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Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [3H]dopamine ([3H]DA) continuously synthesized from [3H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [3H]H2O formation (an index of [3H]tyrosine conversion into [3H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [3H]DA synthesis. In contrast to AA, arachidonic acid, oleic acid, and the methyl ester of AA (all at 10(-4) M) did not modify [3H]DA release. The AA (3 x 10(-5) M)-evoked release of [3H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [3H]DA uptake into synaptosomes, with a complete blockade observed at 10(-4) M. However, AA (10(-4) M) still stimulated [3H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10(-4) M)-evoked release of [3H]DA was not affected by protein kinase A inhibitors (H-89 or Rp-8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).

Serotonin (5-HT) applied at 1, 3, and 10 microM into the striatum of halothane-anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5-HT effect was partially reduced by 1 or 10 microM GR 125,487, a 5-HT4 antagonist, and by 100 microM DAU 6285, a 5-HT3/4 antagonist, whereas the 5-HT1/2/6 antagonist methiothepin (50 microM) was ineffective. In the presence of tetrodotoxin the effect of 1 microM 5-HT was not affected by 5-HT4 antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5-HT4 agonist (S)- zacopride (100 microM). In striatal synaptosomes, 1 and 10 microM 5-HT increased the outflow of newly synthesized [3H]DA up to 163 and 635% of control values, respectively. The 5-HT4 agonists BIMU 8 and (S)-zacopride (1 and 10 microM) failed to modify [3H]DA outflow, whereas 5- methoxytryptamine (5-MeOT) at 10 microM increased it (62%). In prelabeled [3H]DA synaptosomes, 1 microM 5-HT, but not (S)-zacopride (1 and 10 microM), increased [3H]DA outflow. DAU 6285 (10 microM) failed to modify the enhancement of newly synthesized [3H]DA outflow induced by 5-MeOT or 5-HT (1 microM), whereas the effect of 5-HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 microM) alone or in the presence of DAU 6285. These results show that striatal 5-HT4 receptors are involved in the 5-HT-induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.

Arachidonic acid stimulated the release of [3H]-dopamine from striatal microdiscs in a concentration-dependent and partially calcium-dependent manner. Inhibitors of cytosolic and membrane-bound phospholipase A2 were used to determine whether endogenously formed arachidonic acid also contributes to the release of [3H]-DA (previously taken up in tissues or endogenously synthesized from [3H]-tyrosine) evoked by N-methyl-d-aspartate (NMDA) and carbachol alone or in combination. In the presence of magnesium, carbachol was found to remove the magnesium block of NMDA receptors and to facilitate the NMDA-evoked release of [3H]-DA from striatal microdiscs and synaptosomes. In addition, in the absence of magnesium, synergistic responses were induced by both agonists on microdiscs but not on synaptosomes. Responses induced by NMDA, carbachol or both agonists on microdiscs were reduced by phospholipase A2 inhibitors, the most striking effects being observed with mepacrine. Mepacrine was also shown to reduce the oxotremorine, but neither the nicotine- nor the potassium-evoked release of [3H]-DA. Tetrodotoxin decreased the release of [3H]-DA evoked by the co-application of NMDA and carbachol on microdiscs, but mepacrine still decreased this tetrodotoxin-resistant response. Similarly, mepacrine still decreased the release of [3H]-DA evoked by NMDA and carbachol on synaptosomes. Altogether, these results indicate that arachidonic acid which is formed in striatal neurons, and to a lesser extent in DA fibres, under stimulation of NMDA and muscarinic receptors, partially contributes to the presynaptic facilitation of DA release evoked by NMDA and carbachol.

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.

Dopamine (DA) release from nerve terminals of the nigrostriatal DA neurons not only depends on the activity of nigral DA cells but also on presynaptic regulation. Glutamatergic neurons of cortical origin play a prominent role in these presynaptic regulations. The direct glutamatergic presynaptic control of DA release is mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, located on DA nerve terminals. In addition, by acting on striatal target cells, these glutamatergic neurons contribute also to indirect regulations of DA release involving several transmitters such as GABA, acetylcholine and neuropeptides. Diffusible messengers such as nitric oxide (NO) or arachidonic acid (AA) which are particularly formed under the stimulation of NMDA receptors may also participate to the regulation of DA release. In the present study, it will be shown that the co-application of NMDA and carbachol synergistically increases the release of [3H]-DA and that this effect is reduced by mepacrine or 4-bromophenacylbromide (10(-7) M), two inhibitors of PLA2. Therefore endogenously released AA induced by the co-stimulation of NMDA and cholinergic receptors seems to be involved, at least partly, in the release of DA.

In vivo experiments carried out in halothane-anaesthetized cats implanted with push-pull cannulae demonstrated that glutamate (GLU) released from corticostriatal fibers triggers the release of dopamine (DA), even in the absence of activity in nigral DA cells. As shown in vitro, using rat striatal slices or synaptosomes or in vivo in the cat, both NMDA and AMPA receptors subtypes are involved in the GLU-induced release of DA. Beside this direct regulation, GLU also exert several indirect facilitatory and inhibitory controls on DA release, particularly through cholinergic and GABAergic striatal neurons. Indeed, as shown by numerous authors, the GLU-evoked release of DA is markedly reduced in the presence of tetrodotoxin, bicuculline or atropine or by previous kainate- or ibotenate-induced lesion of striatum. Differences in the presynaptic regulation of DA release in striosomal and matrix compartments have also been found with NMDA and acetylcholine. The effect of acetylcholine was of shorter duration in the matrix than in the striosomal-enriched areas. Two opposite indirect regulations of DA release could be demonstrated: one is facilitatory and involves nicotinic receptors, the other is inhibitory, involves muscarinic receptors and mediated, at least in the matrix by dynorphin containing neurons. The NMDA-evoked responses are of larger amplitude and more sensitive to tetrodotoxin in the matrix than in the striosomes. In conclusion, GLU released from corticostriatal fibers, is able to control the release of DA from terminals of nigrostriatal neurons through direct facilitatory mechanisms (NMDA and AMPA receptors), but also through indirect facilitatory and inhibitory local circuits involving cholinergic and GABAergic neurons.

