Serotonergic and noradrenergic lesions suppress the enhancing effect of maternal exercise during pregnancy on learning and memory in rat pups.

The beneficial effects of exercise on learning and memory are well documented but the effects of prenatal exposure to maternal exercise on offspring are not clear yet. Using a two-trial-per-day Morris water maze for five consecutive days, succeeded by a probe trial 2 days later we showed that maternal voluntary exercise (wheel running) by pregnant rats increased the acquisition phase of the pups' learning. Maternal forced swimming by pregnant rats increased both acquisition and retention phases of the pups' learning. Also we found that the rat pups whose mother was submitted to forced-swimming during pregnancy had significantly higher brain, liver, heart and kidney weights compared with their sedentary counterparts. On the other hand we estimated the cell number of different regions of the hippocampus in the rat pups. We found that both exercise models during pregnancy increased the cell number in cornus ammonis subregion 1 (CA1) and dentate gyrus of the hippocampus in rat pups. To determine the role that noradrenergic and serotonergic neurotransmission and N-methyl-D-aspartate (NMDA) receptors hold in mediation of the maternal exercise in offspring, we used N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), p-chloroamphetamine (PCA) and MK-801 to eliminate or block the above systems, respectively. Blocking the NMDA receptors, significantly abolished learning and memory in rat pups from all three experimental groups. Elimination of noradrenergic or serotonergic input did not significantly attenuate the learning and memory in rat pups whose mothers were sedentary, while it significantly reversed the positive effects of maternal exercise during pregnancy on rat pups' learning and memory. The presented results suggest that noradrenergic and serotonergic systems in offspring brain seem to have a crucial specific role in mediating the effects of maternal physical activity during pregnancy on rat pups' cognitive function in both models of voluntary and forced exercise.

Loss of serotonin transporter protein after MDMA and other ring-substituted amphetamines.

We studied in vivo expression of the serotonin transporter (SERT) protein after 3,4-methylenedioxymethamphetamine (MDMA), p-chloroamphetamine (PCA), or fenfluramine (FEN) treatments, and compared the effects of substituted amphetamines to those of 5,7-dihydroxytryptamine (5,7-DHT), an established serotonin (5-HT) neurotoxin. All drug treatments produced lasting reductions in 5-HT, 5-HIAA, and [(3)H]paroxetine binding, but no significant change in the density of a 70 kDa band initially thought to correspond to the SERT protein. Additional Western blot studies, however, showed that the 70 kDa band did not correspond to the SERT protein, and that a diffuse band at 63-68 kDa, one that had the anticipated regional brain distribution of SERT protein (midbrain>striatum>neocortex>cerebellum), was reduced after 5,7-DHT and was absent in SERT-null animals, was decreased after MDMA, PCA, or FEN treatments. In situ immunocytochemical (ICC) studies with the same two SERT antisera used in Western blot studies showed loss of SERT-immunoreactive (IR) axons after 5,7-DHT and MDMA treatments. In the same animals, tryptophan hydroxylase (TPH)-IR axon density was comparably reduced, indicating that serotonergic deficits after substituted amphetamines differ from those in SERT-null animals, which have normal TPH levels but, in the absence of SERT, develop apparent neuroadaptive changes in 5-HT metabolism. Together, these results suggest that lasting serotonergic deficits after MDMA and related drugs are unlikely to represent neuroadaptive metabolic responses to changes in SERT trafficking, and favor the view that substituted amphetamines have the potential to produce a distal axotomy of brain 5-HT neurons.

The acute and long-term neurotoxic effects of MDMA on marble burying behaviour in mice.

When mice are exposed to harmless objects such as marbles in their cage they bury them, a behaviour sometimes known as defensive burying. We investigated the effect of an acute dose of MDMA (èecstasy') and other psychoactive drugs on marble burying and also examined the effect of a prior neurotoxic dose of MDMA or p-chloroamphetamine (PCA) on burying. Acute administration of MDMA produced dose-dependent inhibition of marble burying (EC50: 7.6 micro mol/kg). Other drugs that enhance monoamine function also produced dose-dependent inhibition: methamphetamine PCA paroxetine MDMA GBR 12909 methylphenidate. None of these drugs altered locomotor activity at a dose that inhibited burying. A prior neurotoxic dose of MDMA, which decreased striatal dopamine content by 60%, but left striatal 5-HT content unaltered, did not alter spontaneous marble burying 18 or 40 days later. However, a neurotoxic dose of PCA which decreased striatal dopamine by 60% and striatal 5-HT by 70% attenuated marble burying 28 days later. Overall, these data suggest that MDMA, primarily by acutely increasing 5-HT function, acts like several anxiolytic drugs in this behavioural model. Long-term loss of cerebral 5-HT content also produced a similar effect. Since this change was observed only after 28 days, it is probably due to an adaptive response in the brain.

