Effects of dopamine uptake inhibitor MRZ-9547 in animal models of Parkinson's disease.

MRZ-9547 (d-(2-(2-oxo-4(R)-phenylpyrrolidin-1-yl)-acetamide) is a drug acting at the dopamine transporter (DAT). In the present study, effects of MRZ-9547 alone and in combination with L-3,4-dihydroxyphenylalanine (L-DOPA) were investigated in rodent models predictive for efficacy in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). In rats pre-treated with haloperidol (0.2 mg/kg i.p.), MRZ-9547 (25-100 mg/kg i.p.) dose-dependently attenuated decrease in horizontal locomotion, activity in central zone, and rearings starting at 50 mg/kg i.p. In rats depleted of monoamines by α-methyl-p-tyrosine and reserpine treatment, MRZ-9547 attenuated hypolocomotion starting at 100 mg/kg i.p. At the doses 25-100 mg/kg i.p. the drug induced dose-dependent ipsilateral rotations in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced nigrostriatal system lesions. However, MRZ-9547 enhanced contralateral rotation produced by L-DOPA given at an effective (25 mg/kg i.p.), but not at a sub-effective (6.25 mg/kg i.p.) dose. Microdialysis experiments revealed that MRZ-9547 penetrated well to the brain and did not show any pharmacokinetic interaction with L-DOPA. In unilaterally 6-OHDA-lesioned rats having developed abnormal involuntary movements (AIMs, a rodent correlate of LID) after chronic L-DOPA treatment, MRZ-9547 (50 mg/kg i.p.) did not significantly affect the AIMs expression. The results indicate that MRZ-9547 may by itself have antiparkinsonian activity at early stages of the disease, when some dopaminergic terminals are still intact. It may also enhance antiparkinsonian effect of L-DOPA. MRZ-9547 does not seem to influence the expression of LID in 6-OHDA-lesioned rats. The results support the use of MRZ-9547 in PD patients treated with L-DOPA.

GluN2A and GluN2B NMDA receptor subunits differentially modulate striatal output pathways and contribute to levodopa-induced abnormal involuntary movements in dyskinetic rats.

Dual probe microdialysis was used to investigate whether GluN2A and GluN2B NMDA receptor subunits regulate striatal output pathways under dyskinetic conditions. The preferential GluN2A antagonist NVP-AAM077 perfused in the dopamine-depleted striatum of 6-hydroxydopamine hemilesioned dyskinetic rats reduced GABA and glutamate levels in globus pallidus whereas the selective GluN2B antagonist Ro 25-6981 elevated glutamate without affecting pallidal GABA. Moreover, intrastriatal NVP-AAM077 did not affect GABA but elevated glutamate levels in substantia nigra reticulata whereas Ro 25-6981 elevated GABA and reduced nigral glutamate. To investigate whether GluN2A and GluN2B NMDA receptor subunits are involved in motor pathways underlying dyskinesia expression, systemic NVP-AAM077 and Ro 25-6981 were tested for their ability to attenuate levodopa-induced abnormal involuntary movements. NVP-AAM077 failed to prevent dyskinesia while Ro 25-6981 mildly attenuated it. We conclude that in the dyskinetic striatum, striatal GluN2A subunits tonically stimulate the striato-pallidal pathway whereas striatal GluN2B subunits tonically inhibit striato-nigral projections. Moreover, GluN2A subunits are not involved in dyskinesia expression whereas GluN2B subunits minimally contribute to it.

In vivo evidence for a differential contribution of striatal and nigral D1 and D2 receptors to L-DOPA induced dyskinesia and the accompanying surge of nigral amino acid levels.

