Longitudinal changes in EEG power, sleep cycles and behaviour in a tau model of neurodegeneration.

BACKGROUND: Disturbed sleep is associated with cognitive decline in neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal dementia (FTD). The progressive sequence of how neurodegeneration affects aspects of sleep architecture in conjunction with behavioural changes is not well understood. METHODS: We investigated changes in sleep architecture, spectral power and circadian rhythmicity in the tet-off rTg4510 mouse overexpressing human P301L tau within the same subjects over time. Doxycycline-induced transgene-suppressed rTg4510 mice, tTa carriers and wild-type mice were used as comparators. Spectral power and sleep stages were measured from within the home cage environment using EEG electrodes. In addition, locomotor activity and performance during a T-maze task were measured. RESULTS: Spectral power in the delta and theta bands showed a time-dependent decrease in rTg4510 mice compared to all other groups. After the initial changes in spectral power, wake during the dark period increased whereas NREM and number of REM sleep bouts decreased in rTg4510 compared to wild-type mice. Home cage locomotor activity in the dark phase significantly increased in rTg4510 compared to wild-type mice by 40 weeks of age. Peak-to-peak circadian rhythm amplitude and performance in the T-maze was impaired throughout the experiment independent of time. At 46 weeks, rTG4510 mice had significant degeneration in the hippocampus and cortex whereas doxycycline-treated rTG4510 mice were protected. Pathology significantly correlated with sleep and EEG outcomes, in addition to locomotor and cognitive measures. CONCLUSIONS: We show that reduced EEG spectral power precedes reductions in sleep and home cage locomotor activity in a mouse model of tauopathy. The data shows increasing mutant tau changes sleep architecture, EEG properties, behaviour and cognition, which suggest tau-related effects on sleep architecture in patients with neurodegenerative diseases.

Tissue magnetic susceptibility mapping as a marker of tau pathology in Alzheimer's disease.

Alzheimer's disease is connected to a number of other neurodegenerative conditions, known collectively as 'tauopathies', by the presence of aggregated tau protein in the brain. Neuroinflammation and oxidative stress in AD are associated with tau pathology and both the breakdown of axonal sheaths in white matter tracts and excess iron accumulation grey matter brain regions. Despite the identification of myelin and iron concentration as major sources of contrast in quantitative susceptibility maps of the brain, the sensitivity of this technique to tau pathology has yet to be explored. In this study, we perform Quantitative Susceptibility Mapping (QSM) and T2* mapping in the rTg4510, a mouse model of tauopathy, both in vivo and ex vivo. Significant correlations were observed between histological measures of myelin content and both mean regional magnetic susceptibility and T2* values. These results suggest that magnetic susceptibility is sensitive to tissue myelin concentrations across different regions of the brain. Differences in magnetic susceptibility were detected in the corpus callosum, striatum, hippocampus and thalamus of the rTg4510 mice relative to wild type controls. The concentration of neurofibrillary tangles was found to be low to intermediate in these brain regions indicating that QSM may be a useful biomarker for early stage detection of tau pathology in neurodegenerative diseases.

Application of neurite orientation dispersion and density imaging (NODDI) to a tau pathology model of Alzheimer's disease.

Increased hyperphosphorylated tau and the formation of intracellular neurofibrillary tangles are associated with the loss of neurons and cognitive decline in Alzheimer's disease, and related neurodegenerative conditions. We applied two diffusion models, diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI), to in vivo diffusion magnetic resonance images (dMRI) of a mouse model of human tauopathy (rTg4510) at 8.5months of age. In grey matter regions with the highest degree of tau burden, microstructural indices provided by both NODDI and DTI discriminated the rTg4510 (TG) animals from wild type (WT) controls; however only the neurite density index (NDI) (the volume fraction that comprises axons or dendrites) from the NODDI model correlated with the histological measurements of the levels of hyperphosphorylated tau protein. Reductions in diffusion directionality were observed when implementing both models in the white matter region of the corpus callosum, with lower fractional anisotropy (DTI) and higher orientation dispersion (NODDI) observed in the TG animals. In comparison to DTI, histological measures of tau pathology were more closely correlated with NODDI parameters in this region. This in vivo dMRI study demonstrates that NODDI identifies potential tissue sources contributing to DTI indices and NODDI may provide greater specificity to pathology in Alzheimer's disease.