Regrowth of Serotonin Axons in the Adult Mouse Brain Following Injury.

It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests.

Dual serotonin (5-HT) projections to the nucleus accumbens core and shell: relation of the 5-HT transporter to amphetamine-induced neurotoxicity.

Dopamine release in the nucleus accumbens (NAc) has been implicated as mediating the rewarding effects of stimulant drugs; however, recent studies suggest that 5-HT release may also contribute. In an effort to assess the role of 5-HT in drug-mediated reward, this study analyzed the serotonergic innervation of NAc using immunocytochemistry for 5-HT and the 5-HT transporter (SERT). We report that in control rats the NAc receives two distinct types of 5-HT axons that differ in regional distribution, morphology, and SERT expression. Most regions of the NAc are innervated by thin 5-HT axons that express SERT, but in the caudal NAc shell nearly all 5-HT axons lack SERT and have large spherical varicosities. Two weeks after methamphetamine or p-chloroamphetamine (PCA) treatment, most 5-HT axons in dorsal striatum and NAc have degenerated; however, the varicose axons in the shell appear intact. These drug-resistant 5-HT axons that lack SERT densely innervate the caudal one-third of the accumbens shell, the same location where dopamine axons are spared after methamphetamine. Moreover, 4 hr after PCA, the varicose axons in the caudal shell retain prominent stores of 5-HT, whereas 5-HT axons in the rest of the NAc are depleted of neurotransmitter. The results demonstrate that two functionally different 5-HT projections innervate separate regions of the NAc and that selective vulnerability to amphetamines may result from differential expression of SERT. We postulate that action potentials conducted from the raphe nuclei can release 5-HT throughout the NAc, whereas transporter-mediated release induced by stimulant drugs is more restricted and unlikely to occur in the caudal NAc shell.

BDNF promotes the regenerative sprouting, but not survival, of injured serotonergic axons in the adult rat brain.

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the adult brain and can prevent the severe loss of cortical 5-HT axons caused by the neurotoxin p-chloroamphetamine (PCA). However, it has not been determined whether BDNF promotes the survival of 5-HT axons during PCA-insult or facilitates their regenerative sprouting after injury. We show here that BDNF fails to protect most 5-HT axons from PCA-induced degeneration. Instead, chronic BDNF infusions markedly stimulate the sprouting of both intact and PCA-lesioned 5-HT axons, leading to a hyperinnervation at the neocortical infusion site. BDNF treatment promoted the regrowth of 5-HT axons when initiated up to a month after PCA administration. The sprouted axons persisted in cortex for at least 5 weeks after terminating exogenous BDNF delivery. BDNF also encouraged the regrowth of the 5-HT plexus in the hippocampus, but only in those lamina where 5-HT axons normally ramify. In addition, intracortical BDNF infusions induced a sustained local activation of the TrkB receptor. The dose-response profiles for BDNF to stimulate 5-HT sprouting and Trk signaling were remarkably similar, suggesting a physiological link between the two events; both responses were maximal at intermediate doses of BDNF but declined at higher doses ("inverted-U-shaped" dose-response curves). Underlying the downregulation of the Trk signal with excessive BDNF was a decline in full-length TrkB protein, but not truncated TrkB protein or TrkB mRNA levels. Thus, BDNF-TrkB signaling does not protect 5-HT neurons from axonal injury, but has a fundamental role in promoting the structural plasticity of these neurons in the adult brain.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine.

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphé nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

In vivo [¹8F] FDG PET imaging reveals that p-chloroamphetamine neurotoxicity is associated with long-term cortical and hippocampal hypometabolism.

