Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Molecular and functional analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter.

Kidney proximal tubule cells take up Krebs cycle intermediates for metabolic purposes and for secretion of organic anions through dicarboxylate/organic anion exchange. Alteration in reabsorption of citrate is closely related to renal stone formation. The presence of distinct types of sodium-coupled dicarboxylate transporters has been postulated on either side of the polarized epithelial membrane in the kidney proximal tubule. Using a PCR-based approach, we isolated a novel member of the sodium-dependent dicarboxylate/sulfate transporter called SDCT2. SDCT2 is a 600-amino acid residue protein that has 47-48% amino acid identity to SDCT1 and NaDC-1, previously identified in kidney and intestine. Northern analysis gave a single band of 3.3 kb for SDCT2 in kidney, liver, and brain. In situ hybridization revealed that SDCT2 is prominently expressed in kidney proximal tubule S3 segments and in perivenous hepatocytes, consistent with the sites of high-affinity dicarboxylate transport identified based on vesicle studies. A signal was also detected in the meningeal layers of the brain. SDCT2 expressed in Xenopus oocytes mediated sodium-dependent transport of di- and tricarboxylates with substrate preference for succinate rather than citrate, but excluding monocarboxylates. SDCT2, unlike SDCT1, displayed a unique pH dependence for succinate transport (optimal pH 7.5-8.5) and showed a high affinity for dimethylsuccinate, two features characteristic of basolateral transport. These data help to interpret the mechanisms of renal citrate transport, their alteration in pathophysiological conditions, and their role in the elimination of organic anions and therapeutic drugs.

Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis.

Urea transport in the kidney plays an important role in urinary concentration and nitrogen balance. Recently, three types of urea transporters have been cloned, UT1 and UT2 from rat and rabbit kidney and HUT11 from human bone marrow. To elucidate the physiological role of the latter urea transporter, we have isolated the rat homologue (UT3) of HUT11 and studied its distribution of expression and functional characteristics. UT3 cDNA encodes a 384 amino acid residue protein, which has 80% identity to the human HUT11 and 62% identity to rat UT2. Functional expression in Xenopus oocytes induced a large (approximately 50-fold) increase in the uptake of urea compared with water-injected oocytes. The uptake was inhibited by phloretin (0.75 mM) and pCMBS (0.5 mM) (55 and 32% inhibition, respectively). Northern analysis gave a single band of 3.8 kb in kidney inner and outer medulla, testis, brain, bone marrow, spleen, thymus, and lung. In situ hybridization of rat kidney revealed that UT3 mRNA is expressed in the inner stripe of the outer medulla, inner medulla, the papillary surface epithelium, and the transitional urinary epithelium of urinary tracts. Co-staining experiments using antibody against von Willebrand factor showed that UT3 mRNA in the inner stripe of the outer medulla is expressed in descending vasa recta. These data suggest that UT3 in kidney is involved in counter current exchange between ascending and descending vasa recta, to enhance the cortico-papillary osmolality gradient. In situ hybridization of testis revealed that UT3 is located in Sertoli cells of seminiferous tubules. The signal was only detected in Sertoli cells associated with the early stages of spermatocyte development, suggesting that urea may play a role in spermatogenesis.

Behavioral and anatomical abnormalities in Mecp2 mutant mice: a model for Rett syndrome.

