Blessing’s report (2005). 2.3. Measurement of rectal temperature Rectal temperature of the rats was measured every 30 min. The rats were gently restrained while their body temperature was measured. A thermocouple probe connected to a digital thermometer (Shibaura Electronics Co., Tokyo, Japan) was inserted 6 cm into the rectum, and a steady temperature readout was obtained within 10 s of probe insertion. 2.4. Microdialysis study The DA and 5-HT levels in the rat anterior hypothalamus were measured using a previously described microdialysis method
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(Shioda et al., 2004). In brief, a straight-type cellulose dialysis tubing (length, 1.0 mm; internal diameter, 0.16 mm; molecular weight cutoff, 50,000) was slowly and gently inserted into the right anterior hypothalamus (AP, 1.1. mm; ML, 0.9 mm; DV, 9.2 mm), and fixed in position with acrylic dental cement and three skull screws. The coordinates were selected in relation to the bregma according to the anatomy atlas of Paxinos and Watson (1986). Two days after the surgery, the probe was connected to a microinfusion pump, and the freely moving rats were perfused with Ringer’s solution (147 mM NaCl, 4 mM KCl, and 1.9 mM CaCl2) at a flow rate of 2 mL/min. The perfusate from the right anterior hypothalamic region was collected every 30 min, injected into a high-performance liquid chromatography (HPLC) unit by using an automatic injector (EICOM AS-100; EICOM, Kyoto, Japan), and the DA and 5-HT concentrations were immediately measured. A reverse-phase column (Eicompack CA-5ODS, 150 mm 2.1 mm; EICOM, Kyoto, Japan) was used for separation. The graphite electrode was set at +0.38 V (relative to an Ag/AgCl reference electrode). The mobile phase for the measurements comprised 0.1 M phosphate buffer (pH 6.0), 18% methanol, 80 mg/L sodium 1octanesulfonate, and 50 mg/L ethylenediaminetetraacetic acid. After a 3-h stabilization period, two consecutive dialysate samples were collected to measure the baseline DA and 5-HT levels, following which MDMA (10 mg/kg) was s.c. injected. The DA and 5HT levels were then measured every 30 min. The average DA and 5-HT concentrations before the MDMA injection (two measurements performed every 30 min) were used as the baseline values (=100%). The data are expressed as percentages of the respective baseline values. The average data are shown as means standard error of means (SEMs). 2.5. Histology After the microdialysis experiments, the brains were removed and frozen at 80 8C. In every case, the position of the dialysis probe was macroscopically verified on 150-mm thick serial coronal slices. The data were excluded in the case wherein a blood-filled region was found around a probe site. The data of only those experiments wherein the probe was found to be correctly located were included. 2.6. Statistical analysis Rectal temperature changes from the baseline were calculated at 30 min in each group and statistically analyzed using analysis of variance (ANOVA) with repeated measures, followed by Fisher’s projected least significant difference (PLSD) test or the Bonferroni/ Dunn test. The average data are represented as means SEMs. 3. Results 3.1. Effect of risperidone on MDMA-induced hyperthermia at normal ambient temperature (24 8C) When the rats were s.c. injected with 10 mg/kg MDMA, their rectal temperature increased rapidly; 60 min after MDMA administration, it exceeded 41 8C and then decreased gradually. In contrast, in the group preinjected with risperidone (0.5 mg/kg), the increases in the rectal temperature at 30, 60, 90, and 120 min after MDMA administration were significantly suppressed as compared to that in the saline control group. Further, when risperidone (0.25 and 0.5 mg/kg) was intraperitoneally injected 30 min after MDMA administration, the increase in the rectal temperature from 60 min to 240 min after administration was significantly suppressed as compared to that in the control group (Fig. 1). Administration of
Fig. 1. Effect of risperidone on MDMA-induced hyperthermia (A) Saline and risperidone (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. (B) MDMA (10 mg/kg) was s.c. injected, and 30 min later, risperidone (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of risperidone (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline).
