Methamphetamine (MDMA), P-Chloroamphetamine and Fenfluramine Administration and Effects of Chlormethiazole and Dizocilpine

Br.J. Pharmacol(1993),. lOg,583-589 O MacmillanPressLtd, 1993

5-HT loss in rat brain following 3, 4-methylenedioxymethamphetamine (MDMA), p-chloroamphetamine and fenfluramine administration and effects of chlormethiazole and dizocilpine

_M.I. Colado, T.K. Murray & 2A.R. Green

Astra Neuroscience Research Unit, I Wakefield Street, London WC1N 1PJ

1 The present study has investigated whether the neurotoxic effects of the relatively selective 5- hydroxytryptamine (5-HT) neurotoxins, 3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy'), p-chloroamphetamine (PCA) and fentturamine on hippocampal and cortical 5-HT terminals in rat brain could be prevented by administration of either chlormethiazole or dizocilpine. 2 Administration of MDMA (20 mg kg-t, J.p.) resulted in an approximate 30% loss of cortical and hippocampal 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content 4 days later. Injection of chlormethiazole '(50rog kg-t) 5 rain before and 55 min after the MDMA provided complete protection in both regions, while dizocilpine (1 mgkg -t, J.p.) protected only the hippocampus. 3 Administration of a single dose of chlormethiazole (100mgkg -t) 20min after the MDMA also provided complete protection to the hippocampus but not the cortex. This regime also attenuated the sustained hYperthermia (approx + 2.5'12) induced by the MDMA injection. 4 Injection of PCA (5 mgkg -t, i.p.) resulted in a 70% loss of 5-HT and 5-HIAA content in hippocampus and cortex 4 days later. Injection of chlormethiazole (100rog kg-:, i.p.) or dizociipine (1 mg kg-t, i.p.) 5 min before and 55 rain after the PCA failed to protect against the neurotoxicity, nor was protection afforded by chlormethiazole when a lower dose of PCA (2.5 mg kg -t, i.p.) was given which produced only a 30% loss of 5-HT content. Chlormethiazole did prevent the hyperthermia induced by PCA (5 mg kg-t), while the lower dose of PCA (2.5 mg kg-t) did not produce a change in body temperature. 5 Neither chlormethiazole nor dizocilpine prevented the neurotoxic loss of hippocampal or cortical 5-HT neurones measured 4 days following administration of fentturamine (25 mg kg-z, i.p.). 6 In general, chlormethiazole and dizocilpine were effective antagonists of the 5-HT-mediated behaviours of head weaving and forepaw treading which appeared following injection of all three neurotoxins. 7 Both c..%hlrme_e and dizocilpine have previously been shown to prevent the neurotoxic effects of a high dose ofYnethamphetamine oxxcerebral 5-HT and dopamine pathways. These drugs also prevent MDMA-induced neurotoxicity of 5-HT pathways, but not that induced by injection of PCA or fenfluramine. This suggests that the mechanisms of neurotoxic damage to 5-HT pathways produced by substituted amphetamines cannot be identical. The monoamine loss does not appear to result from the hyperthermia produced by the neurotoxic compounds. Keywords: 5-Hydroxytryptamine; p-chloroamphetamine; fentturamine; 3,4-methylenedioxymethamphetamine; neurotoxicity; hyperthermia; Ecstasy; neurodegeneration; chiormethiazole; dizocilpine