PURPOSE: p-Chloroamphetamine (PCA) is a neurotoxin that selectively degenerates the serotonin (5-HT) axon terminals. In order to study the brain metabolic consequences induced by serotonergic denervation, a single dose of PCA (2.5 or 10 mg/kg i.p.) was administered to male adult rats. PROCEDURES: In vivo regional brain metabolism was evaluated 3 and 21 days after PCA (2.5 or 10 mg/kg; i.p.) injection by 2-deoxy-2-[(18)F] fluoro-D-glucose ([(18)F] FDG) positron emission tomography (PET). At day 22, the following markers of neurotoxicity were determined: (a) 5-HT axon terminal lesion by 5-HT transporter (SERT) autoradiography, (b) reactive gliosis by glial fibrillary acidic protein immunohistochemistry, and (c) eventual neurodegeneration by DAPI/Fluoro-Jade C labeling. RESULTS: An average of 20 % reduction of [(18)F] FDG uptake in most brain areas was observed at day 21 under 10 mg/kg PCA treatment. Instead, 2.5 mg/kg PCA only reduced metabolic activity in neocortex. Likewise, the high dose of PCA exerted a strong decrease (>30 %) in SERT density in several 5-HT innervated regions, but no effect was found in midbrain raphe nuclei, the main source of serotonergic neurons. Although PCA induced astroglial activation both in hippocampus and cortex in response to axotomy, no signs of neuronal death in these areas were detected. CONCLUSIONS: Overall, [(18)F] FDG PET revealed that the reduction of the brain metabolic activity induced by PCA is related to 5-HT axon terminal lesion, with no apparent affectation of neuronal viability.

Dual serotoninergic projections to forebrain in the rat: morphologically distinct 5-HT axon terminals exhibit differential vulnerability to neurotoxic amphetamine derivatives.

The cerebral cortex of the rat and other mammals is innervated by two morphologically distinct classes of serotoninergic (5-HT) axon terminals: fine axons with minute varicosities and beaded axons characterized by large, spherical varicosities. Fine and beaded 5-HT axons exhibit different regional and laminar distributions in forebrain and arise from separate brainstem nuclei, the dorsal and median raphe nuclei, respectively. The present neuroanatomic study, based on immunocytochemical methods to visualize 5-HT axons, demonstrates that the two axon types differ markedly in their vulnerability to the neurotoxic amphetamine derivatives, methylenedioxyamphetamine (MDA), and p-chloroamphetamine (PCA). While both drugs cause extensive degeneration of fine 5-HT axons throughout forebrain, beaded 5-HT axons are consistently spared. Fine 5-HT axons, which richly innervate most regions of dorsal forebrain in control rats, are rarely seen 2 weeks after treatment with MDA or PCA; this loss of fine axons reflects a marked denervation that persists for months after drug administration. The serotoninergic axon terminals remaining after MDA or PCA administration are almost entirely of the beaded type and appear to be unaffected by both drugs. Over a wide range of doses (2.5-40 mg/kg PCA) and survival times (2 weeks to 2 months), these spared 5-HT axons with large, spherical varicosities cannot be distinguished from the normal, beaded 5-HT axons in control rats by morphologic criteria. Moreover, beaded 5-HT axons exhibit a highly characteristic regional distribution which is the same in control as in MDA- and PCA-treated rats: these axons innervate specific zones or layers within parietal and occipital cortex, hippocampus, cingulate cortex, entorhinal cortex, and the olfactory bulb, among other forebrain areas, and they form a dense plexus lining the ventricular system. Taken together, the results of this study demonstrate that fine 5-HT axons are highly vulnerable to the neurotoxic effects of the amphetamine derivatives MDA and PCA, while beaded 5-HT axons are markedly resistant. These findings are consistent with the hypothesis that there are two anatomically and functionally distinct sets of serotoninergic neurons projecting to forebrain. While both of these neuronal systems utilize 5-HT as a neurotransmitter, they differ in several features: 1) origin from separate nuclei in the brainstem (the dorsal and median raphe), 2) two types of morphologically distinct axon terminals, 3) markedly different distribution and innervation patterns in forebrain, and 4) dissimilar pharmacological properties. The results further suggest that psychotropic amphetamine derivatives have a selective action upon fine serotoninergic axons that arise from the dorsal raphe nucleus.

