Involvement of inward rectifier and M-type currents in carbachol-induced epileptiform synchronization

Abstract

Exposure to cholinergic agonists is a widely used paradigm to induce epileptogenesis in vivo and synchronous activity in brain slices maintained in vitro. However, the mechanisms underlying these effects remain unclear. Here, we used field potential recordings from the lateral entorhinal cortex in horizontal rat brain slices to explore whether two different K(+) currents regulated by muscarinic receptor activation, the inward rectifier (K(IR)) and the M-type (K(M)) currents, have a role in carbachol (CCh)-induced field activity, a prototypical model of cholinergic-dependent epileptiform synchronization. To establish whether K(IR) or K(M) blockade could replicate CCh effects, we exposed slices to blockers of these currents in the absence of CCh. K(IR) channel blockade with micromolar Ba(2+) concentrations induced interictal-like events with duration and frequency that were lower than those observed with CCh; by contrast, the K(M) blocker linopirdine was ineffective. Pre-treatment with Ba(2+) or linopirdine increased the duration of epileptiform discharges induced by subsequent application of CCh. Baclofen, a GABA(B) receptor agonist that activates K(IR), abolished CCh-induced field oscillations, an effect that was abrogated by the GABA(B) receptor antagonist CGP 55845, and prevented by Ba(2+). Finally, when applied after CCh, the K(M) activators flupirtine and retigabine shifted leftward the cumulative distribution of CCh-induced event duration; this effect was opposite to what seen during linopirdine application under similar experimental conditions. Overall, our findings suggest that K(IR) rather than K(M) plays a major regulatory role in controlling CCh-induced epileptiform synchronization.

Fig. 1

CCh-induced synchronized field oscillations in EC. A: Field potential recording obtained from EC deep layers during application of CCh. Note the occurrence of frequent, brief interictal-like events (arrows) and of a prolonged ictal-like discharge (dotted line). B: Bin distribution of the duration of CCh-induced events. Note that two components can be identified in the distribution plot, the second of which (indicated by the arrow) represents ictal-like events. C: Suppression of CCh-induced oscillations by pirenzepine indicates that the effect of CCh is primarily mediated by its interaction with muscarinic receptors.

Fig. 2

Ba²⁺ induces the appearance of interictal-like events and influences CCh-induced oscillations. A: Field discharges recorded in the same slice exposed to Ba²⁺ (top) and Ba²⁺ + CCh (bottom). The insets show expanded traces of the boxed events. B: Cumulative frequency distribution of the events duration induced by Ba²⁺ (open circles, n = 5 slices), CCh (filled circles, n = 32) and Ba²⁺ + CCh (open squares, n = 5).

Fig. 3

Linopirdine by itself does not induce field discharges but influences CCh-induced oscillations. A: Field activity recorded during application of linopirdine and after further application of CCh. B: Cumulative frequency of the duration of the events induced by CCh (filled circles, n = 32) and linopirdine + CCh (open circles, n = 6).

Fig. 4

Baclofen suppresses CCh-induced field discharges via GABA_B receptors. A: Baclofen reversibly suppresses CCh-induced events in the EC. B: Concentration-dependence curve of baclofen-induced decrease of the frequency of CCh-evoked events; baclofen IC₅₀ was 2.24 μM and the Hill slope = 1. C: Cumulative frequency curves of the duration of the events recorded in the presence of CCh (filled circles, n = 32) and after addition of baclofen, 1 μM (open circles, n = 6) or 3 μM (open squares, n = 6). D: The GABA_B receptor blocker CGP 55845 prevents baclofen-induced suppression of the field oscillations triggered by CCh. E: Cumulative frequency curves of the duration of the events recorded in the presence of CCh + CGP 55845 (filled circles, n = 6) or CCh + CGP 55845 + baclofen (open circles, n = 6).

Fig. 5

Baclofen suppression of CCh-induced field activity depends on K_IR activation. A: Field discharges recorded in the EC during application of Ba²⁺ + CCh and after addition of baclofen. B: Cumulative frequency curves of the duration of the discharges recorded during perfusion with Ba²⁺ + CCh (filled circles, n = 6) and after baclofen addition (open circles, n = 6). C: Established CCh-induced events change in polarity and become more robust after subsequent application of Ba²⁺ (cf. Figs. 1A and 2A). D: Cumulative frequency curves of the duration of field discharges recorded in Ba²⁺ + CCh (filled circles, n = 6) and after baclofen addition (open circles, n = 6).

Fig. 6

Effects of flupirtine and retigabine on CCh-evoked event duration. Representative traces (left) and cumulative frequency curves of event duration (right) of field recordings obtained in slices exposed to CCh before and after the addition of (A and B) flupirtine (10 μM, n = 6), and (C and D) retigabine 10 μM (n = 8) and (E and F) retigabine 50 μM (n = 6). Note the disappearance of ictal-like discharges in slices exposed to retigabine.

Fig. 7

Effect of linopirdine on CCh-evoked event duration. Representative traces (A) and cumulative event duration frequency curves (B) of field recordings obtained in slices exposed to CCh before and after the addition of linopirdine (30 μM) (n = 6).