In vivo imaging of tau pathology using multi-parametric quantitative MRI.

As the number of people diagnosed with Alzheimer's disease (AD) reaches epidemic proportions, there is an urgent need to develop effective treatment strategies to tackle the social and economic costs of this fatal condition. Dozens of candidate therapeutics are currently being tested in clinical trials, and compounds targeting the aberrant accumulation of tau proteins into neurofibrillary tangles (NFTs) are the focus of substantial current interest. Reliable, translatable biomarkers sensitive to both tau pathology and its modulation by treatment along with animal models that faithfully reflect aspects of the human disease are urgently required. Magnetic resonance imaging (MRI) is well established as a valuable tool for monitoring the structural brain changes that accompany AD progression. However the descent into dementia is not defined by macroscopic brain matter loss alone: non-invasive imaging measurements sensitive to protein accumulation, white matter integrity and cerebral haemodynamics probe distinct aspects of AD pathophysiology and may serve as superior biomarkers for assessing drug efficacy. Here we employ a multi-parametric array of five translatable MRI techniques to characterise the in vivo pathophysiological phenotype of the rTg4510 mouse model of tauopathy (structural imaging, diffusion tensor imaging (DTI), arterial spin labelling (ASL), chemical exchange saturation transfer (CEST) and glucose CEST). Tau-induced pathological changes included grey matter atrophy, increased radial diffusivity in the white matter, decreased amide proton transfer and hyperperfusion. We demonstrate that the above markers unambiguously discriminate between the transgenic group and age-matched controls and provide a comprehensive profile of the multifaceted neuropathological processes underlying the rTg4510 model. Furthermore, we show that ASL and DTI techniques offer heightened sensitivity to processes believed to precede detectable structural changes and, as such, provides a platform for the study of disease mechanisms and therapeutic intervention.

Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment.

Recent studies indicate that brain-derived neurotrophic factor (BDNF) may be implicated in the clinical action of antidepressant drugs. Repeated (2-3 weeks) administration of antidepressant drugs increases BDNF gene expression. The onset of this response as well as concomitant effects on the corresponding BDNF protein is however, unclear. The present study investigated the effects of acute and chronic administration of the selective serotonin reuptake inhibitor, fluoxetine (10mg/kg p.o.), upon regional rat brain levels of BDNF mRNA and protein expression. To improve the clinical significance of the study, fluoxetine was administered orally and mRNA and protein levels were determined ex vivo using the techniques of in situ hybridisation histochemistry and immunocytochemistry respectively. Direct measurement of BDNF protein was also carried out using enzyme-linked immunosorbent assay (ELISA). Four days of once daily oral administration of fluoxetine induced decreases in BDNF mRNA (hippocampus, medial habenular and paraventricular thalamic nuclei). Whilst 7 days of treatment showed a non-significant increase in BDNF mRNA, there were marked and region-specific increases following 14 days of treatment. BDNF protein levels remained unaltered until 21 days of fluoxetine treatment, when the numbers of BDNF immunoreactive cells were increased, reaching significance in the pyramidal cell layer of CA1 and CA3 regions of Ammon's horn (CA1 and CA3) but not in the other sub-regions of the hippocampus. Indicative of the highly regional change within the hippocampus, the ELISA method failed to demonstrate significant up-regulation at 21 days, measuring levels of BDNF protein in the whole hippocampus. In contrast to the detected time dependent and biphasic response of the BDNF gene, activity-regulated, cytoskeletal-associated protein (Arc) mRNA showed a gradual increase during the 14-day course of treatment. The results presented here show that BDNF is expressed differentially depending on length of fluoxetine administration, which could contribute in explaining the slow onset of antidepressant activity observed with selective serotonin reuptake inhibitors.

The role of neuronal nicotinic acetylcholine receptors in acute and chronic neurodegeneration.