Purified striatal synaptosomes were continuously superfused with l ,3,5[3H]tyrosine in order to estimate the synthesis ([3H]water) and release of newly formed [3H]dopamine. In the presence of magnesium, l -glutamate, d , l -α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate, but not N- methyl- d -aspartate (NMDA) and 1-aminocyclopentane-1S,3R-dicarboxylate (t-ACPD), stimulated the release of [3H]dopamine, in a dose-dependent manner. When magnesium was omitted or in the presence of AMPA, NMDA also increased the release of [3H]dopamine. The effects of AMPA and kainate were competitively inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitro-quinoxaline-2,3-dione (DNQX), whereas those of NMDA were reduced by 2-amino-5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5-H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate (MK801). The stimulation of [3H]dopamine release by a high concentration of glutamate resulted from the concomitant activation of AMPA and NMDA receptors since this effect was potentiated by glycine and reduced by 2-amino-5-phosphonovalerate or MK801. This reduction was almost complete in the combined presence of DNQX and MK801. Surprisingly, glutamate and NMDA (in the absence of magnesium) reduced the efflux of [3H]water. The reduction of [3H]dopamine synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Neither AMPA nor kainate affected dopamine synthesis. The inhibition of [3H]dopamine synthesis resulting from the stimulation of NMDA receptors was prevented when synaptosomes were continuously superfused with adenosine deaminase and quinpirole, a combined treatment known to markedly reduce the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase. The opposite effects of a high concentration of glutamate on [3H]dopamine synthesis and release were mimicked by ionomycin. As a working hypothesis, it is proposed that the NMDA-triggered calcium influx could lead to a reduction of tyrosine hydroxylase phosphorylation, possibly through an activation of calcineurin.

Using a new in vitro superfusion device, the release of preloaded [3H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [3H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 microM)-evoked release of [3H]GABA was completely abolished in the presence of tetrodotoxin (1 microM). The selective glutamatergic agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [3H]GABA as well as the basal outflow of [3H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 microM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10 microM), a specific antagonist of N-methyl-D-aspartate receptors. Similarly, N-methyl-D-aspartate (1 mM) enhanced both the potassium (9 mM) and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM)-evoked release of [3H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [3H]GABA. A stimulatory effect of N-methyl-D-aspartate (1 mM) on the basal outflow of [3H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl-D-aspartate (1 mM) observed in the presence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 microM, in the presence of strychnine 1 microM) and the N-methyl-D-aspartate-evoked response seen in the absence of magnesium was not enhanced by D-serine (1 mM), suggesting that endogenous glycine is already acting on N-methyl-D-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl-D-aspartate on the release of [3H]GABA confirming that under our conditions, the glycine site of the N-methyl-D-aspartate receptor is saturated. N-methyl-D-aspartate-evoked responses were all blocked by MK801 (10 microM). Finally, the N-methyl-D-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

It has been suggested that some anesthetic agents could exert their hypnotic/anesthetic effects by selectively blocking receptors involved in the central excitatory neurotransmission mediated by glutamate. In the present study, we analyzed whether riluzole (54274 RP), a novel compound that inhibits both the release and some postsynaptic effects of glutamate in some brain structures, has anesthetic properties in rats. For this purpose, we investigated whether 1) riluzole administered intraperitoneally (ip) at doses ranging from 2.5 to 45 mg/kg induces loss of righting reflex (LRR); 2) riluzole (2.5 and 5 mg/kg) prolongs sleep-times induced by either ketamine (30 or 80 mg/kg ip) or thiopental (25 or 35 mg/kg ip); 3) a 5-mg/kg subanesthetic riluzole dose affects the minimum alveolar concentration of halothane (MACh). Onset of drug action was defined as the period of time from the ip injection to LRR. Sleep-time was considered the period of time from LRR to restoration of righting reflex. Riluzole at doses greater than 15 mg/kg was able to induce LRR (riluzole dose for which LRR was achieved in 50% of the rats [ED50 = 25.6 mg/kg]). A positive correlation was found between the dose of riluzole and sleep-time (r = 0.92, P less than 0.001). A 5-mg/kg (but not 2.5-mg/kg) riluzole dose significantly prolonged sleep-times induced by both ketamine (30 and 80 mg/kg) and thiopental (25 but not 35 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Previously, using purified synaptosomes from the rat striatum, we have shown that agonists of D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors stimulate the release of [3H]dopamine continuously synthesized from [3H]tyrosine. Similar results were obtained with N-methyl-D-aspartate in the absence of magnesium. In the present study, using the same approach, attempts were made to determine whether in the presence of magnesium, the combined stimulation of AMPA receptors allows us to demonstrate the presynaptic facilitation of [3H]dopamine release through N-methyl-D-aspartate receptors. L-Glutamate (10(-3) M) markedly stimulated the release of [3H]dopamine from synaptosomes, this effect being about twice that found with AMPA (10(-3) M) while N-methyl-D-aspartate (10(-3) M) even in the presence of glycine (10(-6) M) was ineffective. In agreement with previous results, a stimulatory effect of N-methyl-D-aspartate and glycine was only observed in the absence of magnesium. This response was blocked by 6,7-dinitro-quinoxaline-2,3-dione (3 x 10(-5) M), confirming that this compound, generally used as an AMPA antagonist, also blocks N-methyl-D-aspartate receptors. The AMPA (10(-3) M)-evoked release of [3H]dopamine was markedly potentiated by the combined application of N-methyl-D-aspartate (10(-3) M) and glycine (10(-6) M) in the presence of strychnine, indicating that the concomitant activation of AMPA receptors removes the voltage-dependent magnesium block of N-methyl-D-aspartate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The N-methyl-D-aspartate (NMDA) receptor-mediated regulation of the release of newly synthesized [3H]dopamine [( 3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2(+)-free medium conditions, the NMDA (5 X 10(-5) M)-evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX-resistant stimulatory effect of NMDA was blocked by either Mg2+ (10(-3) M) or the noncompetitive antagonist MK-801 (10(-6) M). In addition, the TTX-resistant NMDA-evoked response could be potentiated by glycine (10(-6) M) in the presence of strychnine (10(-6) M). The coapplication of NMDA (5 X 10(-5) M) and glycine (10(-6) M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10(-3) M) or MK-801 (10(-5) M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electrophysiological studies for postsynaptic NMDA receptors.

