Muscarinic receptor activation attenuates D2 dopamine receptor mediated inhibition of acetylcholine release in rat striatum: indications for a common signal transduction pathway

In the present investigations, we used a superfusion system to study the effect of simultaneous activation of D2 dopamine receptors and so-called muscarinic "autoreceptors" on the K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. Activation of D2 receptors with the selective agonist LY 171555 (0.01-1 microM) clearly decreased the evoked release of [3H]acetylcholine. This effect was markedly attenuated in the presence of either the selective muscarinic receptor agonist oxotremorine (3 microM) or the cholinesterase inhibitor physostigmine (1 microM). Conversely, D2 receptor activation with LY 171555 (1 microM) completely abolished the muscarinic receptor mediated inhibition of evoked [3H]acetylcholine release induced by oxotremorine (0.03-10 microM). These results show that the inhibitory effects of D2 dopamine receptor and muscarinic receptor activation on striatal acetylcholine release are non-additive and therefore are interdependent processes. In addition, we investigated some aspects of the signal transduction mechanism by which the muscarinic receptor mediates inhibition of K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. It appeared that the effect of muscarinic receptor activation was not significantly influenced either by a lowering of the extracellular Ca2+ concentration from the usual 1.2-0.12 mM or by an increase of the intracellular cyclic adenosine-3',5'-monophosphate content. However, increasing extracellular K+ strongly decreased the inhibition of evoked [3H]acetylcholine release mediated by activation of muscarinic receptors. This set of results indicates that the muscarinic "autoreceptor" mediates the decrease of depolarization induced [3H]acetylcholine release from rat striatum to a large extent through stimulation of K+ efflux (opening of K+ channels) in a cyclic adenosine-3',5'-monophosphate independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo presynaptic control of dopamine release in the cat caudate nucleus--III. Further evidence for the implication of corticostriatal glutamatergic neurons

In confirmation of previous results, experiments in halothane-anaesthetized cats implanted with push-pull cannulae showed that the unilateral application of GABA (10(-5) M for 30 min) into the left thalamic motor nuclei (either ventralis medialis, or ventralis lateralis) markedly stimulated the release of [3H]dopamine continuously synthesized from [3H]tyrosine in both caudate nuclei and in the contralateral substantia nigra. Three types of experiments confirmed that the changes in [3H]dopamine release evoked in both caudate nuclei resulted from a presynaptic facilitation mediated by the bilateral corticostriatal glutamatergic projection: The constant delivery of 2-amino 6-trifluoromethoxy benzothiazole (PK 26124) (10(-5) M) to the left caudate nucleus prevented the increased release of [3H]DA evoked by application of gamma-aminobutyric acid (GABA) (10(-5)M) into ventralis medialis-ventralis lateralis while an enhanced release of [3H]dopamine still occurred in the contralateral caudate nucleus. Since PK 26124 is an antagonist of glutamatergic transmission, the presynaptic facilitation may involve glutamatergic neurons. Single unit recordings of dopamine cells in the contralateral substantia nigra indicated that the increased release of [3H]dopamine from dendrites evoked by the application of GABA (10(-5)M) into ventralis medialis-ventralis lateralis was associated with a reduction in the firing rate of dopamine cells. Thus, the enhanced release of [3H]dopamine in the contralateral caudate nucleus may involve a presynaptic facilitatory process. Finally, the unilateral lesion of the sensory motor cortex made prior to the superfusion of caudate nucleus with [3H]tyrosine prevented the responses evoked in the two caudate nuclei by the application of GABA (10(-4) M) into ventralis medialis-ventralis lateralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

AMPA receptor blockade potentiates the stimulatory effect of L-DOPA on dopamine release in dopamine-deficient corticostriatal slice preparation