Introduction

Administration of several high doses of methamphetamine 1975; Neckers et al., 1976) and 3, 4-methylenedioxymeth- induces neurotoxicity and a subsequent marked decrease in amphetamine (Schmidt, 1987), the last named drug being of dopamine (Koda & Gibb, 1973) and 5-hydroxytryptamine particular current interest because, under the name of 'Ecs- (5-HT) (Hotchkiss & Gibb, 1980) content in several regions tasy' it has become a commonly used drug of abuse (for of rodent brain. Evidence for the neuronal damage following review see McKenna & Peroutka, 1990). methamphetamine has been obtained by use of biochemical The loss in 5-HT content following all three of the more (e.g.Stone et al., 1986; 1988) and morphological (e.g. Jons- 5-HT selective neurotoxins mentioned above follows a two son & Nzanze, 1982; Ricuarto et al., 1982) techniques and phase pattern, an initial rapid release (1-2 h following injec- has been reviewed recently elsewhere (Gibb et al., 1990). tion) followed by recovery by 24 h and then a long term Other substituted amphetamines are also neurotoxic when decrease which is marked by 3-4 days and then lasts for given either as a high single dose or several lower doses several months. These changes have been reported for 3, administered over a relatively short period of time. Some of 4-methylenedioxymethamphetamine (MDMA; Schmidt, 1987), these compounds are relatively selective neurotoxins of 5-HT p-chloroamphetamine (PCJk; Sanders-Bush et al., 1972; Ful- pathways, particularly p-chloroamphetamine (Sanders-Bush let et al., 1975; Neckers et al., 1976) and fenfiuramine et al., 1972; Fuller et al., 1975), fenfluramine (Harvey et al., (Harvey et al., 1975; Neckers et al., 1976). The second phase is marked by loss of monoamine content, and loss of tryp- _ tophan hydroxylaseactivity.Recentimmunocytochemical ] Permanentaddress: Departmento de Farmacologia, Facultad de and visualization studies have reported that neurodegenera- Medicina,Universidad Complutense, Madrid 28040, Spain. tire changes are seen in the second phase and include in- 2Authorfor correspondence, creased 5-HT axon calibre, huge swollen varicosities, frag-

L _4 M.I. COLADO et al. mentfition and dilated proximal axon stumps with forebrain unto (20 x 4.6 mm, ODS, 5 lam). The analytical column was Table I q 5-HT axon terminals being lost (see review of Molliver et al.,, a reversed phase 150 x 4.6 mm, ODSI, 5 btm (Spherisorb) on the 5-1 1990). All indications are therefore that all three drugs, like and detection was by a Coulochem electrochemical cell. The -- methamphetamine (Ricuarto et al., 1982), produce neurode- mobile phase (flow rate I mi min -l) comprised sodium-l- generation and that the biochemical markers reflect the octane sulphonic acid (0.75mM), EDTA (0.1 mM), methanol Neurotoxit neurodegenerative changes. (15%) and KH2PO4 (0. I M), which had been adjusted to MDMA Recently Sonsalla and her colleagues provided evidence pH 3.0 with phosphoric acid, filtered and degassed. Stan- PCA that excitatory amino acids were involved in methampheta- dards contained known concentrations of 5-HT and 5-HIAA Fenfiurami mine-induced neurotoxicity since they found that dizocilpine in the perchlofic acid mixture. (Sonsalla et al., 1989) and other competitive and non-eom- petitive N-methyl-o-aspartate (NMDA) antagonists (Sonsalla Temperature measurement The beha_ et al., 1991)prevented methamphetamine-induced neurotox- (25mgkg- icity of striatal dopamine terminals. Johnson et al. (1989) Temperature was measured by use of a rectal probe inserted Also sho_ extended this work and showed that dizocilpine (or MK 801) 2.5 cm and a thermocouple with digital readout, acnhdlormMDI_ethi also prevented the toxic effect of methamphetamine on Different t striatal 5-HT content. Green et al. (1992) recently confirmed Behavioural assessment the protective effect of dizocilpine against methamphetamine- induced neurotoxicity of striatal dopamine and also showed The two major components of the 5-HT behavioural syn- Table 2 '1 that this drug protected against the neurotoxic loss of 5-HT drome, headweaving and forepaw treading, were scored as cortical an pathways in the cortex and hippocampus. In addition, it was described by Deakin & Green (1978) on a severity score of also shown that chlormethiazole was a very effective drug in 0-3 (O-absent, l-just present, 2-definite, 3-severe) every I0 Injected protecting against methamphetamine-induced neurodegenera- rain for 80 min. The total scores of each animals have been _ tion of striatal dopamine and hippocampal and cortical 5-HT reported. The same scoring system was used for evaluating Saline pathways (Green et al., 1992). the backward walking behaviour seen after fenfluramine. The Chlormetl This work has now been extended to examine whether observer was, where possible, blind to the treatment condi- Dizocilpin dizocilpine and chlormethiazole will protect against the loss tion although in practice this was difficult given the sedative MDMA of 5-HT content in the cortex and hippocampus produced by nature of chlormethiazole. MDMA -_ administration of PCA, MDMA and fenfiuramine. MDMA-_ In general a dose of chlormethiazole of 50 mg kg-i, i.p., Drugs The concer was given since this afforded complete protection against shown in l methamphetamine-induced neurotoxicity (Green et al., 1992). Drugs were obtained from the following (sources in brae- (i mgkg-'_ However, it should be noted that this dose is near the ED_0 kets): p-chloroamphetamine, fenfluramine and 3,4-methylene- *Significan of the drug to protect against ischaemia-induced neuro- dioxymethamphetamine (all from Sigma Chemical Co., not differe_ degeneration in the gerbil (Cross et al., 1991) and as an Poole): dizocilpine (Semat Technical (UK) Ltd, St. Albans), anticonvulsant drug (Green & Murray, 1989). The dizocilpine di-chlormethiazole ethane disulphonate (Astra Areas, S6der- dose of I mgkg -_, J.p., was again chosen because it was an t_ilje, Sweden). Drugs were dissolved in saline (0.9% w/v effective dose against methamphetamine-induced toxicity NaCI), and all doses refer to the concentration of the base. neurotoxic el (Sonsalla et al., 1989; Green et al., 1992) and is neuroprotec- All other chemicals were obtained from Sigma Chemical Co. while dizocilt tive in the gerbil model of ischaemic neurodegeneration (Gill or E. Merck (Dagenham). In the nex et al., 1987; Cross et al., 1991). of chlormett A preliminary account of some of the work has been given Statistics also be prot to the British Pharmacological Society (Colado et al., 1992). Rats were The effet of the neurotoxin on indole concentration has later injected always been compared with the saline (control) treated group kg-I). Injecti Methods using an unpaired Student's t test. The effect of drugs on the 5-HT-media_ neurotoxicity has been likewise compared with the drug quantified. B,