In halothane-anaesthetized cats implanted with several push-pull cannulae the release of dopamine from nerve terminals and dendrites of the two nigrostriatal dopaminergic pathways is shown to be asymmetrically or symmetrically regulated bilaterally when dopaminergic or GABAergic drugs are infused into one side of the nigra. Nigrothalamic GABAergic neurons may intervene in the asymmetrical regulation, as sagittal section of the thalamic massa intermedia blocks the contralateral effects induced by dopaminergic drugs. The role of thalamic nuclei in the bilateral regulation of dopamine and GABA release in the caudate nucleus and substantia nigra is further demonstrated by studies showing that (1) electrical stimulation of thalamic motor nuclei induces bilateral asymmetrical changes in dopamine release resembling the changes evoked by unilateral sensory stimuli or stimulation of cerebellar nuclei; (2) electrical stimulation of intralaminar or some midline thalamic nuclei leads to bilateral (or contralateral) symmetrical changes in dopamine release, some of these effects being comparable to those induced by unilateral stimulation of the motor cortex; (3) unilateral lesions of motor or intralaminar thalamic nuclei reverse the changes in GABA release in the contralateral caudate nucleus or substantia nigra induced by unilateral infusion of muscimol into the nigra; and (4) unilateral infusion of GABA into thalamic motor nuclei induces bilateral symmetrical regulation of dopamine release in caudate nuclei by means of presynaptic facilitatory influences.

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.

When applied locally to the caudate nucleus of the halothane-anaesthetized cat, riluzole (10 −5 M) markedly reduced (−57%) the spontaneous release of glutamate. This effect seems to be specific, since the efflux of the other amino acids, including aspartate was not affected. Indicating further its selective inhibitory effect on the spontaneous release of glutamate, the prolonged (90 min) application of riluzole (10 −5 M) enhanced the size of the potassium-releasable pool of glutamate, but not that of aspartate. This effect of riluzole was not noticed with classical anti-glutamatergic drugs, tested in the same conditions.

The release of 3H-dopamine (3H-DA), continuously synthesized from 3H-tyrosine, using rat striatal synaptosomes, was increased by the activation of two different glutamatergic receptor subtypes. In absence of magnesium, N-methyl-D-aspartate (NMDA) stimulated the release of 3H-DA. This effect was potentiated by glycine and antagonized by MK801. When a normal artificial cerebro-spinal-fluid (CSF) was used, quisqualate (QUI), kainate (KAI) and α-Amino-3-Hydroxy-5-methylisoxazole-4-propionic acid (AMPA) enhanced the release of 3H-DA. The effect induced by QUI was found to desensitize very rapidly. This desensitization could be prevented by concanavalin A (CONA). The effect of KAI was reduced either by 6-nitro-7-cyano-quinoxaline (CNQX) or by a pretreatment with QUI.

Levels of N -acetyl-aspartyl-glutamate measured by high-pressure liquid chromatography were found to be very high in the cat substantia nigra, particularly in the pars compacta, while those in the caudate nucleus were much lower. In halothane-anaesthetized cats implanted with push-pull cannulae, N -acetyl-aspartyl-glutamate (10 −8 M) induced a marked and prolonged release of newly synthesized [ 3 H]dopamine, when infused into the posterior but not into the anterior part of the caudate nucleus. In contrast, in the presence of tetrodotoxin (10 −6 M), N -acetyl-aspartyl-glutamate (10 −8 M) reduced the residual release of [ 3 H]dopamine; this effect was also more pronounced in the posterior than in the anterior part. In the conditions used, as indicated by experiments with [ 3 H] N -acetyl-aspartyl-glutamate no glutamate was formed from the infused N -acetyl-aspartyl-glutamate. Ibotenate (10 −5 M) induced changes in [ 3 H] dopamine release in both the absence and presence of tetrodotoxin, which were closely similar to those observed with N -acetyl-aspartyl-glutamate. Responses induced by either N -acetyl-aspartyl-glutamate or ibotenate were not mediated by N -methyl- d -aspartate receptors since N -methyl- d -aspartate stimulated the release of [ 3 H]dopamine only when used in a high concentration (10 −4 M) and applied in a magnesium-free superfusion medium in both the presence of glycine (10 −6 M) and strychnine (10 −6 M). In addition, the stimulatory effect of N -methyl- d -aspartate persisted in the presence of tetrodotoxin; it was of similar amplitude in both parts of the caudate nucleus and of shorter duration than that evoked by either N -acetyl-aspartyl-glutamate or ibotenate alone. N -Acetyl-aspartyl-glutamate interacted with dopaminergic neurons not only presynaptically in the caudate nucleus but also in the substantia nigra since a marked increase in [ 3 H]dopamine release was observed both from local dendrites and from nerve terminals in the ipsilateral caudate nucleus when N -acetyl-aspartyl-glutamate (10 −7 M) was infused locally into the substantia nigra pars compacta. No effect could be seen in contralateral structures. The isomer of natural N -acetyl-aspartyl-glutamate, β-N-acetyl-aspartyl-glutamate (10 −7 M), had no effect on [ 3 H]dopamine release when applied similarly in the substantia nigra, thus confirming the specificity of the action of N -acetyl-aspartyl-glutamate.

