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Mouse purified striatal synaptosomes were used to study the release of newly synthesised [3H]-dopamine ([3H]-DA) or of previously taken up [3H]-DA. Quinpirole (QP, 10 microM), a D2/D3 dopaminergic agonist, was found to reduce the release of newly synthesised [3H]-DA with a larger amplitude when 4-aminopyridine (100 microM) instead than veratridine (1 microM) or potassium (25 mM) was used to evoke DA release. Among the different D2/D3 dopaminergic agonists tested R(-)-propylnorapomorphine (NPA) and quinpirole were the most potent. These compounds reduced, in a concentration-dependent manner, the 4-aminopyridine-evoked release of [3H]-DA previously taken up by synaptosomes (50% maximal inhibition). In contrast, the D3 agonist PD-128,907 had little effect even when used at 100 nM. The QP (100 nM)-induced response was completely antagonised by sulpiride (1 microM). Strikingly, the NPA (100 nM) and PD-128,907 (100 nM)-evoked responses were completely suppressed in D2 receptor-deficient mice. This data strongly suggest that only D2 but not D3 receptors are involved in the autoreceptor-mediated inhibition of the evoked release of [3H]-DA. Interestingly, while amphetamine-induced release of [3H]-DA was not modified, a slight reduction of [3H]-DA efflux induced by the dopamine (DA) uptake inhibitor cocaine was observed in D2 receptor-deficient mice.

Purified striatal synaptosomes were superfused continuously with L-[3,5-3H]tyrosine to measure simultaneously the synthesis ([3H]water formed during the conversion of [3H]tyrosine into [3H]DOPA) and the release of [3H]dopamine ([3H]DA). Glutamate (10(-3) M) and NMDA (10(-3) M, in the absence of Mg2+) stimulated the release of [3H]DA, but they reduced the efflux of [3H]water. This reduction of [3H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [3H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [3H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [3H]DA synthesis and release were mimicked by ionomycin (10(-6) M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.

Arachidonic acid was shown to stimulate the release of preloaded [3H]GABA from microdiscs of tissue punched out in matrix-enriched areas of the rat striatum. This effect, which was calcium- and dose-dependent, persisted in the presence of inhibitors of arachidonic acid catabolism. Other fatty acids were less or not effective. Arachidonic acid also inhibited [3H]GABA uptake into purified striatal synaptosomes, however the arachidonic acid-evoked release of [3H]GABA persisted following inhibition of the GABA neuronal uptake process. The stimulatory effect of arachidonic acid on GABA release may largely result from the activation of a protein kinase C since the arachidonic acid response was reduced by several protein kinase C inhibitors. Arachidonic acid also dose-dependently stimulated the release of preloaded [3H]GABA from purified striatal synaptosomes. Similar results were obtained when synaptosomes were previously incubated with [3H]glutamine to study the release of endogenously synthesized [3H]GABA. Further indicating a direct action of the fatty acid on GABAergic neurons, the arachidonic acid-induced release of [3H]GABA from microdiscs was not modified in the presence of the D1 dopaminergic antagonist SCH23390 or of glutamatergic antagonists. Finally, the release of [3H]GABA evoked by the combined application of NMDA and carbachol (a treatment known to markedly stimulate arachidonic acid formation) was reduced by inhibitors of phospholipase A2 further indicating that endogenously formed arachidonic acid significantly facilitates the release of GABA in the striatum.

The effects of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 10(-3) M), N-methyl-D-aspartate (10(-3) M, in the absence of magnesium or presence of AMPA) and carbachol (10(-3) M) on the release of preloaded [3H]gamma-aminobutyric acid ([3H]GABA) from microdiscs of tissue punched out from sagittal brain slices in striosome- or matrix-enriched areas of the rat striatum have been compared. Although AMPA stimulated similarly the release of [3H]GABA in both striatal compartments, the release of [3H]GABA evoked by either N-methyl-D-aspartate (in the presence of AMPA) or carbachol was more pronounced in matrix- than in striosome-enriched areas. AMPA- and N-methyl-D-aspartate- (in the absence of magnesium) evoked responses were reduced but not abolished in the presence of tetrodotoxin (10(-6) M) in both compartments while the carbachol-evoked release of [3H]GABA was decreased by tetrodotoxin only in the matrix. The interruption of cholinergic transmission by the combined application of atropine (10(-5) M) and pempidine (10(-4) M) was without effect on the AMPA-evoked release of [3H]GABA, but it reduced the N-methyl-D-aspartate- (in the absence of magnesium or presence of AMPA) evoked release of [3H]GABA in both compartments, these reductions being of similar amplitude than those observed with tetrodotoxin.

