Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

AN ADAPTATION OF THE PUSH-PULL CANNULA METHOD TO STUDY THE IN VIVO RELEASE OF [3H]DOPAMINE SYNTHESIZED FROM [3H]TYROSINE IN THE CAT CAUDATE NUCLEUS: EFFECTS OF VARIOUS PHYSICAL AND PHARMACOLOGICAL TREATMENTS

Abstract— The release of [³H]dopamine ([³H]DA) continuously synthesized from l-[3,5–³H]tyrosine from the caudate nucleus of the cat was estimated in halothane anaesthetized or‘encéphale isolé’animals. For this purpose, an improved superfusion cannula, avoiding tissue damage, was used. The best localization for the tip of the superfusion cannula was found first by determining the topographical distribution of endogenous DA within the caudate nucleus. A rostro-caudal heterogenous distribution of the transmitter was detected. In perfusion experiments, l-[3,5–³H]tyrosine was introduced continuously at a rate of 33μl/min. [³H]DA was the only catecholamine found in serial 15 min fractions as revealed by cochromatography. The spontaneous release of [³H]DA was greater in anaesthetized than in ‘encéphale isolé’ cats; it represented 150 and 100 times the blank value, respectively. Depolarization by K⁺ (30 mm) applied locally in the striatum or by electrical or mechanical stimulation of the substantia nigra caused a transitory increase in [³H]DA release. Conversely, a decrease in nerve activity induced by tetrodotoxin (5 × 10^?-⁷ m) or by electrocoagulation of the substantia nigra was associated with a decline in the amounts of [³H]DA in superfusates. A temporary reduction in [³H]DA release could also be obtained by a short-lasting cooling block of the substantia nigra. As expected, d-amphetamine (10^?-⁵ m) and benzotropine(10^?-⁷ m) added to the superfusing medium increased [³H]DA release. These pharmacological results, as well as the changes in [³H]DA release observed after various manipulations of the activity of dopaminergic neurones, confirms the validity and the high sensitivity of this approach.     

In vivo release of adenosine from cat basal ganglia—studies with a push pull cannula

The release of newly synthesized [³H]adenosine has been studied in vivo in cat caudate nucleus and substantia nigra, using a push pull cannula. In the presence of [³H]adenosine as precursor, spontaneously released [³H]adenosine was easily detectable in superfusates of the push pull cannula. In the caudate nucleus, potassium and veratridine caused a marked and reversible increase in [³H]adenosine release. The effect of veratridine was completely blocked by tetrodotoxin (TTX) although TTX had no action by itself. Ouabain as well as glutamate, also markedly increased the release of [³H]adenosine.The specific 5′ nucleotidase inhibitor α,β-methylene ADP, did not alter the increase in the amount of [³H]adenosine obtained by veratridine, although it diminished the spontaneous release of [³H]adenosine by about 20%.Push pull cannulae were also implanted simultaneously into the caudate nucleus and substantia nigra. Potassium applied into the caudate nucleus increased the local release of adenosine but did not change that observed in the substantia nigra. When potassium was applied into the substantia nigra, it also increased the local release of adenosine but did not change that observed in the caudate nucleus.The results are discussed in term of the possible existence of “purinergic neurons” and of the relation between the adenosine release and central nervous activity.     

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride

In vitro labeling of tissue sections with [³H]sulpiride has been utilized in the present study to autoradiographically localize D₂-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [³H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [³H]sulpiride for the D₂-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.     

Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro

Potassium chloride (25 mM) and (+)-amphetamine (100 microM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4-dihydroxyphenylethylamine [( 3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6-hydroxydopamine (6-OHDA) lesions of one nigrostriatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride- or amphetamine-induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6-OHDA-lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synaptosomal preparations of striatum, nomifensine but not desipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6-OHDA lesions of one nigrostriatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5-hydroxytryptamine ([3H]5-HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5-HT uptake into striatal synaptosomal preparations. Following 6-OHDA lesions of one nigro-striatal pathway the uptake of [3H]5-HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on axonal transport in dopaminergic neurons: effect of neuropharmacologic lesions

Axonal transport of [³H]protein to the nucleus accumbens, olfactory tubercle, septal region, caudate nucleus, and hypothalamic region was investigated in rats after unilateral injection of [³H]lysine into the substantia nigra. Co-injection of 2 μg of colchicine with the [³H]lysine depressed the recovery of [³H]protein from forebrain structures by over 70 percent without altering incorporation into midbrain protein, whereas 1 or 2 μg of cycloheximide decreased the incorporation of labelled lysine into both midbrain and forebrain protein by 69 to 76 percent. Partial 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra decreased striatal dopamine levels by 78 percent and reduced axonal protein transport by 47 to 82 percent. Injecting the [³H]lysine 2 mm dorsal to the substantia nigra decreased transport by 95 percent. Unilateral kainic acid-induced lesions of the caudate, which decreased striatal glutamic acid decarboxylase activity by 44 percent and spared striatal dopamine content, did not alter the transport of [³H]protein. Thus, axonal transport of protein in dopamine-containing systems is dependent upon the site of injection of labelled precursor and upon the integrity of a 6-OHDA sensitive pathway. Further, transport is sensitive to inhibitors of both microtubule assembly and protein synthesis, and insensitive to intrastriatal kainic acid lesions.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study

The effect of a unilateral perinatal hypoxic-ischemic brain injury on dopamine D1 and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2-3 weeks after the insult. We observed significant decreases in the Bmax and KD for [3H]SCH 23390-labeled D1 and in the Bmax for [3H]spiperone-labeled D2 receptors in the lesioned caudate-putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate-putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate-putamen. Pronounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant change in Bmax or KD for [3H]mazindol-labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic-ischemic injury. The absence of changes in [3H]mazindol binding or dopamine levels in the lesioned caudate-putamen indicates that the dopaminergic presynaptic structures are preserved.     

Evidence for D2 Receptor Regulation of Dopamine Release in the Goldfish Retina

The possible existence of a dopamine D₂ receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [³H]dopamine and superfused with D₂ dopamine receptor agonists or antagonists to determine if there was an effect on [³H]dopamine release. The D₂ receptor antagonist sulpiride increased both baseline [³H]- dopamine release and [³H]dopamine release induced by an increase in extracellular potassium concentration. The D₂ receptor agonists LY-171555 and RU-24213 did not reduce baseline [³H]dopamine release but completely inhibited [³H]dopamine release induced by an increase in [K^±]_o. This action of the D₂ agonists was blocked by sulpiride. These studies demonstrate the existence of D₂ receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.     

Neuronal and Glial Release of [3H]GABA from the Rat Olfactory Bulb

Abstract: GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [³H]-γ-aminobutyric acid ([³H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [³H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine-evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine-evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca²⁺-free solution. β-Alanine was strongly exchanged with [³H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The β-alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine-stimulated release of [³H]GABA was not significantly reduced after the β-alanine heteroexchange. Stimulation of the [³H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [³H]GABA release when a high magnesium concentration was added to a calcium-free solution. When D-600 was used as a calcium-flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.     

Neostriatal dopamine receptor loss and behavioral deficits in the senescent rat

This study was carried out to evaluate the behavioral implications of previously reported declines in striatal dopamine receptors sensitive to [³H]-neuroleptic specific binding. Rotational behavior was examined following right intrastriatal dopamine (DA) injections in nialamide pretreated rats that had been previously unilaterally lesioned in the left substantia nigra with 6-hydroxydopamine. Results showed that following DA injections old rats exhibited significant deficits in rotational behavioral response strength when compared to young rats. Results are discussed in terms of relating behavioral alterations in stereotypic behavior that occur with senescence to changes in striatal D₂ receptors.     

