Abstracts

Rationale: Microglia modulate neuronal activity during seizures and post-seizure neuropathology and are thus attractive targets for anti-epileptic interventions (Eyo et al., 2014; Wu et al., 2020). However, microglial cellular and molecular mechanisms in epilepsy are not completely understood. RNA sequencing data from our lab and pharmacological data from others have pointed to a potential role for the volume regulated anion channel (VRAC) in acute seizures. While VRAC classically helps cells regulate their volume under hypo-osmotic stress (Qiu et al., 2014), it has multiple cell-type and context dependent functions. This channel is encoded by the LRRC8 gene family with LRRC8a encoding its obligatory subunit called SWELL1 (Qiu et al., 2014). Here, we investigated the role and mechanisms of microglial VRAC in acute seizures and post-seizure neuropathology using SWELL1 conditional knockout (cKO) mice and primary cells.


Methods: Both male and female mice were used in accordance with guidelines from the Mayo Clinic Institutional Animal Care and Use Committee. We used a tamoxifen inducible Cre-lox system to selectively delete SWELL1 in microglia of adult mice. Acute seizures were induced in these mice through an intra-cerebroventricular (ICV) injection of kainic acid (KA, a glutamate agonist). Seizure severity was quantified using a modified Racine scale (Eyo et al., 2014). Mice brains were collected at various time points after seizure induction to examine pathological changes. Additionally, primary microglial cultures were prepared from 1–7-day old pups for in vitro experiments.


Results: Highly efficient and microglia selective deletion of SWELL1 was confirmed through PCR in the brains of conditional knockout (cKO) mice as well as through functional testing of primary microglia in vitro. Male (but not female) microglial SWELL1 cKO mice showed an increased severity of acute seizures compared to littermate controls after ICV-KA injections. Despite more severe seizures, SWELL1 cKO mice showed relatively lower neuron loss in the hippocampal CA3 compared to the littermate controls at 3 days after seizure induction. On further evaluation we found significantly lower expression of the lysosomal marker CD68 in the SWELL1 cKO group. In line with the neuropathology findings, in vitro investigations of primary cultured microglia showed significantly reduced phagocytosis of opsonized beads by SWELL1 cKO microglia compared to multiple controls.


Conclusions: Our results demonstrate that microglial Swell1 modulates the severity of acute seizures and promotes phagocytosis in post-seizure brains. We thus elucidate novel molecular and cellular mechanisms underlying microglia-neuron cross talk during seizure and post-seizure neuropathology. We continue to investigate the mechanisms by which VRAC/ SWELL1 affects seizure severity and the downstream effect of reduced phagocytosis and relative neuro-preservation on post seizure memory and learning.

Funding: We thank NIH (NINDS, R01NS088627) for funding this research.