Several studies performed on rat striatal slices and in some cases on synaptosomes have indicated that dopamine (DA) and most neurotransmitters found in the striatum regulate presynapticaily the release of DA from nerve terminals of the nigro-striatal DA neurones (chesselet, 1984). These neurotransmitters include excitatory or inhibitory amino-acids, amines and several neuropeptides found either in afferent fibers, interneurones or in collaterals of efferent projections from the striatum. These regulations are either direct or indirect, ie mediated through receptors located on DA nerve terminals or on neurones in contact with these nerve terminals. This has been mainly shown by examining the persistance or disappearance of these presynaptic regulations in the presence of tetrodotoxin (TTX), a neurotoxine which interrupts nerve impulse flow. There is some evidence that some neurotransmitters may diffuse at some distance from their release sites before their inactivation. Nevertheless, the physiological significance of some presynaptic regulations of DA release has been often challenged by neuroanatomists since up to now few axo-axonic contacts have been observed between DA neurones and other neurones innervating the striatum (Bouyer et al., 1984). In vivo studies, in which identified pathways projecting to the striatum can be activated are of great value for determining the physiological relevance of these presynaptic regulations. Therefore, we will first summurize results of in vivo experiments made in halothane anesthetized cats implanted with push-pull cannulae which have allowed to demonstrate the role of the corticostriatal glutamatergic projection in the presynaptic control of DA release. We will also describe more recent experiments made either in vivo or in vitro in which attempts were made to identify the types of glutamatergic receptors involved in the presynaptic control of DA release and to distinguish direct and indirect effects mediated by local circuits.

Experiments were conducted with halothane-anesthetized cats implanted with a push-pull cannula in the caudate nucleus in order to estimate the effects of glutamate (GLU) agonists on the release of 3H-dopamine continuously synthesized from 3H-tyrosine. In the presence of tetrodotoxin (TTX), glutamate (10-8 M, 10-4 M) and kainate (KAI) (10-5 M) stimulated the release of 3H-dopamine while quisqualate (10-5 M) and N-methyl-D-aspartate (NMDA) (10-5 M) were without effect. The stimulatory effect of kainate (10-5 M) on 3H-dopamine release did not seem to be mediated by glutamate released from corticostriatal fibers, as not only kainate, but also quisqualate (QUI) and N-methyl-D-aspartate enhanced the efflux of glutamate through a tetrodotoxin-resistant process. Riluzole (10-5 M), gamma-D-glutamyl-glycine (GDGG) (10-5 M) and glutamine-diethyl-ester (10-5 M) prevented the stimulatory effect of kainate (10-5 M) while 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (10-5 M), kynurenate (10-5 M) and 2-amino-5-phosphonovalerate (APV) (10-5 M) were without effect. In the presence of concanavalin A (CONA) (10-7 M), a lectin which is known to prevent the quisqualate-evoked desensitization of glutamate receptors, quisqualate (10-5 M) stimulated the release of 3H-dopamine. In addition, in the absence of concanavalin A, quisqualate (10-5 M) blocked the stimulatory effects of kainate (10-5 M) or glutamate (10-4 M) on 3H-dopamine release. These results suggest the involvement of receptors of the quisqualate/kainate subtype in the direct glutamate-induced presynaptic facilitation of dopamine release. In contrast to what was observed in the presence of tetrodotoxin, in the absence of the neurotoxin, high concentrations of glutamate (10-4 M) and kainate (10-5 M) reduced rather than stimulated the release of 3H-dopamine. A weak inhibitory effect was also observed with quisqualate (10-5 M) while N-methyl-D-aspartate (10-5 M) was without effect. In the light of previous studies, these latter observations suggest that glutamate can also exert an indirect inhibitory presynaptic influence on the release of dopamine from nerve terminals of the nigrostriatal dopaminergic neurons by acting on receptors of the quisqualate/kainate subtype located on striatal GABAergic neurons.

Studies performed in several in vivo and in vitro conditions have demonstrated that the release of dopamine from nerve terminals of the nigro-striatal dopaminergic neurons depends not only on the activity of dopaminergic cells but also on presynaptic regulations by heterologous fibers. The presynaptic facilitation of dopamine release by the cortico-striatal glutamatergic neurons has been particularly investigated. A quisqualate/kainate receptor subtype is involved in the direct (tetrodotoxine-resistant) presynaptic regulation of dopamine release by glutamate. The respective roles of presynaptic events and nerve activity in the control of dopaminergic transmission are discussed.

The unilateral application of GABA (10(-5) M; 30 min) into thalamic motor nuclei of the cat increases the release of dopamine in both caudate nuclei. This effect has been suggested to be related to an activation of the bilateral corticostriatal glutamatergic projection, glutamate exerting a presynaptic facilitatory influence on dopamine release. To explore this hypothesis further, halothane-anesthetized cats implanted with push-pull cannulae were used in order to examine the effects of such a GABA application on the release of glutamate in both caudate nuclei. Aspartate, alanine, glutamine, serine and tyrosine were also measured in the superfusates. The unilateral application of GABA (10(-5) M; 30 min) into thalamic motor nuclei increased the release of glutamate bilaterally. Although less pronounced, ipsi- or bilateral increases in the efflux of alanine, glutamine and tyrosine were also observed. Contralateral changes in the efflux of glutamate, alanine and tyrosine were prevented following acute section of the corpus callosum. In addition, when applied continuously into one caudate nucleus, 2-amino-5-phosphonovaleric acid, a blocker of N-methyl-D-aspartate receptors, prevented the GABA-induced increase in alanine or tyrosine efflux but did not affect the enhanced release of glutamate. These results confirm that the unilateral application of GABA in thalamic motor nuclei activates a thalamo-cortico-striatal neuronal loop leading to the stimulation of glutamate release in both caudate nuclei. Changes in the efflux of other amino acids could be linked to increased metabolic activity of striatal target cells resulting from the increased release of glutamate and from its effect on N-methyl-D-aspartate receptors.

