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

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 ß-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 -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 µM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 µM) potently blocked INaP with little effect on INaf. Riluzole (>20 µM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 µM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 µM riluzole in the bath solution. These membrane oscillations disappeared with 1 µM TTX, 50 µM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 µM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca(2+)-free medium or in the presence of verapamil were eliminated by blocking INaP with 50 µM Cd(2+). 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 Ca(2+) channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.

A novel GABAergic action mediated by functional coupling between GABAB-like receptor and two different high-conductance K+ channels in cricket Kenyon cells.

The γ-aminobutyric acid type B (GABA(B)) receptor has been shown to attenuate high-voltage-activated Ca(2+) currents and enhance voltage-dependent or inwardly rectifying K(+) currents in a variety of neurons. In this study, we report a novel coupling of GABA(B)-like receptor with two different high-conductance K(+) channels, Na(+)-activated K(+) (K(Na)) channel and Ca(2+)-activated K(+) (K(Ca)) channel, in Kenyon cells isolated from the mushroom body of the cricket brain. Single-channel activities of K(Na) and K(Ca) channels in response to bath applications of GABA and the GABA(B)-specific agonist SKF97541 were recorded with the cell-attached patch configuration. The open probability (P(o)) of both K(Na) and K(Ca) channels was found to be increased by bath application of GABA, and this increase in Po was antagonized by coapplication of the GABAB antagonist CGP54626, suggesting that GABA(B)-like receptors mediate these actions. Similarly, GABA(B)-specific agonist SKF97541 increased the Po of both K(Na) and K(Ca) channels. Perforated-patch recordings using ß-escin further revealed that SKF97541 increased the amplitude of the outward currents elicited by step depolarizations. Under current-clamp conditions, SKF97541 decreased the firing frequency of spontaneous action potential (AP) and changed the AP waveform. The amplitude and duration of AP were decreased, whereas the afterhyperpolarization of AP was increased. Resting membrane potential, however, was not significantly altered by SKF97541. Taken together, these results suggest that GABA(B)-like receptor is functionally coupled with both K(Na) and K(Ca) channels and this coupling mechanism may serve to prevent AP formation and limit excitatory synaptic input.