Patch clamp electrophysiology

Automated voltage-clamp recordings were performed at room temperature using a Syncropatch 768PE (Nanion Technologies, Munich, Germany) as described previously (Vanoye et al., 2018) except that the internal solution contained 5 mM Mg-ATP. The contribution of background currents was determined by recording before and after addition of XE991 (25 μM, TOCRIS, Minneapolis, MN). Only XE991-sensitive currents were used for analysis. Whole-cell currents were elicited from a holding potential of −80 mV using 1000 ms depolarizing pulses (from −80 mV to +40 mV in +10 mV steps every 20 secs) followed by a 300 ms step to 0 mV to analyze tail currents. Cells with seal resistance ≥0.5 GΩ and series resistance ≤20 MΩ (access resistance compensation was set to 80%) were used for analysis. Peak currents were measured 999 ms after the start of the depolarizing voltage pulse and tail currents 5 ms after changing the membrane potential to 0 mV. The time-constant of activation (τ) was determined by fitting currents elicited by voltage steps between −30 mV and +40 mV (50–1000 ms after start of the voltage step) to a single exponential.

Whole-cell current-clamp recordings were made from visually identified GFP-expressing neurons using an inverted Olympus IX51 microscope equipped with a 40X objective. Recording pipettes were made of glass capillaries using a horizontal Sutter P-1000 puller yielding a 2–4 MΩ resistance pipette when filled with standard K-methyl sulfate intracellular solution containing (in mM): 120 K-MeSO₄, 10 KCl, 10 HEPES, 10 Na₂-phosphocreatine, 4 Mg-ATP, 0.4 Na₃-GTP, pH 7.35 adjusted with KOH; osmolality 285–290 mOsm/Kg. Neurons were continuously perfused with oxygenated aCSF bath solution (in mM): 125 NaCl, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgSO₄, 22 glucose, 2 CaCl₂, pH 7.35 at 32–35°C; osmolality 310–315 mOsm/Kg.

Current-clamp recordings were acquired using a Multiclamp 700B amplifier (Molecular Devices, USA) and digitized at 10 kHz (filtered at 3 kHz) with the neurons held at −65 mV (V_h). All reported potential values were corrected for the liquid junction potential, calculated to be −8.2 mV. Resting membrane potential (RMP) was measured immediately after establishing the whole-cell patch clamp configuration. Input resistance (R_N) was calculated as the slope of the voltage-current curve determined using 500 ms current steps from −50 pA to 30 pA in 10 pA steps. Medium (mAHP) and slow (sAHP) afterhyperpolarizations (AHPs) were measured as the difference between V_h and the negative going peak and 1 s after the offset of the last current step, respectively, induced by a 50 Hz train of 25 APs evoked by 2 ms/1.2 nA current injection pulses. Single AP properties, including fast afterhyperpolarization (fAHP), were measured using direct somatic current injection ramps (10–80 pA, 500 ms). AP amplitude was calculated as the difference between V_h to the peak of the first AP of the ramp protocol. AP threshold was calculated where the first derivative of the up phase of the trace equaled 5 mV/ms. Using a 1 ms sliding average, the fAHP measurement was taken when the mean first derivative of the trace reached 0.0 ± 0.5 after initial spike in each sweep. AP half-width measurements were taken at half the AP peak amplitude relative to V_h. Neurons meeting the following criteria were used: series resistance (R_S) <30 MΩ, membrane resistance (R_N) >200 MΩ, resting potential (V_rest) < −45 mV, and AP amplitude >80 mV from V_h. Data were analyzed using custom MATLAB protocols (Simkin et al., 2015). All MATLAB scripts are available for download at github.com/simkind/Patch-clamp-analysis.git (Simkin, 2021; copy archived at swh:1:rev:bde5c7399d9f7c789feec0ee26ab5dad4a661d90). Data collected at three time points in culture defined as week 3 (days 14–16), week 4 (days 22–26), and week 5 (days 32–35; Figure 3A). Data collected from each time-point of 3–5 days were combined for statistical analysis using Statview software.

We tested the action of chronic and acute application of 1 and 20 µM XE991 (Abcam; expected to block 50% and 100% of M-current, respectively; Wang et al., 1998). XE991 (1 µM) was chronically applied to neuronal culture media starting day 12 in differentiation (right before beginning of week three time point) and AP properties were measured on week 4. Acute application of 20 µM XE991 or 500 nM apamin (Alomone) was done during week 4 and AP/AHP properties were measured before and 10 min after continuous perfusion of aCSF with XE991 or apamin.