Postural changes, tremor, and myoclonus in the rat immediately following injections of p-chloromaphetamine.

A single dose of p-chloroamphetamine, 10 mg per kilogram, produced postural abnormalities, tremor, myoclonus, and autonomic signs in rats 5 minutes after intraperitoneal injection. This syndrome lasted 60 to 90 minutes, and its intensity was directly proportional to the amount of p-chloroamphetamine given over a 2 to 10 mg per kilogram range. Whole-brain levels of serotonin and 5-hydroxyindoleacetic acid were not altered during this interval, although both were reduced significantly 1 day later. Pretreatment with drugs that interfere with the uptake of p-chloroamphetamine into terminals of serotonergic neurons (fluoxetine), depress brain serotonin levels (p-chlorophenylalanine), or block serotonin receptors (methiothepin or methergoline) suppressed this syndrome, whereas drugs that antagonize the effects of dopamine, norepinephrine, and acetylcholine did not. These observations implicate serotonergic mechanisms and provide behavioral evidence of p-chloroamphetamine's immediate actions on serotonergic neurons in the central nervous system.

Long-lasting behavioral sensitization to psychostimulants following p-chloroamphetamine-induced neurotoxicity in mice.

Amphetamine analogs such as p-chloroamphetamine (PCA) cause serotonergic and dopaminergic neurotoxicity. The behavioral consequences and the responsiveness to psychostimulants following the neurotoxic insult are unclear. The present study was undertaken to investigate the outcome of neurotoxic and non-neurotoxic PCA pre-treatments on the sensitivity of Swiss Webster mice to the psychomotor stimulating effects of PCA, 3,4-methylenedioxymethamphetamine (MDMA) and cocaine. PCA (15 mg/kg x 2; i.p.) caused 37-70% depletion of dopaminergic and serotonergic markers in mouse brain. Saline and PCA (15 mg/kg x 2) mice were challenged on days 5, 12, 40 and 74 with one of the following drugs: PCA (5 mg/kg), MDMA (10 mg/kg) and cocaine (20 mg/kg). The PCA pre-exposed mice showed marked locomotor sensitization from days 5-74 to all three drugs tested. The time course of the sensitized response coincided with the time course of the neurotoxic insult as determined by reduced densities of striatal dopamine transporter and frontal cortex serotonin transporter binding sites. A single injection of PCA (5 mg/kg) caused neither neurotoxicity nor sensitization to the locomotor stimulating effects of PCA, MDMA and cocaine. Repeated administration of a low non-neurotoxic dose of PCA (5 mg/kg/day; 6 days) caused a transient locomotor sensitization to PCA that dissipated after one month. Results of the present study suggest that PCA-induced serotonergic and dopaminergic neurotoxicity coincides with long-lasting locomotor sensitization to psychostimulants. These findings may be relevant to the psychopathology of amphetamines-induced neurotoxicity.

Novel radiotracers for imaging the serotonin transporter by positron emission tomography: synthesis, radiosynthesis, and in vitro and ex vivo evaluation of (11)C-labeled 2-(phenylthio)araalkylamines.

A series of four 2-(phenylthio)araalkylamines have been radiolabeled with (11)C and evaluated as potential radiotracers for imaging the serotonin transporter (SERT) by positron emission tomography (PET). All four candidates display high affinity for SERT and low affinity for the dopamine or norepinephrine transporters using in vitro binding assays. Biodistribution studies in rats demonstrated that tail-vein injection of the (11)C-labeled radiotracers resulted in high brain uptake of radioactivity with a preferential distribution in brain regions known to be rich in SERT such as hypothalamus and thalamus. The most promising candidate, 16, had hypothalamus-to-cerebellum ratios of 9:1, 1 h postinjection, an indication of high specific to nonspecific binding. Ex vivo pharmacological studies demonstrated that uptake in SERT-rich brain regions was both saturable and selective for SERT. Two of the tested radiotracers, 15 and 16, have highly favorable properties for imaging SERT and will be used in pilot human PET imaging studies.

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.

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 degrees 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 ofa 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.

Tryptophan pretreatment augmentation of p-chloroamphetamine-induced serotonin and dopamine release and reduction of long-term neurotoxicity.

