Abstracts

Rationale: Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of mortality in patients with epilepsy, but the underlying pathophysiological mechanisms leading to death remain poorly defined. Spreading depolarization (SD) is a slow wave of depolarization in neurons and glia that results in transient silencing of brain activity and has been observed in neurological disorders, including migraine and stroke. Our lab previously demonstrated that SUDEP mouse models have a lowered threshold for SD which puts them at risk for cardiorespiratory dysfunction in the peri-ictal period. We reported that a transgenic mouse strain in which transcription of the X-linked 5-HT2C receptor is blocked (loxTB Htr2c) displays a complex epileptic phenotype with increased premature death. We hypothesized that 5-HT2C-null mice have a lowered SD threshold which increases their risk of death following a seizure.  Methods: We prepared 300 µm thick coronal slices from forebrains of mice between postnatal day 25 and 35. These forebrain slices included dorsal hippocampal tissue. Mice were deeply anesthetized with Avertin (250 mg/kg, i.p.) and decapitated. Next brains were harvested in cold dissection solution and sliced with a vibratome. The slices were then incubated in aCSF for 20 minutes before being transferred to a recording chamber and imaged with an upright microscope with a CCD camera. Slices were exposed to oxygen-glucose deprivation (OGD) aCSF for 20 minutes and SD was detected by a band of increased light transmission traveling across the tissue.  Results: We found that in 100% (6/6) of 5-HT2C-null mice, SD was observed in both hippocampus and cortical areas compared to only 50% (2/4) wildtype littermate controls. In addition, the SD observed in 5-HT2C-null mice was more robust and widespread than in their wildtype littermates. For example, SD was observed in all areas of cortex, hippocampus, and subcortical structures imaged in 100% of 5-HT-2C-null mice (6/6) compared to 25% of wildtype littermates (1/4). Of the two wildtype mice that demonstrated SD, one was minor compared to null mice, as it occurred transiently in a localized region and did not propagate to the rest of the tissue. The second wildtype mouse was the only female studied in this preliminary cohort, which may suggest a sex difference in SD threshold. Conclusions: These preliminary data suggest that 5-HT2C-null mice have a lower threshold for SD before EEG abnormalities, seizures, or premature mortality are observed. This may predispose 5-HT2C-null mice to death following seizures later in development. These data suggest that the physiology of the 5-HT2C-null mice is perturbed at an earlier age than we previously observed with in vivo EEG recordings. Future studies will investigate whether a lower SD threshold may be an early biomarker for hyperexcitability during epileptogenesis in this strain. We will test the effects of less severe OGD conditions which may help further distinguish differences between wildtype and 5-HT2C-null mice. Since the 5-HT2C receptor is X-linked and the only wildtype mouse with robust SD was female, we will investigate whether there is a sex difference in SD threshold.  Funding: This work was funded by NINDS U01 NS090340 (JLN) and F32 NS105329 (CAM).