Evidence for an involvement of striatal D1 receptors in levodopa-induced dyskinesia has been presented whereas the contribution of striatal D2 receptors remains controversial. In addition, whether D1 and D2 receptors located in the substantia nigra reticulata shape the response to levodopa remains unknown. We therefore used dual probe microdialysis to unravel the impact of striatal and nigral D1 or D2 receptor blockade on abnormal involuntary movements (AIMs) and striatal output pathways in unilaterally 6-hydroxydopamine lesioned dyskinetic rats. Regional perfusion of D1/D5 (SCH23390) and D2/D3 (raclopride) receptor antagonists was combined with systemic administration of levodopa. Levodopa-induced AIMs coincided with a prolonged surge of GABA and glutamate levels in the substantia nigra reticulata. Intrastriatal SCH23390 attenuated the levodopa-induced AIM scores (~50%) and prevented the accompanying neurochemical response whereas raclopride was ineffective. When perfused in the substantia nigra, both antagonists attenuated AIM expression (~21-40%). However, only intranigral SCH23390 attenuated levodopa-induced nigral GABA efflux, whereas raclopride reduced basal GABA levels without affecting the response to levodopa. In addition, intranigral raclopride elevated amino acid release in the striatum and revealed a (mild) facilitatory effect of levodopa on striatal glutamate. We conclude that both striatal and nigral D1 receptors play an important role in dyskinesia possibly via modulation of the striato-nigral direct pathway. In addition, the stimulation of nigral D2 receptors contributes to dyskinesia while modulating glutamate and GABA efflux both locally and in the striatum.

Pharmacological characterization of MRZ-8676, a novel negative allosteric modulator of subtype 5 metabotropic glutamate receptors (mGluR5): focus on L: -DOPA-induced dyskinesia.

Subtype 5 metabotropic glutamate receptors (mGluR5) are abundant in the basal ganglia, amygdala, septum, hippocampus, peripheral sensory neurones and dorsal horn of the spinal cord. Thus, mGluR5 has been implicated in central processes underlying movement control, emotion, learning, and nociception. Different negative allosteric modulators (NAMs) of mGluR5 were repeatedly shown to be efficacious in models of L: -DOPA-induced dyskinesia (LID), anxiety, and some forms of pain. MRZ-8676 (6,6-dimethyl-2-phenylethynyl-7,8-dihydro-6H-quinolin-5-one) is a novel proprietary, selective, orally bioavailable mGluR5 NAM. MRZ-8676 (8.33, 25 and 75 mg/kg) showed a high efficacy in the rat model of LID, with the maximal effect size reaching ~80%. The antidyskinetic effects of MRZ-8676 (75 mg/kg) did not show tolerance as assessed after repetitive (6 days) treatment. MRZ-8676 (25 or 75 mg/kg) demonstrated moderate efficacy in two rat models of anxiety-contextual fear conditioning and the elevated plus maze. MRZ-8676 (25 mg/kg) was also effective in the formalin test, a rat model of persistent pain. The efficacious doses of MRZ-8676 did not produce any detrimental effects on motor performance of rats as determined by means of automated open field and rotarod. However, high doses of MRZ-8676 (75 or 150 mg/kg) disrupted learning in an aversive learning paradigm of the contextual fear conditioning test. In conclusion, MRZ-8676 is a new investigational agent with an efficacy profile similar to the widely published reference mGluR5 NAMs. The drug was demonstrated to possess a superior antidyskinetic efficacy with a sufficient therapeutic window. MRZ-8676 has also therapeutic potential as an anxiolytic and analgesic drug.

The selective D(3) receptor antagonist, S33084, improves parkinsonian-like motor dysfunction but does not affect L-DOPA-induced dyskinesia in 6-hydroxydopamine hemi-lesioned rats.