Halothane-anaesthetized cats implanted with three push pull cannulae were used to estimate the effects of γ-aminobutyric acid (GABA) application (either 10 −3 M or 10 −5 M ) into the left motor nuclei of the thalamus (either ventralis medialis, or ventralis lateralis) on the firing rate of dopamine cells in the left substantia nigra (caudomedial part) and on the release of [ 3 H]dopamine continuously synthesized from [ 3 H]tyrosine, in the left substantia nigra (caudomedial part) and the left caudate nucleus. Preliminary experiments were performed to establish the electrophysiological characteristics of dopamine cells and non-dopamine cells in the pars compacta (mediocaudal part of substantia nigra) in groups of animals with the electrode inserted within the nigral push pull cannula or with the electrode inserted in the absence of a push-pull cannula. Dopamine and non-dopamine cells were distinguished according to several criteria (shape of the spike, duration of spike, frequency of discharge, conduction velocity estimated following antidromic activation from the caudate nucleus for dopamine cells or from the ventralis medialis for non-dopamine cells). Data obtained from recordings made within the push pull cannula were identical to those obtained in the absence of the cannula. In addition both the intravenous injection of amphetamine or its local application (10 −6 M) in the substantia nigra inhibited the firing rate of dopamine cells. When GABA was applied at 10 −3 M for 30 min into the ventralis medialis ventralis lateralis the multi-unit activity of thalamic cells recorded within the push pull cannula was inhibited. Single unit activity of dopamine cells was also inhibited and [ 3 H]dopamine release was reduced in the caudate nucleus and increased in the substantia nigra. These results suggest that under these conditions, dopamine release from nerve terminals depended upon nerve activity and that dopamine released from dendrites inhibited the activity of dopamine cells. When GABA was applied at 10 −5 M for 30 min into the ventralis medialis-ventralis lateralis, multi-unit activity of thalamic cells was increased, single-unit activity of dopamine cells was inhibited and [ 3 H]dopamine release was enhanced in the ipsilateral caudate nucleus and not affected in the left substantia nigra, demonstrating that in this situation the release of dopamine from nerve terminals was not dependent on the firing rate of dopamine cells. In addition, these results indicated that the activity of dopamine cells was not always dependent on the dendritic release of dopamine. Further experiments performed in cats with an acute hemi-transection (left side) made just in front of the substantia nigra to cut through all dopamine fibres confirmed that the GABA (10 −5 M )-evoked release of [ 3 H]dopamine in the caudate nucleus was mediated by a presynaptic regulation triggered from thalamic-motor nuclei. Indeed, [ 3 H]dopamine release was still enhanced by GABA (10 −3 M) application into the ventralis medialis-ventralis lateralis of such animals. In contrast the application of GABA (10 −3 M) into the ventralis medialis-ventralis lateralis was without effect on [ 3 H]dopamine release; n the caudate nucleus of the hemi-transected animals. Results are discussed in the light of the respective contributions of nerve activity and presynaptic regulation in the control of dopamine transmission in the caudate nucleus.

The role of several motor and intralaminar thalamic nuclei in the regulation of dopamine release from terminals and dendrites of the nigrostriatal dopaminergic neurons was investigated in halothane-anaesthetized cats. For this purpose, the effects of the unilateral electrical stimulation of various thalamic nuclei on the release of newly synthesized [ 3 H]dopamine were simultaneously determined in both substantiae nigrae and caudate nuclei using the push-pull cannula method. The electrical stimulation of the motor nuclei was the only one to induce asymmetric changes in the four structures since [ 3 H]dopamine release was enhanced in the ipsilateral caudate nucleus and reduced in the contralateral structure while opposite responses were observed in the corresponcling substantiae nigrae. A reduction of [ 3 H]dopamine release occurred in the four structures or only in the contralateral substantia nigra and caudate nucleus following the stimulation of the parafascicularis nucleus and the adjacent. posterior part of the nucleus centrum medianum or of the nucleus centralis lateralis and the adjacent paralaminar part of the nucleus medialis dorsalis, respectively. The stimulation of the anterior part of the nucleus centrum medianum, which in contrast to other thalamic nuclei examined, receives few nigral inputs, selectively enhanced [ 3 H]dopamine release in the contralateral substantia nigra. No significant changes in [ 3 H]dopamine release were seen either in the substantiae nigrae or in the caudate nuclei following the stimulation of midline thalamic nuclei. These results indicate that the motor and intralaminar thalamic nuclei exert multiple and selective influences on the release of dopamine from terminals and/or dendrites of the dopaminergic neurons. They also further support a role of thalamic nuclei in the transfer of information from one substantia nigra to the contralateral dopaminergic neurons. The possible involvement of connections between paired thalamic nuclei was underlined by the observations of evoked potentials in contralateral homologous nuclei following unilateral stimulation of motor, or some intralaminar, nuclei. The present report provides new insights on the mechanisms contributing to the reciprocal and/or bilateral regulations of nigrostriatal dopaminergic pathways.

The effect f various opiates on the release of newly synthesized 3H-dopamine (3H.DA) was studied both in vitro in rat striatal slices and in vivo in caudate nuclei and substantiae nigrae of halothane anaesthetized cats, using a push pull cannula technique. The hexapeptide Tyr-D-Ser -Gly-Phe-Leu-Thre (DSTLE) which acts preferentially on δ receptors stimulates 3H.DA release in vitro and produces a biphasic increase of the local 3H-amine outflow when applied in the cat caudate nucleus in vivo . Similar effects were induced by D Ala2-met-enkephalinamide. By contrast, morphine, fentanyl and the potent μ agonist Tyr-D-Ala-Gly-NH-C6-H13 (Trimu 4) did not affect 3H.DA release in vitro . In vivo their effects differ from those of the δ agonists and from each other: morphine produced a delayed long lasting increase in 3H.DA release while fentanyl and Trimu 4 induced an immediate increase of the 3H-amine outflow followed by a delayed inhibition. The local effects of δ and μ agonists also differed as regard to their sensitivity to naloxone but in addition, when co-applied with the opiate antagonist, D-Ala2-met-enkephalinamide and morphine induced changes in 3H.DA release in the contralateral caudate nucleus and the contralateral or ipsilateral substantia nigra respectively. Therefore, in the striatum, some opiates may, under certain circumstances, act directly or indirectly upon striatal efferences involved in the control of the contralateral nigro-striatal dopaminergic neurons. In conclusion, thanks to the use of recently developed opiate agonists exhibiting a high preference for either δ or μ receptors it has been shown that these receptors are involved in different mechanisms regulating the striatal dopaminergic transmission. In addition, these results suggest that less specific agents, by interacting with several types of receptors, induce a unique combination of events responsible for local and distal control of DA release from nigro-striatal dopaminergic neurons.

Dopamine can be released in the substantia nigra for the dendrites of nigrostriatal dopaminergic neurones, to be involved there in the self-regulation of the dopaminergic cells, to control the release of neurotransmitters from nigral afferent fibres and to influence the activity of nigral non-dopaminergic cells.

