Borosilicate glass pipettes

EB cells plated on gelatin-coated glass coverslips were placed in the experimental chamber (23°C), and superfused with Tyrode solution of the following composition (mM): 140 NaCl, 5.4 KCl, 2 CaCl₂, 1 MgCl₂, 5 HEPES, 10 glucose (pH 7.4). Membrane currents or action potentials (AP) from single cells or small clusters of cells were recorded using a computer equipped with pCLAMP 8, a Digidata 1322A series interface and Axopatch 1C amplifier (Molecular Devices). Only cells expressing mCherry fluorescence driven by the MHCα promoter were used for recording. The perforated patch clamp technique was employed. Borosilicate glass pipettes (Sutter Instrument) were filled with (mM) 130 aspartic acid, 146 KOH, 10 NaCl, 2 CaCl₂, 5 EGTA, 10 HEPES, 2 Mg-ATP, 100 μg/ml amphotericin (pH 7.2). After forming a gigaseal, progress in electrical access was evaluated by monitoring capacitance currents induced by 20 ms pulses from -35 mV to -40 mV. AP and I_f were recorded when series resistance was reduced to 40–50 MΩ and 20–30 MΩ respectively. I_f was induced by voltage steps ranging from -35 to -125 mV with duration decrementing with more negative pulses, followed by a 5 s long pulse to -85 mV to measure tail current and 0.5 s deactivating pulse to -5 mV. Holding potential was -35 mV.

Whole-cell patch clamp experiments were performed as described earlier33 (link) on HEK293 cells in an extracellular solution containing 137 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, with the pH adjusted to 7.4, to which 1 mM MgCl₂, 2 mM CaCl₂, or 2 mM EGTA was added, depending on the experimental conditions. Borosilicate glass pipettes (Sutter Instruments) of 2–4-megaohm resistance were filled with a solution containing 135 mM potassium-gluconate, 5 mM KCl, 5 mM EGTA, 1 mM MgCl₂, 2 mM Na₂ATP, 10 mM HEPES (pH 7.2). The cells were kept in extracellular solution containing 1 mM Mg²⁺ but no Ca²⁺ for 20 min before measurements. After formation of gigaohm resistance seals, whole-cell configuration was established, and currents were measured using an Axopatch 200B amplifier (Molecular Devices). Monovalent currents were initiated by switching to a solution without Mg²⁺ and 1 mM EGTA. Ca²⁺-induced inactivation was initiated using a solution containing 2 mM Ca²⁺ and no Mg²⁺, as describe earlier5 (link)33 (link). The data were collected and analyzed with the pCLAMP 9.0 software (Molecular Devices). Measurements were performed without capacitance compensation, but capacitative currents after voltage steps are not shown in the figures. All of the measurements were performed at room temperature (20–25 °C).

Astrocyte cultures from 14 to 42 days in vitro after ND1 transduction were examined using whole-cell patch-clamp recordings on converted cells identified by the morphological phenotype of axonal outgrowth with neurite extension. Recordings were performed using an EPC9 amplifier (HEKA; Elektronik) were performed at 21–23°C. The external solution contained (in mM): 135 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 Glucose at a pH of 7.4. Recording electrodes pulled from borosilicate glass pipettes (Sutter Instrument) had a tip resistance between 5 and 8 MO when filled with the internal solution (in mM): 140 KCl, 2 MgCl₂, 1 CaCl₂, 2 Na₂ATP, 10 EGTA, and 10 HEPES at a pH of 7.2. Action potentials were recorded under the current-clamp mode using the Pulse software (HEKA, Elektronik).

The freshly dissected brainstem-spinal cord was pinned with the ventral side up in Sylgard (Dow Inc. Midland, MI, USA)-coated 6-mL Petri dishes where it was continuously superfused (~7mL/min) with aerated aCSF (98.5% O₂ and 1.5% CO₂) using peristaltic pumps (Watson Marlow, Falmouth, CNL, UK). All preparations were recorded at room temperature ~22°C. Borosilicate glass pipettes were pulled (Sutter Instruments, Novato, CA, USA) and manually adjusted in two sizes using sandpaper and fire polishing. A bigger size was used to ensure a tight seal around the vagal nerve root, and a smaller size was used to record from the lung generator cells. Rhythmic lung activity was recorded by an electrode placed in the area identified as the bullfrog’s lung rhythm generator [38 (link), 39 (link)]. Extracellular signals from both regions were amplified (×1000) and filtered (low pass, 1000 Hz; high pass, 100 Hz) using an AM-Systems 1700 amplifier (Sequim, WA, USA). The signal was digitized using Powerlab 8/35 (ADInstruments, Dunedin, Otago, New Zealand), rectified, and integrated (100 ms τ) using the LabChart data acquisition system (ADInstruments, Dunedin, Otago, New Zealand).