Animals and drug administration Mannalone group.WhitneyBehaviouralrank order data'U' havetest. been evaluated by the agaiconcentratioln protect Adult male Lister Hooded rats (Harlan Olac, Bicester) of 5-HT and weighing 200-300 g were used. They were housed in groups cortex was Il of 5 in conditions of constant temperature (21°C) and a 12 h Results not significar light/dark cycle (lights on 07 h 00 rain) and given free access chlormethiaz_ to food and water. Effect of chlormethiazole and dizocilpine on the loss of than saline-i_ All drugs were given i.p. and because various administra- 5-HT and 5-HIAA in cortex and hippocampus caused by tion protocols were used, these are detailed in the results MDMA administration Effect of chJ section. The rats were always killed 4 days following the drug neurotoxic administration for measurement of brain monoamine concen- Rats were injected with either saline, chlormethiazole (50 mg trations, kg -l) or dizocilpine (lmgkg-I). Five minutes later all In the studj groups were injected with MDMA (20mc kg-l). After a injected with_ Measurement of monoamines further 55 min, the groups were given a further injection of pine (1 mg k_ saline, chlormethiazole (50mc kg-I) or dizocilpine (1 mg After a furth Rats were killed by cervical dislocation and decapitation, the kg-_), dose of the brains removed and cortex and hippocampus dissected out. Administration of MDMA resulted in the appearance of a pine (1 mg k Tissue was homogenized in perchloric acid (0.2 M), con- behavioural syndrome which included forepaw treading and was essential _ taining sodium metabisulphite (0.1%), cysteine (0.1%) and headweaving. Pretreatment with chlormethiazole decreased 25 mg kg-L ethylenediamine tetraacetic acid (EDTA, 0.01%). Homogen- the median 5-HTdnediated behavioural score, albeit not l mgkg -_ thj ates were centrifuged at 13,000 r.p.m, for 10 min in an significantly (Table 1) also producing reduction in the Ioco- as the high d Eppendorf bench centrifuge. A 50 btl sample of the super- motor activity for around 40 rain after administration, while animals also natant was injected into the high performance liquid chroma- dizocilpine abolished both the behavioural changes and loco- Administrt tography (h.p.l.c.) system, motion (Table 1). MDMA administration produced around a weaving and The chromatographic system comprised as ACS isocratic 30% decrease in hippocampal and cortical 5-HT and 5- chlormethiaz pump and Rheodyne 7125 injector with 50 btl loop. Residual HIAA (Table 2). Chiormethiazole when given pre- and post- 1). The beha' micro-particulate material was removed by use of guard col- MDMA injection afforded complete protection against the rather differ_ CHLORMETHIAZOLE PROTECTS AGAINST MDMA TOXICITY 585

