[Simultaneous detection of tryptamine and dopamine in the substantia nigra and raphe nuclei in rats using specific antibodies]. / Détection simultanée par anticorps spécifiques de tryptamine et dopamine dans la substance noire et les noyaux du raphé chez le rat.

Using a double-labelling procedure, morphological relationships existing between dopaminergic and indoleaminergic neuronal systems in rat brain were investigated. Firstly, thanks to a tryptamine (T) antiserum, we visualized this indoleamine in all mesencephalic regions and showed that the T-immunoreactivity (IR) seems to overlap with the staining observed from serotonin (HT) and 5-methoxytryptamine (MT) antisera. Secondly, using a monoclonal anti-dopamine (DA) antibody and our anti-T antibodies, the simultaneous and specific detection of these compounds enabled us to define the chemically relationships existing between the dopaminergic and tryptaminergic neuronal systems from substantia nigra to raphe nuclei. No co-localization exists. But, the intensity of T-IR decreases from the back to the front, whereas the DA-staining decreases in the opposite way, indicating possible interactions at the end of substantia nigra and B9 area.

[Study of characteristics of monoclonal antibody against substance P].

Cross-reaction of a rat monoclonal antibody (BTP-1) against seventeen substance P analogues was studied. The antibody was of IgG type and related to the carboxyl terminal of substance P, especially methionyl in the terminal, but did not depend on the strength of antagonistic effects of these analogues. It did not show cross-reaction with the following nine peptides: glucagon, endorphin, angiotensin I, II, leucine-enkephalin, methionine-enkephalin, bradykinin, oxytocin and dernorthin, indicating its high specificity to substance P. By means of immuno-enzyme histochemical method, it was shown that stained nerve fibers were located in the gelaliternous substance of Rolando, interpeduncular nucleus, substantia nigra and nerve cell bodies in the vestibular nucleus, lateral tegmental nucleus of mesencephalon and ventral region of third ventricle.

Distribution of DARPP-32 in the basal ganglia: an electron microscopic study.

DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, has been studied by light and electron microscopical immunocytochemistry in the rat caudatoputamen, globus pallidus and substantia nigra. In the caudatoputamen, DARPP-32 was present in neurons of the medium-sized spiny type. Immunoreactivity for DARPP-32 was present in dendritic spines, dendrites, perikaryal cytoplasm, most but not all nuclei, axons and a small number of axon terminals. Immunoreactive axon terminals in the caudatoputamen formed symmetrical synapses with immunolabeled dendritic shafts or somata. Neurons having indented nuclei were never immunoreactive. In the globus pallidus and substantia nigra pars reticulata, DARPP-32 was present in myelinated and unmyelinated axons and in axon terminals. The labelled axon terminals in these regions formed symmetrical synaptic contacts on unlabelled dendritic shafts or on unlabelled somata. These data suggest that DARPP-32 is present in striatal neurons of the medium-sized spiny type and that these DARPP-32-immunoreactive neurons form symmetrical synapses on target neurons in the globus pallidus and substantia nigra. The presence of DARPP-32 in these striatal neurons and in their axon terminals suggests that DARPP-32 mediates part of the response of medium-size spiny neurons in the striatum to dopamine D-1 receptor activation.

Expression of a human cocaine-sensitive dopamine transporter in Xenopus laevis oocytes.

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.

An immunohistochemical demonstration of gastrin-releasing peptide (GRP) in the rat substantia nigra.

