Persistent muscarinic excitation in guinea-pig olfactory cortex neurons: Involvement of a slow post-stimulus afterdepolarizing current

Abstract

The persistent excitatory effects of the muscarinic agonist oxotremorine-M were investigated in guinea-pig olfactory cortex neurons in vitro (28–30°C) using a single-microelectrode current-clamp/voltageclamp technique. In 40% of recorded cells (type 1), bath-application of oxotremorine-M (2–10μM; 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 (~ 10–15 mV) lasting ~30s. Injection of steady negative current at the peak of this response produced a slow repolarization of the membrane potential (half-time ~0.6 min) towards a plateau level (“hyperpolarization recovery”); these effects of oxotremorine-M were slowly reversed on washout or by application of atropine (l μ(M). 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 − 100mV, or by raising the extracellular K + concentration from 3 to 9mM. The afterdepolarization current was suppressed by Cd2 + (100 μM) or by removal of external Ca 2+, but not by adding Ba 2+ (500μM) or La 3+ (50–100μM). 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−85 mV for 2 min. These slow relaxations were reduced by Cd2 + (100μM) or in Ca 2+-free medium, and were thought to represent the slow Ca 2+-induced de-activation and re-activation, respectively, of the afterdepolarization current conductance mechanism. Intracellular loading with the Ca 2+ 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 μM), most likely due to increased Ca 2+ 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 Ca 2+ entry during a depolarizing command. We suggest that the de-activation of this conductance by maintained Ca 2+ 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.