Flora Mela

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.

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 Parkinsons 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*)-α-(4-hydroxyphenyl)-β-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 Parkinsons disease.

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.

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 µm) 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 µm) 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 Parkinsons disease.

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

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. N/OFQ concentration‐dependently inhibited K+‐evoked [3H]‐5‐HT and [3H]‐NE overflow with similar potency (pEC50 ∼7.9 and ∼7.7, respectively) and efficacy (maximal inhibition ∼40%). N/OFQ (0.1 μM) inhibition of [3H]‐5‐HT and [3H]‐NE overflow was antagonized by selective NOP receptor antagonists of peptide ([Nphe1]N/OFQ(1‐13)NH2 and UFP‐101; 10 and 1 μM, respectively) and non‐peptide (J‐113397 and JTC‐801; both 0.1 μM) 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 [3H]‐NE overflow. The NOP receptor ligand [Phe1ψ(CH2‐NH)Gly2]N/OFQ(1‐13)NH2 ([F/G]N/OFQ(1‐13)NH2; 3 μM) did not affect K+‐evoked [3H]‐NE but inhibited K+‐evoked [3H]‐5‐HT overflow in a UFP‐101 sensitive manner. [F/G]N/OFQ(1‐13)NH2 antagonized N/OFQ actions on both neurotransmitters. 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 μM) 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. 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)NH2 and time‐dependent sensitivity towards non‐peptide antagonists.

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 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‐NH2 and [Phe1ψ(CH2‐NH)Gly2]N/OFQ(1–13)‐NH2 inhibited the K+‐induced [3H]‐5‐HT overflow with similar maximal effects (≈−35%) but different potencies (pEC50 of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg14,Lys15]N/OFQ also inhibited [3H]‐5‐HT overflow, but the concentration–response curve was biphasic and the efficacy higher (≈−45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg14,Lys15]N/OFQ and [Phe1ψ(CH2‐NH)Gly2]N/OFQ(1–13)‐NH2 but maintained full responsiveness to endomorphin‐1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe1]N/OFQ(1–13)‐NH2 and UFP‐101) and nonpeptide (J‐113397 and JTC‐801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 µm) 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.

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.

RAPID COMMUNICATION: 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.

The selective D3 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 Parkinsons 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.