Introduction:

Astrocytes regulate neuronal excitability through potassium buffering mediated by Kir4.1 inwardly-rectifying potassium channels. Reduced astrocytic Kir4.1 expression has been reported in human temporal lobe epilepsy, yet its causal role in epileptogenesis remains unclear. In epilepsy, spreading depolarisations (SD), slowly propagating waves of neuronal and glial depolarisation, are temporally associated with seizures (seizures+SD) and have been associated with post-ictal period (period immediately following seizure termination) and Sudden Unexpected Death in Epilepsy. However, whether astrocytic Kir4.1 loss is sufficient to drive seizures and SD, and how SD shape post-ictal state, is unknown.

Methods:

We generated focal Kir4.1 knockout mice (Kir4.1-cKO) by deleting the Kcnj10 gene encoding Kir4.1 channels in hippocampal astrocytes of adult Kir4.1-floxed mice using viral Cre recombinase. Ex vivo validation of Kir4.1 loss was performed using western blotting. Network electrophysiological activity and extracellular potassium dynamics were examined in hippocampal slices using local field potential (LFP) and genetically-encoded potassium indicator (GINKO) fluorescence recordings, respectively. To test seizure and SD susceptibility in vivo, optogenetics and DC-coupled graphene micro-transistor arrays were used in awake head-fixed mice. Continuous AC-coupled, and video-DC-coupled ECoG telemetry recordings were employed to characterise the emergence of spontaneous seizures and SD.

Results:

Kir4.1-cKO showed significantly reduced Kir4.1 protein expression (Kir4.1 expression relative to GAPDH: Controls, 0.85±0.11, n=5 mice; cKO, 0.46±0.08, n=4 mice; p=0.03). Electrical stimulation of Schaffer collaterals evoked larger LFP and GINKO fluorescence responses in CA1 stratum radiatum, indicating impaired potassium buffering. Chronic AC-coupled telemetry in Kir4.1-cKO (n=10 mice) revealed the emergence of spontaneous seizures 7–10 days post-viral injection. In awake head-fixed mice, Kir4.1-cKO hippocampal networks show increased susceptibility to optogenetic stimulation-induced seizures and seizures+SD. Optogenetic trains (10 s of 5, 20, and 50 Hz) induced significantly larger DC shifts in Kir4.1-cKO (1.78±0.37 mV; n=15 trials; 5 mice) than in controls (0.30±0.09 mV; n=12 trials, 4 mice; p<0.0001). Larger DC shifts were associated with the occurrence of seizures (seizure: 2.03±0.43 mV, n=12 trials; no seizure: 0.39±0.09 mV, n=15 trials; p=0.003). At higher stimulation frequencies, seizures+SD occurred in the majority of Kir4.1-cKO mice (20 Hz: 60%; 50 Hz: 80%). Seizures+SD profoundly altered the post-ictal state, producing significantly prolonged post-ictal depression and recovery period (seizures: 112.02±25.56s, n=4; seizure+SD: 360.10±75.84s, n=7; p=0.016).

Chronic video-DC-coupled telemetry (six mice monitored for three weeks) revealed spontaneous seizures alone (n=38) and seizures+SD (n=40). Compared with seizures alone, seizures+SD exhibited higher power (25-80 Hz: seizures, 8.91±0.82 a.u.; seizure+SD, 16.49±1.04 a.u.; p<0.0001) and longer intervals to the next seizure event (inter-event intervals: seizures, 113.70±34.29 mins; seizures+SD, 168.01±35.94 mins; p=0.002). Video-DC-coupled ECoG analysis of post-ictal behaviour (120-second period following seizure termination) revealed altered behaviour such as behavioural arrest, loss of posture, and body jerks for ~87% (104.83±5.54 s) of the post-ictal period after seizures+SD (n=12), compared with ~1.7% after seizures alone (2.00±1.13 s, n=12; p<0.0001), indicating severely delayed functional recovery.

Conclusion:

Focal hippocampal astrocytic Kir4.1 loss is sufficient to generate spontaneous seizures with a high incidence of SD, which exacerbate post-ictal impairments. This model provides mechanistic insight into how astrocytic potassium dysregulation drives epileptogenesis and seizure pathology.