Abstracts

Rationale: Neuromodulation through intracranial electrical stimulation reduces seizures in patients with epilepsy. However, the neurophysiological mechanisms through which electrical stimulation leads to clinical benefit is unknown. Further, the stimulation parameters that achieve optimal neuronal responses are not known. Efforts to probe these questions using conventional electrophysiological techniques are limited by electrical artifact. Accordingly, we performed cellular optical voltage imaging to evaluate the impact of electrical stimulation on cortical excitatory or inhibitory neurons in a cortical stroke mouse model with epileptic activity.

Methods: In each mouse, a photothrombosis procedure was first performed to induce a focal stroke in the motor cortex. One week later, we infused AAV1-syn-FLEX-Voltron2 and AAV9-CamKII-Cre in the superficial layers in the C57BL6 mice to selectively express the hybrid voltage sensor Voltron2 in excitatory neurons and placed a 3 mm in diameter glass window and a pair of stimulation electrodes. Similarly, to record from the inhibitory neurons, we infused AAV1-syn-FLEX-Voltron2 into PV-cre mice to express Voltron2 selectively in PV positive fast spiking interneurons. Voltage imaging was performed at 0.8kHz frame rate using a custom confocal microscope, while mice were head-fixed on a treadmill that permitted voluntary locomotion. During each experimental session, 36 electrical stimulation trials were performed, with each trial lasting for 11 seconds, containing 1s baseline and 10s of stimulation. Four stimulation frequencies, 40, 140, 200, or 1000 Hz, in random orders, were tested. The stimulation current amplitude and pulse width were manually determined and kept constant across frequencies in each mouse.


Results: Intracranial electrical stimulation at different frequencies produced robust membrane voltage changes across individual neurons as demonstrated in Figure 1. Our preliminary analysis suggested that some neurons exhibit consistent and transient depolarization during each of the 100ms stimulation periods, which was followed by hyperpolarization (Figure 2).


Conclusions: We demonstrate that cellular membrane voltage imaging allows artifact free analysis of the cellular and circuit effects of intracranial electrical stimulation. By testing the effect of different stimulation pulse patterns on excitatory pyramidal neurons versus fast spiking interneurons, this study will derive a principled understanding of the effect of stimulation pulse parameters on neuronal dynamics that is relevant to epilepsy.

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