Abstracts

Rationale: Neonatal seizures are common and the majority are due to hypoxic-ischemic encephalopathy (HIE). Use of tissue clearing, transgenic mice, and advanced microscopy allow neuronal activity mapping. Circuitry involved in these seizures is unknown and may shed light on the relation between early seizures and later behavioral deficits. Methods: Postnatal day (p)9 mice are implanted with electrodes for electroencephalography (EEG). On p10, HIE is created using modified Vannucci method (unilateral carotid ligation+45 min of 8%O2) in Cre-tamoxifen transgenic mice (TRAP). Injection of 4-hydroxytamoxifen 1hr after hypoxia allows expression of fluorescent protein in active neurons during 1-2 h prior. EEG is performed throughout experiment. Sham mice receive incision+anesthesia without hypoxia/ligation. Mice are perfused and tissue processed using lipid-clearing. Multiphoton imaging and ZeissZen is utilized to obtain/process images (tiling and creation of z-stacks thick tissue), Imaris 8.3 is used for analysis. Continuous video EEG is analyzed for seizures and scored using a behavioral seizure score (BSS) for neonatal rodents. EEG power spectrograms developed using LabChart. Results: 100% of mice exhibited seizures during hypoxia (n=30). Mortality rate was 13%, all following a convulsive seizure (BSS 5-6) during hypoxia. 43% exhibited seizures post-ligation/pre-hypoxia and 47% with post-hypoxia seizures. Tissue clearing/multiphoton microscopy allowed visualization of active neurons during seizures. Activity is robust in the entorhinal cortex, dentate granule cell layer, CA3 hippocampus, lateral thalamus and striatum ipsilateral to the carotid ligation. Both the contralateral hemisphere in HI mice and shams exhibit basal activity in the somatosensory cortex. Hemisphere ipsilateral to carotid ligation exhibited significantly more activated neurons when compared to the contralateral hemisphere (p<0.0001) and to sham mice (p<0.0001). EEG power spectrogram exhibits significant suppression and seizures during hypoxia with prolonged suppression post-hypoxia. Conclusions: Mice exposed to HI exhibit characteristic EEG findings of burst suppression and seizures during hypoxia, re-oxygenation seizures and persistent background suppression. Neuronal activity during seizures suggests ipsilateral activation of the hippocampal-cortical-striatal-thalamic pathway. Future directions will use these techniques to create 3D maps of neuronal activity during seizures and examine chronic EEG changes and neuronal activity in adult mice during learning & memory tasks. Funding: NIH NINDS K08NS101122 (JB)UVA Children's Hospital Funds (JB)