Despite evidence linking dopamine D(3) receptors to the etiology of Parkinson's disease and L-DOPA-induced dyskinesia, the potential therapeutic utility of D(3) receptor ligands remains unclear. In the present study, we investigated whether the selective D(3) receptor antagonist, S33084, affects development and expression of abnormal involuntary movements (AIMs), a behavioural correlate of dyskinesia, in rats hemi-lesioned with 6-hydroxydopamine and chronically treated with L-DOPA. The ability of S33084, alone or in combination with L-DOPA, to attenuate 6-hydroxydopamine induced motor deficits was also investigated employing a battery of behavioural tests. Acute administration of S33084 (0.64 mg/kg, s.c.) did not attenuate the induction of AIMs in dyskinetic rats upon challenge with L-DOPA (6 mg/kg, s.c.). Moreover, S33084 (0.64 mg/kg) did not prevent the development of AIMs affecting axial, limb and orolingual muscles when chronically administered together with L-DOPA (6 mg/kg for 21 days). However, both acute and chronic administration of S33084 enhanced L-DOPA-induced contralateral turning, suggesting potential antiparkinsonian properties. Furthermore, S33084 (0.01-0.64 mg/kg) dose-dependently attenuated parkinsonian disabilities, including bradykinesia, in drag and rotarod tests, although, in these procedures, the combination of S33084 with L-DOPA did not produce synergistic effect. It is concluded that sustained D(3) receptor blockade does not blunt L-DOPA-induced dyskinesia in hemiparkinsonian rats. However, D(3) receptor antagonism may be associated with antiparkinsonian properties. The clinical relevance of these observations will be of interest to explore further.

Pharmacological modulation of glutamate transmission in a rat model of L-DOPA-induced dyskinesia: effects on motor behavior and striatal nuclear signaling.

L-DOPA-induced dyskinesia (LID) in Parkinson's disease has been linked to altered dopamine and glutamate transmission within the basal ganglia. In the present study, we compared compounds targeting specific subtypes of glutamate receptors or calcium channels for their ability to attenuate LID and the associated activation of striatal nuclear signaling and gene expression in the rat. Rats with 6-hydroxydopamine lesions were treated acutely or chronically with L-DOPA in combination with the following selective compounds: antagonists of group I metabotropic glutamate receptors (mGluR), (2-methyl-1,3-thiazol-4-yl) ethynylpyridine (MTEP) for mGluR5 and (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methane sulfonate (EMQMCM) for mGluR1; an agonist of group II mGluR, 1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268); N-methyl-D-aspartate (NMDA)-R2B subunit (NR2B)-selective NMDA receptor antagonists 1-[2-(4-hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl]-4-piperidinol hydrochloride (Ro631908) and (+/-)-(R(*),S(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)1-piperidine propanol (Ro256981); and an L-type calcium channel antagonist, 4-(4-benzofurazanyl)-1,-4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid methyl 1-methylethyl ester (isradipine). Dyskinesia and rotarod performance were monitored during chronic drug treatment. The striatal expression of phospho-extracellular signal-regulated kinase (ERK) 1/2 and mitogen- and stress-activated kinase (MSK)-1, or prodynorphin mRNA was examined after acute or chronic treatment, respectively. In the acute treatment studies, only MTEP and EMQMCM significantly attenuated L-DOPA-induced phospho-ERK1/2 and/or phospho-MSK-1 expression, with MTEP being the most effective (70-80% reduction). In the chronic experiment, only MTEP significantly attenuated dyskinesia without adverse motor effects, whereas EMQMCM and LY379268 inhibited the L-DOPA-induced improvement in rotarod performance. The NR2B antagonist had positive antiakinetic effects but did not reduce dyskinesia. Only MTEP blocked the up-regulation of prodynorphin mRNA induced by L-DOPA. Among the pharmacological treatments examined, MTEP was most effective in inhibiting LID and the associated molecular alterations. Antagonism of mGluR5 seems to be a promising strategy to reduce dyskinesia in Parkinson's disease.

Antagonism of metabotropic glutamate receptor type 5 attenuates l-DOPA-induced dyskinesia and its molecular and neurochemical correlates in a rat model of Parkinson's disease.