Table 1 The effect of administration of 3,4-methylenedioxymethamphetamine (MDMA), p-chloroamphetamine (PCA) or fenfluramine on the 5-hydroxytryptamine (5-HT)-mediated behaviour and the effect on the behaviour of chlormethiazole or dizocilpine Median behavioural score Neurotoxin Behaviour Injected: Saline Chlorrnethiazole Dizocilpine

MDMA 5-HT syndrome 15[ - 7, + 3] 9[ - 5, + 2] 0* PCA 5-HT syndrome 25[ - 1, + 4] 0* 3[ - 2, + 3]* Fenfiuramine 5-HT syndrome 5[ - 2, + 2] 0* 2[ - 2, + l]t Backward walking 13.5[- 2.5, + 3.5] 0* 0*

The behavioural score (see methods) of rats following administration of MDMA (20 mg kg-'), PCA (5 mg kg-I) of fenfiuramine (25 mg kg-l) are shown as a total in the 70 min following the injection of the neurotoxin as a median 4-interquartile range (n = 10). Also shown is the score of animals given either dizociipine (lmgkg -_) 5min before and 55min after the neurotoxin or chlormethiazole on the same time schedule at a dose of either 100mg kg-I (in the PCA experiment) or 50 mg kg-i in the fenfiuramine and MDMA experiments. Different from saline injected groups: *P<0.002; tP<0.05 (Mann Whitney U test).

Table 2 The effect of chlormethiazole and dizocilpine on the neurotoxic action of 3, 4-methylenedioxymethamphetamine (MDMA) on cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations Hippocampus Cortex Injected 5-HT 5-HI,4A S-HT 5-HIA.4

Saline 323 + 9 (8) 325 4- 7 (8) 314 4- 14 (8) 218 _ 17 (8) Chlormethiazole 369 4- 18 (6) 327 _+16 (6) 314 4- 21 (6) 212 4- 11 (6) Dizecilpine 371 4- 6 (6) 327 4- 13 (6) 323 4- 13 (6) 1784- 5 (6) MDMA 2344- 14 (16)* 2494- 13 (16)* 2274- 16 (16)* 1504- 7 (16)* MDMA + chlormethiazole 325 4- 13 (6) 288 4- 16 (6) 294 4- 13 (5) 1824- 10 (6) MDMA + dizocilpine 325 4- 17 (4) 328 + 12 (4) 260 4- 7 (4)_: 1784- 4 (4)_:

The concentration of 5-HT and 5-HIAA in the hippocampus and cortex of rats 4 days following injection of MDMA (20 mg kg- _) is shown in ng g-t tissue (n), together with the effect of these parameters of injection of chlormethiazole (50 mg kg-t) or dizocilpine (1 mgkg -_) given 5 min before and 55min after the MDMA. *Significantly different from saline-injected control rats, P<0.01. MDMA + chlormethiazole and MDMA + dizocilpine groups are not different from the appropriate control group in hippocampus. :[Not different from MDMA alone group.