risperidone (0.5 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.4 8C (data not shown). 3.2. Effect of risperidone on MDMA-induced hyperthermia under low ambient temperature (20 8C) conditions When the rats were s.c. injected with 10 mg/kg MDMA, their rectal temperature initially decreased at 30 min after MDMA administration and then gradually increased up to approximately 38.5 8C under low ambient temperature (20 8C) conditions. These temperature conditions significantly attenuated MDMA-induced hyperthermia as compared with that seen at normal ambient temperature (24 8C) (Fig. 2). When risperidone was administered 30 min after 10 mg/kg MDMA was injected s.c., it completely inhibited MDMA-induced hyperthermia under low ambient temperature (20 8C) conditions. 3.3. Extracellular levels of DA and 5-HT in the hypothalamus after MDMA administration and the effect of risperidone pretreatment on these levels Three rats were excluded because a blood-filled region was found around a probe or the probe was incorrectly located. After the subcutaneous injection of 10 mg/kg MDMA, the DA and 5-HT levels in the hypothalamus increased approximately 10-
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and 50-fold, respectively. In contrast to the findings of the control group, risperidone pretreatment significantly suppressed the increases in both the DA and 5-HT levels (Fig. 3). 3.4. Effect of different types of 5-HT receptor antagonists on MDMAinduced hyperthermia
Fig. 2. Effect of risperidone on MDMA-induced hyperthermia under low ambient temperature conditions (20 8C) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, saline and risperidone (0.5 mg/kg) were injected intraperitoneally at normal and low ambient temperatures (24 and 20 8C, respectively). The statistical differences among the groups are indicated as follows: 60 min, 90 min (p < 0.0001) and 120 min (p < 0.01) (saline + 10 mg/kg MDMA group, 20 8C vs. 0.5 mg/kg risperidone + 10 mg/kg MDMA group, 20 8C); 30 min (p < 0.0001), 60 min (p < 0.001), and 90 min (p < 0.05) (0.5 mg/kg risperidone + 10 mg/kg MDMA group, 24 8C vs. 0.5 mg/kg risperidone + 10 mg/kg MDMA group, 20 8C).
Fig. 3. Extracellular levels of (A) 5-HT and (B) DA in the hypothalamus after MDMA administration and the effect of risperidone pretreatment on these levels Saline and risperidone (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. The average DA and 5-HT concentrations before the MDMA injection (2 measurements performed every 30 min) were used as the baseline values (=100%). The DA and 5-HT values are represented as mean % of control SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline), and ****p < 0.0001 (vs. saline).
Pretreatment with ketanserin (5 mg/kg) and ritanserin (3 mg/ kg), which exert antagonistic effects on the 5-HT2A receptor, significantly prevented MDMA-induced hyperthermia. Pretreatment with R-96544 (1 mg/kg), a 5-HT2A receptor antagonist, also significantly prevented MDMA-induced hyperthermia (Fig. 4A). Post-treatment with ritanserin (3 mg/kg) significantly reversed MDMA-induced hyperthermia. A low dose of ritanserin (1.5 mg/kg) also significantly reversed MDMA-induced hyperthermia. Ritanserin was assumed to reverse MDMAinduced hyperthermia in a dose-dependent manner (Fig. 4B). Administration of ritanserin (3 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.4 8C (data not shown). However, pretreatment with SB 242084 (2.5 mg/kg), a 5-HT2C receptor antagonist, and SB 206553 (3.0 mg/kg), a 5-HT2B/2C receptor antagonist, did not prevent MDMA-induced hyperthermia (Fig. 5). Pretreatment with WAY-100635 (1 mg/kg), a 5-HT1A
Fig. 4. Effect of 5-HT2A antagonists on MDMA-induced hyperthermia (A) Saline, ritanserin (3 mg/kg), ketanserin (5 mg/kg), and R-96544 (1 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline). (B) MDMA (10 mg/kg) was injected s.c., and 30 min later, ritanserin (1.5 and 3 mg/kg) was intraperitoneally injected. For clarity, the results of the administration of ritanserin (3 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline).
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Fig. 5. Effects of the 5-HT2C antagonist SB 242084 and the 5-HT2B/2C antagonist SB 206553 on MDMA-induced hyperthermia Saline, SB242084 (2.5 mg/kg), and SB206553 (3 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs.
receptor antagonist, also did not suppress MDMA-induced hyperthermia (Fig. 6). 3.5. Effect of different types of DA receptor antagonists on MDMAinduced hyperthermia
Fig. 7. Effects of the pretreatments with haloperidol and SCH 23390 on MDMAinduced hyperthermia Saline, haloperidol (0.5 mg/kg), and SCH 23390 (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. saline); **p < 0.01 (vs. saline); ***p < 0.001 (vs. saline); and ****p < 0.0001 (vs. saline).
significantly reversed MDMA-induced hyperthermia. Administration of SCH 23390 (0.5 mg/kg) alone decreased the rectal temperature of the rats by approximately 0.8 8C (data not shown).
