Isoflurane anesthesia

Chronic application of the orthosteric-like mGlu7 antagonist XAP044 (Tocris Bioscience, Bristol, UK; Figure 1B) via s.c.-implanted Alzet^® micro-osmotic pumps (pumping rate: 0.11 µL/h, Alzet^®, Model 1004, Cupertino, CA, USA) connected to an i.c.v. infusion cannula (Brain Infusion Kit 3, Alzet^®, Cupertino, CA, USA) was initiated one week before starting the CSC paradigm (day 6) in order to establish a stable baseline receptor occupancy and was continued until the end of chronic stressor exposure (day 20). XAP044 was formulated as a solution in vehicle (VEH, 5% DMSO in Ringer’s solution, Merck; Darmstadt, Germany) to ensure a continuous substance release of 1 µM, 10 µM or 100 µM of XAP044. As reported by Peterlik et al. 2017 [23 (link)], the micro-osmotic pump was implanted s.c. in the abdominal region through a 1 cm long incision at the lower neck of the mouse under isoflurane anesthesia (Baxter, GmbH, Unterschleißheim, Germany) but additionally connected via a catheter to an i.c.v. infusion cannula. Animals were treated with painkiller (Buprenovet, s.c.; 0.05 mg/kg; Bayer Health Care AG, Leverkusen, Germany) and 100 µL of antibiotics (s.c., Baytril^®, 2.5% Bayer Health Care AG, Leverkusen, Germany). Wound treatment was conducted using betaisodona (Mundipharma GmbH, Limburg, Germany).

Right hind paw inflammation was induced by Freund’s complete adjuvant (FCA, Sigma, USA) containing 1 mg/mL heat-killed Mycobacterium tuberculosis. The mice received a right intraplantar injection of 20 uL of FCA under isoflurane anesthesia (1–3%, Baxter) delivered with oxygen at a flow rate of 1 L/min. The left hind paw remained intact for self-control.

Mice from all experimental and control groups were decapitated under isoflurane anesthesia (Baxter, Deerfield, IL, USA), the brains were removed and cut along the middle sagittal plane. The striatum was isolated from one cerebral hemisphere from bregma 1.70 to bregma 0.14 in the rostrocaudal direction according to the atlas [55 ] and the SN was isolated from bregma −2.54 to bregma −4.04 using a dissecting microscope (Leica M60, Wetzlar, Germany). This procedure was described in detail earlier [28 (link),39 (link)]. The obtained samples from mice of the 1st and 2nd groups of animals (n = 6–8) were weighed, frozen in liquid nitrogen, and stored at −70 °C until the concentration of DA and its metabolites was determined by HPLC-ED.
The second hemisphere of the brain, obtained from mice of the 1st and 2nd groups of animals (n = 4–6 per group) in the experiment and in the control, was fixed by immersion in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.2–7.4) for 12 h at 4 °C. The brain was then washed with 0.02 M phosphate-buffered saline (PBS) (pH 7.2–7.4) at room temperature and incubated in PBS with 20% sucrose at 4 °C for 12 h (all reagents from Sigma-Aldrich, St. Louis, MO, USA). The brain was then frozen in hexane at −40 °C and stored at −70 °C until immunostaining for TH.

A capsaicin (Sigma) injection solution was prepared at a concentration of 0.06% (wt/vol) in 10% DMSO (Thermo Fisher Scientific) and PBS (Thermo Fisher Scientific). Under 2% isoflurane anesthesia (Baxter), 20 µl of the capsaicin solution was injected into the hind paw. Mouse behavior was video recorded over a period of 5 min, and the total time the animal displayed nocifensive behavior (paw lifting, licking, flinching and writhing) was assessed.