The release of [3H]dopamine was measured in rat corticostriatal slice preparations that contained the striatum and the adjacent prefrontal cortex to maintained glutamatergic corticostriatal afferentation. These slices were prepared from either nontreated or 6-hydroxydopamine-pretreated rats. The slices were loaded with [3H]dopamine, submerged in a two-compartment bath so that the cortical region was contained in one compartment, the corpus callosum was passed through a silicone greased slot, and the striatal region was contained in the other compartment. The cortical and the striatal parts were superfused with Krebs-bicarbonate buffer independently. The release of [3H]dopamine was determined from the striatal part at rest and in response to electrical stimulation of the cortical area. Electrical stimulation of the cortical part increased the release of [3H]dopamine from the striatal part of the slices, and this release was found to be higher after lesion of the nigrostriatal dopaminergic pathway with 6-hydroxydopamine. Cortically evoked [3H]dopamine release was even higher in the presence of the dopamine precursor L-DOPA after 6-hydroxdopamine lesion. Perfusion of GYKI-53405, a noncompetitive AMPA receptor antagonist, in combination with L-DOPA further increased both basal and stimulation-evoked [3H]dopamine release, whereas GYKI-53405 by itself did not influence basal [3H]dopamine outflow from striatum. These findings indicate that, in parkinsonian striatum, the stimulatory effect of L-DOPA on dopamine release is potentiated by AMPA receptor blockade, and the antiparkinsonian effect of GYKI-53405 may be due to its L-DOPA sparing effect.     

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein, but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channels

Electrically evoked [3H]acetylcholine ([3H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A1-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1-10 microM. The maximal effect was observed with 1 microM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 microM, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 +/- 0.01% v. 0.10 +/- 0.003% in the control slices), but did not affect stimulated release (0.73 +/- 0.05% vs. 0.74 +/- 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [3H]ACh release in a non-competitive manner. The S2/S1 ratio was 0.92 +/- 0.03 in controls and was reduced to 0.32 +/- 0.02 by 1 microM R-PIA in the control slices and to 0.57 +/- 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (1 microM) and rolipram (30 microM) before the second stimulation (S2) enhanced the S2/S1 ratio by about 30% to 1.26 +/- 0.12, but did not reduce the inhibitory effect of R-PIA (1 microM). The Ca2+-channel agonist Bay K 86(44) (1 microM), a concentration that increases K+-evoked noradrenaline release, did not affect the basal or electrically evoked [3H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and 1 microM) on [3H]ACh release. Our results suggest that the presynaptic inhibitory effects of A1-receptor agonists on [3H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential release of [3H]acetylcholine from the rat phrenic nerve-hemidiaphragm preparation by electrical nerve stimulation and by high potassium

Neuronal transmitter stores of the phrenic nerve were labelled under different conditions. Subsequently, transmitter release evoked by electrical nerve stimulation and by a high potassium-low sodium solution was studied. Incubation of the end-plate preparation with [³H]choline at rest led to the synthesis of [³H]acetylcholine which could not be released by electrical nerve stimulation but it was released by high potassium-low sodium solution, independent of the presence of extracellular calcium. When the end-plate preparation was labelled during stimulation at 1 Hz, prolonged periods of electrical nerve stimulation released 83% of the total releasable [³H]transmitter pool in a completely calcium-dependent manner. After exhaustion of the electrically releasable pool, high potassium-low sodium solution still caused a significant outflow. Without a preceding exhaustion of the [³H]acetylcholine pool, high potassium-low sodium solution released a similar amount in the absence of extracellular calcium or after pretreatment with the intracellular calcium chelating substance, Quin-2. When evoked transmitter release was studied at different temperatures (36, 26 and 16°C) Q 10 values of 1.6 and 1.0 were found for the release caused by electrical nerve stimulation and high potassium-low sodium solution (calcium-independent effect), respectively. After labelling during a short interval (2 min) but at a high stimulation rate (50 Hz), only 72% of the releasable [³H]transmitter could be released by electrical nerve stimulation, whereas the outflow due to the calcium-independent effect of high potassium-low sodium solution increased from 17 (labelling during stimulation at 1 Hz) to 28%.It is suggested that the calcium-independent effect of high potassium-low sodium solution reflects the release of acetylcholine from the cytoplasmic compartment, as this outflow occurred after labelling at rest and increased when cytoplasmic synthesis was enhanced by a high loading stimulation. In contrast to high potassium-low sodium solution, propagated nerve activity cannot release acetylcholine synthesized at rest (presumed to be cytoplasmic), but only [³H]acetylcholine synthesized during quantal release (presumed to be vesicular). The absolute requirement of extracellular calcium for electrically stimulated release suggests an exocytotic release mechanism. The low Q 10 value of 1.6 does not fit into the concept of a carrier- or channel-operated release mechanism. High potassium-low sodium solution triggers both calcium-dependent release (exocytosis) and calcium-independent liberation of acetylcholine, which escapes from the cytoplasmic compartment by a diffusion-like mechanism. Accordingly, it is important to consider that propagated nerve activity and high potassium-low sodium solution can cause the release of transmitter by different mechanisms from different compartments.     

