Abstracts

Rationale: Dravet syndrome (DS) is a severe childhood-onset epilepsy characterized by medically-intractable, temperature-sensitive seizures, developmental delay/intellectual disability, and features of autism spectrum disorder. DS is largely caused by dominant loss-of-function mutations in SCN1A encoding the type 1 neuronal voltage-gated sodium channel (Nav1.1). Prior studies in mouse models (Scn1a+/- mice)suggest that DS pathogenesis involves dysfunction of cerebral cortical GABAergic inhibitory interneurons with a potential contribution of pathological upregulation of non-Nav1.1 Na+ channels. However, much of this work has been performed on dissociated neurons in culture and in acute brain slices prepared from juvenile animals, with only limited in vivo data in the literature. Methods: Here, we perform two-photon (2P) calcium imaging of identified presumed excitatory pyramidal cells as well as defined subsets of interneurons in layer 2/3 sensorimotor neocortex during naturalistic seizures in awake, behaving Scn1a+/- mice in vivo, using the genetically-encoded calcium indicator GCaMP6 (f or s). Mice were head-restrained and free to run on a spherical treadmill. Seizures were induced via passive elevation of core body temperature in a custom-fabricated enclosure compatible with the 2P imaging system. Imaging data was aligned, cell profiles were automatically extracted and confirmed manually post-hoc, and fluorescence intensity was quantified as ΔF/F0 using custom-written Matlab routines. Results: We found that elevated temperature led to increased synchrony of pyramidal cell firing until seizure onset, although individual pyramidal cell profiles were variable. Parvalbumin (PV) positive interneuron firing increased initially with hyperthermia, then decreased immediately prior to seizure onset. All identified cells were recruited by seizure activity. Seizures were frequently followed by cortical spreading depression-like event that preceded a prolonged (30-90 second) period of post-ictal suppression, often leading to death. Conclusions: We show for the first time the structure of seizures across hundreds of identified neurons with cellular-level resolution using in vivo 2P calcium imaging. Our results provide evidence that PV interneurons are dysfunctional in young animals in vivo during awake behavior, and in a temperature-dependent manner. This may contribute to disinhibition of cerebral cortical networks that underlies the temperature sensitivity of seizures in Dravet syndrome as well as mechanisms of sudden unexplained death in epilepsy (SUDEP).   Funding: Burroughs Wellcome Fund Career Award for Medical Scientists; NIH NINDS K08 NS097633