Pretreatment with haloperidol (0.5 mg/kg), which exerts potential antagonistic effects on the D2 and D1 receptors, significantly prevented MDMA-induced hyperthermia. Pretreatment with SCH 23390 (0.5 mg/kg), a selective D1 receptor antagonist, also significantly prevented MDMA-induced hyperthermia (Fig. 7). Post-treatment with haloperidol (0.5 mg/kg) significantly reversed MDMA-induced hyperthermia. Administration of haloperidol (0.5 mg/kg) alone exhibited no effect on the rectal temperature of the rats (data not shown). A low dose of haloperidol (0.25 mg/kg) also significantly reversed MDMA-induced hyperthermia (Fig. 8A). Haloperidol was assumed to reverse MDMA-induced hyperthermia in a dose-dependent manner. Post-treatment with SCH 23390 (0.5 mg/kg) significantly reversed MDMA-induced hyperthermia. (Fig. 8B). A low dose of SCH 23390 (0.25 mg/kg) also
Fig. 6. Effect of the 5-HT1A antagonist WAY-100635 on MDMA-induced hyperthermia Saline and WAY-100635 (1.0 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was injected s.c. Values are represented as means SEMs.
Fig. 8. Effects of the post-treatments with haloperidol and SCH 23390 on MDMAinduced hyperthermia (A) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, haloperidol (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of haloperidol (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline). (B) MDMA (10 mg/kg) was s.c. injected into the rats, and 30 min later, SCH 23390 (0.25 and 0.5 mg/kg) was injected intraperitoneally. For clarity, the results of the administration of SCH 23390 (0.5 mg/kg) alone are not shown. Values are represented as means SEMs. The statistical differences among the groups are indicated as follows: *p < 0.05 (vs. MDMA + saline); **p < 0.01 (vs. MDMA + saline); ***p < 0.001 (vs. MDMA + saline); and ****p < 0.0001 (vs. MDMA + saline).
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Fig. 9. Effect of D2 receptor antagonists on MDMA-induced hyperthermia saline, sulpiride (50 mg/kg), and L-741626 (0.5 mg/kg) were intraperitoneally injected into the rats, and 15 min later, MDMA (10 mg/kg) was s.c. injected. Values are represented as means SEMs.
Pretreatment with sulpiride (50 mg/kg) and L-741626 (0.5 mg/ kg), which are selective D2 receptor antagonists, did not prevent MDMA-induced hyperthermia (Fig. 9). 4. Discussion No established pharmacological treatment for MDMA-induced hyperthermia is available thus far. A muscle relaxant, dantrolene, has been used in some cases. Although treatment with dantrolene has been recommended (Kunitz et al., 2003; Mallick and Bodenham, 1997; Singarajan and Lavies, 1992; Singarajan and Lavies, 1992), its effectiveness remains controversial (Hall and Henry, 2006; Rusyniak and Sprague, 2005). In previous animal experiments, an atypical antipsychotic drug, clozapine (Blessing et al., 2003); a gamma-aminobutyric acid type B receptor (GABAB) agonist, baclofen (Bexis et al., 2004); and an a1 and b1,2,3 adrenergic receptor antagonist, carvedilol (Sprague et al., 2005) have also been reported to be effective against MDMA-induced hyperthermia. On the basis of our previous findings (Nisijima et al., 2000), we hypothesized that risperidone would be effective against MDMA-induced hyperthermia. Further, we demonstrated that risperidone prevents as well as reverses MDMA-induced hyperthermia in an animal model. Risperidone is an atypical antipsychotic drug with a potent 5HT2A receptor blocking effect. Since it blocks other 5-HT receptor subtypes also (Schmidt et al., 2001), we attempted to identify the 5HT receptor subtype that plays the most important role in inhibiting MDMA-hyperthermia. MDMA-induced hyperthermia in rats was found to be significantly prevented by other 5-HT2A receptor antagonists, including ketanserin, ritanserin, and R-96544. Furthermore, ritanserin reversed MDMA-induced hyperthermia. However, SB 206553 (a 5-HT2B/2C receptor antagonist), SB 242084 (a 5-HT2C receptor antagonist), and WAY-100635 (a 5HT1A receptor antagonist) did not prevent MDMA-induced hyperthermia. These results suggest that the inhibitory effect of risperidone on MDMA-induced hyperthermia is mainly due to its potent 5-HT2A receptor antagonism. The 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induces hyperthermia in animals (Mazzola-Pomietto et al., 1995). Moreover, Herin et al. (2005) reported that the selective 5-HT2A receptor antagonist M 100907 prevents MDMA-induced hyperthermia in rats. The findings of these two studies are consistent with those of the present study. DA, as well as 5-HT, is known to be involved in the central control of thermoregulation (Yamada et al., 1988; Yamawaki et al., 1983). In the present study, we investigated the effects of several DA