Inhibition by acetylcholine of the stimulation-evoked release of [3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [³H]choline and then superfused with Tyrode's solution. Exposure to [³H]choline resulted in the formation of [³H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [³H]acetylcholine were studied.In the absence of a cholinesterase inhibitor exogenous acetylcholine (10^?5 M) only slightly inhibited (by 26%) the evoked release of [³H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10^?7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10^?5 M acetylcholine the release was reduced by 76%. Scopolamine (10^?9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA₂ value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [³H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10^?5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [³H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase).It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [³H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.     

Uptake, release, and modulation of release of noradrenaline in rabbit superior colliculus

The noradrenaline content, the uptake of [3H]noradrenaline, and the release of previously incorporated [3H]noradrenaline were studied in slices of rabbit superior colliculus. The concentration of endogenous noradrenaline was higher in superficial than in deep layers of the superior colliculus. Upon incubation with [3H]noradrenaline, tritium was accumulated by a mechanism that was strongly inhibited by oxaprotiline but little inhibited by 6-nitroquipazine. Electrical stimulation at 0.2 or 3 Hz increased the outflow of tritium from slices preincubated with [3H]noradrenaline; the increase was almost abolished by tetrodotoxin or a low calcium medium. Clonidine reduced the evoked overflow of tritium, whereas yohimbine increased it and antagonized clonidine. The evoked overflow was also reduced by the dopamine D2-receptor-selective agonists apomorphine and quinpirole, an effect antagonized by sulpiride. The preferential opioid kappa-receptor agonist ethylketocyclazocine produced an inhibition that was counteracted by naloxone. Nicotine accelerated the basal outflow of tritium; part of the acceleration was blocked by hexamethonium. The muscarinic agonist oxotremorine slightly diminished the electrically evoked overflow, and its effect was abolished by atropine. The oxaprotiline-sensitive uptake of [3H]noradrenaline as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (presumably reflecting the release of [3H]noradrenaline) indicate that noradrenaline is a neurotransmitter in the superior colliculus. The release of [3H]noradrenaline is modulated through alpha 2-adrenoceptors as well as dopamine D2-receptors, opioid kappa-receptors and nicotine and muscarine receptors. No clear evidence was found for modulation through beta-adrenoceptors, D1-receptors, serotonin receptors, opioid mu- or delta-receptors or receptors for GABA or glutamate. Only the alpha 2-adrenoceptors receive an endogenous agonist input, at least under the conditions of these experiments. The pattern of presynaptic modulation resembles that found for noradrenaline release in other rabbit brain regions, suggesting that all noradrenergic axons arising in the locus coeruleus possess similar presynaptic receptor systems.     

Different effects of electrical stimulation of the mesencephalic and pontine reticular formation on the release of dopamine and acetylcholine in the cat caudate nucleus

The effects of unilateral electrical stimulation of the pontine (PRF) and mesencephalic (MRF) reticular formation on the release of acetylcholine (ACh) and of [3H]dopamine continuously synthesised from [3H]tyrosine were examined in both caudate nuclei of halothane-anaesthetised cats implanted with push-pull cannulae. Stimulation of PRF led to a prolonged bilateral increase in the release of [3H]dopamine, whereas a significant reduction in [3H]amine release was observed in the ipsilateral caudate nucleus following stimulation of the MRF. Changes in ACh release were also seen, but they seemed to be independent from those in dopamine release: the release of ACh was enhanced markedly in both caudate nuclei following stimulation of the MRF, whereas a more moderate increase in the release of ACh occurred ipsilaterally following stimulation of the PRF. These data indicate that both the MRF and the PRF are involved in the control of dopaminergic and cholinergic transmission in the basal ganglia.     

Evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus

Acetylcholinesterase staining and studies on the uptake of [3H]choline into the subsequent efflux of tritium from collicular slices were carried out in order to provide evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus. Acetylcholinesterase staining was dense and homogeneous in superficial layers whereas the staining was arranged in patches with slightly higher density caudally than rostrally in the intermediate layers. The accumulation of tritium in slices incubated with [3H]choline depended on time, temperature and concentration, and was inhibited by hemicholinium-3. Accumulation was slightly higher in caudal than in rostral slices. Electrical stimulation enhanced tritium outflow from slices preincubated with [3H]choline. Tetrodotoxin and a low calcium medium inhibited the evoked overflow whereas hemicholinium-3 caused an enhancement. Oxotremorine decreased the evoked overflow; atropine prevented this effect. The opioids [D-Ala2, MePhe4, Glycol5]enkephalin, [D-Ala2, D-Leu5]enkephalin and ethylketocyclazocine caused an inhibition. The effects of the latter two agonists were antagonized by naloxone. The GABAB-receptor-agonist (-)-baclofen decreased the evoked overflow at lower concentrations than GABA, whereas the GABAA-receptor-agonist muscimol was ineffective. Serotonin produced an inhibition which was prevented by metitepin, alpha- and beta-adrenoceptor as well as dopamine-receptor ligands caused no change. It is concluded that in the rabbit superior colliculus the pattern of acetylcholinesterase staining is comparable, but not identical to the distribution in other species. The accumulation of [3H]choline, as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (reflecting presumably release of [3H]acetylcholine) indicate that acetylcholine has a neurotransmitter function in this tissue. The release of [3H]acetylcholine was modulated by various transmitter substances and related compounds. The pattern of modulation of release differed from the pattern in other cholinergically innervated tissues.     

Insulin-like growth factor 1 stimulates the release of acetylcholine from rat cortical slices

The effect of somatomedin, or insulin-like growth factors (IGF-1 and IGF-2), on the basal and potassium induced release of [3H]acetylcholine ([3H]Ach) from rat cortical slices, previously preincubated with [3H]choline ([3H]Ch), was studied in vitro. IGF-1 (1.4 x 10(-9) to 1.4 x 10(-8) M) had no effect on the basal release of [3H]ACh, while IGF-1 (1.4 x 10(-9) to 4.3 x 10(-8) M) increased the potassium induced release of [3H]ACh from rat brain slices in a concentration-dependent manner. However IGF-2 (1.4 x 10(-8) M) had no effect. Insulin (1.8 x 10(-8) to 5.3 x 10(-8) M), similarly, did not have any influence on the release of [3H]ACh, demonstrating that the facilitatory effect of IGF-1 on [3H]ACh release is not mediated via insulin receptors. This report demonstrates for the first time that IGF-1 has an effect on neurotransmission in the adult brain.     

Role of dynorphin-containing neurons in the presynaptic inhibitory control of the acetylcholine-evoked release of dopamine in the striosomes and the matrix of the cat caudate nucleus.

