Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain

Abstract

Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at −40mV, peaked at −26mV, and half-inactivated at −46.7mV. The persistent sodium current (INaP) activated at −51mV, peaked at −23mV, and half-inactivated at −30.7mV. Tetrodotoxin (TTX; 1μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd2+ (50μM) potently blocked INaP with little effect on INaf. Riluzole (>20μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at −30mV, peaked at −11.3mV, and half-inactivated at −34mV. The Ca2+ channel blocker verapamil (100μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca2+-activated K+ (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca2+ or adding verapamil or nifedipine, and increased by adding the Ca2+ agonist Bay K8644, indicating that Ca2+ entry via the L-type Ca2+ channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100μM riluzole in the bath solution. These membrane oscillations disappeared with 1μM TTX, 50μM Cd2+, replacement of external Na+ with choline, and blockage of Na+-activated K+ current (IKNa) with 50μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca2+-free medium or in the presence of verapamil were eliminated by blocking INaP with 50μM Cd2+. Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca2+ channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.