Rompun 20

All mice were anesthetized by using Zoletil 100 (tiletamine HCl 50 mg/mL + zolazepam HCl 50 mg/mL; Virbac, Milan, Italy) and Rompun 20 (xylazine 20 mg/mL; Bayer S.p.A, Leverkusen, Germany) dissolved in a volume of saline of 4.1 mg/mL and 1.6 mg/mL, respectively and intraperitoneally injected in a volume of 7.3 mL/kg. Mice were mounted onto a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) equipped with a mouse adapter and unilaterally implanted with optic fiber (ThorLabs, Newton, NJ, USA) above the PrL part of the right mPFC (AP: +1.8 mm, ML: +0.25 mm, DV: −2.00 mm). The coordinates from bregma were measured according to the atlas of Franklin and Paxinos (1997) and Mouse Brain Atlases (The Mouse Brain Library; www.nervenet.org). Ferrule-terminated implanted optical fibers were secured to the skull using dental acrylic.
Mice were allowed to recover from surgery for 1 week before behavioral testing. During the recovery period, they were habituated to handling and connection of the optic fiber with the optogenetic ferrule. Locations of implanted optical fibers were validated using histology in all experimental mice.

D-Amphetamine sulfate (Amph) and Prazosin hydrochloride (prazosin), were purchased from Sigma (Sigma Aldrich, Milan, Italy). Fluorescently labeled prazosin, BODYPY FL, was purchased by Thermo Fisher Scientific, Italy. Amph (2.5 mg/Kg), was dissolved in saline (0.9% NaCl) and injected intraperitoneally (i.p.) in a volume of 10 ml/kg. Zoletil 100, Virbac, Milan, Italy (tiletamine HCl 50 mg/ml + zolazepam HCl 50 mg/ml) and Rompun 20, Bayer S.p.A Milano, Italy (xylazine 20 mg/ml), purchased commercially, were used as anesthetics. Prazosin and BODYPY FL (1mg/ml) were dissolved in artificial CSF (in mM: NaCl 140.0; KCl 4.0; CaCl₂ 1.2; MgCl₂ 1.0). Artificial CSF was used as Vehicle. The doses of Amph and prazosin were selected on the bases of previous studies (Darracq et al., 1998 (link); Do-Monte et al., 2013 (link); Latagliata et al., 2016 (link)) and preliminary experiments.

Zoletil 100, Virbac, Milano, Italy (tiletamine HCl 50 mg/ml + zolazepam HCl 50 mg/ml) and Rompun 20, Bayer S.p.A Milano, Italy (xylazine 20 mg/ml), purchased commercially, were used as anesthetics, 6-hydroxydopamine (6-OHDA) and GBR 12909 (GBR), were purchased from Sigma (Sigma Aldrich, Milan, Italy). Zoletil (30 mg/kg), Rompun (12 mg/kg) and GBR (15 mg/Kg) were dissolved in saline (0.9% NaCl) and injected intraperitoneally (i.p.) in a volume of 10 ml/kg. 6-OHDA was dissolved in saline containing Na-metabisulfite (0.1 M).

After acclimation, 44 animals were fully anesthetized by intraperitoneal injection of Zoletil 100 (50 mg/mL tiletamine HCl + 50 mg/mL zolazepam HCl; Virbac, Milan, Italy) and Rompun 20 (20 mg/mL xylazine; Bayer S.p.A., Milan, Italy). The right hind limb of each mouse was shaved, and muscle was exposed via a 1 cm long incision in the aseptically prepared skin overlying the biceps femoris muscle. A metallic probe of 4 mm diameter was cooled in liquid nitrogen and then applied vertically to the exposed muscle for 10 s with a moderate uniform pressure to cause severe damage [24 (link),26 (link)]. The skin was sutured with a 6-0 suture string (Assut Europe, Rome, Italy), and mice were kept warm until awake. Buprenorphine (0.1 mg/kg; diluted to 0.015 mg/mL; Sigma-Aldrich, Merck KGaA, Germany) was administered subcutaneously before recovery from anesthesia. In the first few days after the injury, wet food placed onto cage substrate was provided as a supplement in addition to pelleted food and water. This arrangement has made it easier to control the gait and the physiological state of animals. Mice’s weight was measured at each time point to verify animal health conditions. The control group (4 mice) and each experimental group (4 mice) at 1, 2, 4, 7, 12, and 25 days were euthanized using CO₂ asphyxiation. The control group comprises untreated animals.