Inhibition of ACh on the delayed rectifier-like potassium current in acutely isolated cerebral cortical neurons of rats

The modulation of ACh on delayed rectifier-like potassium currents (IK) was studied in freshly dissociated cerebral cortical neurons using the whole-cell patch-clamp technique. Wistar rats between 10- and 14-day old of both sexes were used. After rats were decapitated, their brains were quickly removed, iced, and then manually cut into 400 urn slices. Slices were then incubated for 0.5 h at 32℃ in a buffered artificial cerebrospinal fluid (ACSF) bubbled with 95% O2, 5% CO2. Slices were then removed into buffered ACSF containing protease (0.5 mg/ml) at 32℃. After 30 min of enzyme digestion, tissue was rinsed three times in the buffered saline. Then the enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35 mm dish and placed on the stage of a Olympus inverted microscope. For whole-cell recordings of currents, standard voltage-clamp techniques were used. Neurons were held at -80 mV, and the IK was evoked by 2000 ms depolarizing voltage commands to potential between -40 mV and +60 mV in 10 raV steps applied at a frequency of 0.5 Hz. It was found that the inhibitory effect of ACh (0.1, 1, 10, 100 μmol/L) on IK was dose-dependent. It was also found that ACh affected the activation process of IK significantly, i.e., the activation curve of IK was characterized by half-activation potential of (-41.8±9.7) mV and a slope factor of (30.7±7.2) mV in the cortical neurons and they were changed to (-122.4±38.6) mV and (42.4±7.0) mV, respectively, after giving ACh (10 μmol/L). Tubocurarine (100 μmol/L) antagonized the inhibitory effect of ACh on 7K, and the drop of currents varied from the control value of (36.5±7.8)% to (16.9±13.8)% (n=8, P0.01). 4-DAMP (10 μmol/L) blocked the inhibitory effect of ACh on IK, and the currents reduced from the control value of (36.5±7.8)% to (26.8±4.7)% (n=6, P0.05). Pirenzepin did not antagonize the inhibition of ACh on IK (n=7, P0.05). Chelerythrine (20 μmol/L) blocked the inhibitory effect of ACh on IK and the currents reduced from the control value of (36.5±7.8)% to (11.7±17.3)% (n=6, P0.05). On the contrary, PDBu (10 μmol/L) strengthened the inhibition of ACh on IK and the drop of currents changed from the control value of (36.5±7.8)% to (59.2±14.0)% (n=5, P0.05). PDBu abolished the antagonism of chelerythrine on ACh in cortical neurons. It is suggested that the ACh-induced depolarization of neurons in the cortex is attributed to the inhibition of IK that is most likely evoked by the activation of nicotinic ACh receptors and muscarinic M3 receptor via protein kinase C (PKC) signal transduction pathway.