Using a cup permanently implanted on the antero-lateral surface of the caudate nucleus, attempts were made to study the spontaneous and d -amphetamine induced release of [ 3 H]dopamine (DA), which was continuously synthesised from l -[3,5- 3 H]-tyrosine in the unanaesthetised Macaca mulatta placed in a restraining chair with freedom of movement of the head and members. The spontaneous release of [ 3 H]DA from dopaminergic terminals was detected during the continuous superfusion of the tissue with [ 3 H]amino acid in 8 monkeys. In most cases [ 3 H]DA release reached a steady state level within 30 min after the onset of superfusion. Similar results were obtained when the experiments with [ 3 H]tyrosine were carried out on 3 successive days. In 2 of the cases examined, the quantity of spontaneously released [ 3 H]DA during the last 24 or 48 h of a 5–6 day experiment was markedly increased as compared to that observed during previous days; this effect did not appear to be related to changes in the specific activity of tyrosine in the superfusing medium. In 3 monkeys d -amphetamine ( 10 −5 M ) increased the level of [ 3H H]DA release by 15–20 times that of the normal spontaneous release of the transmitter, the effect was less pronounced in 2 other animals exhibiting a higher level of spontaneous [ 3 H]DA release. The amphetamine effect could be detected every day in longitudinal studies. For example, in one animal the ratio of the amphetamine-induced [ 3 H]DA release to spontaneous [ 3 H]DA release varied only from 10 to 15 during a 5 day experiment. This new approach should make possible long term studies on the regulatory processes involved in DA release from dopaminergic terminals of the nigro-neostriatal system in the unanaesthetised primate.

The effects of diencephalic or telencephalic commissural sectioning on the changes in [ 3 H]dopamine ([ 3 H]DA) release from nerve terminals (in the caudate nucleus, CN) and dendrites (in the substantia nigra, SN) of the two nigro-striatal dopaminergic pathways induced by the application oABA (10 −5 M, 30 min) into the left ventralis medialis (VM) or ventralis lateralis (VL) thalamic nuclei were investigated. Experiments were performed in halothane-anesthetized cats implanted with push-pull cannulae in both CN and SN. In unlesioned cats, GABA application into the left VM-VL increased [ 3 H]DA release in both CN and in the contralateral SN confirming previous results. Sectioning of the thalamic massa intermedia only blocked the GABA-induced increase in [ 3 H]DA release in the contralateral SN, the responses in both CN being preserved. Sectioning of the rostral part of the corpus callosum only prevented the GABA-induced increase in [ 3 H]DA release in the contralateral CN, whereas [ 3 H]DA release in the ipsilateral CN and in the contralateral SN was still enhanced. These results suggest that changes in [ 3 H]DA release evoked in both CN and in the contralateral SN by GABA application into the left VM-VL might involve different mechanisms: (1) those observed in the CN result from potent presynaptic influences mediated by the bilateral cortico-striatal projections; (2) those induced in the contralateral SN are due to other types of messages involving or passing through the thalamic massa intermedia.

The localization of radioactivity was examined in the caudate nucleus of the cat, treated with an MAO inhibitor, following local superfusion of the ventricular surface of the structure with low concentration of 3 H-catecholamines or [ 3 H]5-HT. The caudate nucleus was superfused continuously from 30 to 240 min using a cup technique. Light microscope or high resolution radioautographs revealed: (1) a rather diffuse incorporation of 3 H-catecholamines under the subependymal region which could be hardly attributed to a specific population of nerve terminals. A loose binding of 3 H-catecholamines into dopaminergic terminals could be involved in this effect. (2) An intense and selective uptake of [ 3 H]5-HT in fine scarce varicose nerve fibers localized in the ventricle as in the subependymal layer and in the neuropil of the nucleus. The labeled fibers contained numerous round or elongated large granular vesicles of 80–120 nm and exhibited only very few synaptic contacts suggesting a possible extrasynaptic liberation of 5-HT.