— 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‘encephale isole’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 ‘encephale isole’ 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.

Push-pull cannulae were used to study the release of 3 H-dopamine ( 3 H-DA) continuosly synthetized from L-3,5- 3 H-tyrosine in the caudate nucleus and the substantia nigra of the “encephale isole” cat. GABA (10 −5 M), muscimol (10 −6 M), γ-hydroxybutyrate (GHB, 10 −5 M), GABA-choline (GABA-CH, 10 −5 M) and amino-oxyacetic acid (AOAA, 10 −5 M) stimulated the release of 3 H-DA in the caudate nucleus when introduced for 15 min in the superfusing medium. The initial stimulation of 3 H-DA release was followed by an inhibition of transmitter release when GABA was introduced for a longer time (60 min) into the caudate nucleus. GABA (10 −5 M) had no effect on the release of 3 H-DA in the substantia nigra. However, muscimol (10 −6 M), GHB (10 −5 M) and baclofen (10 −6 M) stimulated the dendritic release of 3 H-DA when each was introduced for 15 min in the substantia nigra. The introduction for 15 min of GABA (10 −5 M), muscimol (10 −6 M), GHB (10 −5 M), GABA-Ch (10 −5 M) or baclofen (10 −6 M) into the substantia nigra simultaneously induced a stimulation of 3 H-DA release in the ipsilateral caudate nucleus. A marked post stimulatory effect was seen with muscimol. When GABA (10 −5 M) was applied for 75 min into the substantia nigra, the initial stimulation of 3 H-DA release (30 min) seen in the caudate nucleus was followed by a short-lasting inhibitory phase (15 min). This inhibition of 3 H-DA release was followed by a second stimulation period. This multiphasic phenomenon was not seen with other compounds. The stimulation of 3 H-DA release in the caudate nucleus induced by a 15 min nigral application of GABA (10 −5 M) was no longer seen when picrotoxin (10 −5 M) was added with GABA in the substantia nigra. On the basis of these results it is proposed that at least two types of GABAergic regulatory processes are involved in the control of the activity of the dopaminergic neurons in the substantia nigra. On the other hand, the interaction of GABA or GABA-related compounds with GABAergic receptors in the striatum may facilitate DA release from dopaminergic terminals through an intrastriatal interneuronal process. The secondary inhibition of 3 H-DA release induced by GABA or GHB could be mediated by a non-GABAergic striato-nigral pathway.

The release of 3H-DA was estimated in superfusates of a superfusion cannula introduced into the caudate nucleus of "encéphale isolé" cats. 3H-DA was continuously formed from L-3,5-3H-tyrosine introduced into the superfusion medium. Injected at the periphery, picrotoxin (2.5 mg/kg, i.p.) markedly enhanced the release of 3H-DA, the effect being particularly pronounced 90 min after the drug injection. The effects of the local introduction of picrotoxin into the substantia nigra and into the caudate nucleus on 3H-DA release were also examined to ensure the specificity of the result. Picrotoxin (10(5) M) added into the superfusing medium of a second superfusion cannula introduced in to the substantia nigra enhanced by 2 to 5 times the release of 3H-DA during the drug application. No effect could be seen when the tip of the superfusion cannula was not exactly localized into the substantia nigra. Although less pronounced, a stimulation of 3H-DA release was also seen when picrotoxin(10(6) M) was added directly into the superfusion medium of the cannula introduced into the caudate nucleus. These results suggest that some gabaergic neurons are involved in the control of DA release by acting on dopaminergic cell bodies or dendrites into the substantia nigra. Other gabaergic neurons may directly or indirectly act on dopaminergic terminals within the caudate nucleus.

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 local effects of various concentrations of L-glutamate (from 10(-8) M up to 10(-3) M) on the release of [3H]dopamine synthesized continuously from [3H]tyrosine were examined in the caudate nucleus of halothane-anaesthetized cats implanted with push-pull cannulae. When used at a concentration of 10(-8) M or 10(-7) M, L-glutamate stimulated the release of [3H]dopamine from nerve terminals of the nigrostriatal dopamine neurons. This effect was still observed in the presence of tetrodotoxin (5 X 10(-7) M) but it was antagonized by 2-amino 6-trifluoromethoxy benzothiazole (PK 26124) (10(-5) M), an antagonist dopamine nerve terminals. While no significant change in the release of [3H]dopamine was observed with 10(-6) M L-glutamate, higher concentrations (from 10(-5) M to 10(-3) M) of the amino acid produced a long-lasting reduction in the [3H]transmitter release. This latter effect was also antagonized by PK 26124 (10(-5) M) but, unlike that observed with 10(-8) M L-glutamate, it did not persist in the presence of tetrodotoxin (5 X 10(-7) M). On the contrary, a marked stimulation of the release of [3H]dopamine was seen in the presence of this neurotoxin. The reduction in the release of [3H]dopamine produced by 10(-4) M L-glutamate was also antagonized by bicuculline (10(-5) M) and moreover a marked stimulation of [3H]dopamine release took place in the presence of this gamma-aminobutyric acid (GABA) antagonist. Therefore, high concentrations of L-glutamate exerted an inhibitory presynaptic control on [3H]dopamine release which seemed to be indirect and mediated partly by GABAergic neurons. Since a sustained reduction in the spontaneous release of [3H]dopamine was seen in the presence of PK 26124, the corticostriatal glutamatergic neurons appeared to exert a tonic facilitatory presynaptic influence on dopamine release. This effect was important since it represented 40% of the tetrodotoxin-sensitive release of the [3H]transmitter. The direct (stimulatory) and indirect (inhibitory) presynaptic controls on dopamine release mediated by corticostriatal glutamatergic fibres are discussed in light of previous findings and of the anatomical organization of the caudate nucleus.

