Abstracts

Rationale: Disorders caused by mutations in the mechanistic target of rapamycin (mTOR) pathway genes, exemplified by Tuberous Sclerosis Complex (TSC), lead to mTOR hyperactivity, brain malformations, and life-long epilepsy in the majority of patients. While partially effective, surgery or pharmacotherapy with mTOR inhibitors are the only available treatments for epilepsy in TSC. A better understanding of epileptogenic mechanisms in the disease is therefore needed. Two modern hypotheses compete to shed light on the underlying mechanisms of epileptogenesis in TSC. 1) the neuronal hypothesis implicates dysplastic neurons and their abnormal activity as the source of seizure activity. 2) the neuro-glial network hypothesis implicates a pathophysiological network of neuro-glial interplay that occurs outside of cortical tubers. To test whether dysplastic neurons in TSC mediates epileptogenesis, we aimed to isolate the seizure focus, measured excitability of dysplastic neurons, and tested the tunability of those neurons alone to modulate seizure severity in a mouse model of cortical tubers with spontaneous epileptogenesis. Methods: Experimental model of cortical tubers that only consists of dysplastic neurons is made by transfecting a small population of cortical pyramidal neurons (PNs) in vivo with 2 µg of a plasmid encoding a constitutively active mTOR activator, ras-homolog enriched in brain (RhebCA). To create a single small seizure focus, we targeted a small population of PNs in the medial prefrontal cortex, using in utero electroporation (IUE). Under the constitutively active promotor CAG, RhebCA effectively hyperactivates mTOR in these neurons, driving dysplasia and causing spontaneous epileptogenesis. To localize seizure focus, we utilized fully independent multi-channel electroencephalogram (EEG) recordings for each of the animals described above. Analyzing 7 days of continuous EEG recordings for each animal, we focused particularly on high frequency activity above 100Hz because those correlated completely with seizure events. To locate the region of initiation, we extracted temporal differences between the channels.To change seizure frequency in each cohort of experimental animals, we varied the amount of DNA concentration used during IUE, while keeping the electroporated region and extent the same.Excitability and biophysics of dysplastic neurons were assessed via whole-cell patch clamp technique, in acute brain slices obtained freshly from experimental and control animals.Suppression of excitability in dysplastic neurons was achieved molecularly by co-transfection of Kir2.1 encoding plasmid with RhebCA plasmid during IUE. Results: We found, after analyzing independent EEG recordings that high frequency oscillations (HFOs), which correlate with cortical seizures, initiate in and around the tubers before traversing to the contralateral hemisphere. We also found that dysplastic neurons express more sodium currents, can sustain longer bursts of regenerative firing, and are more depolarized than normal neurons in and outside of cortical tubers as well as in control animals. Furthermore, suppressing activity in the dysplastic neurons alone, with the expression of an exogenous inward-rectifier potassium channel Kir2.1, mitigates seizure activity. Finally, the escalation of neuronal dysplasia severity, i.e., larger cells and greater migration deficits, leads to greater seizure activity. Conclusions: Together these evidences suggest that dysplastic neurons mediate epileptogenesis in TSC. Funding: NIH Grant NS093510DoD TS150058Swebilius