Impact of 6-hydroxydopamine lesions and cocaine exposure on mu-opioid receptor expression and regulation of cholinergic transmission in the limbic-prefrontal territory of the rat dorsal striatum

Information processing within the striatum is regulated by local circuits involving dopamine, cholinergic interneurons and neuropeptides released by recurrent collaterals of striatal output neurons. In the limbic-prefrontal territory of the dorsal striatum, enkephalin inhibits the NMDA-evoked release of acetylcholine directly through micro-opioid receptors (MORs) located on cholinergic interneurons and indirectly through MORs of output neurons of striosomes. In this territory, we investigated the consequence of changes in dopamine transmission, bilateral 6-hydroxydopamine-induced degeneration of striatal dopaminergic innervation or cocaine (acute and chronic) exposure on (i) MOR expression in both cholinergic interneurons and output neurons of striosomes, and (ii) the direct and indirect enkephalin-MOR regulations of the NMDA-evoked release of acetylcholine. Expression of MORs in cholinergic interneurons was preserved after 6-hydroxydopamine and down-regulated after cocaine treatments. Accordingly, the direct enkephalin-MOR control of acetylcholine release was preserved after 6-hydroxydopamine treatment and lost after cocaine exposure. Expression of MORs in output neurons of striosomes was down-regulated in the 6-hydroxydopamine situation and either preserved or up-regulated after acute or chronic cocaine exposure, respectively. Accordingly, the indirect enkephalin-MOR control of acetylcholine release disappeared in the 6-hydroxydopamine situation but surprisingly, despite preservation of MORs in striosomes, disappeared after cocaine treatment. Showing that MORs of striosomes are still functional in this situation, the MOR agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin inhibited the NMDA-evoked release of acetylcholine after cocaine exposure. Therefore, alteration in the regulation of cholinergic transmission by the enkephalin-MOR system might play a major role in the motivational and cognitive disorders associated with dopamine dysfunctions in fronto-cortico-basal ganglia circuits.     

Effect of selective opiate antagonists on striatal acetylcholine and dopamine release.

We investigated the effect of selective opiate antagonists on striatal acetylcholine (ACh) and dopamine (DA) release. The mu-receptor antagonist beta-funaltrexamine (beta-FNA), the delta-antagonist naltrindole (NTI), and the kappa-antagonist norbinaltorphimine (nor-BNI) were used to selectively block different subtypes of opiate receptors. The experiments were carried out on isolated superfused striatal slices of rats, loaded with [3H]choline or [3H]dopamine. beta-FNA and NTI significantly enhanced the electrical field stimulation-evoked release of ACh but only if the dopaminergic input had been impaired either by chemical denervation or D2 dopamine receptor blockade. By contrast, neither the selective nor nonselective antagonists had any modulatory effect on the release of dopamine. It is concluded, therefore, that the release of ACh is tonically controlled by endogenous opioid peptide(s) through the stimulation of mu- and delta-opiate receptors located on cholinergic axon terminals, in addition to the tonic control by DA.     

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [³H]ACh release was recorded during perifusion of acutely dissociated, [³H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally 300% with EC₅₀=1 μM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K⁺ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.     

Dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine release after neonatal 6-hydroxydopamine: An in vitro analysis

After chemical lesions which destroy the nigrostriatal dopamine pathway, transplants rich in dopamine neurons innervate the striatum and, with appropriate stimulation, drive host motor behaviors normally mediated by dopamine. We wished to determine whether dopamine released from the transplant also reinstated dopaminergic inhibition of striatal acetylcholine release. Three-day-old rat pups received bilateral intraventricular injections of 6-hydroxydopamine. Three days later cell suspensions prepared from embryonic ventral mesencephalon were injected unilaterally into the striatum. Tail pinch and amphetamine were able to elicit contralateral turning in many of these animals. Only those animals which rotated ≥5 turns/min were included for further analysis. Subsequent assays indicated that 6-hydroxydopamine had depleted striatal dopamine to 4% of control and that the transplant had increased dopamine levels to 11% of control. Superfused striatal slices were stimulated (8 Hz, 1 min) and then exposed to amphetamine (10 μM, 3 min). The slice released dopamine, as measured by HPLC, and acetylcholine, as measured by tritium efflux after preincubation with [³H]choline. Moreover, the release of acetylcholine was inhibited by endogenous dopamine as indicated by the ability of sulpiride (1 μM) to increase tritium efflux. Striatal slices prepared from lesioned animals showed a reduction in dopamine overflow in response to both electrical stimulation (0.6% of control) and amphetamine (1% of control), and a decrease in the ability of sulpiride to increase electrically evoked acetylcholine overflow (12% of control). Transplantation partially restored the dopaminergic response to electrical stimulation (21% of control), and amphetamine (15% of control) and fully restored the sulpiride-induced increase in acetylcholine overflow (98% of control). We conclude that transplanted dopaminergic neurons are able to release dopamine and to restore inhibitory control over the release of striatal acetylcholine.     

Dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine release after neonatal 6-hydroxydopamine: an in vitro analysis

After chemical lesions which destroy the nigrostriatal dopamine pathway, transplants rich in dopamine neurons innervate the striatum and, with appropriate stimulation, drive host motor behaviors normally mediated by dopamine. We wished to determine whether dopamine released from the transplant also reinstated dopaminergic inhibition of striatal acetylcholine release. Three-day-old rat pups received bilateral intraventricular injections of 6-hydroxydopamine. Three days later cell suspensions prepared form embryonic ventral mesencephalon were injected unilaterally into the striatum. Tail pinch and amphetamine were able to elicit contralateral turning in many of these animals. Only those animals which rotated greater than or equal to 5 turns/min were included for further analysis. Subsequent assays indicated that 6-hydroxydopamine had depleted striatal dopamine to 4% of control and that the transplant had increased dopamine levels to 11% of control. Superfused striatal slices were stimulated (8 Hz, 1 min) and then exposed to amphetamine (10 microM, 3 min). The slice released dopamine, as measured by HPLC, and acetylcholine, as measured by tritium efflux after preincubation with [3H]choline. Moreover, the release of acetylcholine was inhibited by endogenous dopamine as indicated by the ability of sulpiride (1 microM) to increase tritium efflux. Striatal slices prepared from lesioned animals showed a reduction in dopamine overflow in response to both electrical stimulation (0.6% of control) and amphetamine (1% of control), and a decrease in the ability of sulpiride to increase electrically evoked acetylcholine overflow (12% of control). Transplantation partially restored the dopaminergic response to electrical stimulation (21% of control), and amphetamine (15% of control) and fully restored the sulpiride-induced increase in acetylcholine overflow (98% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotinic receptor-mediated regulation of dopamine transporter activity in rat prefrontal cortex

The objective of this study was to determine whether nicotine could selectively influence dopamine levels in the prefrontal cortex as compared with other dopaminergic areas of brain. Using a superfusion system, we found that nicotine and other agonists at nicotinic acetylcholine receptors enhanced the release of radiolabeled dopamine that was stimulated by 10 microM amphetamine from slices prepared from rat prefrontal cortex. In contrast, nicotine had no effect on amphetamine-stimulated [(3)H]dopamine release from slices of nucleus accumbens nor striatum. Under the conditions used, which included no added calcium to exclude contribution by exocytotic release, nicotine had no effect on basal release of [(3)H]dopamine. The enhancement by nicotine was concentration-dependent, reaching a maximum at 5 microM, and producing less release at higher concentrations. Enhancement by nicotine was fully reversed by 30 microM dihydro-beta-erythroidine, and by 10 microM mecamylamine, but was not affected by alpha-bungarotoxin. The potencies of nicotine, epibatidine, cytisine, and A85380 to enhance amphetamine-stimulated dopamine release, as well as the sensitivity of nicotine enhanced release to antagonists, are consistent with mediation via a high-affinity nicotinic acetylcholine receptor containing alpha 4 and beta 2 subunits, the major species of nicotinic receptor in forebrain. Since low dopaminergic activity in prefrontal cortex is correlated with cognitive deficits in schizophrenia, our findings may help explain why these deficits are improved in schizophrenics by smoking or nicotine administration.     