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 effects of the unilateral application of d-amphetamine, benztropine, haloperidol and thioproperazine to one substantia nigra on the release of 3H-dopamine (3H-DA) in the two caudate nuclei were examined in halothane-anesthetized cats. For this purpose animals were implanted with push-pull cannulae and 3H-DA was estimated in superfusates during the continuous delivery of L-3,5-3H-tyrosine. The nigral application of d-amphetamine (10-6 M) or benztropine (10-6 M) reduced the release of 3-H-DA in in the ipsilateral caudate nucleus and induced an opposite effect in the contralateral side. In contrast, the nigral application of haloperidol (10-6 M) or thioproperazine (10-6 M) slightly increased the release of 3H-DA in the ipsilateral caudate nucleus and induced a reduction of 3H-transmitter release in the contralateral side. These results emphasize the role of the dendritic release of DA in the control of the activity of dopaminergic neurons and confirm our previous findings concerning the existence of a reciprocal control in the activity of the two dopaminergic pathways.

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.

The effects of various sagittal sections on the asymmetric changes in [3H]dopamine release from nerve terminals and dendrites of the two nigrostriatal dopaminergic pathways induced by the application of α-methylparatyrosine ord-amphetamine in the left substantia nigra were examined in halothane-anaesthetized cats implanted with push-pull cannulae in both caudate nuclei and both substantiae nigrae. [3H]tyrosine was continuously delivered to each push-pull cannula and [3H]dopamine was estimated in serial fractions of superfusates. In cats without a lesion, α-methylparatyrosine (10−4M) reduced the release of [3H]dopamine in the left substantia nigra and induced an opposite effect in the left caudate nucleus. In contrast, [3H]dopamine release was enhanced in the right substantia nigra and reduced in the right caudate nucleus. Opposite asymmetric responses were observed in the four structures during application ofd-amphetamine (10−6M) into the left substantia nigra. The sagittal section of the mesencephalic decussations was without effect on the changes in [3H]dopamine release induced either by α-methylparatyrosine ord-amphetamine. The section of the corpus callosum and of the commissura anterior potentiated, whereas the section of the thalamic massa intermedia prevented thed-amphetamine-induced reduction of [3H]dopamine release in the left caudate nucleus. This could indicate that neuronal circuits under the control of contralateral structures may contribute to the regulation of the activity of ipsilateral dopaminergic neurons. Only the section of the thalamic massa intermedia prevented the contralateral responses evoked by the unilateral nigral application of α-methylparatyrosine ord-amphetamine. These results suggest that nigrothalamic neurons and thalamic nuclei are involved in the reciprocal regulation of both nigrostriatal dopaminergic pathways.

The effects of electrical stimulation of midline thalamic nuclei on the release of [3H]dopamine ( [3H]DA) were determined in both substantiae nigrae (SN) and caudate nuclei of halothane-anesthetized cats using the push-pull cannula method. [3H]DA release was increased in the four structures following stimulation of the interanteromedialis nucleus (IAM) but only enhanced in both SN when nucleus reuniens (RE) was stimulated. In contrast, no effect was detected after delivery of the stimuli to nucleus centralis medialis. These results indicate that the anterior part of the thalamic massa intermedia (IAM and RE particularly) is involved in the bilateral regulation of DA release from nerve terminals and dendrites of the nigro-striatal dopaminergic neurons.

1. In vitro studies have indicated that several transmitters present in the striatum can regulate presynaptically the release of dopamine (DA) from nerve terminals of the nigrostriatal DA neurons. 2. The receptors involved in these local regulatory processes are located or not located on DA nerve terminals. 3. Recent in vivo investigations have demonstrated that the corticostriatal glutamatergic neurons facilitate presynaptically the release of DA and have allowed the analysis of the respective roles of presynaptic events and nerve activity in the control of DA transmission.

— A method was developed to measure small amounts of dopamine or norepincphrine in biological samples. It was based on the radioenzymic assay originally described by Engelmanet al. (1968) and improved by other workers: dopamine and norepinephrine are O-methylated by catechol-O- methyl-transferase in the presence of [3H]S-adenosylmethioniiie as la belled methyl donor. O-Methylated derivatives are extracted and estimated. The optimal conditions for the O-methylation and the extractions were determined. The most important improvement in the method consisted in the selective isolation of catecholamines on microcolumns of alumina under conditions that allowed their subsequent enzymic assay. The assay described can thus be used on any size of biological sample. The assay was not affected by the prescnce of various agents (tissue extracts, CSF salts and particularly calcium, drugs etc.) which were shown to interfere with the O-methylation process, and it allowed the measurement of quantities of dopamine and norepinephrine as low as 0.15 and 0.1 pmol respectively, whatever the origin of the sample (CSF superfusates, cerebral tissue).

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.

The effects of the unilateral nigral application of substance P, 4–11 substance P, LHRH and of antibodies against substance P on the release of 3 H-dopamine ( 3 H-DA) in the two caudate nuclei (CN) and the two substantia nigrae (SN) were examined in halothane anaesthesized cats. The animals were implanted with four push-pull cannulae and 3 H-DA was estimated in successive superfusate fractions during the continuous delivery of 3 H-tyrosine. The unilateral nigral application of substance P or of 4–11 substance P resulted not only in a stimulation of 3 H-DA release in the ipsilateral CN, but also in a reduction of the 3 H-transmitter release from dendrites in the SN. As previously reported, the unilateral nigral application of antibodies against substance P induced opposite effects i.e. a decreased release of 3 H-DA from nerve terminals associated to an increase of the 3 H-amine release from dendrites. Surprisingly, in contrast to that previously observed during the unilateral nigral application of dopaminergic glycinergic or GABAergic agonists and antagonists neither substance P (or 4–11 substance P) nor antibodies against substance P affected the release of 3 H-DA from nerve terminlas and dendrites of the contraletaral dopaminergic neurons. LHRH, a similar sized peptide was without effect on ipsilateral as well as on contralateral dopaminergic.