In the last five years there has been a rapid explosion of publications reporting that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in neurodegenerative disorders. Furthermore, there is a well-established loss of nAChRs in post-mortem brains from patients with Alzheimer's disease, Parkinson's disease and a range of other disorders. In the present review we discuss the evidence that nicotine and subtype selective nAChR ligands can provide neuroprotection in in vitro cell culture systems and in in vivo studies in animal models of such disorders. Whilst in vitro data pertaining to a protective effect of nicotine against nigral neurotoxins like MPTP is less robust, most studies agree that nicotine is protective against glutamate and beta-amyloid toxicity in various culture systems. This effect appears to be mediated by alpha7 subtype nAChRs since the protection is blocked by alpha-bungarotoxin and is mimicked by alpha7 selective agonists. In vivo studies indicate that alpha7 receptors play a critical role in protection from cholinergic lesions and enhancing cognitive function. The exact subtype involved in the neuroprotectant effects seen in animal models of Parkinson's disease is not clear, but in general broad spectrum nAChR agonists appear to provide protection, while alpha4beta2 receptors appear to mediate symptomatic improvements. Evidence favouring a protectant effect of nicotine against acute degenerative conditions is less strong, though some protection has been observed with nicotine pre-treatment in global ischaemia models. A variety of cellular mechanisms ranging from the production of growth factors through to inactivation of toxins and antioxidant actions of nicotine have been proposed to underlie the nAChR-mediated neuroprotection in vitro and in vivo. In summary, although the lack of subtype selective ligands has hampered progress, it is clear that in the future neuronal nAChR agonists could provide functional improvements and slow or halt the progress of several crippling degenerative diseases.

Neuroprotective efficacy of AR-A008055, a clomethiazole analogue, in a global model of acute ischaemic stroke and its effect on ischaemia-induced glutamate and GABA efflux in vitro.

We have investigated the neuroprotective properties of AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl-1-phenyl-methylamine], a novel compound structurally related to clomethiazole. Administration (i.p.) of (+/-)-AR-A008055 60 min after 5 min of global cerebral ischaemia in gerbils produced a dose-dependent protection of the hippocampus from damage. Both enantiomers [(R)-(+)-AR-A008055 and (S)-(-)- AR-A008055] at 600 micromol/kg produced similar protection to that following clomethiazole (600& micromol/kg) and both produced similar and sustained neuroprotection, at 4, 7 and 21 days post-insult. When infused intravenously over a 2-h period, both enantiomers produced concentration-dependent neuroprotection, with the enantiomers providing similar protection at every plasma concentration (50-200 nmol/ml). The efficacy of (S)-(-)-AR-A008055 was similar to clomethiazole, but it was slightly less potent. Ischaemia-induced glutamate efflux from rat brain cortical prisms in vitro was inhibited by both isomers (100 microM). The inhibitory effect of (R)-(+)-AR-A008055 was blocked by bicuculline (10 microM) and picrotoxin (100 microM), while the effect of (S)-(-)-AR-A008055 was only antagonised by picrotoxin. This indicated that (S)-(-)-AR-A008055, like clomethiazole, is able to open the GABA(A)-chloride channel in the absence of endogenous GABA. (R)-(+)-AR-A008055 was more potent than (S)-(-)-AR-A008055 in enhancing the concentration of GABA in the medium following 30 min exposure of tissue to the ischaemic conditions, suggesting that it is an effective GABA uptake inhibitor. This action may explain both its effect on glutamate efflux in vitro and its neuroprotective effect in vivo.

The interaction of AR-A008055 and its enantiomers with the GABA(A) receptor complex and their sedative, muscle relaxant and anticonvulsant activity.

AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl)-1-phenylmethylamine] is structurally related to clomethiazole and has been used to probe the mechanism of the neuroprotective effect of clomethiazole. Clomethiazole, (+/-)-AR-A008055 and (S)-(-)-AR-A008055 all displaced [35S]-t-butyl-bicyclophosphorothionate ([35S]TBPS) from rat cerebral cortex tissue (IC50 values: GABA, 8.1+/-0.04 microM; clomethiazole, 130+/-30 microM; (+/-)-AR-A008055, 494+/-7 microM; (S)-(-)-AR-A008055, 221+/-14 microM. (R)-(+)-AR-A008055 was without significant effect (IC50>1000 microM). None of the compounds interacted with NMDA or AMPA receptors or with sodium or calcium (N, P/Q) channels. Brain penetration of both enantiomers following their i.p. administration was excellent, with brain and plasma concentrations being similar. Clomethiazole dose-dependently inhibited spontaneous locomotor activity in rats and was approximately 10 times more sedative than either enantiomer of AR-A008055. Clomethiazole was more potent than (S)-(-)-AR-A008055 in the "pull-up" test (muscle relaxation) and in producing loss of righting reflex, while (R)-(+)-AR-A008055 had little effect. The time animals remained on a Rota-rod was of the order: clomethiazole<(S)-(-)-AR-A008055<(R)-(+)-AR-A008055. (S)-(-)-AR-A008055 (210 micromol/kg) raised seizure threshold to pentylenetetrazole (i.v.) by 119+/-21%. The (R)-(+)- enantiomer was not anticonvulsant. Overall, (S)-(-)-AR-A008055 exhibited a similar pharmacology to clomethiazole. However, its sedative and muscle relaxant effects were substantially less than clomethiazole, emphasising that these properties are not directly related to neuroprotective efficacy. The current data suggest that the proposed GABA uptake inhibitory property of (R)-(+)-AR-A008055 fails to produce significant sedative, myorelaxant or anticonvulsant activity.