Inhibition of striatal acetylcholine release by serotonin and dopamine after the intracerebral administration of 6-hydroxydopamine to neonatal rats

The intraventricular administration of 6-hydroxydopamine (6-OHDA) depletes the striatum of dopamine (DA). When given to rat pups at an early age, the toxin also increases striatal serotonin (5-HT) content. In the accompanying report we observed that endogenous 5-HT, like DA, exerts an inhibitory influence on the release of acetylcholine (ACh) from striatal slices prepared from control animals and that the extent of this inhibition is related to the degree of serotonergic innervation of the region being examined. To determine whether this hyperinnervation was accompanied by an increase in serotonergic influence on ACh release, striatal slices were prepared from adult rats, preincubated with [3H]choline, superfused, and exposed to electrical field stimulation. The efflux of tritium into the superfusate was used as a measure of ACh release. In confirmation of previous reports, we observed that direct and indirect agonists of DA and 5-HT both reduced ACh overflow from control slices, whereas overflow was increased by antagonists of these amines. Slices prepared from rats given 6-OHDA-induced lesions as adults were responsive to each of these pharmacological manipulations, as well. In contrast, ACh overflow from slices prepared from animals lesioned with 6-OHDA as neonates was not modified by either dopaminergic or serotonergic drugs. These results suggest that the serotonergic hyperinnervation of striatum produced by neonatal 6-OHDA is accompanied by a loss of the inhibitory influence of endogenous 5-HT and DA on striatal ACh release and, thus, provide no evidence for a role for either transmitter in the behavioral sparing associated with such lesions.     

Modulation of the endogenous acetylcholine release from rat striatal slices

The dopaminergic modulation of the acetylcholine release from rat striatal slices has been investigated using a chemiluminescent method. Dopamine, more efficiently than apomorphine, decreased the potassium-evoked release of acetylcholine. The effect of dopamine antagonists, haloperidol and sulpiride, has been studied, and haloperidol was a better antagonist than sulpiride to the dopamine effect. Haloperidol elicited an acetylcholine release from striatal slices at 0.1 nM, probably by removing endogenous dopamine from dopaminergic receptors.     

Modulatory action of acetylcholine on striatal neurons: microiontophoretic study in awake,unrestrained rats

Cholinergic interneurons innervate virtually all medium spiny striatal cells, but the relevance of this input in regulating the activity and afferent responsiveness of these cells remains unclear. Studies in anaesthetized animals and slice preparations have shown that iontophoretic acetylcholine (ACh) either weakly excites or inhibits striatal neurons. These differential responses may reflect cholinergic receptor heterogeneity but may be also related to the different activity states of recorded units and different afferent inputs specific in each preparation. Single-unit recording was combined with iontophoresis in awake, unrestrained rats to examine the effects of ACh and selective muscarinic (oxotremorine M or Oxo-M) and nicotinic agonists (nicotine or NIC) on dorsal and ventral striatal neurons. These effects were tested on naturally silent, spontaneously active and glutamate-stimulated units. We found that iontophoretic ACh primarily inhibited spontaneously active and glutamate-stimulated units; the direction of the ACh response, however, was dependent on the firing rate. The effects of ACh were generally mimicked by Oxo-M and, surprisingly, by NIC, which is known to excite units in most central structures, including striatal neurons in anaesthetized preparation. Given that NIC receptors are absent on striatal cells but located primarily on dopamine terminals, we assessed the effects of NIC after complete blockade of dopamine receptors induced by systemic administration of a mixture of D1 and D2 antagonists. During dopamine receptor blockade the number of NIC-induced inhibitions dramatically decreased and NIC had mainly excitatory effects on striatal neurons. Thus, our data suggest that under physiologically relevant conditions ACh acts as a state-dependent neuromodulator, and its action involves not only postsynaptic but also presynaptic cholinoreceptors located on dopamine- and glutamate-containing terminals.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

Physiological release of striatal acetylcholine (in vivo): effect of somatostatin on dopaminergic-cholinergic interaction

The effects of somatostatin (SOM) on the release of acetylcholine (ACh) and dopamine (DA) from striatum of freely moving rats were studied by transversal microdialysis. Acetylcholine (ACh) and dopamine (DA) were detected by high performance liquid chromatography (HPLC) with electrochemical detection. Somatostatin (0.1, 0.5 and 1 microM), administered locally through the microdialysis probe to the striatum, was able to release dose-dependently ACh from the cholinergic neurons of the striatum. The increase in the extracellular levels of ACh produced by 1 microM SOM in the striatum reached a maximum of 200%. ACh-releasing effect of SOM was completely inhibited by tetrodotoxin indicating that neuronal firing is involved in its effect. Local infusion of sulpiride, 10 microM, D(2) receptor antagonist, potentiated (about 100%) the SOM (1 microM)-induced release of ACh. SOM, 1 microM, was more effective in enhancing the release of ACh in the striatum (two-fold increase) after degeneration of the nigrostriatal DA pathway with 6-hydroxydopamine (6-OHDA) (250 microg/animal, i.c.v.). The D(2) receptor agonists bromcriptine, 10 microM, or apomorphine, 10 microM, completely antagonize SOM-induced release. SOM, 1 microM, enhanced the release of DA (about 400%). These findings indicate that SOM is capable of releasing both ACh and DA in the striatum, however, its effect on ACh release is partially masked unless the D(2) receptor-mediated tonic inhibitory effect of released DA from the nigro-striatal pathway is attenuated.     

