A seizure-induced gain-of-function in BK channels is associated with elevated firing activity in neocortical pyramidal neurons

Abstract

A heritable gain-of-function in BK channel activity has been associated with spontaneous seizures in both rodents and humans. We find that chemoconvulsant-induced seizures induce a gain-of-function in BK channel current that is associated with abnormal, elevated network excitability. Action potential half-width, evoked firing rate, and spontaneous network activity in vitro were all altered 24 h following picrotoxin-induced seizures in layer 2/3 pyramidal cells in the neocortex of young mice (P13-P16). Action potential half-width and firing output could be normalized to control values by application of BK channel antagonists in vitro. Thus, both inherited and acquired BK channel gain-of-functions are linked to abnormal excitability. Because BK channel antagonists can reduce elevated firing activity in neocortical neurons, BK channels might serve as a new target for anticonvulsant therapy.

Figure 1

Seizure increases BK channel contribution to AP waveform. (A) Minimal current injection (1 s duration) elicits a single AP from a representative control cell (gray) and 24 hrs post-seizure cell (black). (B) Overlay of AP waveforms aligned to threshold from a control cell (gray) and post-seizure cell (black). (C) Analysis of AP waveform. Rise times (threshold to peak) were not affected by prior seizure (p>0.5) while decay times (peak to baseline) were reduced (p<0.001). Half-width was likewise reduced after seizure (see text). (D) Internally-applied BAPTA (10 mM) normalized AP waveforms aligned to threshold from control (gray) and post-seizure animals (black). Inset shows quantitative comparison between control and seizure cells. (E) Paxilline (10nM) significantly broadened APs in post-seizure (“seizure”) neurons (p<0.05) but had a negligible effect in control cells (p>0.3). Traces from single representative cells before paxilline, gray; after paxilline application, black (within cell comparisons). (F) Paxilline (Pax) significantly broadened only APs from post-seizure animals and had no effect on AP rise times (data not shown). Scale bar in (B) applies to (D) and (E).

Figure 2

BK channel currents are altered after seizure. (A) Representative potassium channel currents from a control neuron (P14) before (left) and after (right) paxilline application. Cells were held at -80 mV and an 80-160 ms voltage step (+100 to +180 mV in 20 mV increments) was applied. Scale is the same for (A-D). (B) Subtracted traces at 100mV holding potential before and after paxilline application yield the BK channel current for control. (C) The same as (A) but in a post-seizure neuron. (D) Subtracted paxilline sensitive current from a post-seizure neuron. (E) Amplitude of paxilline-sensitive steady-state current (measured at the end of an 80ms pulse) in post-seizure neurons was greater than control. *p<0.05.

Figure 3

Evoked firing is increased after seizure in a BK channel -dependent manner. (A) Evoked firing during a 200 pA current injection (1 sec) in control neurons. (B) Same as (A) but after paxilline application. (C) Evoked firing as in (A) but in a post-seizure neuron. (D) Same as (C) but after paxilline application. (E) IFF for different amplitudes of current injection. In control, IFF at all current amplitudes (closed gray squares) is not altered after paxilline application (data not shown). After seizure, IFF is elevated (closed black circles) and can be reduced to control levels by paxilline application (open circles). (F) Total number of spikes elicited during a 200 pA current injection (see (A-D) above) show that total spike output is increased in post-seizure neurons and can be reduced to control levels by paxilline application, p<0.05 vs seizure, No Drug. (G) ISIs for later spikes in the spike train are not affected by BK channel antagonists in control (closed gray squares, control No Drug; open gray squares, control+paxilline). (H) After seizure, all ISIs are reduced and can be increased by BK channel antagonists (post-seizure No Drug, closed black circles; post-seizure+paxilline, open black circles). All statistical comparisons were carried out by ANOVA between cell groups.

Figure 4

BK channels do not contribute to short-term facilitation. (A-B) Paired pulse synaptic currents evoked by layer 2/3 stimulation (interstimulus interval = 50 ms) in the presence of 50 uM picrotoxin before (baseline) and after bath application of 10 nM paxilline (post-paxilline), for control animals (A) and 24 hrs post-seizure (B). (C) Averaged PPR before (gray) and after paxilline application (black) for control and post-seizure cells.

Figure 5

BK channel antagonists reduce spontaneous activity after seizure. (A) Spontaneous firing activity over the course of ~8 minutes from a representative control cell. (B) Representative example of spontaneous firing in the presence of paxilline in a control cell. (C) Representative example showing increased spontaneous firing in a post-seizure cell. (D) Representative example of spontaneous firing in the presence of paxilline in a post-seizure cell. (E) Summary of spontaneous firing rates in control and post-seizure cells in either modified ACSF or with Ibtx or paxilline application. Bath application of glutamate and GABA_A antagonists (N, A, P; last column in (E)) abolished all firing. * p<0.05 and ** p<0.01 for seizure versus all other groups by ANOVA.