The presence of gastrin-releasing peptide-like immunoreactivity in the rat brain was investigated by use of the indirect peroxidase-antiperoxidase technique. A high density of gastrin-releasing peptide-like immunoreactive terminals in the ventral pallidum, the interpenduncular nucleus and in substantia nigra, pars reticulata, was observed. Moreover, gastrin-releasing peptide-like immunoreactive perikarya were observed in the hypothalamic suprachiasmatic nucleus. Antisera raised against gastrin-releasing peptide have been shown to cross-react with substance P, another peptide highly concentrated in the substantia nigra, the ventral pallidum and the interpenduncular nucleus, and gastrin-releasing peptide-immunoreactivity in these areas has therefore been regarded as substance P immunoreactivity. To determine the antigenic epitope recognized by the antiserum raised against gastrin-releasing peptide, specificity studies were performed with known peptides fixed to nitrocellulose filter strips as well as preabsorptions with the same peptides on fixed brain sections containing the substantia nigra. From these experiments, it could be deduced that the antiserum recognizes an epitope within the peptide sequence: Val-Gly-His-Leu-Met-NH2. The antiserum cross-reacts with bombesin and alytesin, but not with substance P, allowing us to conclude that gastrin-releasing peptide or a very closely related peptide is present in areas of the rat central nervous system in which substance P has previously also been shown to be present.

Localization of ARPP-90, a major 90 kiloDalton basal ganglion-enriched substrate for cyclic AMP-dependent protein kinase, in striatonigral neurons in the rat brain.

Cyclic AMP-regulated phosphoproteins with specific cellular localizations in brain represent important targets through which this second messenger system can mediate or modulate distinct neurotransmitter signals. This study reports that two cyclic AMP-regulated phosphoproteins (Mr 90,000 and 93,000) found in brain share several properties, including similar isoelectric points and similar phosphopeptide maps. This protein doublet is particularly enriched in the forebrain basal ganglia, but it can also be found in the substantia nigra, a brainstem region which is a major target for fibers from the forebrain basal ganglia. Quinolinic acid lesions of neurons in the neostriatum decrease the levels of the 90/93 kDa phosphoprotein doublet to about the same extent as they reduce the levels of DARPP-32, a phosphoprotein specifically enriched in striatonigral medium-sized spiny neurons. These reductions are seen in both the neostriatum and the substantia nigra. Therefore, within the basal ganglia, the 90/93 kDa phosphoprotein doublet, termed adenosine 3':5'-monophosphate-regulated phosphoprotein, Mr = 90,000 (ARPP-90), is largely, if not solely, present in striatonigral cells and fibers. The specific localization in these neurons suggests that ARPP-90 could be important in receptor-regulated, cyclic AMP-mediated functions in the striatonigral neurons.

Cholecystokinin system in striatal-nigral neuronal networks: infusion of quinolinic acid into rat striatum.

Unilateral infusions of quinolinic acid (QUIN) into the rat striatum led to an increase in cholecystokinin octapeptide sulfate-like immunoreactivity (CCK8S-LI) in the striatum and substantia nigra 4 days later. These changes were suppressed by the injection of gamma-aminobutyric acid into substantia nigra 30 min before sacrifice. Intraperitoneal administration of haloperidol 40 min before sacrifice also suppressed the effect of QUIN on CCK. These results suggest that nigrostriatal dopaminergic neurons regulate CCK neurons via presynaptic sites in the striatum, and also that striatonigral GABAergic neurons interact with CCK neurons in the substantia nigra.

Regional distribution of ubiquinones and tocopherols in the mouse brain: lowest content of ubiquinols in the substantia nigra.

C57 black mice of 3 months of age were sacrificed, and their brain regionally dissected according to a protocol that strickly control for the death-freezing interval of each region. HPLC measurements of tocopherols and oxidized and reduced ubiquinones demonstrated significant regional variations. The substantia nigra had the lowest content of Q10 and a skewed ratio in favor of its oxidized form. Forebrain cholinergic nuclei had also more oxydized than reduced Q10 and in addition the lowest content of tocopherols. These findings suggest that nuclei that show neuronal depletion with age are the ones prone to oxidative stress.

Rate of cell death in parkinsonism indicates active neuropathological process.

It has been hypothesized that idiopathic parkinsonism might be due to age-related attrition of dopamine neurons occurring long after an initial acute episode. We present evidence against this hypothesis, based on our finding of at least six times as many HLA-DR-positive microglia phagocytosing dopamine neurons in parkinsonian brains as in control brains. This difference indicates an active pathological process.

