Evaluation of pre- and post-synaptic events involved in the development of L-DOPA-induced dyskinesia in Parkinson’s Disease.

L-DOPA-induced dyskinesia (LID) is a severe motor side effect frequently observed in parkinsonian patients receiving dopamine replacement therapies. Parkinson’s disease (PD) and LID have been correlated with dramatic changes affecting several Basal Ganglia (BG) structures. Alterations in the neuronal connectivity, in the production and release of neurotransmitters and in striatal medium spiny neurons (MSNs) activity have been frequently reported in the Caudate-Putamen nucleus (CPu), the major BG component. Importantly, in advanced PD, striatal serotoninergic terminals, rather than dopaminergic ones, are the major site of L-DOPA metabolism and dopamine (DA) release. In this respect, preclinical findings have demonstrated as mixed serotoninergic 5-HT1A/1B receptor agonists suppress dyskinetic behaviors at the expense of the anti-parkinsonian effects of L-DOPA. Interestingly, blockade of the Gs-coupled A2A receptors for adenosine was found to be an efficacious approach to boost L-DOPA efficacy without worsening the severity of LID. Moreover, striatal DA D2 receptors (D2R) play an important role both in the manifestation of parkinsonian symptoms as well as in the generation of LID. Indeed, D2R modulate the release of several neurotransmitters and are vital for the expression of long-term forms of synaptic plasticity. Starting from these bases, two studies were planned to evaluate whether: (1) the combined administration of a mixed 5-HT1A/1B receptor agonist (eltoprazine) with a A2A receptors antagonist (preladenant) may prevent the onset of LID in a rat model of PD; (2) cell-specific deletion of D2R either in indirect MSNs (iMSNs) or in cholinergic interneurons (ChIs) may differently affect the expression of LID in a mouse model of PD. Study (1) was performed in unilateral 6-OHDA-lesioned drug naive rats. Animals were pre-treated for 14 days with L-dopa (4mg/kg), alone on in combination with eltoprazine (0.6mg/kg) and/or preladenant (0.3mg/kg). Abnormal involuntary movements (AIMs) as index of dyskinesia, were evaluated. Four days after the last administrations, all rats were treated with L-dopa (4 mg/kg) for the pharmacological challenge. Immunoreactivity for the immediate-early gene zif-268 was evaluated in the dorsal CPu. Behavioural and immunohistochemical results point out as the combine pre-administration of eltoprazine, preladenant and L-DOPA significantly reduce the onset of LID and lower zif-268 activation in DA-depleted rats challenged with L-DOPA. Study (2) was performed in unilateral 6-OHDA-lesioned mice carrying a cell-specific deletion of D2R either in iMSNs (iMSN-D2RKO), ChIs (ChIs-D2RKO) or WT controls. Mice were treated either with: i) saline; ii) 15 mg/kg of L-DOPA (once a day for 11 consecutive days), iii) an ascending-dose regimen of L-DOPA (1.5, 3, 6 mg/kg; once a day for 9 consecutive days), or iv) acutely with L-DOPA alone or in combination with the selective M1 antagonist VU 0255035. AIMs as index of dyskinesia were evaluated. Afterward, electrophysiological and immunohistochemical analyses were performed to evaluate the expression of long-term depression (LTD) and the activation of established biomarkers correlated with LID. Results showed as stimulation of D2R located on iMSN, but not on ChIs, were implicated in the modulation of dyskinetic behaviours induced by L-DOPA. Indeed, iMSN-D2RKO mice expressed more severe AIMs than WT and ChIs-D2RKO mice. On the other hand, no differences were observed between ChI-D2RKO and WT mice in LID severity. Immunohistochemical results further support behavioural observations since levels of c-fos and pERK were found significantly increased in iMSN-D2RKO mice when compare to both WT and ChI-D2RKO mice. Moreover, electrophysiological data revealed deficit in the endocannabinoid-mediated LTD generation in the dorsolateral CPu of iMSN-D2RKO mice.