Morphological and Electrophysiological Assessment of Pediatric Tuberous Sclerosis: The Spatial impact of Brain Tubers in an Ex Vivo Human Brain Slice Model

Introduction: Tuberous Sclerosis Complex(TSC) causes benign tumors in multiple organs including the brain and are associated with significant neurological manifestations including medically intractable epilepsy requiring neurosurgical intervention. Current models lack the ability to conduct long-term electrophysiologic and pharmacologic studies. Using our novel long-term brain slicing assay protocol we assessed electrophysiologic changes, further confirming our brain organoid TSC model findings.

Methods: en-bloc resections of three tubers from two patients for epilepsy was conducted. Slices were acutely oxygenated in sucrose/pyruvate enriched artificial CSF(aCSF), vibratome sectioned (250microns), and cultured with neuroprotective media using ROCK-inhibitor. Slices were transduced with GFP-fluorescent EF1-A, SYNAPSIN1, gCAMP6 to assess viability, neuronal health, and Ca2+-influx of Action Potentials respectively. Patch-clamping, Multielectrode Array, Immunofluorescence(I.F.) were used at various timepoints up to 30 days. Slices were treated with Rapamycin vs vehicle and assessed for electrophysiologic changes. I.F., Transmission Electron Microscopy(TEM), and single nuclear RNA sequencing were conducted from patient samples comparing tuber core, edge, and surrounding parenchyma.

Results: TEM demonstrated dysregulated mitochondria in TSC specimens compared to normal control. I.F. showed Neurofilament light-chain(NfL) upregulation in the tuber core, significantly decreasing at the border, and even further outside of the tuber(P < 0.05) although this region still contained higher NfL vs normal control (P < 0.001). Brain slice TSC-GFP+ neurons demonstrated significant enlargement of somas/dendritic spines compared to control(P <.005). Patch-clamping showed increased firing frequency, resting membrane potential, and action potential amplitude compared to control(P < 0.05). Additionally, TSC neurons failed to return to resting membrane potential baseline, further enhancing neuronal network activity. gCAMP6-TSC neurons Ca2+ imaging demonstrated increased action potential frequency compared to normal(P < 0.05) which was decreased with Rapamycin(P < 0.05).

Conclusion : Our novel brain slice assay as well as spatial analysis of TSC tubers has for the first time demonstrated ex-vivo electrophysiological/morphological characteristics of TSC neurons, serving as a foundation for understanding ¬¬cellular changes of this disease, and opening avenues for therapeutic intervention.