Over 90% of Rett syndrome (RTT) cases have a mutation in the X-linked gene encoding methyl CpG binding-protein 2 (MeCP2). A mouse model that reprises clinical manifestations of the disease would be valuable for examining disease mechanisms. Here, we characterize physical and behavioral measures, as well as brain region volumes in young adult mice that have mutations in mouse methyl CpG binding-protein 2 gene (Mecp2) to serve as a baseline for other studies. Hemizygous males, which produce no functional protein, exhibit hypoactivity and abnormalities in locomotion, stereotypies, and anxiety reminiscent of the clinical condition. The mutant males also exhibit cognitive deficits in fear conditioning and object recognition relative to wildtypes. Volumetric analyses of male brains revealed a 25% reduction in whole brain volume in mutants relative to wildtypes; regional differences were also apparent. Mutants had decreased volumes in three specific brain regions: the amygdala (39%), hippocampus (21%), and striatum (29%). Heterozygous females, which produce varying amounts of functional protein, displayed a less severe behavioral phenotype. The mutant females exhibit abnormalities in locomotion, anxiety measures, and cognitive deficits in object recognition in an open field. This study provides the first evidence that the abnormal motor and cognitive behavioral phenotype in Mecp2 mice is consistent with specific volume reductions in brain regions associated with these behaviors, and shows how these data parallel the human condition. The Mecp2 mutant mice provide a very good model in which to examine molecular and behavioral mechanisms, as well as potential therapeutic interventions in RTT.

Differential distribution of the glutamate transporters GLT-1 and GLAST in tanycytes of the third ventricle.

The ventral one-third of the ventricular lining in the hypothalamus is formed by specialized ependymal cells called the tanycytes. These cells may serve a neuroendocrine transport function because of their structural specializations, which include apical microvili on the ventricular surface and long basal processes that terminate on blood vessels or on the glia limitans. Here, we describe the expression of mRNA and protein for the glutamate transporters GLT-1 and GLAST in unique tanycyte populations of the third ventricle in rat brain. Using nonisotopic in situ hybridization, we demonstrate GLAST mRNA labeling in tanycytes of the ventral floor and lateral walls in the tuberal and mammillary recess portions of the third ventricle. This GLAST mRNA labeling had a higher intensity than the labeling intensity observed in regular ependymal cells throughout the ventricular system. Furthermore, we have identified strong GLT-1 mRNA labeling in a population of tanycytes situated in the dorsolateral walls of caudal tuberal and mammillary recess portions. Immunocytochemical staining indicates that both GLT-1 and GLAST protein are expressed in the tanycyte populations as well. These data corroborate previous findings that third ventricle tanycytes are functionally heterogeneous. Furthermore, the GLT-1-expressing tanycytes represent a population of tanycytes that, to date, has not been recognized as functionally distinct. The strong GLAST expression by the ventral tanycytes in the hypophysiotropic area suggests a role of tanycyte-mediated glutamate transport in neuroendocrine activity. The functional role of GLT-1 in dorsal wall tanycytes remains to be explored.

Distribution of the glutamate transporters GLAST and GLT-1 in rat circumventricular organs, meninges, and dorsal root ganglia.

The glial glutamate transporters GLAST and GLT-1 are primarily responsible for the removal of glutamate from brain extracellular fluid. This study compares the distribution of GLAST and GLT-1 expression in the circumventricular organs of the brain, in the meninges, and in the dorsal root ganglion. By using a highly sensitive nonisotopic in situ hybridization method and immunostaining, we demonstrate marked differences in the expression patterns for the two transporters. In the three sensory circumventricular organs that contain neuronal elements, i.e., the subfornical organ, the vascular organ of the lamina terminalis, and the area postrema, GLAST is strongly expressed, whereas GLT-1 is faintly expressed or absent. Both transporters are absent from the choroid plexus, and only GLAST mRNA is found in the subcommisural organ. In the pineal gland, GLAST is expressed by astrocytic cells near the pineal stalk, whereas GLT-1 is expressed by pinealocytes throughout the gland. In the pituitary gland, GLAST is likely expressed by folliculo-stellate cells in the anterior lobe, by a group of astrocyte-like cells and by marginal cells in the intermediate lobe, and by pituicytes in the posterior lobe, whereas GLT-1 is expressed only by the astrocyte-like cells in the intermediate lobe. Finally, GLAST, but not GLT-1, is expressed by specific layers of the meninges, and by satellite cells in the dorsal root ganglion. These results show that GLAST is the primary glutamate transporter in the circumventricular organs. The data provide further evidence that these two glutamate transporters fulfill markedly different functions in the nervous system.

