Halothane

300 μm thick acute coronal brain slices (≈1.7 mm posterior to bregma) were made after the deep anesthesia of the mice with halothane (Sigma). Isolated brain was sliced by Vibratome (VT-1200, Leica, Nussloch, Germany) in a cold, high-sucrose, artificial cerebrospinal fluid (ACSF) solution containing (in mM): 240 Sucrose, 26 NaHCO₃, 2.5 KCl, 1.0 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, and 10 D(+)-glucose, pH 7.4, by gassing with 95% O₂ / 5% CO₂. Slices were then placed in an interface chamber filled with ACSF oxygenated with 95% O₂ / 5% CO₂.Slices were incubated at 36°C for at least 1 hour. During the experiment, a slice was placed on the microscope stage chamber with continuous perfusion of ACSF at 1–2 ml/min. The temperature of ACSF on the recording chamber was adjusted to 33 ± 0.5 °C by a temperature controller (TC-344B, Warner Instruments, CT) throughout the experiment. When required, the following drugs were dissolved in perfused ACSF for perfusion: 50 μM D-AP5, 40 μM CNQX, 40 μM bicuculline and 1 μM tetrodotoxin (TTX) (all from Sigma). A minimum of 10 minutes of drug perfusion was done to ensure proper delivery.

P0-P2 Dat1-eGFP mice were cryoanesthetized and
decapitated for brain tissue collection. For older mice, animals were
anaesthetized with halothane (Sigma-Aldrich, Canada) and decapitated. Primary
cultures of mesencephalic DA neurons were prepared according to a previously
described protocol (Fasano et
al., 2008). Mesencephalic cells were plated onto a
pre-established monolayer of astrocytes. Cultures were prepared at a density of
5,000 cells/ml of FACS-purified mesencephalic DA neurons from
Dat1-eGFP mice, as previously reported (Mendez et al., 2008 (link)).

Amoxicillin, atropine, carbamazepine, dicloxacillin, digoxin, erythromycin, estradiol, furosemide, halothane, streptomycin, ticlopidine and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Allopurinol, azathioprine, diclofenac, diphenhydramine, flutamide, ibuprofen, imipramine, indomethacin, isoniazid, kanamycin, ketoconazole, metronidazole, nifedipine, phenobarbital, phenytoin, pioglitazone, sulfamethoxazole, troglitazone and valproic acid were purchased from Wako Pure Chemical (Osaka, Japan). Primidone was purchased from the Tokyo Chemical Industry (Tokyo, Japan). Primers were commercially synthesized at Life Technologies (Carlsbad, CA, USA). HaCaT cells were purchased from CLS Cell Lines Service (Eppelheim, Germany). CnT-Prime (CnT-PR) Epithelial Culture Medium and CnT-Prime 2D Diff (CnT-PR-D) Epithelial Culture Medium were from CELLnTEC Advanced Systems (Bern, Switzerland). All other chemicals and solvents were of analytical grade or the highest grade commercially available.