Biocytin

All chemicals were obtained from either Sigma Aldrich (Munich, Germany) or Carl Roth (Karlsruhe, Germany). Biocytin was obtained from Life Technologies (Dunfermline, UK). Pharmacological agents were obtained from Abcam Biochemicals (Cambridge, UK) or Tocris Bioscience (Bristol, UK). Drugs were stored as 1000-fold concentrated stocks at −80°C until used. Working solutions were prepared fresh on the day in normal ACSF at final concentrations given in the text.

Chemicals were obtained from either Sigma Aldrich (Munich, Germany) or Carl Roth (Karlsruhe, Germany). Biocytin was obtained from Life Technologies (Dunfermline, UK). Drugs were obtained from Abcam Biochemicals (Cambridge, UK) or Tocris Bioscience (Bristol, UK). Drugs were stored as 1000-fold concentrated stocks at –80°C. Working concentrations were prepared fresh on the day in normal ACSF: DNQX 10 μM, DL-APV 50 μM, gabazine (SR-95531)10 μM, Rubi-GABA 20 μM, CGP-55845 5 μM, and baclofen 10 μM.

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).