The effects of unilateral focal electrical stimulation of the deep cerebellar nuclei on the activity of the nigrostriatal dopaminergic neurons on both sides of the brain were examined in halothane anaesthetized cats. For this purpose, push-pull cannulae were inserted into both caudate nuclei and both substantia nigrae, and the release of [3H] dopamine ([3H]DA) continuously formed from [3,5-3H]L-tyrosine was estimated in superfusates. The unilateral electrical stimulation of the right cerebellar dentate nucleus induced a long-lasting increase in the release of [3H]DA in the left caudate nucleus and a simultaneous decrease in the release of [3H]transmitter in the right caudate nucleus. These changes were associated with opposite fluctuations in the release of [3H]DA from the corresponding substantia nigrae. Thus, the electrical stimulation of the right dentate nucleus induced a pronounced decrease in the release of the [3H]-amine in the [3H]transmitter in the corresponding substantia nigra, whereas the activity of the contralateral substantia nigra, whereas the release in the ipsilateral substantia nigra was simultaneously increased. In contrast, the unilateral electrical stimulation of the right cerebellar fastigial nucleus resulted only in an increased release of [3H]DA in the ipsilateral (right) caudate nucleus, associated with a decreased release of the [3H]transmitter in the corresponding substantia nigra, whereas the activity of the contralateral (left) dopaminergic system was not significantly affected. These results support a direct functional interaction between the cerebellum and the basal ganglia. They also suggest that the release of DA from dopaminergic axonal terminals is inversely correlated to the extent of the transmitter release from dendrites.

The effect of (−) isoproterenol on the spontaneous release of [3H]dopamine ([3H]DA) continuously formed from [3H]tyrosine has been studied both in vitro using rat striatal slices and in vivo in halothane-anaesthetized cats implanted with push-pull cannulae in each caudate nucleus and each substantia nigra. In vitro, (−) isoproterenol (10−6M) stimulated [3H]DA release. This effect was stereospecific since (+) isoproterenol (10−6M) was without effect. (±) Propranolol (10−6M), which did not block the acetylcholine (10−5M)-induced release of [3H]DA, completely prevented the stimulatory effect of (−) isoproterenol on [3H]DA release. (±) Practolol (10−5M) significantly reduced the (−) isoproterenol-induced release of [3H]DA whereas phentolamine (10−5M) was uneffective. These results suggest that (−) isoproterenol acts on [3H]DA release through β1-receptors. Since the adrenergic agonist still stimulated [3H]DA release in the presence of tetrodotoxin, the β-receptors involved may be located on DA terminals. Confirming in vitro results, (−) isoproterenol (10−6M) enhanced the local release of [3H]DA when applied into one caudate nucleus. This effect was biphasic and prevented by (±) propranolol (10−6M). The β-antagonist by itself slightly reduced [3H]DA spontaneous release suggesting that noradrenergic neurons which may innervate the caudate nucleus could exert a tonic facilitatory influence on DA release. No change in [3H]DA release occured in other structures studied during the application of (−) isoproterenol into one caudate nucleus. These various results indicate that striatal β-adrenergic receptors play a role in the regulation of DA release from DA nerve terminals and suggest an interaction between noradrenergic and dopaminergic neurones in the caudate nucleus.

Using an indirect immunoperoxidase technique, the localization of enkephalin-like cell bodies in the thalamus of the cat was carried out. Enkephalin-like cell bodies are widely distributed in the cat thalamus. However, immunoreactive cells may be regrouped in 4 clusters which do not exactly correlate with the anatomical subdivisions of the thalamus. One is located in the dorsocaudal aspect of the thalamus, another in the midline area, and the others are formed by the nuclei geniculatum mediale and laterale.

The effects of unilateral sensory stimuli on dopamine (DA) release from nerve terminals and dendrites of the two nigrostriatal dopaminergic pathways were estimated in halothane-anaesthetized cats without or with sagittal transections. In control animals, the electrical stimulation of the right forelimb enhanced DA release in the right caudate nucleus (CN) and decreased DA release in the right substantia nigra (SN). Opposite effects were observed in the contralateral structures. Sagittal transections of the corpus callosum and commissura anterior, or of the mesencephalic decussations or of the thalamic massa intermedia were made to investigate mechanisms involved in the reciprocal regulation of the two dopaminergic pathways. These sections were without effect on the spontaneous release of DA from nerve terminals and dendrites. The transection of the thalamic massa intermedia was the only one which interrupted the asymmetric changes in DA release induced by unilateral sensory stimuli; an increased dendritic release of DA was only seen in the left SN but it was significantly less pronounced than that observed in control cats. The other transections did not prevent the asymmetric changes in DA release evoked by the sensory stimulation. However, the mesencephalic sagittal transection significantly reduced the stimulatory effect on DA release induced in the left SN. These results suggest that the thalamus is involved in the transfer of information implicated in the reciprocal regulation of the two dopaminergic pathways. In the light of electrophysiological data, the role of nigrothalamic neurones in this phenomenon is discussed.

