T.K. Murray

In vivo evidence for free radical involvement in the degeneration of rat brain 5‐HT following administration of MDMA (‘ecstasy’) and p‐chloroamphetamine but not the degeneration following fenfluramine

Administration of 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) to several species results in a long lasting neurotoxic degeneration of 5‐hydroxytryptaminergic neurones in several regions of the brain. We have now investigated whether this degeneration is likely to be the result of free radical‐induced damage. Free radical formation can be assessed by measuring the formation of 2,3‐ and 2,5‐dihydroxybenzoic acid (2,3‐DHBA and 2,5‐DHBA) from salicylic acid. An existing method involving implantation of a probe into the hippocampus and in vivo microdialysis was modified and validated. Administration of MDMA (15 mg kg−1, i.p.) to Dark Agouti (DA) rats increased the formation of 2,3‐DHBA (but not 2,5‐DHBA) for at least 6 h. Seven days after this dose of MDMA, the concentration of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) was reduced by over 50% in hippocampus, cortex and striatum, reflecting neurotoxic damage. There was no change in the concentration of dopamine or 3,4‐dihydroxyphenylacetic acid (DOPAC) in the striatum. p‐Chloroamphetamine (PCA), another compound which produces a neurotoxic loss of cerebral 5‐HT content, when given at a dose of 5 mg kg−1 also significantly increased the formation of 2,3‐DHBA (but not 2,5‐DHBA) in the dialysate for over 4.5 h. post‐injection starting 2 h after treatment. In contrast, fenfluramine administration (15 mg kg−1, i.p.) failed to increase the 2,3‐DHBA or 2,5‐DHBA concentration in the dialysate. A single fenfluramine injection nevertheless also markedly decreased the concentration of 5‐HT and 5‐HIAA in the hippocampus, cortex and striatum seven days later. When rats pretreated with fenfluramine (15 mg kg−1, i.p.) seven days earlier were given MDMA (15 mg kg−1, i.p.) no increase in 2,3‐DHBA was seen in the dialysate from the hippocampal probe. This indicates that the increase in free radical formation following MDMA is occurring in 5‐HT neurones which have been damaged by the prior fenfluramine injection. Administration of the free radical scavenging agent α‐phenyl‐N‐tert‐butyl nitrone (PBN; 120 mg kg−1, i.p.) 10 min before and 120 min after an MDMA (15 mg kg−1, i.p.) injection prevented the acute rise in the 2,3‐DHBA concentration in the dialysate and attenuated by 30% the long term damage to hippocampal 5‐HT neurones (as indicated by a smaller MDMA‐induced decrease in both the concentration of 5‐HT and 5‐HIAA and also the binding of [3H]‐paroxetine). These data indicate that a major mechanism by which MDMA and PCA induce damage to 5‐hydroxytryptaminergic neurones in rat brain is by increasing the formation of free radicals. These probably result from the degradation of catechol and quinone metabolites of these substituted amphetamines. In contrast, fenfluramine induces damage by another mechanism not involving free radicals; a proposal supported by some of our earlier indirect studies. We suggest that these different modes of action render untenable the recent suggestion that MDMA will not be neurotoxic in humans because fenfluramine appears safe at clinical doses.

A study of the neurotoxic effect of MDMA ('ecstasy') on 5-HT neurones in the brains of mothers and neonates following administration of the drug during pregnancy

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) is neurotoxic and produces long term degeneration of cerebral 5‐hydroxytryptamine (5‐HT) nerve terminals in many species. Since MDMA is used extensively as a recreational drug by young people, it is being ingested by many women of child bearing age. We have therefore examined the effect of administering high doses of MDMA to rats during pregnancy on the cerebral content of both the dams and the neonates. MDMA (20 mg kg−1, s.c.) was injected twice daily on days 14–17 of the gestation period. The initial dose produced a marked hyperthermic response in the dam which was progressively attenuated in both peak height and area under the curve following further doses of the drug. The body weight of the dams decreased during the period of treatment. There was a modest decrease in litter size (−20%) of the MDMA‐treated dams. The concentration of 5‐HT and its metabolite 5‐HIAA was decreased by over 65% in the hippocampus and striatum and 40% in the cortex of the dams 1 week after parturition. In contrast, the content of 5‐HT and 5‐HIAA in the dorsal telencephalon of the pups of the MDMA‐treated dams was the same as that seen in tissue from pups born to control animals. Administration of MDMA (40 mg kg−1, s.c.) to adult rats increased thiobarbituric acid reacting substances (TBARS) in cortical tissue 3 h and 6 h later, indicating increased lipid peroxidation. No increase in TBARS was seen in the cortical tissue of 7–10 day neonates injected with this dose of MDMA 3 h or 6 h earlier. The data suggest that exposure to MDMA in utero during the maturation phase does not produce damage to 5‐HT nerve terminals in the foetal rat brain, in contrast to the damage seen in the brains of the mothers. This may be due to MDMA being metabolized to free radical producing entities in the adult brain but not in the immature brain or, alternatively, to more effective or more active free radical scavenging mechanisms being present in the immature brain.