Metabotropic glutamate receptor type 5 (mGluR5) modulates dopamine and glutamate neurotransmission at central synapses. In this study, we addressed the role of mGluR5 in l-DOPA-induced dyskinesia, a movement disorder that is due to abnormal activation of both dopamine and glutamate receptors in the basal ganglia. A selective and potent mGluR5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine, was tested for its ability to modulate molecular, behavioural and neurochemical correlates of dyskinesia in 6-hydroxydopamine-lesioned rats treated with l-DOPA. The compound significantly attenuated the induction of abnormal involuntary movements (AIMs) by chronic l-DOPA treatment at doses that did not interfere with the rat physiological motor activities. These effects were paralleled by an attenuation of molecular changes that are strongly associated with the dyskinesiogenic action of l-DOPA (i.e. up-regulation of prodynorphin mRNA in striatal neurons). Using in vivo microdialysis, we found a temporal correlation between the expression of l-DOPA-induced AIMs and an increased GABA outflow within the substantia nigra pars reticulata. When co-administered with l-DOPA, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine greatly attenuated both the increase in nigral GABA levels and the expression of AIMs. These data demonstrate that mGluR5 antagonism produces strong anti-dyskinetic effects in an animal model of Parkinson's disease through central inhibition of the molecular and neurochemical underpinnings of l-DOPA-induced dyskinesia.

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.

Blockade of nociceptin/orphanin FQ transmission attenuates symptoms and neurodegeneration associated with Parkinson's disease.

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are expressed in the substantia nigra (SN), a brain area containing dopamine neurons that degenerate in Parkinson's disease. Endogenous N/OFQ facilitates nigral glutamate release and inhibits nigrostriatal dopamine transmission and motor behavior. Here, we present evidence suggesting that endogenous N/OFQ may contribute to Parkinson's disease. Pharmacological blockade of the SN N/OFQ-NOP receptor system attenuated parkinsonian-like akinesia/hypokinesia in 6-hydroxydopamine hemilesioned or haloperidol-treated rats, whereas deletion of the NOP receptor gene conferred mice partial protection from haloperidol-induced motor depression. The antiparkinsonian action of NOP receptor antagonists was associated with reduction of glutamate release in the SN. In 6-hydroxydopamine hemilesioned rats, enhancement of N/OFQ expression and release was detected in the lesioned compared with the unlesioned SN, indicating that parkinsonism may be associated with overactivation of the N/OFQ-NOP receptor system in the SN. Finally, deletion of the N/OFQ gene conferred mice partial protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of SN dopamine neurons. Based on these data, we propose that NOP receptor antagonists may represent a novel approach for combined (symptomatic and neuroprotective) therapy of Parkinson's disease.

[(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor.

A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release.

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.

Blockade of nociceptin/orphanin FQ receptor signaling in rat substantia nigra pars reticulata stimulates nigrostriatal dopaminergic transmission and motor behavior.

A multidisciplinary approach was followed to investigate whether the opioid-like peptide nociceptin/orphanin FQ (N/OFQ) regulates the nigrostriatal dopaminergic pathway and motor behavior. Nigrostriatal dopaminergic cells, which express N/OFQ peptide (NOP) receptors, are located in the substantia nigra pars compacta and extend their dendrites in the substantia nigra pars reticulata, thereby modulating the basal ganglia output neurons. In vitro electrophysiological recordings demonstrated that N/OFQ hyperpolarized the dopaminergic cells of the substantia nigra pars compacta and inhibited their firing activity. In vivo dual-probe microdialysis showed that N/OFQ perfused in the substantia nigra pars reticulata reduced dopamine release in the ipsilateral striatum, whereas UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ(1-13)-NH2) (a selective NOP receptor peptide antagonist) stimulated it. N/OFQ microinjected in the substantia nigra pars reticulata impaired rat performance on a rotarod apparatus, whereas UFP-101 enhanced it. Electromyography revealed that N/OFQ and UFP-101 oppositely affected muscle tone, inducing relaxation and contraction of triceps, respectively. The selective NOP receptor nonpeptide antagonist J-113397 (1-[3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one), either injected intranigrally or given systemically, also elevated striatal dopamine release and facilitated motor activity, confirming that these effects were caused by blockade of endogenous N/OFQ signaling. The inhibitory role played by endogenous N/OFQ on motor activity was additionally strengthened by the finding that mice lacking the NOP receptor gene outperformed wild-type mice on the rotarod. We conclude that NOP receptors in the substantia nigra pars reticulata, activated by endogenous N/OFQ, drive a physiologically inhibitory control on motor behavior, possibly via modulation of the nigrostriatal dopaminergic pathway.

Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine release in the mouse neocortex.

A synaptosomal preparation was employed to pharmacologically characterize the role of presynaptic nociceptin/orphanin FQ (N/OFQ) receptors (NOP receptors) in the regulation of 5-hydroxytryptamine release in the Swiss mouse neocortex. In the present study, the NOP receptor ligands N/OFQ, Ac-RYYRWK-NH(2) and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) inhibited the K(+)-induced [(3)H]-5-HT overflow with similar maximal effects ( approximately -35%) but different potencies (pEC(50) of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg(14),Lys(15)]N/OFQ also inhibited [(3)H]-5-HT overflow, but the concentration-response curve was biphasic and the efficacy higher ( approximately -45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg(14),Lys(15)]N/OFQ and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) but maintained full responsiveness to endomorphin-1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe(1)]N/OFQ(1-13)-NH(2) and UFP-101) and nonpeptide (J-113397 and JTC-801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 microm) N/OFQ concentrations. This phenomenon was prevented by the protein kinase C inhibitor, bisindolylmaleimide. Moreover, N/OFQ-induced desensitization did not affect mu opioid receptor responsiveness. Finally, it was observed in a similar preparation of rat cerebrocortical synaptosomes, although it was induced by higher N/OFQ concentrations than that used in the mouse. Together, these findings indicate that presynaptic NOP receptors inhibit 5-hydroxytryptamine release in the mouse neocortex. Based on present and previous studies, we conclude that NOP receptors in the mouse are subtly different from the homologous receptor population in the rat, strengthening the view that there exist species differences in the pharmacology of central NOP receptors.

Differential responsiveness of rat striatal nerve endings to the mitochondrial toxin 3-nitropropionic acid: implications for Huntington's disease.

Rat striatal synaptosomes and slices were used to investigate the responsiveness of different populations of nerve terminals to 3-nitropropionic acid (3-NP), a suicide inhibitor of the mitochondrial enzyme succinate dehydrogenase, and to elucidate the ionic mechanisms involved. 3-NP (0.3-3 mm) stimulated spontaneous gamma-aminobutyric acid (GABA), glutamate and [3H]-dopamine efflux but left unchanged acetylcholine efflux from synaptosomes. This effect was associated with a >70% inhibition of succinate dehydrogenase, as measured in the whole synaptosomal population. The facilitation was not dependent on extracellular Ca2+ but relied on voltage-dependent Na+ channel opening, because it was prevented by tetrodotoxin and riluzole. 3-NP also elevated spontaneous glutamate efflux from slices but in a tetrodotoxin-insensitive way. To investigate whether energy depletion could change the responsiveness of nerve endings to a depolarizing stimulus, synaptosomes were pretreated with 3-NP and challenged with pulses of KCl evoking 'quasi-physiological' neurotransmitter release. 3-NP potentiated the K+-evoked GABA, glutamate and [3H]-dopamine release but inhibited the K+-evoked acetylcholine release. The 3-NP induced potentiation of GABA release was Ca2+-dependent and prevented by tetrodotoxin and riluzole whereas the 3-NP-induced inhibition of acetylcholine release was tetrodotoxin- and riluzole-insensitive but reversed by glipizide, an ATP-dependent K+ channel inhibitor. We conclude that the responsiveness of striatal nerve endings to 3-NP relies on activation of different ionic conductances, and suggest that the selective survival of striatal cholinergic interneurons following chronic 3-NP treatment (as in models of Huntington's disease) may rely on the opening of ATP-dependent K+ channels, which counteracts the fall in membrane potential as a result of mitochondrial impairment.

