Unilateral 6-OHDA lesion
Rats (150 g) were unilaterally injected in the (right) medial forebrain bundle with 8 µg 6-OHDA hydrobromide (dissolved in 0.02% ascorbate-saline), according to the following stereotaxic coordinates from bregma and the dural surface (in mm): antero-posterior (AP) -4.4, medio-lateral (ML) 1.2, ventro-dorsal (VD) -7.8, tooth bar at -2.4 mm (Paxinos et al., 1986), as previously described (Mela et al., 2012; Morari et al., 1996; Paolone et al., 2015). Animals were pretreated with antibiotics (SynuloxTM, 50 µl kg-1, i.p.). The wound was sutured and infiltrated with 2% lidocaine solution (EsteveTM). Two weeks later, rats were screened by assessing the motor asymmetry score in two different ethological tests (the bar and drag tests) (Pisanò et al., 2020). Rats showing immobility time at the contralateral paw in the bar test >20 sec and a number of steps at the contralateral paw <3 (or alternatively a contralateral/ipsilateral paw ratio <50%) were enrolled in the study (Pisanò et al., 2020).
Microdialysis experiments .
Dual probe microdialysis was performed as previously described (Mela et al., 2012; Morari et al., 1996; Paolone et al., 2015). Concentric microdialysis probes were constructed using AN69 (Gambro Industries, Meyzieu, France) semipermeable hollow membranes (65 kDa molecular weight cut-off, 340 µm outer diameter). Ninety-six (96) 6-OHDA fully dyskinetic rats (AIMs > 100) were used in the study. One microdialysis probe was stereotactically implanted under isoflurane anesthesia in the DA-depleted dorsolateral striatum and another in the ipsilateral SNr. Implantation coordinates (in mm, from bregma and the dural surface) (Paxinos et al., 1986), and dialysis membrane lengths were: dorsolateral striatum, AP +1.0, ML ±3.5, DV -6.0 (3 mm), SNr, AP -5.5, ML ±2.2, DV -8.3 (1 mm). Twenty-four hours after surgery, probes were perfused with a modified Ringer solution (CaCl2 1.2 mM; KCl 2.7 mM; NaCl 148 mM; MgCl2 0.85 mM) at a 3 µl min-1 flow rate. Sample collection (every 20 min) started after 6 h rinsing. At least four baseline samples were collected, then treatments were administered in a randomized fashion. At the end of experiments, animals were sacrificed by an overdose of isoflurane, and the correct placement of the probes was verified histologically. Severe (93%) dopamine depletion was randomly confirmed by post-mortem analysis of tyrosine hydroxylase (TH) levels in Western blot assay (optical density of TH band normalized over α-tubulin: 0.22±0.04 vs 3.13±0.39 ipsilateral vs contralateral striatum, respectively, t=7.37 df=19, Student’s t-test, two-tailed for unpaired data; n=20 rats; Supplementary Figure 1).