Altered Hippocampal Activation in Seizure-Prone CACNA2D2 Knock-out Mice

Granule cell activation after handling-induced convulsions. A, Images of c-fos expression (red) 1 h after gentle handling in representative WT and KO mice. Cell nuclei were visualized using DAPI stain (blue). B, Gentle handling caused convulsive seizures in CACNA2D2 KO, but not WT, mice (n = 4, 4; ***p < 0.0001). C, Granule cell c-fos expression was reduced in CACNA2D2 mice after handling-induced convulsions (n = 4, 4; *p = 0.023). D, Images of ΔFosB expression (green) 1 h after gentle handling in representative WT and KO mice. E, Granule cell ΔFosB expression was increased in CACNA2D2 mice after handling-induced convulsions (n = 4, 4; **p = 0.009).

CACNA2D2 KO mice have a reduced threshold for PTZ -induced granule cell activation. A, Dentate c-fos expression (red) after low-dose (30 mg/kg) PTZ administration in WT and KO mice. B, Maximum seizure intensity after PTZ (n = 8, 8; ***p = 0.005). C, c-fos+ cell density after PTZ (n = 8, 8; *p = 0.028).

Hilar mossy cell and CA3 cell densities in CACNA2D2 KO mice. A, Hilar mossy cells were identified as calretinin-positive cells (red; arrows) located within the hilar space and distinct from the small calretinin-positive immature granule cells in the SGZ. Mossy cell axons appear as a calretinin-stained band in the IML, just outside of the GCL (blue, DAPI). B, Quantification of hilar mossy cell density (n = 8, 8; *p = 0.037). C, CA3 pyramidal cell layer seen in cross section, visualized using DAPI nuclear stain (blue). White lines designate three separate locations at which CA3 pyramidal layer width was measured. D, Quantification of CA3 pyramidal cell layer width (n = 6, 6; p = 0.94). E, Quantification of CA3 pyramidal cell layer density (n = 6, 6; p = 0.46).

Mossy fiber sprouting is absent in CACNA2D2 KO mice. A, Granule cell mossy fiber axon terminals in WT and KO mice were identified by staining for the mossy fiber terminal marker, ZnT3 (magenta). GCL highlighted with DAPI (blue). B, ZnT3-stained terminals were exclusively found within the hilar space, and not within the IML, which would have indicated pathological retrograde sprouting.

Hippocampal cell proliferation and granule cell neurogenesis are unchanged in juvenile CACNA2D2 KO mice. A, Mitotic cells in the dentate SGZ identified by the proliferation-associated nuclear marker Ki67 (red) in WT and KO mice; GCL nuclei were counterstained with DAPI (blue). B, Immature granule cell neurons identified by expression of the microtubule-associated protein Dcx (green) in WT and KO dentate sections. C, Quantification of Ki67+ cell density (n = 6, 6; p = 0.11). D, Quantification of Dcx+ cell density using staining fluorescence intensity in the GCL (n = 6, 6; p = 0.58).

Parvalbumin interneuron staining in WT and CACNA2D2 KO mice. A, Confocal image projections of parvalbumin-stained (PV, red) dentate gyrus tissue from WT and KO mice. B, C, Quantification of PV+ cell density within both the GCL (B; n = 6, 6; p = 0.29) and dentate hilus (C; n = 6, 6; p = 0.97). D, Confocal image projections of PV-stained hippocampal CA3 pyramidal cell layers from WT and KO mice. E, Quantification of PV+ cell density in the CA3 pyramidal cell layer (n = 6, 6; p = 0.40).