Whole-cell recording for voltage-gate channel current in acutely isolated hippocampal pyramidal neurons

Qing CAI; Zhongliang ZHU; Xiaoli FAN; Zhuanli BAI; Ning JIA; Xia LI

ABSTRACT

BACKGROUND:

Hippocampus is an important region involved in learning and memory. Some methods for freshly isolated hippocampal pyramidal neurons have been described. These methods need multiple enzymes or the procedure is complex.

OBJECTIVE:

To acquire large quantities of hippocampal neurons from rats suitable for patch clamp study.

DESIGN:

An animal experimental observation.

SETTING:

Department of Physiology and Pathophysiology, School of Medicine, Xi'an Jiao Tong University.MATERIALS The experiments were carried out in the Medical Experimental Center, School of Medicine, Xi'an Jiao Tong University between March and October 2004. The animals were the 10-15-day-old SpragueDawley rats, bothmale and female.

METHODS:

Hippocampal pyramidal neurons were freshly isolated. Delayed rectifier potassium current and voltage-gated Ca2 + currents were recorded by whole-cell patch clamp configuration.MAIN OUTCOME

MEASURES:

Acute isolated neurons were observed with a Leica microscope. Recordings of delayed rectifier potassium currents and voltage-gated Ca2+ currents were obtained.

RESULTS:

① Under the inverted microscope, the acute isolated neurons had a smooth and glassy surface, a three-dimensional contour, a pyramidal shape with a longer apical dendrite and several basal dendrites. ② After the whole cell recording configuration was formed, voltage-gated barium currents through calcium channels were elicited by 200 ms depolarizing potential from -70 mV to +20 mV, with an increment of 10 mV, a holding potential was -90 mV. Delayed rectifier potassium current (Ikd) was elicited by a protocol consisting of a multiple depolarizing pulses (-60 mV to +50 mV),preceded by a single prepulse step to -50 rmV, with inactivated transient outward potassium currents from a holding potential of -80 mV.

CONCLUSION:

This method is simple and ideal for the dissociation of neurons from rat hippocampus, and offers a powerful tool for functional analysis of ion channels by patch clamp.