400 neurotoxic effect of MDMA in both brain regions (Table 2) while dizocilpine only protected the hippocampus. # In the next experiment we examined whether a single dose of chlormethiazole, given shortly after the MDMA would # also be protective. Rats were injected with MDMA (20 mg kg -l) and 20 min 300 later injected with a single dose of chlormethiazole (100 mg = kg-I). Injection of chlormethiazole at this time inhibited the ._ 5-HT-mediated behaviour and locomotion but this was not -;- quantified. Both groups were killed and regional brain indole concentrations examined 4 days later. Chlormethiazole was again protective in the hippocampus as indicated by the lack § of 5-HT and 5-HIAA loss (Figure 1). The protection of the .--. 200 cortex was less clear. While the 5-HIAA concentration was _, not significantly different from control values in the MDMA/ t_ chlormethiazole treated rats, the 5-HT content was lower Oz than saline-injected animals (Figure 1). o O "O Effect of chlormethiazole and dizocilpine on the -_ loo neurotoxic effects of PCA and fenfluramine In the study on the neurotoxic effect of PCA, rats were injected with saline, chlormethiazole (100 mg kg -l) or dizocilpine (1 mg kg -l) followed 5 rain later by PCA (5 mg kg-l). After a further 55 min the animals were injected with another dose of the saline, chlormethiazole(100mgkg-')ordizocil- 1 2 3 1 2 3 1 2 3 1 2 3 pine (1 mgkg-_). In the case of fenfluramine, the protocol 5-HT S-HIAA 5-HT 5-HIAA was essentially as above with the dose of fenfluramine being Cortex Hippocampus 25 mg kg -_. However while the dizocilpine dose remained at I mg kg-_ the dose of chlormethiazole given was 50 mg kg -t Figure I The effect of a single dose of chlormethiazole (100 mg as the high dose of chlormethiazole was not well-tolerated in kg-2) given 20 rain after 3, 4-methylenedioxymethamphetamine animals also given fenfluramine. (MDMA, 20mg kg-z) on the 5-hydroxytryptamine (5-HT) and 5- Administration of PCA produced clear and marked head andhydrocxyindortexoleac4 daysetic latacider: (5-HIAA)(1) Saline;co(2)ntentMDMA;of the (3)rat MDMAhippocampusplus weaving and forepaw treading which were abolished by chlormethiazole. *Significantly different from saline injected control chlormethiazole or markedly reduced by dizocilpine (Table group, P<0.01 or better; tsignificantly different from MDMA- !/. The behavioural changes seen following fenfluramine were treated group P<0.01 or better; #not different from saline-treated ther different. There was a weak expression of head weav- group. 586 M.I. COLADO et al. ing and forepaw treading (Table l) which was reduced with Effect ofchlormethiazole on the hyperthermia produced 4o dizocilpine and abolished by chlormethiazole but the pre- by p-chloroamphetamine administration dominant behaviour was backward-walking which was abolished by both drugs (Table I). Groups of rats were injected with either saline or chlor- Neither chlormethiazole nor dizocilpine protected against methiazole (50 mg kg-l). Both groups were injected 5 min the neurotoxic consequences of fentturamine or PCA in either later with PCA (5 mg kg-_). After a further 55 min the 30 brain region, as judged by the loss of 5-HT or 5-HIAA animals were injected with, saline or chlormethiazole (50 mg content (Table 3). The decrease in 5-HT content produced by both fenfiuramine and PCA was considerably greater than that 400 _' seen after MDMA administration (Tables 2 and 3). In order F _ 33 merely because chlormethiazole and dizocilpine were ineffective against drugs producing a substantial monoamine loss, we next lowered the dose of PCA to achieve 5-HT losses similar to those seen after MDMA and mimic the MDMA _z 37 therefore to confirm that the lack of protection was not [ ; : _- study as closely as possible. Rats were injected with chlor- A 3001- methiazole (50mgkg -_) followed 5min later by PCA (2.5 =a' mg kg-l) with a further dose of chlormethiazole (50 mg kg-t) after a further 55 min. There was no indication for chlorme- 7 36 thiazole producing any neuroprotection in this study (Figure Co_9 2). = :

The effect of chlormethiazole on the hyperthermia o 20{_ produced by MDMA administration _ r 3s' MDMA administration to rats results in marked hyperther- c .... Chic mia (Nash et al., 1988). We therefore examined whether this o° effect was altered by chlormethiazole injection. Rats were _ Figure 3 Rec injected with saline or MDMA (20 mg kg- l). Twenty minutes _ : 3,4_methylene, later both groups were injected with either saline or chlor- - 100 chlormethiazo methiazole (100mgkg-X), the protocol was thus exactly as later by CM: described in the second neuroprotection experiment (results MDMA or of which are shown in Figure 1). . marked, by tlc The rectal temperature of the saline injected group declined bars). modestly through the course of the experiment (Figure 3). Chlormethiazole administration produced a temperature decrease of approximately0.5'C greater than that seen in the 0 I 2 3 4 1 2 3 4 _ 2 3 4 1 2 3 4 control (saline-injected) animals. The further decrease during S-HT 5-HIAA S-HT S-HIAA the experiment thus paralleled that of the control animals Cortex Hippocampus (Figure 3). MDMA administration produced a rapid hyper- Figure 2 The effect ofp-chloroamphetamine (PCA, 2.5 mg kg -_) on 40.5 thermia of approximately + 2.5°C above saline-injected rats the 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5- and this hyperthermia continued for the next 3 h (Figure 3). HIAA) content of hippocampus and cortex 4 days later, together Administration of chlormethiazole (100 mg kg- l) 20 min al- with the lack of effect of chlormethiazole (50 mg kg-l) or dizocilpine ter the MDMA injection attenuated the temperature rise (1 mg kg -l) given 5min before and 55min after the PCA injection: producing a fairly rapid temperature decrease so that rectal (I) Saline; (2) PCA; (3) PCA plus chlormethiazole; (4) PCA plus temperature was the same as control values 40 rain after the grodizocilpine.up, P_0.001.*SignifiTchantlye 5-HdifferentT and 5-fromHIAA saliconnecenintrajectiotedns cinontrothlc 39.5 chlormethiazole injection (Figure 3). chlormethiazole and dizocilpine-treated rats were not in general The 5-HT-mediated behavioural changes were also observ- significantly different from the PCA alone groups, although hip- ed to be abolished immediately following the chlormethiazole pocampal 5-HT values were lower than PCA alone group injection. (P < 0.005). en E 38.5

