Na2 phosphocreatine

Patch clamp pipettes filled with internal solution were used to obtain blind in vivo patch clamp recordings. Pipettes were pulled from borosilicate capillaries (1B120F-4, World Precision Instruments, Inc., Sarasota, FL, USA) with a horizontal puller (Model P-87, Sutter Instrument Co., Novato, CA, USA). Electrode resistances were 5–7 MΩ when filled with internal solution and measured in cerebrospinal fluid. The internal solution contained (in mM) 115 K gluconate (Sigma); 4.42 KCl (Fisher); 10 Na₂ phosphocreatine (Sigma); 10 HEPES (Sigma); 0.5 EGTA (Sigma); 4 Mg-ATP (Sigma); 0.3 Na-GTP (Sigma); and 0.1–0.2% biocytin (Invitrogen). pH was brought to 7.30 with KOH (Sigma) and osmolality to 300 mmol/kg with sucrose (Sigma). A patch clamp amplifier (BC-700A; Dagan, Minneapolis, MN, USA) was used to obtain membrane potential recordings, where the analog signal was low-pass filtered (cut-off frequency 5 kHz) and digitized at 50–100 kHz (ITC-18, HEKA, Ludwigshafen/Rhein, Germany; RX8, Tucker-Davis Technologies, Alachua, FL, USA). Series resistance was 61.3 ± 3.3 MΩ (mean ± SEM; N = 23, excluding one outlier with a series resistance >100 MΩ). Opening resting membrane potential was −56.3 ± 0.69 mV (mean ± SEM, N = 23).

Patch clamp electrodes were pulled from borosilicate glass capillaries (1B120F-4, World Precision Instruments, Inc., Sarasota, FL, USA) with a horizontal puller (Model P-87, Sutter Instrument Co.). The electrode resistances were 5–8 MΩ when filled with the solution. The internal solution contained 115 mM K gluconate (Sigma), 4.42 mM KCl (Fisher), 10 mM Na₂ phosphocreatine (Sigma), 10 mM HEPES (Sigma), 0.5 mM EGTA (Sigma), 4 mM Mg-ATP (Sigma), 0.3 mM Na-GTP (Sigma) and 0.1 or 0.2% biocytin (Invitrogen), with pH 7.30 (adjusted with KOH, Sigma) and osmolality 300 mmol/kg (adjusted with sucrose, Sigma) (Roberts et al., 2014 (link)). Patch clamp recordings were obtained using the blind in vivo method as described before (Margrie et al., 2002 (link); Franken et al., 2015 (link)). Membrane potential recordings were obtained in current clamp using a patch clamp amplifier (BVC-700A; Dagan, Minneapolis, MN, USA). The analog signal was low-pass filtered (cut-off frequency 5 kHz), digitized at 50–100 kHz and saved using scripts in MATLAB (The Mathworks) or IgorPro (WaveMetrics). Series resistance was 51.7 ± 10.8 MΩ (mean ± SEM; N = 8; excluding one outlier with a series resistance >100 MΩ). Initial resting membrane potential was –54.6 ± 1.95 mV (mean ± SEM; N = 10).