The impact of tryptophan (TRP) pretreatment on the neurochemical effects of p-chloroamphetamine (PCA) was investigated. The neurotoxic effects of PCA on serotonin (5-HT) neurons, the acute effects of PCA on extracellular 5-HT and dopamine (DA), and the displacement by PCA of whole blood 5-HT were examined. TRP pretreatment (400 mg/kg of the methyl ester) significantly reduced the long-term (1 week) decrease in tissue 5-HT resulting from PCA (2 mg/kg, i.p., of the hydrochloride salt) in the prefrontal cortex and striatum, but not in the dorsal hippocampus. Microdialysis studies in awake animals showed that this pretreatment regimen resulted in augmented PCA-induced increases in extracellular 5-HT (4-fold) and DA (2-fold). TRP pretreatment also resulted in increased displacement of 5-HT from whole blood. The implications of these results toward possible mechanisms of action of PCA-induced neurotoxicity are discussed.

Metabolic activation of the serotonergic neurotoxin para-chloroamphetamine to chemically reactive intermediates by hepatic and brain microsomal preparations.

Para-chloroamphetamine (PCA) is selectively toxic to serotonergic neurons in laboratory animals. Acute, reversible neurotoxicity is followed by long-term effects which include inactivation of tryptophan hydroxylase and destruction of neurons. We have studied the metabolic formation of reactive intermediates that might be involved in long-term PCA neurotoxicity. Incubation of [3H]PCA with rat hepatic microsomes resulted in NADPH-dependent and oxygen-dependent covalent binding of radioactivity to microsomal protein. Addition of SKF-525A and glutathione to incubation mixtures inhibited [3H]PCA covalent binding 30% and 92% respectively. No inhibition of radiolabeled covalent binding was observed in an atmosphere of carbon monoxide/oxygen (80/20). 7,8-Benzoflavone was more effective than metyrapone in inhibiting [3H]PCA covalent binding. The extent of [3H]PCA covalent binding to microsomal protein was unchanged after phenobarbital pretreatment of rats, whereas 3-methylcholanthrene pretreatment increased [3H]PCA covalent binding (175%). NADPH-dependent and oxygen-dependent covalent binding of radioactivity was also observed when [3H]PCA was incubated with rat brain microsomal preparations. Addition of SKF-525A and glutathione to incubation mixtures inhibited covalent binding 10 and 40% respectively. There were no significant differences in total, NADPH-independent or NADPH-dependent covalent binding of radiolabeled R,S(+/-)-, R(-)-, or S(+)-PCA to rat hepatic microsomal protein. Less covalent binding was observed when [3H]amphetamine was incubated with rat liver microsomal preparations as compared to results with [3H]PCA. Minimal covalent binding was observed when [3H]PCA was incubated with liver microsomal preparations from rabbits, a species resistant to PCA neurotoxicity. Results of these metabolism studies are consistent with the hypothesis that oxidative metabolic activation of PCA to reactive and toxic metabolites is related to the long-term neurotoxicity of this agent.

Studies on the mechanism of p-chloroamphetamine neurotoxicity.

Studies were conducted to investigate the sensitivity of p-chloroamphetamine (PCA)-induced neurochemical changes to various pharmacological manipulations known to block the neurochemical effects of 3,4-methylenedioxymethamphetamine (MDMA). The monoamine oxidase-B (MAO-B) inhibitor L-deprenyl (2 mg/kg) given 4 hr before a nonneurotoxic dose of PCA (2 mg/kg) was shown not to alter the amount of [3H]paroxetine bound to serotonin (5-HT) uptake sites 7 days after treatment. L-Deprenyl 4 hr before a neurotoxic dose of PCA (10 mg/kg) did not change the acute hyperthermia. Further, neither L-deprenyl nor another selective MAO-B inhibitor, MDL-72,974 (1.25 mg/kg), given 30 min before or daily for 4 days before a single dose of PCA attenuated or potentiated the decrease in the number of [3H]paroxetine binding sites measured 7 days after PCA treatment. The combination of the MAO-A inhibitor clorgyline (2.5 mg/kg) or a nonspecific dose of L-deprenyl (10 mg/kg) with the selective 5-HT releasing agent 5,6-methylenedioxy-2-aminoindan did not lead to changes in the levels of 5-HT, 5-hydroxyindoleacetic acid or dopamine 7 days after treatment. Finally, the 5-HT2A receptor antagonist MDL-11,939 (5 mg/kg) did not protect against the neurotoxicity of PCA. By comparing the present work with previous studies of MDMA, these results can be interpreted to suggest that the mechanism of the neurotoxicity induced by PCA is not identical to that induced by MDMA. The relationship of these results to the neurotoxicity induced by MDMA is also discussed.

