Neuronal chloride transients during spontaneous ictal-like discharges.

(a) Simultaneous extracellular field potential recording and two-photon fluorescence chloride imaging in the organotypic hippocampal slice at DIV18. Example of spontaneous recurrent ictal-like epileptiform discharges (ILDs) and corresponding [Cl-]i transients in the CA1 pyramidal cells (n=6), and the mean [Cl-]i. [Cl-]i progressively increased during “crescendo” phase of sustained ictal-tonic discharges, was relatively stable during “forte” phase of intermittent ictal-clonic discharges, and progressively decreased during “decrescendo” phase of secondary after-discharges and post-ictal depression (decay time constant (t1) 80-111 s). (b) Expansion of ILDs and mean [Cl-]i changes normalized to values between 0 and 1 ([0, 1]). [Cl-]i rapidly increased during hyper-synchronous onset of ILD (“subito crescendo”) and subsequent sustained ictal-tonic discharges (“poco a poco crescendo”) and was relatively stable during intermittent epileptiform discharges. (b1-2) Expansion of sustained ictal-tonic discharges and intermittent ictal-clonic epileptiform discharges.

WNK-SPAK/OSR1 inhibitor WNK463 facilitated neuronal chloride extrusion.

(a) An illustration of the designed experiment to induce neuronal chloride transients in the CA1 pyramidal cells expressing sCLM. The puff micropipette filled with extracellular solution containing GABA (50 µM) and L-Glu (50 µM) was placed above the dendritic area of the neurons. Merged CFP (red) and YFP (green) fluorescent signals in subpopulation of neurons before (6 s), during (10 s) and after (24 s) GABA/L-Glu puff application. Alexa Fluo-594 (blue) was added for visualization. Corresponding [Cl-]i transients induced by GABA/L-Glu puff application. Exponential decay fit (y=y0+A1exp(-(x-x0)/t1)) was used to measure decay time constant (t1) of [Cl-]i recovery in individual cells (1-4; solid curves). (b) Linier regression analysis revealed that decay time constant of [Cl-]i recovery in individual cells was independent of the baseline [Cl-]i and corresponding EGABA. (c-h) [Cl-]i transients, corresponding [Cl-]i changes normalized to values between 0 and 1 and exponential decay curves in individual cells and averaged means (mean±SD) in control (c, d), before and during 1 µM VU0463271 application (e, f) and 1 µM WNK463 application (g, h). (d) In control, exponential decay time constant was not significantly different between two consecutive Cl- transients. (f) The KCC2 antagonist VU0463271 significantly increased the mean decay time constant (*P<0.05, paired t-test). (h) The WNK-SPAC/OSR1 inhibitor WNK463 significantly decreased the mean decay time constant of chloride recovery (**P<0.01, paired t-test).

WNK-SPAK/OSR1 inhibitor WNK463 abolished recurrent ictal-like epileptiform discharges.

(a) Extracellular field potential recording in the CA1 pyramidal cell layer in the organotypic hippocampal slice at DIV21 before (control), during and after application of 1 µM WNK463 for 30 min. Expansion of recurrent ictal (ILDs) and interictal (IEDs) epileptiform discharges before, during and after application of WNK463. (b, c) Corresponding summary plots of the frequency and duration of recurrent ILDs (b) and IEDs (c) in individual slice cultures (DIV14-22, open symbols) and corresponding power of electrical activity in 10 min windows. Filled symbols indicate group mean ± SD. WNK463 (1 µM) progressively decreased the mean frequency, duration, and power of recurrent ILDs and abolished ILDs (*P<0.05; **P<0.01; ***P<0.001, One Way RM ANOVA, Tukey Test). (c) WNK463 (1 µM) did not significantly change the mean frequency and duration of periodic IEDs (P>0.05).

WNK463 progressively reduced activity-induced elevations in interictal [Cl-]i and abolished neuronal Cl- transients.

(a) Two-photon microscopy images of Clomeleon in the CA1 pyramidal cell layer in the organotypic hippocampal slice in vitro (DIV19). Merged CFP (red) and YFP (green) fluorescent signals before, during and after WN463 (1 µM for 30 min) application. (b) Corresponding [Cl-]i changes in individual cells are plotted as a function of time. WNK463 abolished recurrent ILDs and corresponding Cl- transients. (c) Baseline [Cl−]i distribution in subpopulations of neurons before (IED and ILD phases), during and after application of WNK463. Box (left) + data (right) plots correspond to median (25%–75%) [Cl−]i in individual cells (filled symbols) and their Gaussian distribution curves; open squares and whisker range indicate mean±SD. WNK463 significantly reduced the median interictal [Cl−]i (*P<0.05; Friedman RM ANOVA on Ranks). (d) Corresponding interictal [Cl−]i changes induced by WNK463. Data were fitted with a linier regression fit. (e) WNK463 (1 µM) did not change baseline [Cl-]i when synaptic activity had already been suppressed with the sodium channel blocker TTX (1 µM). (f) Baseline [Cl−]i distribution and corresponding [Cl−]i changes induced by TTX and WNK463 in the presence of TTX (*P<0.05; Friedman RM ANOVA on Ranks). (g) Corresponding interictal [Cl−]i changes in individual cells induced by TTX (black symbols and line) and baseline [Cl−]i changes induced by WNK463 in the presence of TTX (red symbols and line). Data were fitted with a linear regression fit.

Effects of the WNK-SPAK/OSR1 blocker WNK463 on neuronal chloride transients during recurrent ictal-like epileptiform discharges.

