Animal subjects
One hundred ten (110) male Sprague-Dawley rats (150 g; Charles River Lab, Calco, Lecco; Italy) were used in this study. Seven (7) naïve rats were kept for WB analysis of RGS4 levels (Fig. 7) whereas 103 underwent 6-OHDA lesioning. Eighty-five (85) 6-OHDA rats passed the selection criteria (see below): 7 were used for WB studies (Figs. 5-7) and 78 underwent chronic treatment with L-Dopa. At the end of L-Dopa treatment, we obtained 59 dyskinetic rats (ALO AIM score ≥100), of which 35 were kept for WB studies and 24 for behavioral analysis.
Sixteen (16) RGS4-/- and 16 C57BL/6J wild-type male mice (24-26 g, 10-12 weeks old) were used for the analysis of RGS4 selectivity of CCG-203920 and RGS4 antibody specificity (Fig. S1). Specifically, 10 RGS4-/- mice and 10 C57BL/6J controls were used for behavioral studies with CCG-203920, and other 6 RGS4-/- mice and 6 C57BL/6J controls were used for Western blot analysis. RGS4-/- mice, originally developed by Dr S Heximer (University of Toronto) (Cifelli et al. , 2008), were provided by Dr RR Neubig and colony raised at the University of Ferrara. Animals were housed in a specific pathogen-free (SPF) standard facility (LARP) of the University of Ferrara with free access to food (4RF21 standard diet; Mucedola, Settimo Milanese, Milan, Italy) and water, and kept under regular lighting conditions (12 hr dark/light cycle). Animals were housed in groups with environmental enrichments. Adequate measures were taken to minimize animal pain and discomfort. Rats were sacrificed with an overdose of isoflurane or with isoflurane anaesthesia followed by decapitation (WB studies), mice with isoflurane anaesthesia followed by cervical dislocation. Experimental protocols were approved by the Ethical Committee of the University of Ferrara and the Italian Ministry of Health (licenses 714/2016-PR and 368/2018). Ten (10) time-mated pregnant female Sprague-Dawley rats were used to generate primary cultures of the striatum. There were housed in the Biological Service Unit, at University College Cork under regular conditions of lights (12h light/dark cycle) with ad libitum access to food and water. On embryonic day (E) 14, embryos (106 in total) were removed by laparotomy from euthanized dams and used to generated primary cultures of the rat striatum as outlined below (license number AE19130/ I304). Seven 12-week-old male C57BL6 mice were used for ERK studies in slices in vitro. Mice were housed in a standard facility at Cardiff University, under regular conditions of light (12 h light/dark cycle), with food and water ad libitum.