Dual stage glass micropipette puller

mIPSCs were recorded using a Multiclamp 700B amplifier (Molecular Devices) and analyzed as previously described (46 (link)). Briefly, syt1 KO hippocampal neurons expressing WT, Juxta K, F349A, or Juxta K + F349A at day in vitro (DIV) 14 through 19 were transferred to a recording chamber with a bath solution containing the following (in mM): 128 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 30 D-glucose, 25 Hepes, and 1 μM tetrodotoxin, pH 7.3 (305 mOsm). Borosilicate glass pipettes (Sutter Instruments) were pulled by a dual-stage glass micropipette puller (Narishige) and filled with an internal solution containing (in mM) 130 KCl, 1 EGTA, 10 Hepes, 2 ATP, 0.3 GTP, 5 QX-314 (Abcam), and 5 sodium phosphocreatine, pH 7.35 (295 mOsm). mIPSCs were pharmacologically isolated by bath applying D-AP5 (50 µM, Abcam) and cyanquixaline (20 µM, Abcam) and acquired using a Digidata 1440B analog-to-digital converter (Molecular Devices) and Clampex 10 software (Molecular Devices) at 10 kHz. Neurons were held at −70 mV. All cells were equilibrated for ∼1 min after break in before recordings started. Series resistance was compensated, and traces were discarded if the access resistance exceeded 15 ΜΩ for the entire duration. The collected miniature events were detected in Clampfit 11.1 (Molecular Devices) using a template matching search.