The roles of acetylcholine and dynorphin (1-13) in the presynaptic control of the release of [3H]dopamine continuously synthesized from [3H]tyrosine were examined in a prominent striosomal enriched area and in an adjacent matrix enriched area of the cat caudate nucleus. This was achieved using microsuperfusion devices applied vertically onto coronal slices of cat brain. These devices were placed in a striosomal enriched area located in the core of the structure (acetylcholinesterase-poor zone) and in an adjacent matrix enriched area (acetylcholinesterase-rich zone). [3H]Tyrosine was delivered continuously to each microsuperfusion device and [3H]dopamine released was estimated in the superfusate. As previously shown, in the presence of tetrodotoxin (1 microM), acetylcholine (50 microM) induces a prolonged stimulation of [3H]dopamine release in both compartments through an interaction with muscarinic receptors. Our present study indicates that both dynorphin 1-13 (1 microM) and the selective kappa agonist trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]benzeneace tamine (U50488) (1 microM) inhibit the tetrodotoxin-resistant acetylcholine-evoked release of [3H]dopamine, these effects being slightly more pronounced in the matrix than in the striosomal enriched area. Naloxone (1 microM) reversed the inhibitory effect of U50488 in both areas. These results suggest that dynorphin exerts an inhibitory presynaptic control of dopamine release through kappa opioid receptors located on dopamine nerve terminals in the striosome as well as in the matrix. However, the presynaptic cholinergic control of dopamine release is much more complex in the matrix than in the striosomal enriched area. Besides its tetrodotoxin-resistant stimulatory effect, acetylcholine exerts two opposing tetrodotoxin-sensitive effects on [3H]dopamine release, one facilitatory and the other inhibitory. We demonstrate here that in the superfused matrix enriched area, the indirect acetylcholine inhibitory response is mediated by dynorphin-containing neurons. Indeed, the short-lasting stimulatory effect of acetylcholine on [3H]dopamine release was converted into a long-lasting response in the presence of naloxone (1 microM), and, in this latter condition, the co-application of dynorphin 1-13 (1 microM) restored the short-lasting stimulatory effect.     

Effects of oxiracetam on neurotransmitter release from rat hippocampus slices and synaptosomes.

The effects of the nootropic drug oxiracetam on the K(+)-evoked overflow of [3H]D-aspartic acid ([3H]D-ASP), [3H]acetylcholine ([3H]ACh), [3H] gamma-aminobutyric acid ([3H]GABA), [3H]noradrenaline ([3H]NA) and [3H]5-hydroxytryptamine ([3H]5-HT) have been studied in superfused rat hippocampal slices. The overflow of [3H]D-ASP was enhanced by low concentrations of oxiracetam (0.01-1 microM) but not by high concentrations (10-100 microM) which showed some tendency to inhibit it. Similarly, low concentrations of oxiracetam increased, although less effectively, the depolarization-evoked overflow of [3H]ACh, whereas higher concentrations were without effect. At the concentrations active on [3H]D-ASP and [3H]ACh overflow oxiracetam did not affect that of [3H]GABA, [3H]NA or [3H]5-HT. The oxiracetam effects present in slices could not be observed in hippocampal synaptosomes. Thus oxiracetam may selectively increase the release of glutamate and acetylcholine in hippocampus by a mechanism which appears not to be sited in the releasing nerve terminals.     

Differential release of coexisting neurotransmitters: frequency dependence of the efflux of substance P, thyrotropin releasing hormone and [3H]serotonin from tissue slices of rat ventral spinal cord

In few systems has the release of coexisting classical and peptide neurotransmitters been studied. Here the release of substance P-like immunoreactivity (SP-LI), thyrotropin releasing hormone-like immunoreactivity (TRH-LI) and [3H]serotonin ([3H]5-HT) from tissue slices of rat ventral spinal cord was investigated in a superfusion system. The slices were stimulated electrically with field stimulation (900 pulses, 2 ms duration, 36 V) at frequencies between 0.25 Hz and 40 Hz. The evoked fractional release of SP-LI increased significantly from 0.46 to 1.24% of the total tissue store when the frequency of stimulation was changed from 3 to 10 Hz, while the evoked fractional release of TRH-LI increased significantly from 0.28 to 0.71% of the total tissue store with increasing frequency of stimulation between 0.5 and 3 Hz. The evoked fractional release of [3H]5-HT did not show any significant change when the frequency of stimulation was changed in the frequency range of 0.25-40 Hz but remained between 5.6 and 7.2% of the total tissue store. It appears that at frequencies lower than 0.5-1 Hz these 5-HT/SP/TRH neurons may function predominantly as serotonergic neurons. At 3 Hz stimulation with 900 pulses the extracellular Ca2+ concentration required for half-maximal release of [3H]5-HT was 1.2 mmol l-1, while for half-maximal release of SP-LI significantly higher concentrations of Ca2+ (4.2 mmol l-1) would be required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of unilateral electrical stimulation of various thalamic nuclei on the release of dopamine from dendrites and nerve terminals of neurons of the two nigrostriatal dopaminergic pathways