ARL 17477, a selective nitric oxide synthase inhibitor, with neuroprotective effects in animal models of global and focal cerebral ischaemia.

In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.

Clomethiazole is neuroprotective in models of global and focal cerebral ischemia when infused at doses producing clinically relevant plasma concentrations.

We investigated the neuroprotective effect of infusing various doses of clomethiazole in models of global and focal cerebral ischemia. In a model of global ischemia, gerbils were infused with clomethiazole (intravenous), attaining steady state plasma concentrations of between 1 and 13 microM for 24 h. In a transient middle cerebral artery occlusion model in rats, clomethiazole was administered subcutaneously over 22.75 h using osmotic minipumps producing steady state plasma concentrations of between 1 and 13 microM. Clomethiazole was protective in these models at plasma concentrations of respectively 6.1 microM and above and 3.5 microM and above. Clomethiazole is thus neuroprotective in both global and focal ischemia at plasma concentrations known to be well tolerated in stroke patients.

The metabolism of clomethiazole in gerbils and the neuroprotective and sedative activity of the metabolites.

A single dose of clomethiazole (600 micromol kg(-1) i.p.) has previously been shown to be neuroprotective in the gerbil model of global ischaemia. In gerbils, clomethiazole (600 micromol kg(-1)) injection produced a rapid appearance (peak within 5 min) of drug in plasma and brain and similar clearance (plasma t(1/2): 40 min) from both tissues. The peak brain concentration (226+/-56 nmol g(-1)) was 40% higher than plasma. One major metabolite, 5-(1-hydroxyethyl-2-chloro)-4-methylthiazole (NLA-715) and two minor metabolites 5-(1-hydroxyethyl)-4-methylthiazole (NLA-272) and 5-acetyl-4-methylthiazole (NLA-511) were detected in plasma and brain. Evidence suggested that clomethiazole is metabolized directly to both NLA-715 and NLA-272. Injection of NLA-715, NLA-272 or NLA-511 (each at 600 micromol kg(-1)) produced brain concentrations respectively 2.2, 38 and 92 times greater than seen after clomethiazole (600 micromol kg(-1)). Clomethiazole (600 micromol kg(-1)) injected 60 min after a 5 min bilateral carotid artery occlusion in gerbils attenuated the ischaemia-induced degeneration of the hippocampus by approximately 70%. The metabolites were not neuroprotective at this dose. In mice, clomethiazole (600 micromol kg(-1)) produced peak plasma and brain concentrations approximately 100% higher than in gerbils, drug concentrations in several brain regions were similar but 35% higher than plasma. Clomethiazole (ED(50): 180 micromol kg(-1)) and NLA-715 (ED(50): 240 micromol kg(-1)) inhibited spontaneous locomotor activity. The other metabolites were not sedative (ED(50) >600 micromol kg(-1)). These data suggest that the neuroprotective action of clomethiazole results from an action of the parent compound and that NLA-715 contributes to the sedative activity of the drug. British Journal of Pharmacology (2000) 129, 95 - 100

The effect of excitotoxic hippocampal lesions on simple and conditional discrimination learning in the rat.