(1) The spontaneous and pharmacologically induced release of3H-DA, synthesized from [3H]tyrosine, has been studied in vivo on the cat caudate nucleus. A new technique has been developed that enables to label and superfuse a limited area of the ventricular surface of the caudate nucleus. [3H]Tyrosine was applied for a short time (acute labelling) or superfused continuously (continuous labelling). Superfusates obtained after or during the labelling process were collected in serial fractions. (2) 3H-DA released spontaneously in the resting state was identified and estimated quantitatively in collected fractions. (3) Changes in the decline of3H-DA after acute labelling, observed in anaesthetized animals (N2OO2penthrane), are attributed to an inhibiting effect of the anaesthetic on the release of the newly synthesized3H-DA. (4) In unanaesthetized cats, marked and rapid increases in the3H-DA release (5–10 times the resting state level) were induced repetitively by K+, catron and amphetamine during continuous labelling of DA terminals with [3H]tyrosine. Bothd- andl-amphetamine were potently active when applied topically or injected intravenously. (5) Results obtained in continuous labelling experiments emphasize the importance of the newly synthesized transmitter in release studies.

The effects of unilateral stimulation of the cerebral motor cortex and of the visual cortical area on the activity of the nigrostriatal dopaminergic neurons were examined in halothane-anesthetized cats. For this purpose, one push-pull cannula was inserted in each caudate nuclei and another one in the substantia nigra ipsilateral to the stimulated side. In all cases, the release of [3H]dopamine ([3H]DA) continuously formed froml-[3,5-3H]tyrosine was estimated in superfusates. Unilateral electrical stimulation of the cerebral motor cortex (area 4) induced a long-lasting and similar activation of [3H]DA release in both caudate nuclei. The activation of [3H]DA release in the contralateral side was selectively abolished after acute transection of the rostral part of the corpus callosum. This transection also suppressed the flexion of the contralateral forelimb induced by the stimulation. The activation of [3H]DA release could be related to the stimulation of corticostriatal neurons which may interact directly or indirectly with dopaminergic terminals in both caudate nuclei. Unilateral electrical stimulation of the visual cortex (areas 18 and 19) markedly stimulated the release of [3H]DA in the ipsilateral caudate nucleus. A slight effect was seen in the contralateral structure 20 min after the stimulation. These results are consistent with the existence of a main ipsilateral pathway originating from the visual cortex and projecting directly to the striatum. Both types of electrical stimulation immediately activated the release of [3H]DA in the ipsilateral substantia nigra. These effects were still seen 20 min after the stimulations. The activation of the dendritic release of [3H]DA could be related to the stimulation of a corticonigral projection. These results further indicate that the nigrostriatal dopaminergic neurons may be involved in sensory motor integration.

Summary Topographical variations in the uptake of [3H]dopamine (DA) and in the endogenous content of DA were estimated in the striatum of the rat. For this purpose, microdiscs were punched out in serial 500 μm sections. [3H]DA uptake was measured in 0.25 M sucrose homogenates prepared from microdiscs punched out from frozen slices (−7 °C). This uptake was similar to that observed in fresh tissues. It was unaffected by desmethylimipramine (5 × 10−7M), inhibited by benztropine (10−6M) and no longer detectable after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopaminergic pathway. Both [3H]DA uptake and DA content decreased regularly from the rostral to the caudal part of the structure. In contrast, no important differences could be found in the dorso-ventral plane. These results suggest that the extent of dopaminergic innervation is heterogenous within the structure.

The release of [3H]DA synthesized from [3H]tyrosine was estimated in the two caudate nuclei (CN) during the unilateral nigral application of glycine and GABA-related compounds in 'encéphale isolé' cats using push-pull cannulae. Glycine (10(-5) M) reduced the release of [3H]DA in both CN and these effects were antagonized by strychnine (10(-5) M). A decrease in [3H]DA release was also seen in both CN during the unilateral nigral application of diazepam (10(-5) M). In contrast, muscimol (10(-6) M) and GABA (10(-5) M) stimulated [3H]DA release on both sides. The effect of GABA was blocked by picrotoxin (10(-5) M). Picrotoxin alone stimulated the release of [3H]DA in the ipsilateral CN and was without effect in the contralateral side. Bicuculline (10(-5) M) stimulated [3H]DA release only in the contralateral CN. A symmetric increase in [3H]DA release in both CN was also observed during the unilateral nigral application of potassium (30 mM). A model involving a facilitatory polysynaptic pathway originating from the substantia nigra (SN) and acting presynaptically on ther terminals of the contralateral DA neurons is proposed to explain the changes in [3H]DA release induced in the contralateral CN in these various situations. The results are discussed taking into account previous data on the reciprocal control of the two dopaminergic pathways induced by the unilateral nigral application of dopaminergic drugs.