Pclamp 9

Data were transferred to a computer hard disk after digitization with an A/D converter (Digidata 1322, Molecular Devices). Data acquisition (digitized at 10 kHz and filtered at 2 kHz) was performed with pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA).
In voltage clamp mode, cells were stimulated with a 100 ms lasting hyperpolarizing stimulus (10 mV), then, in the recording, area below the capacitative transients was measured and normalized for voltage transient amplitude to calculate cellular capacitance; input resistance was obtained through Ohm's law, by measuring the amplitude of steady state current generated by voltage transient. Spontaneous postsynaptic currents were analyzed using pClamp 9 (Molecular Devices, Sunnyvale, CA, USA). This program uses a detection algorithm based on a sliding template. The template did not induce any bias in the sampling of events because it was moved along the data trace by one point at a time and was optimally scaled to fit the data at each position. All the collected events were averaged and the amplitude of current was calculated as that of the mean trace.
Statistical significance was tested using unpaired Student t-test (Origin, Northampton, MA, USA). A p < 0.05 was considered as statistically significant. Values are given as mean ± SEM.

Whole-cell patch-clamp recordings of neurons were performed at room temperature using an inverted microscope and an EPC-10 amplifier (HEKA). Cells in different groups were maintained in a bath solution of 150 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4, 300 mOsm). Patch pipettes were pulled and polished to yield a resistance of 3–4 MΩ when filled with the intracellular solution [130 mM K-gluconate, 6 mM KCl, 3 mM NaCl, 0.2 mM EGTA, 10 mM HEPES, 4 mM ATP-Mg, 0.4 mM GTP-Na, and 14 mM phosphocreatine-di(Tris) (pH 7.2, 285 mOsm)]. During recordings, the pipette capacitance was neutralized, and access resistance was continuously monitored. In the current-clamp recording mode, action potentials were elicited by a depolarizing step current from − 60 pA to 120 pA at 20pA increments and 800 ms in duration. In the voltage-clamp mode, whole-cell currents were evoked with voltage steps ranging from − 60 to 30 mV at 10-mV increments. Then, the tetrodotoxin (TTX, 1 μM) was applied to the chamber, and the voltage steps were repeated to examine TTX-sensitive currents. Currents were filtered and digitized at 3 and 10 kHz, respectively, and the data were analyzed using pClamp 9.0 (Axon Instruments).

In voltage-clamp experiments, the total currents were recorded using an extracellular solution containing 130 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, and 10 mM HEPES-HCl, pH 7.4). The patch pipette was filled with 130 mM KCl, 2 mM MgCl₂, 10 mM glucose, 5 mM EGTA and 10 mM HEPES-NaOH (pH 7.4). The electrophysiological recordings were carried out at room temperature using the whole-cell configuration of the patch-clamp technique [25] (link), [26] (link). Stimulation, acquisition, and data analysis were performed using the pCLAMP 9.0 and Clampfit software (Axon Instruments, Burlingame, CA, USA). The cells were clamped to a holding potential of −90 mV and the currents recorded from −100 mV to +65 mV, in increments of 5 mV. The components of the leak and capacitive currents were cancelled using the P/N method. The patch pipettes had a resistance of 3 MΩ to 6 MΩ, and to reduce their capacitance, their tips were coated with Sylgard. A sampling interval of 25 µs point⁻¹ was used, and the currents were filtered at 5 kHz.

Conventional whole-cell configurations of the patch-clamp technique were used in the electrophysiological study. Signals were amplified using an Axopatch 200B amplifier (Axon Instruments) and filtered at 1 kHz. Data acquisition and analysis were carried out using pClamp 9.0 (Axon Instruments, San Jose, CA, USA) software. Patch electrodes were pulled from a horizontal micropipette puller (P-1000, Sutter Instruments, Novato, CA, USA) and fire polished to a final tip resistance of 4–6 MΩ when filled with internal solutions. For whole-cell recording on HEK-293 cells, the pipette solution contained (in mM) 140 KCl, 2 MgCl₂, 10 HEPES, 0.1 EGTA, 4 K2-ATP, and pH 7.3 (adjusted with KOH); the bath solution contained (in mM) 120 NaCl, 5 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, and pH 7.4 (adjusted with NaOH). The data were acquired at 20 kHz and low-pass filtered at 5 kHz. During post-analysis, data were further filtered at 200 Hz.

In voltage-clamp experiments, Ca²⁺ currents were recorded using an extracellular solution containing 10 mM CaCl₂, 5 mM glucose, 120 mM tetraethylammonium chloride, 10 mM Hepes, 1 mM MgCl₂, 0.1 mM ethylene glycol-bis-b-aminoethyl ether-N,N,N',N'-tetraacetic acid (EGTA), pH 7.4. The patch pipette was filled with 130 mM CsCl, 1 mM EGTA, 0.5 mM MgCl₂, 10 mM Hepes, pH 7.4. During recordings, the bath contained 2 μM tetrodotoxin, to prevent Na⁺ current activation. The electrophysiological recordings were carried out at room temperature using the whole-cell configuration of the patch-clamp technique. Stimulation, acquisition, and data analysis were performed with the pCLAMP 9.0 and Clampfit version 10.0 software (Axon Instruments, Burlingame, USA). The linear components of leak and capacitive currents were canceled using the P/N4 method. The patch pipettes had resistances of 3–6 MΩ. To abolish the T-type contribution and select L-type Ca²⁺ currents, the cells were clamped from the holding potential of −90 to −30 mV for 750 ms, then a ramp was applied from −60 to +60 mV, with 10 mV increments and of 500 ms duration. A sampling interval of 50 kHz (20 μs/point) was used, and the currents were filtered at 2 kHz (Bessel filter).

The data were presented as the Mean ± SD. Dosage curves were fitted with pCLAMP 9.0 (Axon Instruments) and software Origin 7.0 (OriginLab, USA). The statistical significance was determined using a t-test when comparing two groups and ANOVA when comparing multiple groups. A value of P < 0.05 was considered statistically significant.