Glutamate carboxypeptidase II is expressed by astrocytes in the adult rat nervous system.

The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Müller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells.

The immunocytochemical localization of N-acetylaspartyl glutamate, its hydrolysing enzyme NAALADase, and the NMDAR-1 receptor at a vertebrate neuromuscular junction.

Although glutamate is thought to be the neurotransmitter at the invertebrate neuromuscular junction, acetylcholine is accepted as the primary neurotransmitter of the vertebrate motoneurons. N-acetylaspartylglutamate, a dipeptide localized in putative glutamatergic neurons in brain, is also found in high concentrations (> mM) in mammalian motoneurons and the ventral roots of spinal cord. N-acetylaspartylglutamate, which is released from neurons by depolarization in a Ca(2+)-dependent fashion, is implicated in glutamatergic transmission in two ways: it is a partial agonist at NMDA receptors, and it is cleaved to yield extracellular glutamate and N-acetylasparate by the specific peptidase N-acetylated alpha-linked acidic dipeptidase. Given the localization of N-acetylaspartylglutamate in motor neuronal perikarya and axons, we wondered whether N-acetylaspartylglutamate or glutamate cleaved from N-acetylaspartylglutamate by N-acetylated alpha-linked acidic dipeptidase may also play a role in neuromuscular transmission. Here we describe the immunocytochemical detection at the rat neuromuscular junction of N-acetylaspartylglutamate in terminals of motoneurons, of N-acetylated alpha-linked acidic dipeptidase in perisynaptic Schwann cells, and of the NMDAR-1 glutamate receptor subunit on postsynaptic muscle membranes. These results point to a potential role for N-acetylaspartylglutamate at the rat neuromuscular junction. Further, this is the first demonstration of a glutamate receptor protein at vertebrate neuromuscular synapses. Together with other recent findings, our results suggest that glutamate-like molecules are involved in neuromuscular transmission not only in invertebrates but also in veretebrates where they may modulate signaling by acetylcholine.

Quantitative in vivo 1H nuclear magnetic resonance spectroscopic imaging of neuronal loss in rat brain.

The aim of this research was to determine whether in vivo nuclear magnetic resonance spectroscopic measurement of N-acetyl aspartate, a neuron specific brain marker, provides a quantitative index of neuronal loss. Five rats were injected unilaterally in the corpus striatum with kainic acid, an analogue of glutamate that causes excitotoxic degeneration of intrinsic neurons, and were subjected to nuclear magnetic resonance imaging and spectroscopic imaging. Measurements of N-acetyl aspartate were determined in vivo and compared to post mortem nuclear magnetic resonance spectroscopic measures of N-acetyl aspartate and choline acetyl transferase and glutamate decarboxylase activities, biochemical markers for striatal intrinsic neuronal integrity. Mean per cent neuronal survival of hemispheres with lesion versus the contralateral hemispheres measured 72 for glutamate decarboxylase and 71 for N-acetyl aspartate (in vivo), 74 for N-acetyl aspartate (in vitro), and 62 for choline acetyl transferase, respectively. Our studies in rats have shown that estimates of neuronal loss through nuclear magnetic resonance spectroscopic measurements of N-acetyl aspartate are equivalent to traditional neuronal enzyme activity assays. The results unequivocally demonstrate that N-acetyl aspartate is a valid and sensitive neuronal marker with the capability of providing accurate assessments of neuronal loss in vivo.

Neurotoxicity of MDMA and related compounds: anatomic studies.

