A Multiclamp 700B amplifier from Axon Instruments was used to conduct whole-cell patch clamp recordings from Purkinje cells in current clamp and voltage clamp modes. Purkinje cells were identified using the established criteria listed below. Signals were digitized using a 1440 A/D converter (Axon Instruments). Electrophysiological data were sampled at 10 kHz and filtered at 20 kHz [21 (link)]. Patch pipettes had a resistance of 5–9 MΩ when filled with an internal solution comprising (in mM) 140 potassium gluconate, 5 KCl, 10 HEPES, 2 MgCl2, 0.2 EGTA, 2 Na2ATP, and 0.4 Na2GTP. The internal solution's pH and osmolarity were adjusted to 7.3 (by KOH) and 290 mOsm, respectively. The perfusion rate of the recorded slides was 1.9–2 ml/min. Following the formation of a giga seal on the cell membrane, a brief suction was used to rupture the membrane, allowing the cell to be clamped to a holding voltage of -60 mV. The test seal function was continuously used throughout the recording to make sure the seal was stable. The number of action potentials generated after negative current injection when the cell rebounded to the resting membrane potential, and the spike latency of the first action potential were measured. In spontaneously firing neurons, action potential parameters including action potential (AP) half-width, AP frequency, AP amplitude, AHP amplitude, voltage threshold (the membrane potential from the base and start of the action potential), interspike interval (ISI), and coefficient of variation (CV) (regularity of action potential and dispersion of a probability/frequency) were measured [22 (link)].
The sag voltage in response to hyperpolarizing current pulses (amplitude of -100 pA to -500 pA) was calculated. The peak voltage deviation was divided by the amplitude of the steady-state voltage deviation using the following formula:
Sag voltage = Vpeak − Vsteady state.
Furthermore, the sag was calculated as [(peak response—steady state response) / peak response] 100. To examine the effect of a CB1R agonist/antagonist on Ih in Purkinje cells, I-V activation curves were obtained in voltage clamp mode using 520 ms of hyperpolarizing steps (50 to 140 mV in increments of 10 mV).
The sag voltage in response to hyperpolarizing current pulses (amplitude of -100 pA to -500 pA) was calculated. The peak voltage deviation was divided by the amplitude of the steady-state voltage deviation using the following formula:
Sag voltage = Vpeak − Vsteady state.
Furthermore, the sag was calculated as [(peak response—steady state response) / peak response] 100. To examine the effect of a CB1R agonist/antagonist on Ih in Purkinje cells, I-V activation curves were obtained in voltage clamp mode using 520 ms of hyperpolarizing steps (50 to 140 mV in increments of 10 mV).
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