Table 3 The effect of chlormethiazole and dizocilpine on the neurotoxic effect of p-chloroamphetamine (PCA) and fenfiuramine on e·r' cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations Hippocampus . Cortex Injected 5-HT 5-Ilia A S-HT 5-HIAA 37.5-

Saline 323 _+ 9 (8) 325 +_ 7 (8) 314 4- 14 (8) 218 _+17 (8) PCA 93 4- 5 (10)* 1224- 6 (10)* 72 4- 3 (10)* 67_+ 5 (10)* PCA + chlormethiazole 81 4- I (4)1. 102+ 12 (4)1. 77 4- 3 (4)1. 68 4- 8 (4)1. PCA + dizocilpine 77 4- 11 (3)t 138+_18 (3)t 76 4- 7 (3)? 82 4- 11 (3)1. Fenfiuramine 97 4- 5 (9)* 138+- 9 (10)* '76 4- 4 (10)* 79 4- 5 (10)* Fenfluramine + chlormethiazole 82 4- 5 (6)t 92 4- 5 (6)1. 59 4- 4 (6)1. 52 + 3 (6)t 36.5 Fenfluramine + dizocilpine 108+ 13 (3)1' 1554- 23 (4)1' 110+ 20 (4):1: 84 + 9 (4)1'

The concentration of 5-HT and 5-HIAA in the hippocampus and cortex of rats 4 days following injection of either PCA (5 mg kg -_) or fenfiuramine (25 mg kg-_) is shown in ng g-_ tissue (n), together with the effect on these parameters of injection of chlormethiazole Figure 4 [ (100mg kg-l in the PCA experiments; 50 mg kg-l in the fenfluramine experiment) or dizocilpine (1 mg kg-l) given 5 min before and amphetamir 55min after the neurotoxin. 5mgkg-I v · Significantly different from saline-injected control rats, P<0.001. 1'Not significantly different from neurotoxin alone treated group, and 55min $Different from neurotoxin alone group, P

-- ! 40. kg -t) respectively. The dose protocol was exactly aa in the i neuroProtective study shown in Table 2. Administration of I PCA (5 mg kg -il resulted in an increase of rectal temperature f of around +2.5'C, peaking 60m in after administration (Figure 4). Chlormethiazole administration abolished this 39 hyperthermic response (Figure 4). The lower dose of PCA (2.5 mg kg-:_ which also produced monoamine loss (Figure 25 did not have a significant effect on rectal temperature "_ (Figure 4).

