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).