Digidata 1550b

Whole cell recordings were performed on DIV 18–20. Upright microscope (Olympus BX51WI, Olympus Optics, Japan) equipped with infrared-differential interference contrast optics was used to locate neurons in μ3D cultures. The recordings were conducted at 32–34°C, and the resistance of the recording pipette (1.5 mm borosilicate glass, Sutter Instruments) was 4 to 7 MΩ. The cultures were held in a perfusion chamber with constant flow of recording solution composed of: 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl₂, 0.9 mM MgCl₂, 25 mM NaHCO₃, 1.23 mM NaH₂PO₄, and 10 mM glucose, bubbled with 5% CO₂ 21% O₂, and balanced with N₂. In some experiments, 3 mM kynurenic acid (KYNA) was added to recording solution to prevent network bursts. The recording pipette was loaded with intracellular solution containing 130 mM K-gluconate, 5 mM KCl, 4 mM ATP-Mg, 0.3 mM GTP, 10 mM HEPES, and 10 mM phosphocreatine (pH 7.2, adjusted with KOH). Up to 2 cells were patched from each culture. Current-voltage curves were recorded in current clamp mode using MultiClamp 700B (Molecular Devices) and acquired at sampling frequency of 20 kHz through DigiData 1550B and pCLAMP 10 software (Molecular Devices). The data was analyzed in pCLAMP 10, without correction for liquid junction potential.

Ionic currents were recorded through cell-attached patches using a standard patch clamp technique in voltage clamp mode. Patch pipettes had a resistance of 4–6 megaohm when filled with pipette solution. An ionic solution composed of 145 mm CsCl, 2 mm MgCl₂, 10 mm HEPES, 5 mm ATP, 0.1 mm GTP, and 1 mm EGTA (titrated to pH 7.2 using CsOH) was used in the pipette. The bath solution was SBS. Recordings were at a constant holding potential of +80 mV (applied to the patch pipette). 200-ms pressure steps were applied to the patch pipette with an interval of 10 s using a high-speed pressure clamp HSPC-1 system (ALA Scientific Instruments). All recordings were made with an Axopatch-200B amplifier (Axon Instruments, Inc.) equipped with Digidata 1550B and pClamp 10.6 software (Molecular Devices) at room temperature. Currents were filtered at 2 kHz and digitally sampled at 20 kHz. Data were analyzed using pClamp 10.6 and the MicroCal Origin 2018 (OriginLab Corp.) software packages. All recordings were made blind (i.e. without knowledge of which cells had been transfected with control or Piezo1 siRNA).

Two-electrode voltage clamp (TEVC) recordings were performed as described previously (Robinson et al., 2018 (link); Spiers et al., 2019 (link)). Sharp-electrode recordings were made from ventral longitudinal muscle 6 (m6) in abdominal segments 2 and 3 of third instar larvae using pClamp 10.5, an Axoclamp 900A amplifier, and Digidata 1550B (Molecular Devices, USA) in hemolymph-like solution 3 (HL-3; Stewart et al., 1994 (link)). Recording electrodes (20–50 MΩ) were filled with 3 M KCl. All synaptic responses were recorded from muscles with input resistances ≥4 MΩ, holding currents <4 nA at −60 mV and resting potentials more negative than −60 mV at 25°C. Holding potentials were −60 mV. The extracellular HL-3 contained (in mM): 70 NaCl, 5 KCl, 20 MgCl₂, 10 NaHCO₃, 115 sucrose, 5 trehalose, 5 HEPES, and 1.5 CaCl₂. Average single evoked excitatory junction potential (eEJC) amplitudes (stimulus: 0.1 ms, 1–5 V) were based on the mean peak eEJC amplitude in response to 10 presynaptic stimuli (recorded at 0.2 Hz). Nerve stimulation was performed with an isolated stimulator (DS2A, Digitimer). All data were digitised at 10 kHz and for miniature event recordings, 60-s recordings were analysed to obtain mean miniature EJC (mEJC) amplitudes. Both, mEJC and eEJC recordings were off-line low-pass filtered at 500 Hz and 1 kHz, respectively. Materials were purchased from Sigma-Aldrich (UK).

Patch pipettes were pulled from borosilicate glass capillaries with filament (1.5 mm outer diameter and 1.1 mm inner diameter; Sutter Instruments, BF150-110-7.5HP) with a resistance of 2–3 MΩ. The pipette recording solution contained (in mM) 10 KCl, 130 K-gluconate, 1.8 NaCl, 0.2 EGTA, 10 HEPES, 2 Na-ATP, 0.2% Biocytin, pH 7.3 adjusted with KOH; 290–300 mOsm. Whole-cell recordings were made with Axopatch 700B amplifier (with Clampex 10.7 and Axoclamp1.1, Molecular Devices) using an upright microscope (Nikon Eclipse FN1, with × 40, 0.8 NA water immersion objective lens) equipped with differential interference contrast (DIC) optics. DIC images were captured with an Andor Zyla 5.5 sCMOS camera. All recordings were performed at 32 °C in ACSF bubbled with 95% O₂ and 5% CO₂. For AP parameter determination experiments, 1 μM CNQX (Sigma-Aldrich, C-127) was applied in the bath solution to eliminate EPSPs. Cells with < 20 MΩ access resistance (continuously monitored) were accepted for analysis. Signals were low-pass filtered at 5 kHz and digitized at 20 kHz (Digidata 1550B, Molecular Devices). In vitro data analysis was performed with the help of Clampfit 10.7 (Molecular Devices) and Origin 8.6 (OriginLab Corporation). When it is indicated 30 μM 9-phenanthrol (Sigma-Aldrich, 211281) was applied into the bath solution.

The whole-cell recording experiments have been described previously (Pan et al., 2019 (link)). The internal solution contained (in mM) 130 potassium gluconate, 5 KCl, 4 Na₂ATP, 0.5 NaGTP, 20 HEPES, 0.5 EGTA, pH 7.28 with KOH, and measured osmolality at 310–320 mOsm. We acquired data using the pClamp 10.0 software (Molecular Devices, San Jose, CA, United States) with the MultiClamp 700B patch-clamp amplifier and Digidata 1550B (Molecular Devices, San Jose, CA, United States). We low-pass-filtered responses online at 2 kHz and digitized them at 5 kHz. We constructed phase plots from the first derivative of the somatic membrane potential (dV/dt) vs. the instantaneous somatic membrane potential.