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

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 fenfluramine 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−1, i.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 (50 mg kg−1) 5 min before and 55 min after the MDMA provided complete protection in both regions, while dizocilpine (1 mg kg−1, i.p.) protected only the hippocampus. 3 Administration of a single dose of chlormethiazole (100 mg kg−1) 20 min after the MDMA also provided complete protection to the hippocampus but not the cortex. This regime also attenuated the sustained hyperthermia (approx +2.5°C) induced by the MDMA injection. 4 Injection of PCA (5 mg kg−1, i.p.) resulted in a 70% loss of 5‐HT and 5‐HIAA content in hippocampus and cortex 4 days later. Injection of chlormethiazole (100 mg kg−1, i.p.) or dizocilpine (1 mg kg−1, i.p.) 5 min before and 55 min 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−1, 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−1), while the lower dose of PCA (2.5 mg kg−1) 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 fenfluramine (25 mg kg−1, 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 chlormethiazole and dizocilpine have previously been shown to prevent the neurotoxic effects of a high dose of methamphetamine on cerebral 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.

A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066

The in vitro and in vivo effects of the novel acetylcholinesterase inhibitors donepezil and NXX-066 have been compared to tacrine. Using purified acetylcholinesterase from electric eel both tacrine and donepezil were shown to be reversible mixed type inhibitors, binding to a similar site on the enzyme. In contrast, NXX-066 was an irreversible non-competitive inhibitor. All three compounds were potent inhibitors of rat brain acetylcholinesterase (IC50 [nM]; tacrine: 125 +/- 23; NXX-066: 148 +/- 15; donepezil: 33 +/- 12). Tacrine was also a potent butyrylcholinesterase inhibitor. Donepezil and tacrine displaced [3H]pirenzepine binding in rat brain homogenates (IC50 values [microM]; tacrine: 0.7; donepezil: 0.5) but NXX-066 was around 80 times less potent at this M1-muscarinic site. Studies of carbachol stimulated increases in [Ca2+]i in neuroblastoma cells demonstrated that both donepezil and tacrine were M1 antagonists. Ligand binding suggested little activity of likely pharmacological significance with any of the drugs at other neurotransmitter sites. Intraperitoneal administration of the compounds to rats produced dose dependent increases in salivation and tremor (ED50 [micromol/kg]; tacrine: 15, NXX-066: 35, donepezil: 6) with NXX-066 having the most sustained effect on tremor. Following oral administration, NXX-066 had the slowest onset but the greatest duration of action. The relative potency also changed, tacrine having low potency (ED50 [micromol/kg]; tacrine: 200, NXX-066: 30, donepezil: 50). Salivation was severe only in tacrine treated animals. Using in vivo microdialysis in cerebral cortex, both NXX-066 and tacrine were found to produce a marked (at least 30-fold) increase in extracellular acetylcholine which remained elevated for more than 2 h after tacrine and 4 h after NXX-066.