Release and Uptake Rates of 5-Hydroxytryptamine in the Dorsal Raphe and Substantia Nigra Reticulata of the Rat Brain

Abstract: Fast scan cyclic voltammetry with carbon fiber electrodes has been used to investigate the dynamics of the neurotransmitter 5-hydroxytryptamine (5-HT) in the extracellular fluid of two brain regions: the dorsal raphe and the substantia nigra reticulata. The method used previously was shown to be optimized to allow the time course of 5-HT concentration changes to be measured rapidly. Measurements were made in slices prepared from the brains of rats with the carbon fiber electrode inserted into the tissue and a bipolar stimulating electrode placed on the slice surface. Identification of 5-HT as the detected substance in both regions was based on voltammetric, anatomical, physiological, and pharmacological evidence. Autoradiography using [³H]paroxetine revealed highest 5-HT transporter binding densities in the regions in which voltammetric measurements were made. Evaluation of the pharmacological actions of tetrodotoxin and tetrabenazine, as well as the effects of calcium removal, suggested that 5-HT storage was vesicular and that the release process was exocytotic. The effects of fluoxetine (0.5 µM) were typical of a competitive uptake inhibitor, changing K_m with little effect on V_max. Release of 5-HT was found to be maximal with wide (2-ms) stimulus pulses in both regions, as expected for release from small unmyelinated processes, and to increase linearly with the number of pulses when high frequencies (100 Hz) were used. At lower frequencies, the concentration observed was a function of both release and uptake. Kinetic simulations of the data revealed that the major difference in 5-HT neurotransmission between the two regions was that release and uptake rates are twice as large in the dorsal raphe ([5-HT] per pulse = 100 ± 20 nM, V_max = 1,300 ± 20 nM/s for dorsal raphe; [5-HT] per pulse = 55 ± 7 nM, V_max = 570 ± 70 nM/s for substantia nigra reticulata). When normalized to tissue content, uptake rates in both regions were identical and similar to rates previously reported for dopamine in dopamine terminal regions. Nonetheless, compared with dopaminergic transmission in terminal regions such as the striatum, the absolute clearance rates in the substantia nigra reticulata and dorsal raphe were lower, resulting in a longer lifetime of 5-HT in the extracellular fluid and allowing long-range interactions.     

Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.     

Investigations concerning the cellular origin of dopamine receptors

The cellular investigation of dopamine receptors was examined using glial cell fractions and synaptic fractions prepared from bovine caudate. Binding of the dopamine agonist apomorphine and antagonist spiroperidol was examined. A fractionation of bovine substantia nigra was developed. No evidence of haloperidol binding was found in neuronal, glial or synaptosomal fractions from this tissue. Finally, an autoradiographic localication of [³H]-haloperidol was undertaken. The results support the astroglial localization of a significant portion of the dopamine receptors.     

Functional Identification and Molecular Cloning of a Human Brain Vesicle Monoamine Transporter

A vesicle monoamine transporter was functionally identified, molecularly cloned, and characterized from a human substantia nigra cDNA library. The ATP-dependent transport of 5-[³H]hydroxytryptamine ([³H]5-HT) by digitonin-permeabilized fibroblasts expressing the vesicle monoamine/H^± antiporter in culture exhibited a K_m of 0.55 μM. Reserpine and tetrabenazine, inhibitors of two monoamine binding sites, effectively blocked [³H]5-HT accumulation with K₁ values of 34 and 78 nM, respectively. Pretreatment of cells with as little as 10 nM reserpine in the presence of ATP abolished uptake. The rank order for substrate inhibition of [³H]5-HT uptake for both the previously reported rat vMAT1 and the human transporter clone followed the order 5-HT > dopamine > epinephrine > norepinephrine > 1 -methyl-4-phen- ylpyridinium > 2-phenylethylamine > histamine. The virtually identical transport characteristics of rvMATI and hvMAT1 confirm the relevance of neuropharmacological studies of rat brain biogenic amine uptake and storage to human brain neurochemistry.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Halothane Increases Non-vesicular [<Superscript>3</Superscript>H]dopamine Release from Brain Cortical Slices

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [³H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [³H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [³H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na⁺ channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [³H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [³H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [³H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [³H]DA into rat brain cortical slices. A decrease on halothane-induced release of [³H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na⁺/K⁺ ATPase pump inhibitor, which induces DA release through reverse transport, decreased [³H]DA release induced by halothane. It is suggested that halothane increases [³H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.     