Activation by nicotine of striatal neurons receiving excitatory input from the substantia nigra via dopamine release

An electrophysiological study was performed to elucidate the role of nicotinic receptors in the striatal neurons in chloral hydrate-anesthetized rats. The effects of microiontophoretic application of nicotine and other drugs were examined on the caudate nucleus (CN) neurons activated monosynaptically by stimulation of the substantia nigra pars compacta (SN). Application of nicotine facilitated spontaneous firing. The nicotine-induced firing of the CN neurons was inhibited by concomitant application of domperidone or hexamethonium. These findings suggested that nicotine enhances dopamine release from the SN-derived dopaminergic nerve terminals by activating the neurons via D2 receptors.     

Action of apomorphine,bromocriptine and lergotrile onγ-aminobutyric acid and acetylcholine release in nucleus accumbens and corpus striatum

The effect of three dopamine agonists, apomorphine, bromocriptine and lergotrile, was tested on the release of gamma-aminobutyric acid, (GABA) and acetylcholine (ACh) from tissue slices of rat nucleus accumbens and striatum. All three agents in vitro caused a dose dependent depression of the K+-evoked release of [14C]-GABA in corpus striatum. This effect was also obtained following in vivo drug application and when endogenous GABA release was determined. A similar depression of GABA release was obtained in the nucleus accumbens. Both dopamine and dibutyryl adenosine-3':5'-cyclic monophosphoric acid inhibited the K+-evoked release of [14C]-GABA in corpus striatum. This inhibitory effect was not reversed by sulpiride. Bromocriptine and lergotrile also depressed the K+-evoked release of [3H]-acetylcholine from tissue slices of corpus striatum but not nucleus accumbens, as has previously been demonstrated for dopamine and apomorphine. In contrast, sulpiride enhanced the release of [3H]-acetylcholine and molindone reversed the apomorphine inhibition of [3H]-acetylcholine release. These results indicate that dopaminergic agents may influence the release of both GABA and ACh in the corpus striatum but only GABA in the nucleus accumbens.     

Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites

The main objective of the present work was to determine whether the regulation of ACh release by nicotinic and muscarinic autoreceptors is compromised in the aged rat brain. For this, the effects of the nicotinic agonist N-methylcarbamylcholine (MCC) and the muscarinic-M2 antagonist AF-DX 116 on ACh release from brain slices of young (3-month-old), adult (9-month-old), and aged (27-month-old) rats were tested. The ability of MCC to enhance spontaneous ACh release in hippocampal, cerebral cortical, and cerebellar slices was only modestly altered with age. In contrast, the sensitivity of muscarinic autoreceptors in the aged hippocampus and cerebral cortex, but not the striatum, to blockade by the muscarinic-M2 antagonist AF-DX 116 was severely attenuated. To assess whether the age-related changes in cholinergic autoreceptor function may be due to deficits in presynaptic cholinergic markers, we tested whether choline acetyltransferase (ChAT) activity, basal and evoked ACh release, and nicotinic and muscarinic binding sites are altered in the aged rats. ChAT activity in forebrain regions was decreased in the aged compared to the young and mature adult rats. Furthermore, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in striatal, hippocampal, and cortical slices of aged rats. The densities of nicotinic and muscarinic-M2 binding sites, assessed using 3H-MCC and 3H-AF-DX 116 as selective ligands, respectively, were markedly reduced in homogenates of the striatum, hippocampus, cerebral cortex, and thalamus of aged rats. In contrast, muscarinic-M1 sites, selectively labeled with 3H-pirenzepine, were not affected. Therefore, it appears that age-related decrements in ChAT activity and in muscarinic-M2, but not nicotinic, binding sites in the rat brain are reflected in a decreased function of muscarinic-M2 autoreceptors. However, the positive correlation between loss of ChAT activity, decreased muscarinic-M2 binding sites, and impaired muscarinic autoreceptor function is clearly tissue dependent.     

