Abstracts

Rationale: Tuberous sclerosis complex (TSC) is a genetic disease typically involving severe epilepsy. Neuropathological studies of TSC demonstrate abnormalities in astrocytes in cortical tubers, suggesting that astrocyte dysfunction is important for epileptogenesis. Alterations in gap junction coupling and spatial potassium buffering by astrocyte networks could represent a mechanism for promoting seizures. We previously showed that mice with Tsc1 gene inactivation primarily in glia (Tsc1^GFAPCKO mice) develop progressive epilepsy associated with abnormalities in astrocyte proliferation and function. Here we evaluated the possibility that seizures in Tsc1^GFAPCKO mice result from impaired gap junction coupling and spatial potassium buffering of Tsc1-deficient astrocytes. Methods: Brain expression of gap junction proteins localized to astrocytes (Cx26, 30, 43) and neurons (Cx36) was assayed in control and Tsc1^GFAPCKO mice (KO mice) by Western blotting. Astrocytic gap junction coupling was evaluated by immunohistochemical staining of astrocytes after diffusion of biocytin from a single biocytin-filled astrocyte for a fixed period of whole-cell recording in hippocampal slices. Potassium-buffering in hippocampal slices in response to electrical stimulation was assessed with potassium-selective microelectrodes. Results: The expression of Cx43, an astrocytic specific gap junction protein, by Western blotting was decreased in KO mice, whereas other astrocytic and neuronal gap junction proteins were unchanged. Biocytin staining found that the number of coupled astrocytes was significantly less in hippocampal slices from KO mice compared to controls. In control mice, biocytin spread from single injected astrocytes to 243±87 encircling cells (n=16 injections) after 20 minutes of whole-cell recording, as opposed to 118±57 cells in KO mice (n=20 injections), indicating impairment of astrocytic gap junction coupling in the KO mice. Finally, potassium-selective electrode recordings demonstrated a significantly larger rise in [K⁺]₀ in response to electrical stimulation in hippocampal slices from KO mice compared with control mice, indicating a deficiency in potassium buffering in KO mice. To assess the role of astrocytic gap junctions in this defective K⁺ regulation, we compared stimulation-induced [K⁺]₀ changes in the hippocampal slices before and after perfusion with the gap junction inhibitor, carbenoxolone. Carbenoxolone caused an additional increase in evoked [K⁺]₀in control mice but not in KO mice, suggesting that the impaired potassium buffering in KO mice is due to deficient astrocyte gap junction coupling. Conclusions: These findings suggest that Tsc1 inactivation in astrocytes causes defects in astrocytic gap junction coupling and potassium clearance, which may contribute to hyperexcitability and seizure development in Tsc1^GFAPCKO mice. Supported by NIH R01 NS056872 and the Tuberous Sclerosis Alliance. The authors thank Julian Meeks for guidance on potassium-selective electrode methods.