The cytotoxic effects of amphetamine derivatives were studied by immunocytochemistry to identify the cellular compartments affected by these drugs, to obtain morphologic evidence of neuronal degeneration, and to assess the potential for regeneration. The substituted amphetamines, MDA, MDMA, PCA, and fenfluramine, all release serotonin and cause acute depletion of 5-HT from most axon terminals in forebrain. (1) Unequivocal signs of axon degeneration were seen at 36-48 hour survivals: 5-HT axons exhibited increased caliber, huge, swollen varicosities, fragmentation, and dilated proximal axon stumps. (2) Fine 5-HT axon terminals were persistently lost after drug administration, while beaded axons and raphe cell bodies were spared. These two types of 5-HT axons, which arise from separate raphe nuclei and form distinct ascending projections, are differentially vulnerable to psychotropic drugs. (3) From 2-8 months after treatment, there was progressive serotonergic re-innervation of neocortex along a fronto-occipital gradient. Longitudinal 5-HT axons grew into layers I and VI from rostral to caudal, before sprouting into middle cortical layers; this bilaminar pattern of growth simulates perinatal development of 5-HT innervation. This study demonstrates differential vulnerability of 5-HT projections, evidence for axonal degeneration, and sprouting of 5-HT axons leading to re-innervation of forebrain. While the sprouting axons are anatomically similar to the type that was damaged, it is not known whether a normal pattern of innervation is re-established.

The vitamin C transporter SVCT2 is expressed by astrocytes in culture but not in situ.

Ascorbic acid (vitamin C) is known to be selectively accumulated by brain cells through sodium-dependent vitamin C transporters. It is unclear however, whether this uptake occurs in neurons, astrocytes or both. Using Northern analysis we demonstrate that the recently cloned ascorbate transporter isoform SVCT2 is expressed by cultured astrocytes. In contrast, in situ hybridization experiments reveal that SVCT2 mRNA is expressed only in neurons and not in normal astrocytes or astrocytes stimulated by an intrastriatal injection of the neurotoxin quinolinic acid. We conclude that SVCT2 is neuron specific and that the majority of ascorbate storage occurs in neurons. Furthermore, we propose that the observed sodium-dependent ascorbate transport in cultured astrocytes may be due to artificial upregulation of SVCT2 during cell culturing.

The neurotoxic effects of p-chloroamphetamine in rat brain are blocked by prior depletion of serotonin.

Systemic administration of p-chloroamphetamine (PCA) causes degeneration of serotonergic (5-HT) axons, but recent data indicate that this drug itself is not neurotoxic when applied directly to 5-HT axons. The present study was designed to test whether the toxic effects of PCA in the brain are dependent on release of endogenous 5-HT and to identify which stores of 5-HT are involved. The long-term effects of PCA on brain levels of 5-HT and on central 5-HT axons were determined in rats that had been initially depleted of 5-HT by administration of p-chlorophenylalanine and reserpine. The results show that transient depletion of 5-HT provides substantial protection against subsequent PCA-induced degeneration of 5-HT axon terminals; the neurotoxicity induced by PCA thus appears to be dependent on the presence of endogenous stores of 5-HT. In addition, the protective effect of 5-HT depletion is found only after pretreatment regimens that deplete peripheral as well as central stores of 5-HT. We interpret this finding as evidence that release of 5-HT from peripheral storage sites may be necessary for the expression of PCA-induced toxicity. Based on these results, we propose that central neurotoxicity is not induced by a direct action of PCA alone but may require or be augmented by a toxic metabolite of 5-HT.

The substituted amphetamines 3,4-methylenedioxymethamphetamine, methamphetamine, p-chloroamphetamine and fenfluramine induce 5-hydroxytryptamine release via a common mechanism blocked by fluoxetine and cocaine.

The abilities of the substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, p-chloroamphetamine (PCA) and fenfluramine to induce synaptosomal [3H]serotonin (5-HT) release were compared using a novel microassay system. The rank order of release potencies was found to be (+/-)PCA congruent to (+)-fenfluramine greater than (+)-MDMA much greater than (+)-methamphetamine. Combination of two drugs at their EC50 did not cause more release than either drug alone at an equivalent concentration. In addition, the 5-HT uptake blockers fluoxetine and cocaine inhibited the release induced by MDMA, methamphetamine, PCA and fenfluramine to the same percentage. However, threshold concentrations of the substituted amphetamines known to inhibit uptake did not attenuate the release caused by higher concentrations of these compounds. These results suggests that MDMA, methamphetamine, PCA and fenfluramine cause 5-HT release via a common mechanism. Furthermore, these results indicate that the 5-HT uptake blockade induced by these substituted amphetamines in vitro is different from that induced by either fluoxetine or cocaine.