Lesion with the neurotoxin AF64A alters hippocampal cholinergic receptor function

The effect of selective lesion of cholinergic inputs to the hippocampus on the function of hippocampal cholinergic receptors was examined. Hippocampal cholinergic neurons were lesioned in the rat by administration of the selective cholinergic neurotoxin AF64A (ethylcholine mustard azirtdinium). Cholinergic receptor function was examined by assessing the ability of cholinergic agonists and antagonists to modulate the evoked release of radiolabelled acetylcholine (ACh) from hippocampal slices. Nicotine enhanced release, with a bell-shaped dose-response curve. The dose-response curve and EC₅₀ for nicotine was shifted 10-fold to the left in lesioned rats, suggesting an increased sensitivity to nicotine. However, there were no differences in either the number or affinity of nicotinic receptors as determined with binding studies. The muscarinic agonist oxotremorine inhibited the evoked release of ACh in control tissues, but had much less effect in AF64A-lesioned tissues. Binding to the M1 receptor subtype was not changed. However, the K_d for binding to the high affinity subtype of the M2 receptor was increased 10-fold, suggesting that the receptor has become less sensitive to stimulation. Loss of M2 function may allow an increase in the effect of stimulating nicotinic receptors that modulate ACh release.     

Group-II metabotropic glutamate receptors negatively modulate NMDA transmission at striatal cholinergic terminals: role of P/Q-type high voltage activated Ca++ channels and endogenous dopamine

Striatal cholinergic nerve terminals express functional group-II metabotropic (mGlu) and NMDA glutamate receptors. To investigate whether these receptors interact to regulate ACh release, LY354740 (a group-II mGlu receptor agonist) and NMDA were co-applied in striatal synaptosomes and slices. LY354740 prevented the NMDA-evoked [3H]-choline release from synaptosomes and ACh release from slices. In synaptosomes, this modulation was prevented by omega-agatoxin IVA, suggesting that it was mediated by P/Q-type high voltage activated Ca++ channels. In slices, LY341495 (a group-II mGlu receptor antagonist) enhanced the NMDA-induced ACh release, suggesting that group-II mGlu receptor activation by endogenous glutamate inhibits NMDA transmission. Co-immunoprecipitation studies excluded direct group-II mGlu-NMDA receptor interactions. Finally, group-II mGlu negative modulation of NMDA transmission was abolished in dopamine-depleted synaptosomes and slices, suggesting that it relied on endogenous dopamine. We conclude that group-II mGlu receptors attenuate NMDA inputs at striatal cholinergic terminals via Ca++ channel modulation and dopamine-sensitive pathways.     

Reduced striatal acetylcholine efflux in the R6/2 mouse model of Huntington's disease: an examination of the role of altered inhibitory and excitatory mechanisms

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by the progressive onset of cognitive, psychiatric, and motor symptoms. In parallel, the neuropathology of HD is characterized by progressive loss of projection neurons in cortex and striatum; striatal cholinergic interneurons are relatively spared. Nonetheless, there is evidence that striatal acetylcholine (ACh) function is altered in HD. The present study is the first to examine striatal ACh function in awake, behaving animals, using the R6/2 mouse model of HD, which is transgenic for exon 1 of the mutant huntingtin gene. Physiological levels of extracellular striatal ACh were monitored in R6/2 mice and wild type controls using in vivo microdialysis. Results indicate that spontaneous ACh release is reduced in R6/2 mice relative to controls. Intrastriatal application of the GABA_A antagonist bicuculline methiodide (10.0 μM) significantly elevated ACh levels in both R6/2 mice and wild type controls, while overall ACh levels were reduced in the R6/2 mice compared to the wild type group. In contrast, systemic administration of the D₁ dopamine receptor partial agonist, SKF-38393 (10.0 mg/kg, IP), elevated ACh levels in control animals, but not R6/2 mice. Taken together, the present results suggest that GABA-mediated inhibition of striatal ACh release is intact in R6/2 mice, further demonstrating that cholinergic interneurons are capable of increased ACh release, whereas D₁ receptor-dependent activation of excitatory inputs to striatal cholinergic interneurons is dysfunctional in R6/2 mice. Reduced levels of extracellular striatal ACh in HD may reflect abnormalities in the excitatory innervation of cholinergic interneurons, which may have implications ACh-dependent processes that are altered in HD, including corticostriatal plasticity.     