(a, c) Neuronal Cl- changes in individual cells as a function of time and corresponding mean [Cl−]i transients in control ACSF (a1-2), and before and during WNK463 (1 µM) application (c1-2). (b, d) Corresponding rise time and decay time constant of [Cl−]i transients in individual cells (open symbols) as a function of time in control (b), and before and during WNK463 application (d). Filled symbols indicate group mean ± SD. WNK463 progressively decreased the mean decay time constant of [Cl−]i transients (*P<0.05, **P<0.01, ***P<0.001; One Way RM ANOVA). (e)[Cl−]i rise time and decay time constant of neuronal chloride transients in individual cells before and during WNK463 application (n/s corresponds to P>0.05, ***P<0.001, One Way RM ANOVA, Tukey’s test). Box (right) + data (left) plots correspond to median (25%–75%) [Cl−]i in individual cells (open symbols); open squares and whisker range indicate mean±SD. (f) The mean ILD duration, corresponding [Cl-]i rise time and decay time constant of neuronal chloride transients, and corresponding normalized parameters before and during WNK463 application. WNK463 progressively reduced the mean duration of ILDs (black symbols) in line with enhanced Cl-extrusion rate (blue symbols).

WNK463 had no effects in the presence of the GABAA-receptor antagonist and CCCs inhibitors

(a, d) Extracellular field potential recordings in the CA1 pyramidal cell layer in the organotypic hippocampal slices in vitro. Expansion of recurrent interictal (IEDs) and ictal (ILDs) epileptiform discharges in control and during drug applications. (b, c) Application of the GABAA-R antagonist SR95531 (10 µM) abolished spontaneous ILDs and induced large amplitude IEDs. Subsequent application of WNK463 (1 µM) in the presence of SR95531 did not change the mean frequency and duration of epileptiform discharges and corresponding power of electrical activity (n/s – P>0.05; *P<0.05; Friedman RM ANOVA on Ranks, Dunn’s test). (e) Cation-chloride cotransporter blocker bumetanide (0.5 mM) reduced the mean frequency of ILDs, corresponding power of electrical activity, and prevented the anti-ictal effects of WNK463 (n/s – P>0.05; *P<0.05; ***P<0.001; One Way RM ANOVA, Tukey test). (f) The mean effect of WNK463 on the power of epileptiform activity (percent of treatment-preceding power) was significantly different as the corresponding effects of WNK463 in the presence of 10 µM SR95531 or 0.5 mM bumetanide (***P<0.001; One Way ANOVA, Holm-Sidak Test).

Anti-ictal effects of WNK463 in the presence of NKCC1 blocker bumetanide and KCC2 blocker VU0463271.

(a, c) Extracellular field potential recordings in the CA1 pyramidal cell layer in the organotypic hippocampal slices in vitro. WNK463 (1 µM) was applied in the presence of (a) NKCC1 blocker bumetanide (10 µM) and (c) KCC2 blocker VU0463271 (1 µM). Expansion of epileptiform discharges before and during drugs applications. (b) WNK463 application in the presence of bumetanide significantly reduced the mean frequency of recurrent ILDs in control and the mean power of electrical activity. (d) WNK463 in the presence of VU0463271 significantly reduced the mean frequency of ILDs in control and the mean power of electrical activity in the presence of VU0463271 alone (*p<0.05, **p<0.01, ***p<0.001; One-Way RM ANOVA, Tukey test).

siRNA targeted Slc12A2 (NKCC1) and Slc12a5 (KCC2) gene silencing did not prevent the anti-ictal effects of WNK463.

(a) Cellular penetration by Accell green (fluorescein) non-targeting siRNA in the organotypic hippocampal slice in vitro at DIV12. (b) Simultaneous extracellular field potential recordings in the CA1 pyramidal cell layer revealed spontaneous multiple unit activity (MUA), interictal epileptiform discharges (IEDs) and ictal-like epileptiform discharges (ILDs). Expansion of recurrent ILD during recordings. (c, d) NKCC1 (green) and KCC2 (red) antibodies overlaid with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) in control (N/T siRNA) and NKCC1 and KCC2 siRNA treated organotypic hippocampal slices. Optical density of NKCC1 and KCC2 immunostaining relative to DAPI staining revealed significant siRNA targeted Slc12A2 and Slc12a5 gene silencing and protein expression (*P<0.05;***P<0.001; Mann-Whitney test on ranks). (e, f) WNK463 (1 µM) abolished spontaneous ILDs (marked by asterisks) in control slices treated with non-targeting (N/T) siRNA. (g, h) WNK463 (1 µM) abolished spontaneous ILDs (marked by asterisks) in slices treated with Slc12a2 (NKCC1) siRNA. (i, j) WNK463 (1 µM) abolished spontaneous ILDs (marked by asterisks) in slices treated with Slc12a5 (KCC2) siRNA (*P<0.05, **P<0.01, ***P<0.01, One-Way RM ANOVA, Tukey test).

Simultaneous NKCC1 and KCC2 inhibition reduced the anti-ictal effects of WNK463.

(a) Extracellular field potential recordings in the CA1 pyramidal cell layer in the organotypic hippocampal slices in vitro. WNK463 (1 µM) was applied in the presence of NKCC1 and KCC2 blockers bumetanide (10 µM) and VU0463271 (1 µM). Expansion of recurrent ILDs before and during drug applications. (b) WNK463 application in the presence of bumetanide in composition with VU0463271 did not change the frequency of ILDs but significantly reduced the mean power of epileptiform discharges preceding WNK463 application (*p<0.05; One-Way RM ANOVA, Tukey test). (c) Summary data of the effects of WNK463 on the mean power of electrical activity in control ACSF, in the presence of bumetanide (10 µM), in the presence of VU0463271 (1 µM), and in the composition of bumetanide (10 µM) and VU0463271 (1 µM). Simultaneous NKCC1 and KCC2 inhibition significantly reduced the anti-ictal effects of WNK463 (**P<0.01; One Way ANOVA, Holm-Sidak test).