? 3B' o. Discussion E The current study demonstrates that chlormethiazole or dizocilpine when given immediately before and shortly after _: the MDMA injection both protect against the delayed neurotoxic loss of hippocampal 5-HT and 5,HIAA. Chlor- methiazole, but not dizocilpine, also protected the cortex. Additionally it was demonstrated that a single dose of chlormethiazole, given shortly after the MDMA, provided 36 total protection to the hippocampus and some protection to the cortex (Figure 1). These results are therefore somewhat analogous to our earlier observation that both chlormethiazole and dizocilpine protect both cortical and hippocampal 5-HT neurones from neurotoxic damage due to methamphet- 3_ _, 3'0 J0 00 120 160 180 amine administration (Green et al., 1992). Chlormethiazole lime (min) In contrast, neither chlormethiazole nor dizocilpinc were ' or saline able to prevent the neurotoxic damage to the cortex or Rgure 3 Rectal temperature in rats (n = 5) injected with saline (O); hippocampus induced by injection of the neurotoxins PLA 3,4-methylenedioxymethamphetamine (MDMA), 20 mg kg-_ (0); and fenfluramine. However, as the loss of 5-HT induced by ¢hlormethiazole (CMZ), 100mgkg -_ (r-I); MDMA followed 20min PCA and fenfluramine was considerably greater (around later by CMZ (l). Temperatures shown immediately before the 75%) than that seen following MDMA (around 30%) it MDMA or saline injection with the time of the CMZ injection seemed possible that chlormethiazole or dizocipline were raarkedby the arrow. Results shown as mean 4-s.e.mean (vertical unable to protect the brain from this considerable insult, but bars), might protect if the 5-HT depletion were less, although this seemed unlikely given that both compounds were active * against methamphetamine-induced toxicity when amine losses of around 75% were also observed (Green et al., 1992). Since we found that chlormethiazole was also ineffective in protecting against the effects of a smaller dose of PCA which 40.s lowered cerebral 5-HT content by only 30% (and thus to the same degree as MDMA) our results suggest strongly that chlormethiazole is ineffective against PCA and fenfluramine- induced neurotoxieity. It has recently been suggested that the mechanism of the initial 5-HT loss induced by injection of methamphetamine, 39.5 MDMA, PCA and fenfluramine is the same (Berger et al., 1992). Our data nevertheless indicate that the neurotoxic effects of these drugs are unlikely to be via a common mechanism given the protection from the neurotoxic effects o_ of methamphetamine and MDMA by chlormethiazole and dizocilipine, but failure of these drugs to protect against the consequences of administration of PCA and fenfluramine.

current lack of information on other drugs that do, or do a: not, provide protection and the conflicting information in the literature concerning studies that have been performed. While there is general agreement that dizocilpine protects _ 38'5 _i/_ _ Howeagainstver, methamphetamine-inducedit is difficult to state this lossunequofivocallystriatal dopaminegiven the 37.5 and 5-HT in several brain receptors (Sonsalla et al., 1989; 1991; Gibb et al., 1990; Green et al., 1992), there are conflicting data regarding the effect of N-methyl-D-aspartate (NMDA) antagonists on MDMA neurotoxicity. Johnson et al. (1989) have reported that dizocilpine does not protect against MDMA-induced toxicity of 5-HT, while Finnegan et 36.5 ...... al. (1990) have shown that these effects of MDMA are -10 20 s0 80 ll0 140 1_0 prevented by dextromethorphan. Our data are clearly consis- Time (rain) tent with those of Finnegan et al. (1990) since we found Figure4 Rectal temperature in rats (n= 5) injected with p-chlor- dizocilpine to be an effective protective agent against MDMA amphetamine(PCA), 5mgkg -_ (FI); PCA 2.5mgkg -_ (O)or PCA, toxicity. However this same group has also reported that 5ragkg-_ with chlormethiazole 50 mg kg-I being given 5min before dextromethorphan (which also blocks neuronal cell damage and 55min after the PCA injection (Il). Results shown as mean induced by NMDA in cell cultures) prevented PCA-induced 4-s.e.raean (vertical bars), neurotoxicity in vivo in hippocampus and cortex (Finnegan et 588 M.I. COLADO et al.

