Eighteen male Wistar rats were used in this experiment. The measurement of oxytocin from PVN was performed accordingly to previous reports [8 ,25 (link)]. Briefly, Wistar rats aged eight weeks were deeply anesthetized by a mixture of three types of anesthetic agents, decapitated, and their brains were removed. Three 400 μm thick slices were prepared using a vibrating microtome, and the PVN-containing area was punched out.
These processes were performed in a buffer composed of (in mM) 229 mannitol, 3 KCl, 26 NaHCO3, 1H3PO4, 0.5 CaCl2, 7 MgCl2, 7 glucose, and 1 kynurenate at pH 7.4 with 95% O2 and 5% CO2 mixed gas.
Prepared PVN slices were incubated at 37 °C for 30 min in artificial cerebrospinal fluid (aCSF) composed of (in mM) 126 NaCl, 3 KCl, 26 NaHCO3, 1H3PO4, 2 CaCl2 and 1 MgSO4 at pH 7.4, with 95% O2 and 5% CO2 mixed gas for recovery. Next, the PVN slices were incubated in aCSF with or without KKT (500 μg/mL) for 1 h. The concentrations of KKT (500 μg/mL) for Oxt secretion from brain slice were decided based on the concentration of KKT used in brain slice patch clamp experiments (500 μg/mL). These incubations were performed in pairs (control and KKT). Secreted Oxt in the supernatant was determined using an oxytocin ELISA kit (Enzo Life Sciences) [10 (link)].
These processes were performed in a buffer composed of (in mM) 229 mannitol, 3 KCl, 26 NaHCO3, 1H3PO4, 0.5 CaCl2, 7 MgCl2, 7 glucose, and 1 kynurenate at pH 7.4 with 95% O2 and 5% CO2 mixed gas.
Prepared PVN slices were incubated at 37 °C for 30 min in artificial cerebrospinal fluid (aCSF) composed of (in mM) 126 NaCl, 3 KCl, 26 NaHCO3, 1H3PO4, 2 CaCl2 and 1 MgSO4 at pH 7.4, with 95% O2 and 5% CO2 mixed gas for recovery. Next, the PVN slices were incubated in aCSF with or without KKT (500 μg/mL) for 1 h. The concentrations of KKT (500 μg/mL) for Oxt secretion from brain slice were decided based on the concentration of KKT used in brain slice patch clamp experiments (500 μg/mL). These incubations were performed in pairs (control and KKT). Secreted Oxt in the supernatant was determined using an oxytocin ELISA kit (Enzo Life Sciences) [10 (link)].