In vivo dopaminergic activity from nucleus accumbens, substantia nigra and ventral tegmental area in the freely moving rat: basal neurochemical output and prolactin effect.

In this paper, by means of a push-pull perfusion technique, we have examined the local effect of ovine prolactin (oPRL) on the spontaneous in vivo dopaminergic (DAergic) activity of the nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) of living male rats. First, we found a 2-fold spontaneous rise of 3,4-dihydroxyphenylacetic acid (DOPAC) output during the afternoon hours of the photoperiod only in the NAcc. In addition, native oPRL (10 ng/microliters for 20 min), but not boiled oPRL, perfused into the NAcc caused a significant elevation in DOPAC output without changes in homovanillic acid or 5-hydroxyindoleacetic acid. In a similar preparation, male rats bearing push-pull cannulae in the SN or VTA were infused locally with oPRL (10 or 50 ng/microliters) for 20 min. The hormone did not alter the efflux of DOPAC from these DA cell body/dendrite areas. These results indicate that oPRL can activate the mesolimbic DA system through local actions on DA presynaptic terminals of the NAcc without affecting the SN or VTA.

Alterations of the central noradrenergic system in MPTP-induced monkey parkinsonism.

Alterations of the coeruleo-cortical adrenergic system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced monkey parkinsonism were examined through neuropathological observation and radioreceptor assay. Neuronal degeneration was found not only in the substantia nigra but also in the locus coeruleus of all the MPTP-treated monkeys. From radioreceptor assay, decreases of alpha 1 and alpha 2 receptors in the parietal and temporal cortices were observed in parkinsonian monkeys. In addition, beta receptors increased in the temporal cortex. These changes in monkey parkinsonism are very similar to those in human Parkinson's disease.

Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains.

We detected large numbers of HLA-DR-positive reactive microglia (macrophages), along with Lewy bodies and free melanin, in the substantia nigra of all cases studied with Parkinson's disease (5) and parkinsonism with dementia (PD) (5). We found similar, but less extensive, pathology in the substantia nigra of six of nine cases of dementia of the Alzheimer type (DAT) but in only one of 11 age-matched nonneurologic cases. All dementia cases with a premortem diagnosis of DAT or PD showed large numbers of HLA-DR-positive reactive microglia and significant plaque and tangle counts in the hippocampus, as well as reduced cortical choline acetyltransferase activity. One of 11 nondemented controls showed mild evidence of similar cortical pathology. These data indicate that HLA-DR-positive reactive microglia are a sensitive index of neuropathologic activity. They suggest a frequent coexistence of DAT- and Parkinson-type pathology in elderly patients.

Topographical organization of efferent projections from the cat substantia nigra pars reticulata.

The topographical organization of the efferent projections from the cat substantia nigra (SN) to the thalamus and the superior colliculus was examined using the anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) and of labelled proteins. HRP-WGA or a mixture of [14C]amino acids was injected into various areas of the SN and the transported material visualized on coronal brain sections by histochemistry or autoradiography, respectively. The retrograde transport of [14C]gamma-aminobutyric acid ([14C]GABA) injected into thalamic nuclei was used also to determine the identity of the nigrothalamic projections. Identical results were found using either the anterograde transport of HRP-WGA or of labelled proteins. In the thalamus, dense nigral projections were observed in the nucleus ventralis medialis (VM) and in the rostromedioventral part of the nucleus ventralis lateralis (VL) whilst more limited projections were seen in the nuclei centralis lateralis (CL) and paracentralis (PC) as well as in the paralamellar zone of the nucleus medialis dorsalis (MD-Il). In addition, a patchy distribution of HRP-WGA or of radioactivity was found in the intermediate layer of the superior colliculus. More precisely, labelling of the VM was dense following injection of [14C]amino acids into the intermediate part of the SN pars reticulata (SNR) regardless of the depth of the injection site, whilst the intralaminar nuclei were labelled preferentially following injections made into the dorsal part of the intermediate SNR. Nigral projections to the intermediate layer of the superior colliculus were visualized over the whole mediocaudal and laterorostral extent when [14C]amino acids were injected into the rostral part of the SNR. Labelling of the superior colliculus was also seen following injection of [14C]amino acids into the intermediate part of the SNR but, in this case, ventral injections led to a more intense labelling than dorsal ones. Both the SNR and the SN pars compacta (SNC) were labelled when [14C]GABA was injected into the VM nucleus of the thalamus, confirming that the nigro VM projection is GABAergic and showing that recurrent collaterals of these GABAergic cells innervating the SNC also contained the transported radioactive material. In this condition ([14C]GABA injection into the VM), the thalamic reticularis nucleus also exhibited a dense labelling.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium binding protein in the basal ganglia system of a non-mammalian vertebrate: an immunohistochemical study in the reptile Caiman crocodilus.

