Ionic mechanism of muscarinic cholinergic depolarization of mouse spinal cord neurons in cell culture

Abstract

Muscarinic cholinergic actions were investigated in a population of large multipolar spinal cord neurons in primary dissociated cell culture using conventional intracellular recording and single-microelectrode voltage-clamp techniques. Cholinergic agonists were applied to the surface of neuronal somata by pressure ejecting drug-containing bathing medium from small blunt (2-10 .mu.m) glass micropipettes. Atropine was applied by diffusion from large (20-30 .mu.m) blunt micropipettes positioned near the soma. Muscarine increased action-potential firing and evoked slow sustained membrane depolarization. Action potentials but not slow membrane depolarizations were eliminated by the sodium channel blocker, tetrodotoxin. Muscarine-induced depolarizing responses were unaffected by the Ca channel blocker, Ca. Depolarizing responses evoked by selective and nonselective muscarinic cholinergic agonists were dose dependent, reversibly antagonized by atropine, and did not desensitize. Muscarine depolarized neurons and decreased membrane conductance during recording with both 3 M KCl- and 4 M potassium acetate filled intracellular recording micropipettes. When membrane potential was held constant using the single-electrode voltage-clamp technique (KCl-filled micropipettes), muscarine and GABA evoked inward currents at resting membrane potential. GABA-induced inward current responses were decreased by depolarization and had reversal potentials near -30 mV, consistent with GABA increasing Cl- conductance. Muscarine-induced inward current responses were increased by depolarization and had extrapolated reversal potentials near -80 mV, consistent with muscarine decreasing a K conductance. Unlike GABA-induced currents, muscarine-induced currents evoked in normal Tris-buffered saline (5 mM K) did not vary as a linear function of membrane potential and did not reverse polarity in 6 of 7 neurons near K equilibrium potential. In high-K medium (15 mM) muscarinic responses did reverse polarity and current was linearly related to membrane potential. The apparent voltage dependence of muscarine responses was probably due to voltage dependency of the potassium conductance and not due to K channel rectification. Preliminary evidence (37) indicates that muscarine decreases a time- and voltage-dependent K current in some cultured spinal cord neurons. Whether reduction of m current can completely account for muscarine postsynaptic actions in these cells remains unclear. Muscarine may also block a small population of non-voltage-dependent K channels in addition to reducing m current.