The effect of excitotoxic lesions of the hippocampus on acquisition and reversal of simple and conditional tasks was investigated using a Y-maze. Hippocampal-lesioned rats were severely impaired on acquisition and reversal of a conditional visuo-spatial task (where different pairs of visually distinctive choice arms indicated whether a left or right arm choice was correct on that trial) and were unable to acquire a visuo-visual conditional discrimination (where the appearance of the start arm indicated which of the visually distinctive choice arms was correct irrespective of their left/right position). They were not impaired on acquisition or reversal of a simple spatial left/right discrimination task (where all arms had the same visual appearance) nor on acquisition of a visual discrimination (where the correct, visually distinctive, choice arm varied in its left/right position). Hippocampal-lesioned rats were, however, impaired on reversal of this visual discrimination task and on acquisition and reversal of another visual discrimination task in which the visually distinctive choice arms were less different from each other than in the first version of this task. The degree of impairment in the lesioned rats was related to task difficulty for the sham-operated rats and was not specific to tasks requiring spatial choices, visual discrimination or conditional responding. The impairment on conditional tasks was greater than the impairment on those non-conditional tasks which happened to be matched for task difficulty for the sham-operated rats, suggesting that the conditional demand may target the function of the hippocampus rather closely. Statistically worse than chance performance by hippocampal-lesioned (and sham-operated) rats at the beginning of reversal testing, which was given 24 h after achieving criterion on acquisition of that task, indicated that hippocampal-lesioned rats simultaneously exhibited good memory but impaired learning for the type of information required for those tasks.

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.

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.

1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. 7. Administration of the free radical scavenging agent alpha-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). 8. 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. 9. 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.

1. 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. 2. 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. 3. There was a modest decrease in litter size (-20%) of the MDMA-treated dams. 4. 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. 5. 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. 6. 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.

The effect of dizocilpine (MK-801) on conditional discrimination learning in the rat.

The effect of the non-competitive N-methyl-D-aspartate (NMDA)-receptor antagonist, dizocilpine (MK-801) on the acquisition and reversal of conditional tasks was investigated using a Y-maze. Dizocilpine-treated rats were severely impaired on acquisition of a visuo-spatial task and were unable to learn the reversal of this task. 'Worse than chance' performance by dizocilpine-treated (and saline-treated) rats at the beginning of reversal testing, which was given 24 h after achieving criterion on acquisition, indicated that dizocilpine-treated animals simultaneously exhibited good memory but impaired learning for the type of information required for this task. Dizocilpine-treated rats were unable to acquire a visuo-visual conditional discrimination and therefore could not be assessed on reversal of this task. These very substantial impairments were larger than those found in comparable experiments using non-conditional tasks.

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.

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.

The effect of dizocilpine (MK-801) on spatial and visual discrimination tasks in the rat.

The present experiments investigated the effect of the non-competitive N-methyl-d-aspartate (NMDA)-receptor antagonist, dizocilpine (MK-801) (0.075mg/kg) on acquisition and reversal of either a spatial or a visual, appetitively rewarded, simultaneous discrimination task in a Y-maze. The experimental design for the spatial and the visual discriminations was identical, the only difference between the tasks being the nature of the stimulus. Dizocilpine had not effect on acquisition of the spatial task although, when this task was reversed, dizocilpine-treated rats took significantly more trials to reach criterion. On the visual task, dizocilpine impaired both acquisition and reversal. Thus, systemic administration of dizocilpine did not produce a specific spatial learning impairment. The fact that dizocilpine impaired reversal but not acquisition in the spatial task argues against a global performance deficit. Although both the acquisition and reversal phases of the experiment make equal sensory and motor demands on the animal, reversal involves specific learning processes that may be disrupted by dizocilpine.

Characterisation of learning and memory deficits following NMDA receptor antagonism.

The effects of the non-competitive NMDA antagonist dizocilpine in tests of cognitive function have been compared with its effects on motor function in rats. Severe motor impairments were observed at doses above 0.1 mg/kg. Dizocilpine (0.075 mg/kg) had no effect on the acquisition of a spatial discrimination task in a Y-maze, but disrupted reversal learning. Both the acquisition and reversal of a visual discrimination task were impaired following dizocilpine (0.075 mg/kg). Dizocilpine (0.04 mg/kg) also disrupted performance of a fivechoice visual reaction time task. It is clear that dizocilpine can impair cognitive function at doses which do not induce pronounced motor dysfunction. The impairment induced by dizocilpine includes a disruption of spatial discrimination learning and a deficit in tasks with sustained attentional demands.