Harnessing Calcium Influx to Visualize and Regulate Seizure Networks

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PROJECT SUMMARY. Epilepsy, with a prevalence in the United States of 7 per 1000 people, is the fourth most common neurological disorder. While many patients achieve relief from disabling seizures through medication, about one third of epilepsy cases are pharmacoresistant. For many of these medically intractable cases, surgical intervention is indicated and while seizure freedom is obtained in as many as three quarters of these patients, the surgical approaches have substantial limitations and drawbacks. Open resection and ablation are destructive and permanent, and neurostimulation, necessitating implanted hardware, presents a risk for hardware malfunction and infection. Furthermore, these methods lacking cell-type specificity impact all tissue in the region targeted, carrying a risk for off target effects. Hence there is a need for an epilepsy treatment that is specific to the neurons initiating or propagating the pathologic neuronal activity. Development of such a treatment would be greatly informed by a better understanding of seizure activity dynamics, especially at a cellular resolution. The majority of the current understanding of seizure dynamics come from electrophysiology, including EEG, which only offers population resolution of activity, and single unit recordings. While single units record activity from individual neurons the density of neurons recorded is sparse and it is exceedingly difficult to know what subtype of neuron is being recorded. Genetically encoded calcium indicators (GECI) circumvent many of these drawbacks and allow for observation of the activity of individual neurons through the use of two-photon microscopy in animal models. While this approach has been taken to visualize acute seizures in a rodent model, this work has yet to capture seizures in spontaneous seizure models, which better approximate epilepsy. Thus, we first aim to examine spatiotemporal firing of neurons within seizure networks in a chronic mouse model of neocortical seizures and parse the activity by neuronal subtype (Aim 1). The light emitted during this activity will then be harnessed to develop an activity responsive neuromodulatory agent to allow networks to self-regulate. Our lab developed opto-chemogenetic agents, luminopsins, which we have used for cell subtype specific, hardware independent in vitro and in vivo neuromodulation. Specifically, luminopsins are light responsive ion channels or pumps fused with their own light source, a bioluminescent enzyme. We aim to modify the luminopsin construct, exchanging the bioluminescent enzyme with a bioluminescent GECI, which will result in a luminopsin whose functionality is contingent upon sufficient intracellular calcium and thus activity. The calcium binding affinity for these responsive luminopsins will be selected such that they are only responsive to high intracellular levels of calcium, such as those experienced during seizures, which would allow for preservation of non- pathologic neuronal activity. Responsive luminopsins will then be introduced in vivo in a spontaneous seizure model to observe if they are able to enable these networks to self-regulate (Aim 2). Ultimately the development of such knowledge and tools could be used to inform development of future treatments for epilepsy.