al., 1991) while we found no neuroprotective effect of dizocil- fenfluramine is associated with neurodegenerative changes if GREEN, A.R. pine. Studies do not appear to have been made on the effect The preserved monoamine concentrations which occur in_i mediated of dizocilpine on fenfiuramine-induced neurotoxicity; MDMA-treated rats given chlormethiazole and dizocilpim 7' ed. Green although there is at least some evidence that long term effects may therefore reflect a neuroprotective action of these com.! GREEmenN,t A.R.of 5 of this compound on the 5-hydroxytryptaminergic system pounds. While this cannot be stated with authority unt_ by amphe may differ from MDMA, PCA and methamphetamine morphological studies have been performed, it would not b_ raaco!., $_ (Schmidt et al., 1991). surprising in view of the fact that both chlormethiazole and GREEN,A.R. The present experiments appear to rule out any direct dizocilpine are effective neuroprotective agents in the gerbil method in relationship between the neurotoxic effects of the substituted model of global forebrain ischaemia (Gill et al., 1987; 1988;: acting thr amphetamines studied and their acute effects on either body Cross et al., 1991). ; 41, 879-8 temperature or behaviour. In the previous study (Green et Chlormethiazole and dizocilpine share a similar profile in GREEN, A.R. al., 1992)a relationship between methamphetamine-induced protecting against neurotoxic damage induced by either ConRO5-hydr_SS, A. hyperthermia and neurotoxicity appeared to be unlikely as methamphetamine or MDMA but not that produced by protective the hyperthermia was antagonized by both chlormethiazole PCA or fenfluramine administration. It would seem likely 315-321. and diazepam, while only the former drug prevented neuro- therefore that the neurotoxicity induced by both MDMA and HARRISON, toxicity. The current study reinforces the view that body methamphetamine involves excitotoxic damage since dizocil, tions of bi temperature and neurotoxicity are unrelated. MDMA (20 mg pine is a non-competitive NMDA antagonist (Wong et al.. tor comph kg -_) did produce a sustained hyperthermia of about 2.5°C 1986; Kemp et al., 1987). While there is no evidence for 387-394. above normal and this effect was antagonized by chlor- chlormethiazole acting at any site on the NMDA receptor HARVEY, J.A. methiazole given at a dose and administrative regime that complex (Cross et al., 1992), it is nevertheless an effective a neuroto: was protective (Figures I and 3). However PCA (5 mg kg-l) antagonist of NMDA-induced convulsions (Cross et al., HEMRIserotConiK-LUn. produced a similar degree of hyperthermia which chlormeth- 1992) so can clearly antagonize NMDA function in some (1992). MI iazole prevented, while not altering the neurotoxieity (Table 3 way, possibly through potentiating _/-aminobutyric acid dopamine and Figure 4). Additionally the lower dose of PCA of (GABA) function (Harrison & Simmonds, 1983; Cross et al.. PL31-PL2 2.5 mgkg -_ had no effect on rectal temperature (Figure 4) 1989; Green & Murray, 1989). One is also !eft with the HOTCHKISS,, but was neurotoxic (Figure 2). conclusion that the neurotoxic damage produced to 5-HT doses of m Administration of PCA resulted in the appearance of the pathways by PCA and fenfiuramine is unlikely to involve sine hydro ' 5-HT-induce d behavioural syndrome (for review of the syn- NMDA receptor-induced changes. 257-262. drome, see Green & Heal, 1985), confirming several earlier There is reasonable evidence to support the proposition JOHNSON, M. reports (e.g. Green & Kelly, 1976; Curzon et al., 1979). These that dopamine release in the striatum occurs after adminis- neurotoxici behaviours were antagonized by both chlormethiazole and tration of the neurotoxic compounds under study, and thal methamph4pie agent dizocilpine, but the PCA-induced monamine loss still occur- prevention of this change can prevent the degeneration of hay., 40, 9 red. MDMA-treated animals also weakly displayed the 5- 5-HT pathways not only in the striatum but also elsewhere i_ JOHNSON,M., HT-mediated behavioural syndrome together with marked the brain (Schmidt et al., 1985; 1991; Schmidt & Kehne. on the dec hyperactivity. Fenfluramine induced a rather different behav- 1990; Johnson et al., 1991). Nevertheless the current data phetamine ioural syndrome with a pronounced backward walking com- indicate that this cascade of events cannot be identical in the 165, 315-2 ponent, confirming the report of Curzon et al. (1979). These case of PCA and fenfluramine, compared with MDMA and JONSSON, G. behaviours were again prevented by dizocilpine and chlor- methamphetamine and also possibly amphetamine, given the neurotoxici methiazole while the neurotoxicity still occurred. It was, how- recent report (Hemrick-Luecke et al., 1992) that dizocilpine Br. J. PhcL ever, difficult to assess accurately the behavioural changes prevents the neurotoxic effect of this compound on dopamine KEMP.antagJ.A.onist, F(s since chlormethiazole was immediately sedative and dizocil- neurones, macol. Sci. pine itself induced many abnormal behaviours (see Trick- KODA, L.Y. & lebank et al., 1989). droxylase Recent work of Molliver et al. (1990) has demonstrated M.I.C. thanks the European Science Foundation for a fellowship Ther., 185, that the monoamines loss following PCA, MDMA and and DGICYT (Spain)for financial support. MCKENNA, D. neurotoxicil 'Ecstasy').. 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