Abstracts

Rationale: Epilepsy, a disease plaguing more than 50 million people worldwide, is characterized by recurrent, unprovoked seizures associated with a three-fold increase in mortality. Current anti-epileptic medications (AEM) inhibit neurons, consequently dimming cognitive function. We aim to find non-neuronal targets for AEMs to circumvent the deleterious side effect of current treatments. A potential target is astrocytic volume regulated anion channel (VRAC), an outward rectifier channel. Activated by swelling, VRAC expels anions and organic osmolytes—including glutamate—into the synapse after a synaptic event. We hypothesize that inhibition of VRAC will decrease glutamate efflux into the synaptic space, thereby reducing neuronal excitation and diminishing neurotoxicity. In this study, we aim to answer the key question: Is VRAC regulated by seizures in epilepsy?

Methods: 8-10-week-old male C57BL6 mice received a 46 nL intrahippocampal kainic acid (IHKA) injection (20 mM kainic acid, Tocris) using a microinjector to induce status epilepticus (SE). Controls were treated with saline. Mice were euthanized 1, 7, 14, and 30 days post-IHKA for Western blot and IHC. WB: blots were incubated O/N at 4°C in 5% milk with primaries against LRRC8A (Cell Signal #24979) and β-Actin (Sigma-Aldrich A2228), then stained at RT for 1 hr in 5% milk with secondaries (LI-COR IRDye Goat anti-Rabbit and Donkey anti-Mouse). IHC: sections were incubated O/N at 4°C in 10% BSA with primary antibodies against LRRC8A (Abcam 157489) and GFAP (Abcam ab7260), then incubated with secondaries conjugated with Alexa 488-Tyramide or Alexa 594.

Results: Changes in expression levels of LRRC8A, the essential subunit of VRAC, were determined at various time points in the epileptogenic process. WB quantification demonstrated a significant increase in LRRC8A expression in the ipsilateral (n=8, p=0.0023) and contralateral (n=8, p=0.0026) dorsal hippocampus 14 days post-IHKA. IHC quantification corroborates this finding, as there was an increase in the immunoreactivity of LRRC8A in both the ipsilateral (n=5, p< 0.0001) and contralateral (n=5, p=0.0003) dorsal hippocampus at 14 days post-IHKA. Layer-specific quantification demonstrated upregulation of LRRC8A in all layers of the ipsilateral hippocampus except S. Lacunosum Moleculare (p ≤ 0.0086). In the contralateral hippocampus, LRRC8A was upregulated in all layers except S. Lacunosum Moleculare, the molecular layer, the granule cell layers, and the hilus (p ≤ 0.0213).