Distribution of peptide transporter PEPT2 mRNA in the rat nervous system.

The signaling action of neuropeptides in the brain is terminated by breakdown through extracellular peptidases and subsequent removal of the peptide fragments from the extracellular fluid via specific transporter proteins. Here we describe the anatomical distribution in the rat nervous system of the recently isolated high affinity peptide transporter PEPT2. Using nonisotopic in situ hybridization we demonstrate that PEPT2 mRNA is expressed in brain by astrocytes, subependymal cells, ependymal cells and epithelial cells of choroid plexus. Furthermore, PEPT2 is expressed in retina by Müller cells and in dorsal root ganglia by satellite cells. The mRNA levels of PEPT2 in astrocytes are moderate and relatively homogenous throughout the brain except for an area in ventral forebrain where PEPT2 levels are below average. PEPT2 mRNA expression is weakly upregulated in reactive astrocytes that were stimulated through an injection of the glutamatergic neurotoxin quinolinic acid. These data suggest that removal of neuropeptide fragments from brain extracellular fluid occurs via PEPT2 expressed in astrocytes, ependymal cells and choroid plexus epithelial cells.

Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization.

This study compares the mRNA expression pattern for the three glutamate transporters EAAC1, GLT1 and GLAST in rat brain, using a sensitive non-radioactive in situ hybridization technique. The results confirm the predominantly neuronal localization of EAAC1 mRNA, the astroglial and ependymal localization of GLAST mRNA and the astroglial and neuronal localization of GLT1 mRNA. Further, we demonstrate, using a novel differential double hybridization protocol, that the presence of GLT1 mRNA in neurons is more widespread than previously thought, and that it encompasses the majority of neurons in the neocortex, neurons in the external plexiform layer in the olfactory bulb, neurons in dorsal and ventral parts of the anterior olfactory nucleus, the majority of neurons in the anteromedial thalamic nuclei, the CA3 pyramidal neurons in the hippocampus and neurons in the inferior olive. In addition, we demonstrate marked variations in the expression levels of GLT1 and GLAST mRNAs in different brain areas, suggesting that their mRNA levels are regulated by different mechanisms. Finally, for EAAC1 we demonstrate also a widespread distribution and a marked heterogeneity in the expression levels. EAAC1 is strongly expressed by a heretofore unrecognized group of cells in white matter tracts such as the corpus callosum, fimbria-fornix or anterior commissure. Also, strong EAAC1 expression is present in groups of scattered cells in grey matter areas of much of the forebrain and the cerebellum. These results provide more detailed information about the precise cellular localization of these three glutamate transporters and their regulation at the mRNA level.

Distribution of mRNA for the facilitated urea transporter UT3 in the rat nervous system.

Recently, the cDNA encoding the rat urea transporter UT3 has been cloned from rat kidney. Here we describe the cellular localization of this transporter in the brain as detected by non-radioactive in situ hybridization. UT3 is expressed in astrocytes throughout the central nervous system as well as in Bergmann glia in the cerebellum. The expression in astrocytes was verified by double staining using the astrocytic marker GFAP. UT3 mRNA is also strongly expressed by the ependymal cells lining the cerebral ventricles and by Müller cells in the retina. Furthermore, UT3 expression was detected in subgroups of neurons in the inferior colliculus and dorsal root ganglia, as well as in cells in the anterior pituitary gland. Other types of brain cells, including oligodendrocytes, microglia, tanycytes, endothelial cells of blood vessels, and epithelial cells in the choroid plexus were devoid of UT3 mRNA. Northern blot analysis confirmed that the mRNA species in the brain and in dorsal root ganglia are identical, and that cultured astrocytes and C6 cells also express the UT3 mRNA. UT3 mRNA expression by astrocytes is markedly upregulated in quinolinic acid-induced gliosis, possibly as a result of increased urea levels during gliosis induced polyamine formation. We propose that UT3 in astrocytes represents a mechanism to control urea formed in the brain by equilibrating it throughout the astrocyte network and guiding it to blood vessels and the CSF for disposal.