The neurotoxic effects of methamphetamine on 5-hydroxytryptamine and dopamine in brain: Evidence for the protective effect of chlormethiazole

Studies were undertaken in mice and rats on the neurotoxic effects of methamphetamine on dopaminergic and 5-hydroxytryptaminergic neurones in the brain and the neuroprotective action of chlormethiazole. In initial studies, mice were injected with methamphetamine (5 mg/kg, i.p.) at 2 hr intervals, to a total of 4 times. This procedure produced a 66% loss of striatal dopamine and a 50% loss of tyrosine hydroxylase activity 3 days later. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each dose of methamphetamine, totally prevented the methamphetamine-induced loss of tyrosine hydroxylase activity and partly prevented the loss of dopamine. Phencyclidine (20 mg/kg, i.p.), given in place of chlormethiazole, also prevented the loss of tyrosine hydroxylase. Administration to rats of 4 doses of methamphetamine (15 mg/kg, i.p.) at 3 hr intervals resulted in a 75% loss of striatal dopamine 3 days later and a similar loss of 5-HT and 5-HIAA in cortex and hippocampus. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each injection of methamphetamine, protected against the loss of dopamine and indoleamine content, in the respective regions. Pentobarbital (25 mg/kg, i.p.) also provided substantial protection but diazepam (2.5 mg/kg, i.p.) was without effect. Confirming earlier studies, dizocilpine (1 mg/kg) also provided substantial protection against the methamphetamine-induced neurotoxicity. Preliminary data indicated that chlormethiazole was not neuroprotective because of a hypothermic action. These data therefore demonstrate that chlormethiazole is an effective neuroprotective agent against methamphetamine-induced neurotoxicity and extend the evidence for the possible value of this drug in preventing neurodegeneration.

The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro

1 The effect of tetrahydroaminoacridine (THA) on cholinergically mediated behaviour in the rat and mouse has been investigated. In addition the actions of this compound on cholinesterase activity and on muscarinic and nicotinic receptors has also been examined. 2 Administration of THA (5–20 mg kg−1, i.p.) produced a dose‐dependent increase in tremor, hypothermia and salivation in both rats and mice. A similar profile of activity was seen following physostigmine (0.1‐0.6 mg kg−1) administration. 3 THA was approximately fifty fold less potent than physostigmine in inducing behavioural change but its effects persisted for over twice as long as those of physostigmine. For example THA‐induced hypothermia was still present at 4 h in the mouse and 8 h in the rat. 4 In vitro THA was a potent non‐competitive inhibitor of rat brain cholinesterase (IC50: 57 + 6 nM) and bovine erythrocyte acetylcholinesterase (IC50: 50 + 10 nM) but was a more potent inhibitor of horse serum butyrylcholinesterase (IC50: 7.2 + 1.4 nM). 5 Radioligand binding studies indicated that THA binds non‐selectively but with moderate potency to both M1 (Ki: 600 nM) and M2 (Ki: 880 nM) muscarinic receptors. THA also interacted with the allosteric site present on cardiac M2 receptors. 6 It is concluded that THA is a reversible non‐competitive inhibitor of cholinesterase with a long half life (compared with physostigmine). It also may antagonize muscarinic receptors at high doses. The long half life may account for its reported efficacy in the treatment of Alzheimers disease.

Striatal dopamine release in vivo following neurotoxic doses of methamphetamine and effect of the neuroprotective drugs, chlormethiazole and dizocilpine

1 Administration to rats of methamphetamine (15 mg kg−1, i.p.) every 2 h to a total of 4 doses resulted in a neurotoxic loss of striatal dopamine of 36% and of 5‐hydroxytryptamine (5‐HT) in the cortex (43%) and hippocampus (47%) 3 days later. 2 Administration of chlormethiazole (50 mg kg−1, i.p.) 15 min before each dose of methamphetamine provided complete protection against the neurotoxic loss of monoamines while administration of dizocilpine (1 mg kg−1, i.p.) using the same dose schedule provided substantial protection. 3 Measurement of dopamine release in the striatum by in vivo microdialysis revealed that methamphetamine produced an approximate 7000% increase in dopamine release after the first injection. The enhanced release response was somewhat diminished after the third injection but still around 4000% above baseline. Dizocilpine (1 mg kg−1, i.p.) did not alter this response but chlormethiazole (50 mg kg−1, i.p.) attenuated the methamphetamine‐induced release by approximately 40%. 4 Dizocilpine pretreatment did not influence the decrease in the dialysate concentration of the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) produced by administration of methamphetamine while chlormethiazole pretreatment decreased the dialysate concentration of these metabolites still further. 5 The concentration of dopamine in the dialysate during basal conditions increased modestly during the course of the experiment. This increase did not occur in chlormethiazole‐treated rats. HVA concentrations were unaltered by chlormethiazole administration. 6 Chlormethiazole (100–1000 μm) did not alter methamphetamine (100 μm) or K+ (35 mm)‐evoked release of endogenous dopamine from striatal prisms in vitro. 7 Several NMDA antagonists prevent methamphetamine‐induced neurotoxicity; however chlormethiazole is not an NMDA antagonist. Inhibition of striatal dopamine function prevents methamphetamine‐induced toxicity of both dopamine and 5‐HT pathways. Therefore the attenuation of the enhanced dopamine release which occurs in animals given chlormethiazole may be associated with the protective action of this drug against methamphetamine‐induced neurotoxicity.