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 [3H]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 [3H]dopamine release was enhanced in the ipsilateral caudate nucleus and reduced in the contralateral structure while opposite responses were observed in the corresponding substantiae nigrae. A reduction of [3H]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 [3H]dopamine release in the contralateral substantia nigra. No significant changes in [3H]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.     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.     

Failure of omega-conotoxin to block L-channels associated with [3H]5-HT release in rat brain slices.

The effects of the mixed N- and L-type voltage-sensitive calcium channel (VSCC) antagonist, omega-conotoxin GVIA and the L-type VSCC agonist Bay K-8644 on calcium-dependent, potassium evoked release of [3H]5-hydroxtryptamine ([3H]5-HT) were investigated in slices of rat hippocampus. Bay K-8644 (1 microM) enhanced, whilst omega-conotoxin (10-30 nM) attenuated, but did not abolish, evoked release of [3H]5-HT. The facilitatory actions of Bay K-8644 on evoked release were unaffected by concentrations of omega conotoxin that significantly inhibited [3H]5-HT release. The experiments indicate that concentrations of omega-conotoxin which inhibit neurotransmitter release by blockade of N-type VSCC, may leave L-type calcium channel activity unaffected.     

Inhibition of the electrically induced release of [3H]dopamine by serotonin from superfused rat striatal slices

The effect of serotonin (5-hydroxytryptamine) on the electrically induced release of [3H]dopamine from superfused slices of the rat striatum has been studied. It was observed that serotonin produced a concentration dependent decrease in the field stimulation-induced release of [3H]dopamine with the threshold concentration being 10(-6) M or lower. Methysergide, in a concentration which did not alter the evoked release, antagonized the inhibitory effect of serotonin. The present results suggest that serotonin should be added to the list of endogenous substances that can influence dopaminergic transmission in the striatum.     

Evidence for calcium-dependent vesicular transmitter release insensitive to tetanus toxin and botulinum toxin type F

Whether exocytosis evoked by a given releasing stimulus from different neuronal families or by different stimuli from one neuronal population occurs through identical mechanisms is unknown. We studied the release of [3H]noradrenaline, [3H]acetylcholine and [3H]dopamine induced by different stimuli from superfused rat brain synaptosomes pretreated with tetanus toxin or botulinum toxin F, known to block exocytosis by cleaving VAMP/synaptobrevin. The external Ca2(+)-dependent [3H]transmitter overflows evoked by KCl were similarly inhibited by tetanus toxin or botulinum toxin F; the toxins cleaved similar amounts of synaptosomal synaptobrevin, as determined by western blot analysis, suggesting prevalent involvement of synaptobrevin-II. GABA uptake-mediated release of the three [3H]transmitters was that differentially sensitive to the toxins: only the release of [3H]noradrenaline, which is dependent on external Ca2+, but not of [3H]acetylcholine and [3H]dopamine was blocked. Neither toxin affected the [3H]transmitter overflows evoked by the Ca2(+) ionophore ionomycin. Cadmium blocked the K(+)-evoked release of all [3H]transmitters and the GABA-evoked release of [3H]noradrenaline; the GABA-evoked releases of [3H]acetylcholine and [3H]dopamine and those elicited by ionomycin were insensitive to cadmium. The results suggest that tetanus toxin and botulinum toxin F selectively affect exocytosis linked to activation of voltage-sensitive Ca2(+) channels; the Ca2(+)-dependent, exocytotic-like release induced by stimuli not leading to activation of voltage-sensitive Ca2+ channels seems insensitive to these clostridial toxins.     

Functional heterogeneity of the matrix compartment in the cat caudate nucleus as demonstrated by the cholinergic presynaptic regulation of dopamine release.