N-acetylated alpha-linked acidic dipeptidase may be involved in axon-Schwann cell signalling.

N-Acetylated alpha-linked acidic dipeptidase is a membrane-bound peptidase that cleaves the neuropeptide N-acetyl-aspartyl-glutamate to N-acetyl-aspartate and glutamate. Previously, we have shown that in adult rat this enzyme is expressed by the non-myelinating Schwann cells in peripheral nerve. In the present study, we have determined the expression pattern of this peptidase in rat sciatic nerve during late embryonal and early postnatal development, using double-label immunofluorescence, enzyme assays and immunoblotting. We demonstrate that N-acetylated alpha-linked acidic dipeptidase is expressed by all Schwann cell precursor cells on embryonal day 14/15 and by all undifferentiated Schwann cells on embryonal days 16/17 and 20/21 and postnatal day 1. Moreover, we show that during the first postnatal week, the peptidase expression is down-regulated in the myelinating Schwann cells while the total enzyme activity levels and the enzyme amounts present in the nerve are transiently increased. To determine whether Schwann cell peptidase expression is dependent on axonal contact, we performed immunofluorescence experiments in cultured Schwann cells. These in vitro experiments demonstrate that the expression of this enzyme is maintained in culture for several weeks without axonal contact. Furthermore, they confirm previous suggestions that this peptidase is expressed on the extracellular side of the Schwann cell membrane. These findings support the notion that N-acetylated alpha-linked acidic dipeptidase takes part in signaling between peripheral axons and Schwann cells. The temporary increase in peptidase activity during the first postnatal week strongly implicates a role for this enzyme in the process of axon ensheathment and/or axon myelination.

Unlike systemic administration of p-chloroamphetamine, direct intracerebral injection does not produce degeneration of 5-HT axons.

Systemic administration of the amphetamine derivative p-chloroamphetamine (PCA) causes degeneration of 5-HT axon terminals in rat brain. The present study was designed to determine whether PCA induces neurotoxic effects by a direct action on 5-HT axon terminals. PCA was administered by microinjection directly into the cerebral cortex of rats. Continuous intracerebral infusions were made over extended time periods (10 min-48 h) to explore whether the induction of neurotoxicity requires a prolonged exposure of axon terminals to the drug. Two weeks after drug administration, brain sections that passed through the injection site were processed for 5-HT immunohistochemistry. The 5-HT innervation of cerebral cortex in PCA-injected animals was compared with that after intracortical injection of saline or of 5,7-dihydroxytryptamine. The results demonstrate that, in the concentrations used, direct application of PCA into the neocortex does not elicit axonal degeneration, even after a continuous infusion for 2 days. This finding suggests that PCA itself is not directly toxic to 5-HT axons.

Depletion of serotonin using p-chlorophenylalanine (PCPA) and reserpine protects against the neurotoxic effects of p-chloroamphetamine (PCA) in the brain.

The present study attempts to determine whether the neurotoxicity of p-chloroamphetamine (PCA) is dependent on a releasable pool of serotonin (5-HT). Rats treated with PCA alone or with reserpine and PCA exhibit a profound loss of 5-HT innervation in cerebral cortex after a 2-week survival period. However, depletion of 5-HT by combined treatment with p-chlorophenylalanine (PCPA) and reserpine provides substantial protection against the neurotoxic effects of PCA. These results indicate that release of 5-HT is a necessary step in the neurotoxicity of PCA and that a peripheral source of 5-HT is involved. We suggest that 5-HT release from platelets into the peripheral circulation may result in the formation of a neurotoxic 5-HT metabolite.