Sections of hCS, hSS (day 96–141) or fused hSS-hCS (29–53 daf) for physiological recordings were obtained using an approach we previously described11. Briefly, spheroids were incubated in bicarbonate buffered artificial cerebrospinal fluid (aCSF) at 23°C and equilibrated with a mixture of 95% O2 and 5% CO2. The aCSF solution contained: 126 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, and 2 mM CaCl2. Slicing was performed using a Leica VT1200 vibratome. Immediately after sectioning, slices were moved to a circulation chamber containing oxygenated aCSF at 32°C. For patch-clamp recording, cells were identified by the presence of a fluorescent reporter using an upright Axoscop II microscope (Zeiss). Recording electrodes of borosilicate glass had a resistance of 4–6 MΩ when filled with internal solution. A low Cl− internal solution was used to distinguish between EPSCs and IPSCs containing: 145 mM K+ gluconate, 0.1 mM CaCl2, 2.5 mM MgCl2, 10 mM HEPES, 0.2 mM EGTA, 4 mM Na+ phosphocreatine. Cl− reversal was calculated to be at −91 mV according to the Nernst equation. A