Previously, using a new in vitro microsuperfusion procedure, we have demonstrated marked differences in the cholinergic presynaptic regulation of the release of [3H]dopamine continuously synthesized from [3H]tyrosine in two close striosomal- and matrix-enriched areas of the cat caudate nucleus. A tetrodotoxin-resistant stimulatory effect of acetylcholine mediated by muscarinic receptors was observed in both compartments. However, in addition, two opposing types of tetrodotoxin-sensitive acetylcholine-evoked regulation of [3H]dopamine release were only seen in the matrix: one facilitatory, involving nicotinic receptors located on as yet unidentified neurons, and the other inhibitory, mediated by muscarinic receptors located on dynorphin-containing neurons. In the present study, using the same approach, a functional heterogeneity was demonstrated in the matrix. Indeed, in various conditions the effects of acetylcholine (50 microM) on the release of [3H]dopamine were different in a matrix-enriched area (matrix 2) distinct from that previously investigated (matrix 1); these areas being characterized by the presence or absence of islands of striatonigral cells, respectively. As in matrix 1, acetylcholine induced a short-lasting stimulation of [3H]dopamine release in matrix 2 but, in contrast to that observed in matrix 1, the acetylcholine-evoked response in matrix 2 was not modified in the presence of tetrodotoxin (1 microM). Experiments made in the presence of the tetrodotoxin and atropine (1 microM) indicated that both muscarinic and nicotinic receptors are located on dopaminergic nerve terminals in matrix 2 while muscarinic receptors are only present in matrix 1. In the absence of tetrodotoxin, the short-lasting stimulation of [3H]dopamine release was transformed into a long-lasting response in the presence of pempidine (50 microM), in matrix 2 but not in matrix 1 while prolonged responses were seen in both matrix areas in the presence of atropine. Finally, the acetylcholine short stimulatory effect on [3H]dopamine release was transformed into a long stimulatory response in the presence of bicuculline (50 microM) but not naloxone (1 microM) in matrix 2 while the reverse was observed in matrix 1. By providing further evidence for a functional heterogeneity of the matrix, our results suggest that depending on the matrix area investigated, dynorphin- or GABA-containing neurons are involved in the indirect cholinergic inhibitory control of dopamine release.     

Release and turnover of noradrenaline in isolated median eminence: lack of negative feedback modulation

A low volume (tissue holder, 100 microliter; dead space, 300 microliter) perfusion system has been developed for measuring [3H]noradrenaline release from isolated median eminence, where supramaximal electrical field stimulation can be applied. In tissue preloaded with [3H]noradrenaline, the resting release (0.4-2% of the content) was enhanced by electrical stimulation (2-10-fold increase). That the released radioactivity in response to electrical stimulation is mainly due to release of [3H]noradrenaline was confirmed by high pressure liquid chromatography combined with radiochemical detection. Evidence has been obtained that of the stimulation-evoked release of radioactivity 70-80 percent originates from noradrenergic neurons, however, the release observed at rest was not affected by 6-hydroxydopamine pretreatment. 6-Hydroxydopamine pretreatment selectively reduced the concentration of noradrenaline of the median eminence without affecting its dopamine content. The release evoked by electrical stimulation was [Ca2+]- and tetrodotoxin-sensitive. 4-Aminopyridine enhanced both the resting and stimulation-evoked release. The ratio between the amount of [3H]noradrenaline released by two consecutive stimulation periods at 2 Hz (120 shocks) was constant, 0.94 +/- 0.08. In contrast with other noradrenergic axon terminals, the release of [3H]noradrenaline in the median eminence was not subject to negative feedback modulation, yohimbine and xylazine had no effect. This conclusion was substantiated by in vivo study showing that yohimbine, an alpha2-adrenoceptor antagonist enhanced the turnover rate of noradrenaline in the cortex but not in the median eminence. Since noradrenergic axon terminals in the median eminence do not make synaptic contact and the released noradrenaline does not modulate its own release via alpha2-adrenoceptors, it is an interesting anatomical arrangement: the modulatory alpha2-adrenoceptors are located exclusively on the terminals of the hormone-containing neurons.