In Caiman, calbindin D28K immunoreactivity (CaBP) was observed within many neurons of the substantia nigra (SN) but only in the caudal portion of the area ventralis of Tsai (AVT). A dense CaBP fiber plexus showing some regional inhomogeneity was observed in the dorsolateral portion of the telencephalic ventrolateral area (VLA). These results are consistent with previous reports that the SN and AVT project to the dorsolateral and medial portions of the VLA, and strongly support the theory that the caiman VLA contains cell populations homologous to those found in the mammalian corpus striatum.

Loss of matrix calcium-binding protein-containing neurons in Huntington's disease.

In post-mortem brain specimens from patients dying with a clinical diagnosis of Huntington's disease (HD) immunohistochemistry showed a substantial loss from the neostriatum of neurons containing the calcium-binding protein calbindin 28K. These calbindin neurons, and the straital compartment in which they are sited, are particularly damaged in HD, suggesting that a failure of calcium buffering or homeostasis may contribute to cell death in HD.

Substance P-dopamine relationship in the rat substantia nigra: a light and electron microscopy study of double immunocytochemically labeled materials.

The synaptic relationships between substance P-containing terminals and dopaminergic neurons (immunoreactive for tyrosine hydroxylase) in the substantia nigra were studied at both light and electron microscopic levels using a pre-embedding double-labeling immunocytochemical method. Many substance P-containing terminals were found to form synapses directly with dendrites and somata of nigral dopaminergic neurons. Since most of the substance P-containing axon terminals arise from the striatum, this result suggests that striatal substance P neurons can have monosynaptic influence on nigral dopaminergic neurons.

Effects of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine isomers on central serotonergic, dopaminergic and nigral neurotensin systems of the rat.

This study demonstrates that the isomers of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) are different in their ability to induce changes in serotonergic parameters and nigral concentrations of neurotensin-like immunoreactivity. With five successive doses (3.5 mg/kg) the d-MDA isomer was more potent than the l-MDA in its ability to decrease the concentrations of serotonin in the frontal cortex and hippocampus. The same difference occurred in the ability to decrease the hippocampal activity of tryptophan hydroxylase as well as the hippocampal and neostriatal 5-hydroxyindoleacetic acid concentrations. However, both isomers of MDMA were equipotent in their ability to decrease serotonergic parameters in the brain areas examined. When the doses were increased to 5 and 10 mg/kg, both isomers of MDA were equipotent in their effects on the serotonin system, whereas the l-MDMA was significantly less potent than its d isomeric counterpart in causing a decrease in serotonergic parameters of the different brain areas. In contrast, treatments with any of the isomers appeared to have a minimal impact on neostriatal dopaminergic parameters. However, treatment with MDA or MDMA caused increases in the nigral concentrations of neurotensin, with the d isomer of both compounds having substantially greater effects on this neuropeptide system. These increases are suspected to result from drug-released dopamine. This study demonstrates that at selected doses, the d isomers of MDA and MDMA are more potent than their l forms in affecting neurochemical systems, whereas high doses of either isomer of MDA share a common ability to induce changes in the serotonergic system that are likely associated with neuronal damage.

Subcellular distribution of mammalian tachykinins in rat basal ganglia.

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.