Reversal by tetrahydroaminoacridine of scopolamine-induced memory and performance deficits in rats

The effects of the cholinesterase inhibitors physostigmine and tetrahydroaminoacridine (THA) on memory and performance deficits induced by scopolamine were studied using an operant delayed non-matching to position task. No effect was seen on the performance of rats when treated with either physostigmine (0.1 mg/kg IP) or THA (1 mg/kg IP) alone. However, the performance deficits induced in the task by scopolamine (0.03 mg/kg SC) were reversed by the same doses of the cholinesterase inhibitors.

The spin trap reagent PBN attenuates degeneration of 5-HT neurones in rat brain induced by p-chloroamphetamine but not fenfluramine

Dark Agouti rats injected with either p-chloroamphetamine (PCA; 2.5 mg/kg i.p.) or fenfluramine (15 mg/kg i.p.) had substantial decreases (approximately 50%) in the concentration of 5-HT and 5-HIAA and binding of [3H]paroxetine in the cerebral cortex 7 days later. This indicates that both compounds had produced neurodegeneration of 5-HT axon terminals. Two doses of alpha-phenyl-N-tert-butyl nitrone (PBN; 150 mg/kg i.p.) 130 min apart had no effect on cortical 5-HT content or [3H]paroxetine binding. However, when PBN (150 mg/ kg) was given 10 min before and 120 min after PCA (2.5 mg/kg) it attenuated the PCA-induced neurodegeneration. In contrast, PBN was without significant effect on the fenfluramine-induced damage. Changes in rectal temperature following either the neurotoxins or neurotoxins+ PBN were no more than +/-1 degree C of saline-injected control rats. These data indicate that PCA, like MDMA, probably induces neurotoxic degeneration because of the formation of catechol or quinone metabolites and subsequent reactive tree radical formation. Such a mechanism does not appear to explain fenfluramine-induced damage to 5-HT neurones.

A behavioural and neurochemical study in rats of the pharmacology of loreclezole, a novel allosteric modulator of the GABAA receptor.

Loreclezole is an anticonvulsant and anxiolytic compound which has been reported to potentiate GABA via a novel allosteric site on the beta-subunit of the receptor. We have now studied in rats both the in vivo and in vitro pharmacology of the compound. The dose of loreclezole required to increase by 50% the dose of intravenous pentylenetetrazol eliciting a seizure was comparable to that of barbiturates and chlormethiazole (in mg/kg): diazepam, 1.3; pentobarbitone, 16; chlormethiazole, 22; loreclezole, 25; pentobarbitone, 36. Loreclezole dose-dependently decreased locomotion (dose to decrease locomotion by 50% (in mg/kg): chlormethiazole, 9; pentobarbitone, 16; loreclezole, 25). Loreclezole, chlormethiazole and pentobarbitone all failed to displace [3H]muscimol and [3H]flunitrazepam binding from a rat cortical membrane preparation. All three compounds fully displaced [35S]TBPS binding (IC50 values: loreclezole, 4.34 +/- 0.68 microM; pentobarbitone, 37.39 +/- 3.24 microM; chlormethiazole, 82.10 +/- 8.52 microM). Addition of bicuculline (10 microM) produced a major rightward shift in the loreclezole and pentobarbitone displacement curves, increasing IC50 values for [35S]TBPS binding by 25 times (loreclezole), 6 times (pentobarbitone) and 2.7 times (chlormethiazole), suggesting a greater involvement of GABA in the interaction of loreclezole with the chloride channel than in the case of chlormethiazole. Anticonvulsant activity of the compounds did not appear to relate to [35S]TBPS binding activity. Other binding data suggested that although the evidence of others indicates that loreclezole interacts with a specific allosteric site on the beta-subunit, it nevertheless also alters the binding characteristics of other modulatory sites.