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antagonists on MDMA-induced hyperthermia. Haloperidol and the D1 receptor antagonist SCH 23390 significantly attenuated and reversed MDMA-induced hyperthermia, but the D2 receptor antagonists – sulpiride and L-741626 – did not attenuate MDMAinduced hyperthermia at all. Although haloperidol is a typical antipsychotic drug with a potent D2 receptor blocking effect, it also has a relatively strong D1 receptor blocking effect (Schmidt et al., 2001). In fact, its inhibitory effect on MDMA-induced hyperthermia may be due to its D1 receptor blocking effect. These results suggest that the induction of hyperthermia by MDMA may be related to the activities of both the 5-HT2A and D1 receptors. Risperidone exerts a weak antagonistic effect on the D1 receptor (Ki = 75 nM) as compared to haloperidol (Ki = 25 nM) (Bymaster et al., 1996). This suggests that MDMA-induced hyperthermia may not only be inhibited by the D1 receptor blocking effect of risperidone but also by its potent 5-HT2A receptor blocking effect. The acute toxicity of MDMA is considered to be due to the release of a neurotransmitter by the 5-HT and other neurons, including the dopaminergic neurons (Parrott, 2002). In fact, in vivo microdialysis experiments demonstrate that MDMA increases the extracellular levels of 5-HT and DA in several regions of the rat brain; however, the effects of MDMA on 5-HT are greater than on DA (Gough et al., 2002; Mechan et al., 2002). In the present study, we found that the 5-HT and DA levels in the anterior hypothalamus of the rat increased 50- and 10-fold, respectively, after the subcutaneous administration of 10 mg/kg MDMA. The rate of increase in the DA and 5-HT levels appeared to be markedly higher in the present study than in previous studies. The changes in the 5HT and DA levels in the anterior hypothalamus have not been measured previously. However, in the present study, the increase in the 5-HT levels was observed to be greater than that in the DA levels, which corroborates the previously reported findings. As compared to the increases in the 5-HT and DA levels in the control group, those in the group pretreated with risperidone were significantly suppressed. Ago et al. (2007) reported that ritanserin suppressed methamphetamine-induced 5-HT release in the prefrontal cortex. They assumed that activation of the 5-TH2A receptor might lead to an enhancement in the methamphetamine-induced 5HT release. However, in the present study, the drug investigated by us (i.e., MDMA) and the region in which the DA and 5-HT levels were measured (i.e., the anterior hypothalamus) differed from those considered in the study conducted by Ago et al. The MDMA-induced increase in the 5-HT level was suppressed by pretreatment with risperidone may be due to the potent antagonistic effect of risperidone on the 5-HT2A receptor. However, the exact underlying mechanism remains unclear. Nash (1990) and Schmidt et al. (1992) reported that 5-HT2A receptor antagonists, including ketanserin and MDL 100907, reduce MDMA-induced DA release in the striatum. Activation of 5-HT2A receptors has been reported to increase DA synthesis in the brain (Huang and Nichols, 1993). These previous findings are consistent with our results; the MDMA-induced increase in the DA levels was attenuated by risperidone pretreatment. Our experiments demonstrated that the MDMA-induced increase in body temperature at a low ambient temperature (20 8C) was less than that at normal ambient temperature (24 8C). Our study results confirm those of the previous studies (Gordon et al., 1991; Green et al., 2005), and our result is reasonable because acute MDMA injection has been shown to inhibit the normal heatloss mechanism in rats (Mechan et al., 2002). Moreover, risperidone significantly reversed MDMA-induced hyperthermia in rats at normal ambient temperature (24 8C) and completely inhibited it at a low ambient temperature (20 8C). These results indicate that body cooling together with risperidone administration may be more effective against MDMA-induced hyperthermia in humans.
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