Neostriatal dopaminergic terminals prevent the GABAergic involvement in the μ- and δ-opioid inhibition of KCl-evoked endogenous acetylcholine release

Endogenous acetylcholine (ACh) release from rat neostriatal slices were inhibited by the μ-opioid agonist [d-Ala²,Gly(ol)⁵]-enkephalin (DAGO) both in 6-hydroxydopamine (6-OHDA)-lesioned and non-lesioned neostriatum. However, the δ-opioid agonist [d-Pen², d-Pen⁵]-enkephalin (DPDPE) could not inhibit KCl-evoked ACh release in the 6-OHDA-lesioned striatum. This results suggests that δ-opioid agonist act on dopaminergic terminals to inhibit the cholinergic neurons. In unlesioned rats, GABA_A or GABA_B antagonists (bicuculline or phaclofen, respectively) prevented μ- or δ-opioid inhibition of endogenous ACh release evoked by glutamate, but not by potassium. However, in the 6-OHDA-lesioned side, DAGO inhibition of KCl-evoked ACh release was antagonized by either of the GABA antagonists. Our results suggest that the dopaminergic neurotransmission, favored by KCl, blocks the GABAergic involvement in the μ- and δ-opioid inhibition of endogenous ACh release.     

Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: territorial specificity and diurnal variation

Striatal cholinergic interneurons play a crucial role in the control of movement as well as in motivational and learning aspects of behaviour. Neuropeptides regulate striatal cholinergic transmission and particularly activation of mu opioid receptor (MOR) inhibits acetylcholine (ACh) release in the dorsal striatum. In the present study we investigated whether this cholinergic transmission could be modulated by an enkephalin/MOR direct process. We show that mRNA and protein of MORs are expressed by cholinergic interneurons in the limbic/prefrontal territory but not by those in the sensorimotor territory of the dorsal striatum. These MORs are functional because potassium-evoked release of ACh from striatal synaptosomes was dose-dependently reduced by a selective MOR agonist, this effect being suppressed by a MOR antagonist. The MOR regulation of cholinergic interneurons presented a diurnal variation. (i) The percentage of cholinergic interneurons containing MORs that was 32% at the beginning of the light period (morning) increased to 80% in the afternoon. (ii) The MOR-mediated inhibition of synaptosomal ACh release was higher in the afternoon than in the morning. (iii) While preproenkephalin mRNA levels remained stable, enkephalin tissue content was the lowest (-32%) in the afternoon when the spontaneous (+35%) and the N-methyl-d-aspartate-evoked (+140%) releases of enkephalin (from microsuperfused slices) were the highest. Therefore, by acting on MORs present on cholinergic interneurons, endogenously released enkephalin reduces ACh release. This direct enkephalin/MOR regulation of cholinergic transmission that operates only in the limbic/prefrontal territory of the dorsal striatum might contribute to information processing in fronto-cortico-basal ganglia circuits.     

differential effect of systemic administration of bromocriptine andl-DOPA on the release of acetylcholine from striatum of intact and 6-OHDA-treated rats

A presumed balance between striatal dopaminergic and cholinergic systems forms a major theoretical framework for the development of new agents for the treatments of Parkinson's disease. We therefore studied the effect of two drugs currently used as anti-parkinsonian agents, bromocriptine (BROMO) andl-β-3-,4-dihydroxyphenylalanine (l-DOPA), on the release of striatal acetylcholine (ACh) in intact and 6-hydroxy-dopamine-treated rats using in vivo microdialysis. Lesioned rats with a 90% tissue depletion of striatal dopamine (DA) had a significantly higher output of striatal ACh than unlesioned rats (88 fmol/min vs. 52 fmol/min; 0.3 μmol/l neostigmine in perfusate). BROMO (4 mg/kg) inhibited the output of striatal ACh in both groups. Whereas the lowest dose ofl-DOPA (50 mg/kg) potently stimulated ACh output in lesioned rats, unlesioned rats were significantly less responsive. A higher dose ofl-DOPA (100 mg/kg) stimulated ACh output to the same extent in both groups. At the highest dose tested,l-DOPA (200 mg/kg) given to intact rats did not further increase striatal ACh output. Thus, BROMO decreases whereasl-DOPA increases striatal ACh release after systematic application. Therapeutic as well as side effects ofl-DOPA may therefore be mediated by neurochemical alterations that are more complex than previously thought.