Potentiation of oxotremorine-induced hypothermia by alaproclate in PCA lesioned and non-lesioned rats.

Stimulation of muscarinic cholinergic receptors with the highly potent and selective receptor agonist oxotremorine produced hypothermia in rats. Alaproclate, a purported selective serotonergic reuptake inhibitor, potentiated this response. Destruction of central presynaptic serotonergic terminals with the potent cytotoxin p-chloroamphetamine (PCA) failed to attenuate the hypothermic response to oxotremorine in alaproclate-pretreated animals. These results could be taken to suggest that alaproclate may act, at least in part, via a non-serotonergic mechanism to potentiate the oxotremorine-induced hypothermic response.

Increased behavioural activity of rats in forced swimming test after partial denervation of serotonergic system by parachloroamphetamine treatment.

The present study aimed at characterizing the effect of partial 5-HT denervation by parachloroamphetamine (PCA), a 5-HT selective neurotoxin, on forced swimming behaviour and monoamine levels in several rat brain regions. PCA was administered intraperitoneally in two independent experiments in doses of 2, 4 and 6 mg/kg and in doses 1, 2, 4 mg/kg, respectively. PCA (2 mg/kg) reduced immobility in the forced swimming test in the Experiment 1 and according to Experiment 2 this is explained by increased swimming time. Dose-dependent reductions in 5-HT and 5-HIAA levels were found in all brain regions studied, and the maximal effects were of a similar magnitude. In septum, the effect of PCA took more time to develop. The effects of the lowest dose of PCA suggest that the neurotoxin affects not only the dorsal raphe projection areas but also the fine axons which arise from the median raphe. alpha2-Adrenoceptors and beta-adrenoceptors in cerebral cortex were not affected by the PCA treatment. Binding affinity of the 5-HT(1A) receptors was higher after all doses of PCA. On the second exposure to the forced swimming the time spent in swimming was found to be negatively and the time spent in immobile posture positively correlated with serotonin turnover in frontal cortex. The time spent in struggling on the second exposure to test was found to be negatively correlated with KD of beta-adrenoceptor binding in cerebral cortex. These data suggest that partial 5-HT denervation with low doses of PCA, which elicits a specific pattern of neurodegeneration, results in an increased behavioural activity, and that the traditional interpretation of the measures in forced swimming test, despite of the test's predictive power in revealing antidepressants acting on monoaminergic systems, is not adequate for studies on the neurochemical basis of depression.

Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis.

The role of monoamines in the functional regulation of the septo-hippocampal cholinergic system was studied using in vivo microdialysis of acetylcholine (ACh) release in the hippocampus of awake unrestrained rats. Systemic administration of the dopamine receptor agonist apomorphine (2.0 mg/kg) resulted in a 170% increase in hippocampal ACh overflow. Similarly the catecholamine-releasing agent amphetamine (2.5 mg/kg) produced a 400% increase in ACh overflow. The effect induced by amphetamine, but not that of apomorphine, was blocked in animals pretreated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). The effect of amphetamine on ACh release was reduced by 75% after a 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA) but was not affected by 6-OHDA lesions of the noradrenergic dorsal and ventral bundles. However, baseline ACh overflow was increased by 130% by the dorsal and ventral bundle lesions. The serotonin-releasing agent p-chloroamphetamine (2.5 mg/kg) produced a 160% increase in hippocampal ACh release, and this effect was enhanced after a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the serotonin projection system. The results show that surgical or pharmacological manipulations of the ascending brainstem monoaminergic systems, which innervate wide areas of the forebrain, including the septum and the hippocampal formation, have pronounced effects on septo-hippocampal cholinergic activity. Thus, the present data provide support for the view that information regarding behavioral state and arousal is conveyed to the septo-hippocampal system via ascending monoaminergic systems.