Microm hm 650v vibratome

Approval (# 338/2016A) of the ethics committee of the University of Tübingen as well as written informed consent was obtained from all patients, allowing spare tissue from resective surgery to be included in the study. Tissue preparation was performed according to published protocols (Verhoog et al., 2013 (link)). Cortex was carefully microdissected and resected with only minimal use of bipolar forceps to ensure tissue integrity, directly transferred into ice-cold artificial cerebrospinal fluid (aCSF) (in mM: 110 choline chloride, 26 NaHCO₃, 10 D-glucose, 11.6 Na-ascorbate, 7 MgCl₂, 3.1 Na-pyruvate, 2.5 KCl, 1.25 NaH₂PO₄, und 0.5 CaCl₂) equilibrated with carbogen (95% O₂, 5% CO₂) and immediately transported to the laboratory. Tissue was kept submerged in cool and carbogenated aCSF at all times. After removal of the pia, tissue chunks were trimmed perpendicular to the cortical surface and 250–350 µm thick acute slices were prepared using a Microm HM 650V vibratome (Thermo Fisher Scientific Inc) (Figure 1—figure supplement 2A). Afterwards the slices were kept in aCSF equilibrated with carbogen for 0.5 hr at room temperature before they were transferred either to patch setups for acute electrophysiological recordings and biocytin fillings or onto culture membranes for cultivation.

20 days after virus injection into the RMS (20 dpi), 250 µm thick sagittal OB slices were cut on Microm HM 650 V vibratome (Thermo Fisher Scientific, Dreieich, Germany). After an incubation period of up to 1 h, slices were transferred into the experimental setup and perfused with the extracellular solution containing (in mM): 125 NaCl, 3.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, and 20 glucose, pH 7.4 when bubbled with 95% O₂ and 5% CO₂. Experiments were carried out at ~ 33 °C. Twitch-2B-positive control or Kv1.2/Kir2.1 overexpressing abJGCs were recorded in whole-cell configuration using an EPC-10 patch-clamp amplifier (HEKA, Lambrecht, Germany). The intracellular pipette solution contained (in mM): K-Gluconate 140, NaCl 10, HEPES 10, EGTA 0.2, Mg-ATP 4, Na-GTP 0.4, Alexa Fluor 594 0.05, pH = 7.3. Current and voltage traces were acquired at a 20 kHz sampling rate. For current-clamp recordings, currents of different polarity and amplitude (usually from − 40 pA to + 100 pA, 100 or 200 ms long) were injected stepwise to assess the cell’s firing ability. The holding current was adjusted to maintain basal membrane potential at − 70 mV.

All procedures involving animals were conducted in compliance with Dutch regulations and were approved by the animal experimental committee (“Dier ethische commissie (DEC)”; license number: INF 13-02) of the Vrije Universiteit. Animals were housed and bred according to institutional and Dutch governmental guidelines and regulations.
C57BL/6 males aged 6–8 days (1 week), 13–16 days (2 weeks) or 26–30 days (4 weeks) were rapidly decapitated and their brains dissected out in ice cold cutting solution containing (in mM): 110 choline chloride, 26 NaHCO₃, 10 D-glucose, 11.6 sodium ascorbate, 7 MgCl₂, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH₂PO₄, and 0.5 CaCl₂ (Bureau et al., 2006). 300 µm thick coronal slices containing the prelimbic cortex were obtained using a Microm HM 650 V vibratome (Thermo Scientific, Waltham, MA, USA), and allowed to recover at room temperature in aCSF containing (in mM): 125 NaCl, 26 NaHCO₃, 10 D-glucose, 3 KCl, 2.5 MgSO₄, 1.6 CaCl₂, and 1.25 NaH₂PO₄, with an osmolality of ±300 mOSm, which was continuously bubbled with carbogen gas (95% O2, 5% CO₂).

All experimental procedures were approved by and carried out in accordance with University de La Laguna (ULL) Ethics Committee, Spanish (RD 53/2013) and European Commission (2010/63/EU) animal care guidelines. C57BL/6J mice (The Jackson Laboratory no. 000664) were housed in standard laboratory cages with ad libitum access to water and food in temperature- and humidity-controlled rooms under a 12:12 h light–dark cycle at the ULL animal facilities (registry no. ES380230024514). C57BL/6J adult male mice (postnatal 28 36) were slightly anesthetized with isoflurane, decapitated, and the brain quickly removed and immersed in an ice-cold high-sucrose solution (in mM: 189 sucrose, 10 glucose, 26 NaHCO₃, 3 KCl, 5 MgSO₄, 0.1 CaCl₂, and 1.25 NaH₂PO₄) continuously bubbled with carbogen (95% oxygen/5% carbon dioxide). Coronal brain slices (350 µm) were obtained using a Microm HM650V vibratome (Thermo Scientific). Slices were maintained for 1 to 1.5 h in ACSF (in mM: 124 NaCl, 2.69 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 10 glucose, 2 CaCl₂, and 0.4 ascorbic acid; pH 7.35) bubbled with carbogen at room temperature (22 to 24 °C).