Establishment Of Patch-clamp Recording Technique For Acutely Isolated Spinal Ventral Horn Neurons And Related Receptor Analysis

Objective: To establish a method of acute isolation of spinal ventral horn neurons combined with single-cell patch-clamp recording technique for deep study of the various receptors in spinal ventral horn neurons,to lay the foundation for uncovering the motion modulation of the spinal cord,and provide new ideas for the treatment on motor neuron disease.Methods: The neonatal SD rats aged 7-12 days were anesthetized and lumbosacral enlargement of the spinal cord was isolated.After removing nerve roots,the 400-500 ?m-thick transverse slices were prepared,and placed in artificial cerebrospinal fluid for 1 hour of incubating,then digested in enzyme solution in constant temperature for 20-35 mins.After cutting out bilateral dorsal horns,the ventral horns were moved into a Petri dish filled with standard extracellular fluid,and triturated with fire-polished Pasteur pipettes until the single cells were isolated and attached to the bottom.Drugs were given by a rapid delivery system,current responses were recorded and photographs of the neurons were taken by an imaging system.Results:(1)The isolated ventral horn neurons were large cells with spindle shaped,triangular or polygonal soma,and the edges were smooth.The neurites emanated from the poles,and the morphology was complete.(2)The main electrophysiological parameters of spinal ventral horn neurons of neonatal rat: resting potential,-65.34 ± 13.63 m V;threshold,-46.11 ± 8.96 m V;spike potential amplitude,54.51 ± 15.17 m V;overshoot,8.39 ± 7.70 m V;discharge frequency,19.93 ± 9.22 Hz.(n=14)(3)The glutamate-induced current had a concentration dependence,EC50 was 0.24 m M;the average equilibrium potential was 17.63 ± 10.04 m V.(4)The nicotine-induced current had a concentration dependence,EC50 was 0.08 m M;the average equilibrium potential was 29.06 ± 14.88 m V.(5)The recorded glutamate-induced current had a short peak time,and the receptor activation was faster and the desensitization was slower compared to nicotine receptor.The average rise slope,the peak time,the average decrease slope,the loss of time,all have significant difference(p<0.05).(6)6 neurons were given ?7-nAChR selectively antagonist MLA,there was no significant change in nicotine-induced current.Another 6 neurons were given ?4?2-nAChR selectively blocker DH?E,nicotine current amplitudes of 2 cells were reduced by 45%;and for the other 4 neurons,nicotinic currents did not change,even given MLA at the same time.Conclusion: According to the method established by us on acute isolation of spinal ventral horn neurons,the morphology of the isolated neurons was complete.The acutely isolated spinal ventral horn neurons had stable and continuous spontaneous action potentials.Whole-cell currents could be induced by glutamate or nicotine,and the equilibrium potentials could be measured,respectively.The nicotine-induced currents in some neurons recorded could be suppressed by DH?E,and the currents in most of the neurons were not sensitive to DH?E and MLA.Nicotinic currents of acutely dissociated spinal ventral horn neurons were partly mediated by ?4?2-nAChRs,while the other part may be mediated by non ?4?2-and non ?7-nAChRs.