Validating vlPAG OTR Neuron Functionality

To validate the functionality of vlPAG OTR neurons in OTR-IRES-Cre rats using electrophysiology, 12–13-week-old female OTR-IRES-Cre rats (n = 4) received injections of the Cre-dependent reporter virus, rAAV_1/2-pEF1α-DIO-GFP, into the vlPAG. Following a 4–8 week recovery period, rats were anesthetized by administering i.p. ketamine (Imalgene 300 mg/kg) and paxman (Rompun, 60 mg/kg). Transcardial perfusions were performed using an ice-cold, NMDG-based aCSF was used containing (in mM): NMDG (93), KCl (2.5), NaH₂PO₄ (1.25), NaHCO₃ (30), MgSO₄ (10), CaCl₂ (0.5), HEPES (20), D-Glucose (25), L-ascorbic acid (5), Thiourea (2), Sodium pyruvate (3), N-acetyl-L-cysteine (10), and Kynurenic acid (2). The pH was adjusted to 7.4 using either NaOH or HCl, after bubbling in a gas comprised of 95% O₂ and 5% CO₂. Rats were then decapitated, brains were removed and 350 µm thick coronal slices containing the hypothalamus were obtained using a Leica VT1000s vibratome. Slices were warmed for 10 min in 35 °C NMDG aCSF then placed in a room temperature holding chamber filled with normal aCSF for at least 1 h. Normal aCSF was composed of (in mM): NaCl (124), KCl (2.5), NaH₂PO₄ (1.25), NaHCO₃ (26), MgSO₄ (2), CaCl₂ (2), D-Glucose (15), adjusted to pH 7.4 with HCL or NaOH and continuously bubbled in 95%-O₂ 5%-CO₂ gas. Osmolarity of all aCSF solutions were maintained between 290 and 310 mOsm/L. Finally, slices were transferred from the holding chamber to an immersion-recording chamber and superfused at a rate of 2 ml/min.