Cell-attached patch clamp electrophysiology

Primary rat and mouse hippocampal neurons were used on 7–10 DIV. Cell-attached patch clamp recordings were performed on an Olympus IX70 inverted microscope in a 15 mm culture coverslip at room temperature (22–25°C). Signals were recorded at 10 kHz and low-pass filtered at 2 kHz with an Axopatch 200B amplifier and digitized with a Digidata 1440 (Molecular Devices). Recording pipettes were pulled from borosilicate capillary glass (0.86 OD) with a Flaming micropipette puller (Model P-97, Sutter Instruments) and polished (polisher from World Precision Instruments). Pipette resistances were strictly maintained between 6–7 MΩ to ameliorate variations in number of channels in the patch pipette. The patch transmembrane potential was zeroed by perfusing cells with a high K⁺ extracellular solution containing (in mM) 145 KCl, 10 NaCl, and 10 HEPES, pH 7.4 (NaOH). The pipette solution contained (in mM) 20 tetraethylammonium chloride (TEA-Cl), 110 BaCl₂ (as charge carrier), and 10 HEPES, pH 7.3 (TEA-OH). This pipette solution was supplemented with 1 µM ω-conotoxin GVIA and 1 µM ω-conotoxin MCVIIC to block N and P/Q-type Ca²⁺ channels, respectively, and (S)-(-)-BayK-8644 (500 nM) was included in the pipette solution to promote longer open times and resolve channel openings as previously performed by our group and others (Shen et al., 2018; Davare et al., 2001; Wang et al., 2001; Qian et al., 2017; Hess et al., 1986; Schuhmann et al., 1997; Costantin et al., 1998; Yue and Marban, 1990; Dzhura and Neely, 2003; Navedo et al., 2005). In a subset of experiments, BayK was left out of the pipette solution. Note that ISO and CAR had similar effects on channel activity whether BayK was included or not in the pipette solution. To examine the effects of β-adrenergic stimulation on the L-type Ca_V1.2 single-channel activity, 1 μM isoproterenol was added to the pipette solution in independent experiments. Note that we have previously used the L-type Ca_V1.2 channel blocker nifedipine (1 μM) to confirm the recording of L-type Ca_V1.2 currents under control conditions and in the presence of isoproterenol (Patriarchi et al., 2016). Single-channel activity was recorded during a single pulse protocol (2 s) from a holding potential of −80 mV to 0 mV every 5 s. An average of >50 sweeps were collected with each recording file under all experimental conditions. The half-amplitude event-detection algorithm of pClamp10 was used to measure overall single-channel L-type Ca_V1.2 activity as nPo, where n is the number of channels in the patch and Po is the open probability. Because the variability of nPo can be a critical element to interpret single channel data due to overstating open probability based on a high n number, we corrected this parameter by the number of channels (n) describing channel open probability and availability as well as calculating the mean ensemble average current. Data were pooled for each condition and analyzed with GraphPad Prism software.