Dantrolene inhibits the calcium plateau and prevents the development of spontaneous recurrent epileptiform discharges following in vitro status epilepticus

Abstract

Status epilepticus is a clinical emergency that can lead to the development of acquired epilepsy following neuronal injury. Understanding the pathophysiological changes that occur between the injury itself and the expression of epilepsy is important in the development of new therapeutics to prevent epileptogenesis. Currently, no anti-epileptogenic agents exist; thus, the ability to treat an individual immediately after status epilepticus to prevent the ultimate development of epilepsy remains an important clinical challenge. In the Sprague-Dawley rat pilocarpine model of status epilepticus-induced acquired epilepsy, intracellular calcium has been shown to increase in hippocampal neurons during status epilepticus and remain elevated well past the duration of the injury in those animals that develop epilepsy. This study aimed to determine if such changes in calcium dynamics exist in the hippocampal culture model of status epilepticus-induced acquired epilepsy and, if so, to study whether manipulating the calcium plateau after status epilepticus would prevent epileptogenesis. The in vitro status epilepticus model resembled the in vivo model in terms of elevations in neuronal calcium concentrations that were maintained well past the duration of the injury. When used following in vitro status epilepticus, dantrolene, a ryanodine receptor inhibitor, but not the N-methyl-D-aspartic acid channel blocker MK-801 inhibited the elevations in intracellular calcium, decreased neuronal death and prevented the expression of spontaneous recurrent epileptiform discharges, the in vitro correlate of epilepsy. These findings offer potential for a novel treatment to prevent the development of epileptiform discharges following brain injuries.

Fig. 1

In vitro SE causes significant, long-lasting elevations in [Ca²⁺]_i (Ca²⁺ plateau). Time course comparing sham controls treated with pBRS (●) vs. neurons treated with low Mg²⁺ (○) at the end of in vitro SE (acute) and at various time points following in vitro SE (10, 30, 60 min, 3, 6, 24, 48 h). All data represented as average 340 / 380 ratio ± SEM. *P < 0.05, Student’s t-test, n = 6 or more plates with at least 60 neurons imaged per condition at each time point studied.

Fig. 2

Dantrolene lowers [Ca²⁺]_i to baseline levels following in vitro SE. (A) Pseudocolor ratiometric images of representative neurons in culture. (a) Phase bright reference image depicting typical pyramidal-shaped hippocampal neurons that lay above the focal plane of glia. (b–e) Representative ratiometric (340 / 380) images of neurons from control (b), low Mg²⁺ (c), MK-801 (d) and dantrolene (e) treated cultures. All the ratiometric images are at the 30-min time point following respective treatments. (B) Following 3 h of in vitro SE (time = 0), cells were treated with vehicle (0.1% DMSO, ●), MK 801 (10 µm, ■) or dantrolene (50 µm, Δ). 340 / 380 ratios were recorded every 30 s for 30 min and normalized to percentage of the peak ratio observed at time = 0. Dotted lines represent data fitted using exponential decay function [y = a*exp(b / (x+c))] using parameter estimates reported in Table 1. n = 6 plates per treatment group with ~60 neurons imaged per condition.

Fig. 3

In vitro SE is not inhibited by dantrolene. Representative current-clamp trace from control neuron showing occasional action potentials (A), neuron in low Mg²⁺ or in vitro SE showing high-frequency spiking (B), and neuron in low Mg²⁺ plus dantrolene (50 µm) showing no effects of dantrolene on high-frequency spiking (C). n = 15 culture plates.

Fig. 4

Dantrolene maintains baseline Ca²⁺ levels 24 and 48 h after SE. Following in vitro SE, the effect of dantrolene (50 µm) (gray bar) vs. vehicle (black bar) on 340 / 380 ratios 24 h after treatment (A) and 48 h after treatment (B). Data represented as percentage control ± SEM. *P < 0.05, one-way anova, Fisher’s post-hoc test, n = 10 or more plates with ~100 cells imaged per treatment group for 24-h time point; n = 6 plates with ~60 cells imaged per treatment group at 48-h time point.

Fig. 5

Dantrolene prevents the development of SREDs 24 and 48 h after SE but does not inhibit SREDs acutely. Representative current-clamp trace from control neuron (A), low Mg²⁺ -treated neuron displaying characteristic SREDs (B), and neurons treated with low Mg²⁺ then dantrolene (50 µm) at both 24 h (C) and 48 h (D) after treatment and were devoid of SREDs. Dantrolene did not inhibit SRED activity when added acutely to neurons displaying SREDs (n = 12–15 culture plates with a minimum of threee neurons recorded from each plate).

Fig. 6

Dantrolene is neuroprotective. Cell death was assessed at 24 (A) and 48 (B) h after treatment with pBRS (sham, black bars), low Mg²⁺ then vehicle (0.1% DMSO) (low Mg²⁺, gray bars), and low Mg²⁺ then dantrolene (50 µm) (dantrolene, white bars). No significant difference was observed between the sham and drug-treated group at either of the time points. Low Mg²⁺ neurons demonstrated significantly more cell death than the other two groups at both 24 and 48 h. Data represented as mean fraction cell death ± SEM. *P < 0.05, one-way anova, Fisher’s post-hoc test, n = 7 or more plates per treatment and time point.