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors.

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.

Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex.

1 The pharmacological profiles of presynaptic nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) modulating 5-hydroxytryptamine (5-HT) and noradrenaline (NE) release in the rat neocortex were characterized in a preparation of superfused synaptosomes challenged with 10 mM KCl. 2 N/OFQ concentration-dependently inhibited K(+)-evoked [(3)H]-5-HT and [(3)H]-NE overflow with similar potency (pEC(50) approximately 7.9 and approximately 7.7, respectively) and efficacy (maximal inhibition approximately 40%). 3 N/OFQ (0.1 micro M) inhibition of [(3)H]-5-HT and [(3)H]-NE overflow was antagonized by selective NOP receptor antagonists of peptide ([Nphe(1)]N/OFQ(1-13)NH(2) and UFP-101; 10 and 1 microM, respectively) and non-peptide (J-113397 and JTC-801; both 0.1 microM) nature. Antagonists were routinely applied 3 min before N/OFQ. However, a 21 min pre-application time was necessary for J-113397 and JTC-801 to prevent N/OFQ inhibition of [(3)H]-NE overflow. 4 The NOP receptor ligand [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]N/OFQ(1-13)NH(2); 3 microM) did not affect K(+)-evoked [(3)H]-NE but inhibited K(+)-evoked [(3)H]-5-HT overflow in a UFP-101 sensitive manner. [F/G]N/OFQ(1-13)NH(2) antagonized N/OFQ actions on both neurotransmitters. 5 The time-dependency of JTC-801 action was studied in CHO cells expressing human NOP receptors. N/OFQ inhibited forskolin-stimulated cAMP accumulation and JTC-801, tested at different concentrations (0.1-10 microM) and pre-incubation times (0, 40 and 90 min), antagonized this effect in a time-dependent manner. The Schild-type analysis excluded a competitive type of antagonism. 6 We conclude that presynaptic NO receptors inhibiting 5-HT and NE release in the rat neocortex have similar pharmacological profiles. Nevertheless, they can be differentiated pharmacologically on the basis of responsiveness to [F/G]N/OFQ(1-13)NH(2) and time-dependent sensitivity towards non-peptide antagonists.

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors.

Dual probe microdialysis was employed in conscious rats to investigate whether endogenous dopamine is involved in the stimulation of glutamate release in the substantia nigra pars reticulata following striatal NMDA receptor activation. Intrastriatal perfusion with NMDA (1 and 10 microm) facilitated nigral glutamate release (dizocilpine- and tetrodotoxin-sensitive). The D2 dopamine receptor antagonist raclopride increased spontaneous nigral glutamate release and caused a leftward shift in the NMDA sensitivity, lowering NMDA effective concentrations to submicromolar levels. Conversely, the D1 antagonist SCH23390 prevented the effect of NMDA (1 microm) and caused a rightward shift in the NMDA sensitivity. It was tested whether the antagonist effects were due to dopamine receptor blockade or increased tone on D1/D2 receptors. SCH23390 prevented the raclopride-induced enhancement of spontaneous but not NMDA-evoked glutamate release while raclopride left unchanged the SCH23390-induced inhibition. The physiopathological relevance of the dopaminergic modulation was strengthened by perfusing NMDA in the dopamine-depleted striatum of hemiparkinsonian rats. Nigral glutamate responsiveness to NMDA was enhanced as with raclopride. We conclude that endogenous striatal dopamine regulates both spontaneous and NMDA-induced nigral glutamate release via an opposite control mediated by D1 facilitatory and D2 inhibitory receptors. Alterations of this control may subserve the motor symptoms of Parkinson's disease.