Release of γ-[3H]Aminobutyric Acid from Rat Olfactory Bulb and Substantia Nigra: Differential Modulation by Glutamic Acid

We have studied the glutamate modulation of gamma-[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N-Methyl-D-aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2-amino-4-phosphonobutyric acid and the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid significantly inhibited the K+-glutamate- and the kainate-induced [3H]GABA release. Mg2+ (5 mM), which blocks the N-methyl-D-aspartate receptors, significantly inhibited the K+-glutamate-induced but not the kainic acid-induced [3H]GABA release. The K+-glutamate-stimulated release, but not the K+-stimulated [3H]GABA release, was strongly inhibited by Na+-free solutions or by 300 nM tetrodotoxin. Apparently the glutamate-induced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine Receptors in the Human Substantia Nigra as Defined by [3H]Strychnine Binding

Abstract: Specific [³H]strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity K d (3–8 n m ) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 n m , respectively) and rat substantia nigra (12 n m ). In the human brain the distribution of [³H]strychnine binding (at 10 n m ) was: spinal cord – substantia nigra, pars compacta > substantia nigra, pars reticulata = inferior olivary nucleus > cerebral cortex. The binding capacity ( B _max) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. [ ³ H]Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in a preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, [³H]strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. [³H]Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.     

Axoplasmic transport of dopamine in nigro-striatal neurons

The possibility that dopamine is transported in the nigro-striatal system was investigated by the stereotaxic injection of labelled tyrosine or l -DOPA into the substantia nigra of tranylcypromine-pretreated rats. At various intervals thereafter (2-48 h), significant quantities of labelled material were recovered from the ipsilateral substantia nigra, globus pallidus and caudate-putamen, The activity in the substantia nigra consisted of DOPA, dopamine, methoxytyramine, acid metabolites and other unidentified metabolites. In the caudate-putamen, however, nearly all of the activity (85 per cent) was recovered in the dopamine fraction, the remainder being distributed among some of the metabolites. No DOPA was recovered from the caudate-putamen. On the basis of time-course studies after the injection of [¹⁴C]DOPA into the substantia nigra, we calculated the transport rate of dopamine in the nigro-striatal bundle to be 0.8 mm/h. Electrolytic lesions of the nigrostriatal bundle at the level of the lateral hypothalamus, pretreatment with 6-hydroxydopamine, or injections of [¹⁴C]DOPA dorsal to the substantia nigra each produced profound reductions in the amount of activity subsequently recovered from the caudate-putamen. These data suggest that the activity recovered from the caudate-putamen after injections of [¹⁴C]DOPA into the or substantia nigra reflected axonal transport rather than other processes such as diffusion or transport via the circulation. Pretreatment with the DOPA decarboxy-lase inhibitor, R_o 4-4602, significantly reduced the amount of activity recovered in the caudate-putamen, an indication that decarboxylation of DOPA to dopamine was a prerequisite for transport. Pretreatment with reserpine also severely reduced the transport of dopamine in the nigro-striatal bundle, an observation suggesting that dopamine was transported by binding to the amine storage granules. There was no evidence of retrograde transport of dopamine in the nigrostriatal bundle. Injections of larger than tracer quantities of labelled tyrosine into the substantia nigra did not produce the degree of transport of dopamine that was obtained after injections of DOPA, a result suggesting that the amine storage granules may not normally be filled during axonal transport.