Digidata 1550b interface

SAN cells were harvested in custom-made chambers with glass bottoms for cell attachment and superfused with Tyrode solution warmed at 36 °C before recording. All electrophysiological signals, APs and ion currents, were measured by perforated patch-clamp technique and recorded using a Multiclamp 700 A patch clamp amplifier connected to Digidata 1550B interface (Molecular Devices). Electrodes had a resistance of 3–4 MΩ when filled with a solution containing (in mM): 80 K-aspartate, 50 KCl, 1 MgCl₂, 2 CaCl₂, 5 EGTA, 5 HEPES, and 3 ATP-Na (adjusted to pH 7.2 with KOH). Perforated patch-clamp was performed by adding 30 µM β-escin to the intracellular solution. Seal resistances were in the range of 2–5 GΩ. Perfusion of pre-warmed (36 °C) experimental solutions was achieved by using a multi-MPRE8 heating pen (Cell Micro Controls). AP parameters were measured as previously described (see Supplementary Table S1 inset in ref. [27 (link)]). For ion current measurements, dormancy or firing of SAN cells at baseline were recorded in current-clamp for at least 30 s before switching to voltage-clamp.

Extracellular recordings were obtained from retinal ganglion cells (RGCs) (eight WT mice) in all retinal quadrants. Whole-cell recordings were performed by using an Axopatch 700B amplifier connected to a Digidata 1550B interface and pCLAMP 10 software (Molecular Devices, San Jose, CA, United States). Cells were visualized with near-infrared light (>775 nm) at 40× magnification with a Nuvicon tube camera (Dage-MTI, Michigan, IN, United States) and differential interference optics on a fixed-stage microscope (Eclipse FN1; Nikon, Tokyo, Japan). Retinas were superfused at a rate of 1 to 1.5 mL min^–1 with Ringer solution, composed of (in mM) 120 NaCl, 2.5 KCl, 25 NaHCO₃,0.8 Na₂HPO₄, 0.1 NaH₂PO₄, 1 MgCl₂,2 CaCl₂, and 5 D-glucose. The bath solution was continuously bubbled with 95% O₂–5% CO₂ at 32°C (Pan et al., 2016 (link)).
Electrodes were pulled to 5 to 7 MΩ resistance, with internal solutions consisting of (in mM) 120 potassium gluconate, 12 KCl, 1 MgCl₂, 5 EGTA, 0.5 CaCl₂, and 10 HEPES (pH adjusted to 7.4 with KOH). This internal solution was used in experiments in which spiking was not blocked. Spike trains were recorded digitally at a sampling rate of 10 kHz with Axoscope software, which were sorted by using Off-line Sorter (Plexon, Dallas, TX, United States) and NeuroExplorer (Nex Technologies, Littleton, MA, United States) software.

Extracellular recordings were obtained from RGCs (41 WT mice) in all retinal quadrants by using whole-cell recordings. Whole-cell recordings were performed by using an Axopatch 700B amplifier connected to Digidata 1550B interface and pCLAMP 10 software (Molecular Devices). Cells were visualized with near infrared light (>775 nm) at ×40 magnification with a Nuvicon tube camera (Dage-MTI, Michigan City, IN, USA) and differential interference optics on a fixed-stage microscope (Eclipse FN1; Nikon, Tokyo, Japan). Retina were superfused at a rate of 1–1.5 mL min⁻¹ with a Ringer solution composed of (mM): 120 NaCl, 2.5 KCl, 25 NaHCO₃, 0.8 NaHPO₄, 0.1 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂ and 5 D-glucose. The bath solution was continuously bubbled with 95% O₂–5%CO₂ at 32°C.
Electrodes were pulled to 5−7 MΩ resistance, with internal solutions consisting of (mM): 120 potassium gluconate, 12 KCl, 1 MgCl₂, 5 EGTA, 0.5 CaCl₂, 0.2GTP and 10 HEPES (pH adjusted to 7.4 with KOH). This internal solution was used in experiments where spiking was not blocked. Spike trains were recorded digitally at a sampling rate of 20 kHz with Axoscope software, with sorting by using Off-line Sorter (Plexon, Dallas, TX, USA) and NeuroExplorer (Nex Technologies, Littleton, MA, USA) software.