Submerged recording chamber

BRS3-Cre mice (5–10 weeks) received bilateral 50 nl injections of AAV1-Ef1a-DIO-ChR2-EYFP in the anteroventral POA (AP: 0.5; ML: +/− 0.3; DV: −5.25, mm from bregma). Four to 6 weeks later, brain slices were obtained and stored at 30 °C in a heated, oxygenated chamber containing aCSF (in mmol/l) 124 NaCl, 4.4 KCl, 2 CaCl₂, 1.2 MgSO₄, 1 NaH₂PO₄, 10.0 glucose, and 26.0 sodium bicarbonate before being transferred to a submerged recording chamber maintained at 30 °C (Warner Instruments, Hamden, CT). Recording electrodes (3–5 MΩ) were pulled with a Flaming-Brown Micropipette Puller (Sutter Instruments, Novato, CA) using thin-walled borosilicate glass capillaries.
Light evoked excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) were measured in voltage-clamp mode using electrodes filled with an intracellular recording solution containing (in mM): 135 Cs-methanesulfonate, 10 KCl, 10 HEPES, 1 MgCl₂, 0.2 EGTA, 4 Mg-ATP, 0.3 GTP, 20 phosphocreatine, 2 QX314. Neurons were held at −55 mV to isolate glutamatergic synaptic transmission and record EPSCs, or +10 mV to isolate GABAergic synaptic transmission and record spontaneous IPSCs within individual neurons. Tetrodotoxin (TTX, 500 nM) and 4-aminopyridine (4-AP, 100 μM) were included in the bath aCSF.

Eight weeks old male Cx43^f/f and Cx43 cKO^GFAP mice were anaesthetized with isoflurane, and after decapitation, the brain was rapidly removed and put in ice-cold oxygenated (O₂ 95%/CO₂ 5%) artificial cerebrospinal fluid (ACSF) containing the following (in mM): 120 NaCl, 3.2 KCl, 1 NaH₂PO₄, 26 NaHCO₃, 1 MgCl₂, 2 CaCl₂, 2.5 glucose (osmolarity adjusted to 300 mOsm with sucrose, pH 7.4). After removal of the cerebellum, the brain was glued and coronal hypothalamic slices containing the LHA were cut using a vibratome (VT1200S; Leica). Before recording, slices were incubated at 35°C for a recovery period of 1 h. After recovery, slices were placed in a submerged recording chamber (31°C; Warner Instruments) and continuously perfused (2 ml/min) with oxygenated ACSF. The glucose concentration used for recordings was 2.5 mM unless otherwise stated.
All drugs were applied to the perfusing system (bath application) to obtain the final concentrations indicated unless otherwise stated. Alpha-cyano-4-hydroxycinnamic acid (4-CIN), carbenoxolone disodium salt, sodium oxamate, sodium pyruvate, and tolbutamide were obtained from Sigma, sodium L-lactate from Alfa Aesar and tetrodotoxin citrate (TTX) from Tocris.