(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 effect of the acute administration of morphine (60 mg/kg) on the metabolism of dopamine (DA) and tyrosine in the rat striatum were investigated using in vivo and in vitro methods. 30 min after morphine injection, the initial accumulation of 3H-DA in tissues seen after the i.v. injection of L-3,5-H-tyrosine was enhanced as well as the accumulation of 3HH2O and 3H-DA in tissues and medium of striatal slices incubated with the 3H precursor. These results and the estimation of the conversion index of tyrosine into DA (3H-DA/tyrosine specific activity) indicated that morphine stimulated DA synthesis. This effect was not seen 2 hr later. The enhanced DA synthesis was associated with a parallel increase in the release of newly synthesized 3H-DA as indicated by the greater accumulation of 3H-DA in incubating medium of slices of morphine-pretreated rats. Therefore the increased endogenous levels of DA seen after morphine are related to the increased synthesis of the transmitter. Apart from its effect on dopaminergic neuron activity, morphine also induced marked changes in tyrosine metabolism, particularly at 2 or 2.5 hr after its injection. Tyrosine levels in plasma and in striatum were about 160% of the control levels at this time: these changes in the size of the precursor pool may explain the enhanced accumulation of 3H-tyrosine seen in plasma and striatum 10 min after the 3H-amino acid injection. Marked changes in endogenous levels of DA and tyrosine occurred in the striatum of control animals from 9.00 to 12.00 am. Acute treatment with morphine significantly affected these diurnal variations.

Amphetamine and DMI pretreatment in rats increased 3 H-CA quantities in incubating medium of medulla oblongata slices simultaneously incubated with 3 H-tyrosine. Amphetamine but not DMI induced a similar effect on 3 H-DA newly synthesized and released from striatal slices. Thus both drugs enhanced the synthesis of 3 H CA in the two structures examined.

Acetylcholinesterase (AChE), which is known to inactivate acetylcholine (ACh), is present in great abundance in the substantia nigra, although ACh levels and choline acetylase activity in this region are relatively low1. Nigral dopaminergic cell bodies and their dendrites also contain AChE2–4. The functional significance of this enzyme in nigro-striatal dopaminergic neurones has been questioned1,4,5. Earlier studies demonstrated an evoked release of AChE from unidentified central neurones into cerebrospinal fluid (CSF) in cats6, rabbits7,8 and dogs9. Later experiments have provided indirect evidence that the substantia nigra may contribute to a substantial amount of AChE detected: a unilateral nigral lesion in rabbits reduced AChE levels in the CSF, whereas electrical stimulation of the substantia nigra induced the opposite effect10. To investigate the possible release of AChE from dopaminergic dendrites and terminals we measured the in vivo release of this enzyme from the substantiae nigrae and caudate nuclei of cats implanted with four push–pull cannulae and compared it with that of dopamine (DA). DA is released from dendrites in the substantia nigra11,12 as well as nerve terminals in the caudate nucleus. Spontaneous AChE release was observed in the substantia nigra and in the caudate nucleus. Moreover, the application of potassium (30 mM) in one substantia nigra increased the local release of AChE. This was accompanied by remote changes in the enzyme release from the other three structures which differed from that seen for DA. The different patterns of responses observed for AChE and DA suggest that AChE may also originate from other neurones in both the substantia nigra and the caudate nucleus.

amine is released spontaneously and can be easily determined in superfusates after its separation from labelled tyrosine and metabolites. Simultaneously, information about the rate of conversion of ~-[3,5-~H]tyrosine into 'H-labelled dopa (3,4-dihydroxyphenylalanine) can be obtained continuously and by measuring 'HzO in superfusates. Indeed one molecule of 'HzO is produced for each molecule of 'H-labelled dopa synthesized, and large amounts of labelled water diffuse into superfusates. The measurement of jHzO is a specific index of the rate of tyrosine hydroxylation; this is substantiated by the fact that experiments carried out in rats indicated that the formation of jHzO could no longer be detected following the inhibition of the amine synthesis with a-methylparatyrosine or the complete degeneration of the nigrostriatal dopaminergic pathway induced by injection of 6-hydroxydopamine into the substantia nigra (Besson et al., 19716; Javoy et al., 1974).

Halothane-anaesthetized cats were implanted with push-pull cannulae to demonstrate the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) in the substantia nigra and the ipsilateral caudate nucleus. The spontaneous and the calcium-dependent potassium-evoked release of CCK-LI were observed in both structures. In addition, the local application of tetrodotoxin (10-6 M) reduced the spontaneous release of the peptide. 6-OHDA lesions made in the substantia nigra pars compacta led to a complete destruction of nigrostriatal dopaminergic neurons. CCK-LI levels were not affected in the caudate nucleus but were reduced substantially in the substantia nigra. The activation of dopaminergic cells induced by the nigral application of alpha-methyl-para-tyrosine (10-4 M) stimulated the release of CCK-LI and dopamine in the ipsilateral caudate nucleus, whilst opposite effects were seen in the substantia nigra. Similar results were obtained when dopaminergic transmission was blocked in the caudate nucleus suggesting that the evoked release of CCK-LI by the alpha-methyl-para-tyrosine treatment originates from dopaminergic nerve terminals and not from other CCK-LI containing fibres in response to released dopamine. Dopamine (10-7 M) as well as the D1 agonist SKF 38393 (10-5 M) stimulated CCK-LI release when applied into the caudate nucleus while the D2 agonist, LY 171555 (10-6 M) slightly reduced peptide release. The local application of cholecystokinin-8 sulfate (CCK-8S) (10-8 M, for 30 min) into the substantia nigra pars compacta increased the firing rate of dopaminergic cells and stimulated the release of newly synthesized 3H-dopamine from dendrites and nerve terminals. These results suggest, but do not definitively prove, that, in the cat, CCK-LI and dopamine are coreleased from nigrostriatal mixed dopaminergic/CCK-LI neurons and that CCK-LI released from dendrites is, like dopamine, involved in the regulation of the activity of these cells.

"Encéphale isolé" cats were implanted with three push-pull cannulae. The two caudate nuclei (CN) were superfused continuously with 3H-tyrosine to measure the release of 3H-DA in serially collected fractions. Three hours after the beginning of the experiment, d,l-baclofen, d- or l-baclofen or gamma-hydroxybaclofen were introduced for 15 min in the medium used to superfuse the left substantia nigra. d,l-Baclofen (10(-6) M) stimulated 3H-DA release in the ipsilateral as well as in the contralateral CN but the effect was more pronounced in the ipsilateral CN. Similar effects were observed with l-baclofen. d-Baclofen and gamma-hydroxybaclofen were inactive. The results obtained are discussed in the light of effects previously observed during the nigral application of gamma-aminobutyric acid (GABA) and related compounds.