PERSISTENT MUSCARINIC EXCITATION IN GUINEA-PIG OLFACTORY CORTEX NEURONS - INVOLVEMENT OF A SLOW POSTSTIMULUS AFTER DEPOLARIZING CURRENT

The persistent excitatory effects of the muscarinic agonist oxotremorine-M were investigated in guinea-pig olfactory cortex neurons in vitro (28-30-degrees-C) using a single-microelectrode current-clamp/voltage-clamp technique. In 40% of recorded cells (type 1), bath-application of oxotremorine-M (2-10 muM; 1-2 min) induced a strong membrane depolarization, an increase in input resistance and a sustained neuronal discharge lasting over 30 min following agonist washout. A large depolarizing stimulus applied during the action of oxotremorine-M, evoked a slow post-stimulus afterdepolarization (approximately 10-15 mV) lasting approximately 30 s. Injection of steady negative current at the peak of this response produced a slow repolarization of the membrane potential (half-time approximately 0.6 min) towards a plateau level (''hyperpolarization recovery''); these effects of oxotremorine-M were slowly reversed on washout or by application of atropine (1 muM). In a second population of neurons (type 2; 39% of total), oxotremorine-M produced a large depolarization, a resistance increase and repetitive firing that did not persist after agonist washout; these neurons failed to generate a prominent slow afterdepolarization on stimulation, and showed no hyperpolarization recovery effect. Their resting membrane properties were not significantly different from those of type 1 cells. The remaining proportion of cells (type 3) elicited little or no muscarinic response to oxotremorine-M and no slow afterdepolarization; these cells showed characteristic spike fractionation (pre-potentials) during an evoked train of action potentials. In type 1 cells, the inward tail current underlying the slow afterdepolarization was revealed under voltage-clamp; the afterdepolarization current had a slow time to peak, an exponential decay, and its amplitude was reduced (but not reversed) by hyperpolarization between -60 and -100 mV, or by raising the extracellular K+ concentration from 3 to 9 mM. The afterdepolarization current was suppressed by Cd2+ (100 muM) or by removal of external Ca2+, but not by adding Ba2+(500 muM) or La3+ (50-100 muM). In oxotremorine-M, voltage-clamp commands from -70 to -40 mV, evoked slowly-decaying outward current relaxations followed by slow inward tail currents. A slowly-relaxing outward current underlying the hyperpolarization recovery phenomenon was also revealed on stepping from - 40 to - 8 5 mV for 2 min. These slow relaxations were reduced by Cd2+ (100 muM) or in Ca2+-free medium, and were thought to represent the slow Ca2+-induced de-activation and re-activation, respectively, of the afterdepolarization current conductance mechanism. Intracellular loading with the Ca2+ chelators EGTA or BAPTA failed to reduce the afterdepolarization current; however, the tail current evoked under ''hybrid'' voltage-clamp, was dramatically enhanced after pre-treatment with tetrabutylammonium (500 mu M), most likely due to increased Ca2+ entry. These results add further support to the view that the afterdepolarization tail current and slow outward current relaxations revealed under voltage-clamp during muscarinic receptor stimulation reflect the slow re-activation of a novel K+ conductance that is de-activated by Ca2+ entry during a depolarizing command. We suggest that the de-activation of this conductance by maintained Ca2+ influx at depolarized membrane potentials (e.g. during repetitive neuronal firing) could contribute to the prolonged muscarinic excitation of olfactory and perhaps other mammalian cortical neurons.