Abstracts

Rationale: Interictal epileptiform discharges (epileptic spikes), high-frequency oscillations (pathological ripples, 80-250 Hz), and spike ripples (SR, the co-occurrence of spikes and ripples) are all well-known epileptic biomarkers in cortical local field potential (LFP) recordings. While much research has been done on their utility to detect epileptogenic tissue, the cellular and network mechanisms that generate these biomarkers remain poorly understood. Experimental testing of these hypotheses has been difficult, as traditional electrophysiological techniques lack cell type specificity and cellular calcium imaging is too slow and an indirect measure of membrane voltage. To examine the contribution of excitatory and inhibitory neurons to epilepsy biomarkers, we performed ultrafast (800 Hz) cellular voltage imaging using a hybrid voltage sensor Voltron2 from many individual neurons of these two cell types in a cortical stroke mouse model.

Methods: We first introduced unilateral strokes in the motor cortex of mice using a well-established photothrombotic procedure to generate epileptiform activity. One week later, to express the voltage sensor Voltron2 in excitatory pyramidal cells, we infused AAV1-syn-FLEX-Voltron2 and AAV9-CamKII-Cre into the superficial layers of the cortex next to the stroke site in C57BL6 mice, and then placed a 3 mm diameter glass window and a pair of LFP electrodes. To image parvalbumin (PV) positive fast spiking interneurons, AAV1-syn-FLEX-Voltron2 was infused into PV-cre mice instead. As Voltron2 is a hybrid voltage sensor requiring chemical cofactors, JF-552 HaloTag ligand was systemically administered at least 24 hours prior to imaging. Voltage imaging was performed while mice were awake and head-fixed on a treadmill permitting voluntary locomotion. Membrane voltage (Vm) of individual cortical neurons were imaged using a custom confocal microscope (Fig. 1), and simultaneous LFP was collected using an OpenEphys system.

Results: Our previous study showed that photothrombosis induced cortical stroke results in selective increases of spikes, ripples, and SRs in the injured hemisphere (bioRxiv 2024.03.01.582958). To examine changes of Vm around LFP spikes, ripples, and SRs, we aligned Vm to the corresponding epileptic biomarkers (Fig. 2). Our preliminary analysis revealed evidence of transient Vm changes in both excitatory and inhibitory neurons around these biomarkers. For example, we observed pyramidal cells with robust Vm hyperpolarization preceding SRs, then depolarization at and following SRs. Changes in inhibitory neuron activity were less organized around the LFP events. Continued analysis of single neuron activity from this mouse model will yield informative results on the role of excitatory and inhibitory cells in supporting LFP spikes, ripples, and SRs.

Conclusions: Voltage imaging analysis of excitatory and inhibitory neurons in a cortical stroke mouse model with increased seizure risk provides a powerful platform to study the physiological mechanisms of epilepsy biomarkers.

Funding: R01NS119483, R01NS110669, AES Predoctoral Fellowship, 1R01MH122971, R01EB029171, NSF 1955981-CIF, 1RF1NS129520