Nickel chloride

Following previous protocols from our lab (Zombeck et al., 2008 (link)),a 1-in-6 series of free-floating sections were pretreated with 0.6% hydrogen peroxide in PBS for 20 min at room temperature, then washed in PBS containing 0.2% Triton X-100 (PBS-X) and blocked with 6% goat serum for 1 h at room temperature. Sections were then incubated in monoclonal primary antibody against mouse Fos made in rabbit (Calbiochem, San Diego, CA) at a dilution of 1:20,000 in PBS-X containing 3% NGS for 48 h at 4 °C. Sections were subsequently washed in PBS-X and incubated in secondary biotinylated antibody against rabbit immunoglobulin made in goat (Vector Labs, Burlingame, CA) at a dilution of 1:200 in PBS-X with 3% NGS for 90 minutes at room temperature. Sections were then treated using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (DAB), enhanced with 0.008% nickel chloride (Sigma, St. Louis, MO).

Mice were anesthetized using isofluorane and then decapitated. Brains were removed, cut into left and right hemispheres, and then 300 μm coronal slices were made at 1-4°C in oxygenated (95% v/v O₂, 5% v/v CO₂) dissecting solution (208 mM sucrose, 2.5 mM KCl, 1 mM CaCl₂, 4 mM MgCl₂, 4 mM MgSO₄, 1.6 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, and 3 mM Na-pyruvate) using a Vibratome 3000 (The Vibratome Company). Typically, a total of 5-7 striatal hemi-slices (left/right hemisphere combined) were obtained from each mouse brain (1.1 to 0.14 mm from Bregma). Slices were allowed to recover on a nylon mesh for 1 h at 30°C in oxygenated ACSF (113 mM NaCl, 2.5-5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1 mM NaH₂PO₄, 26 mM NaHCO₃, 20 mM glucose, and 3 mM Na-pyruvate) followed by addition of picrotoxin (50 μM) for 30 min. Slices were then transferred to oxygenated 30°C ACSF solutions supplemented with vehicle or drug for 1-30 min, as defined in the figure legends. The following drugs were used and dissolved in water or DMSO based on manufacturer's instructions: BAPTA (Sigma), (S)-BayK8644 (Tocris), FPL64176 (Tocris), isradipine (National Institute of Mental Health Chemical Synthesis and Drug Supply Program), KCl (Sigma), mibefradil (Tocris), Nickel Chloride (Sigma), nimodipine (MP Biomedicals), SNX-482 (Peptides International), and TTX (Tocris).