Toward a Mechanistic Framework of Infrared Neural Stimulation: Methods for Linking Subcellular Calcium Responses of Cortical Neurons to Spatiotemporal Photothermal Gradients

Abstract

Neuromodulation therapies have transformed the treatment of neurological disorders. Current devices apply an electric field to induce stimulation, but the current spread between electrodes can lead to off-target stimulation. Therefore, alternative neuromodulation approaches are under investigation to improve the precision of stimulation. Infrared neural stimulation (INS) uses short-wave infrared laser pulses delivered via optical waveguides to spatially confine stimulation to the irradiated tissue. Yet the effects of INS within the cortex remain poorly understood, limiting its translation for clinical and research applications. The objective of this dissertation is to link the thermal dynamics of photothermal laser-tissue interactions to cortical neuron responses to elucidate how laser parameters influence the underlying mechanisms. Optical microscopy combined with fluorescent calcium indicators enabled monitoring of cellular and subcellular responses to varying laser parameters, including beam diameter and profile, pulse duration, and pulse energy. Using a previously overlooked imaging artifact caused by thermal heating, I present a methodology that extracts the spatial distribution of the laser beam to improve optical dosing precision and correlate it with neuronal responses. The laser beam size influences these responses; reducing it, and therefore the thermal relaxation time of the irradiated spot, reveals a bimodal calcium response, indicating that mechanisms recruited by INS depend on the spatiotemporal thermal gradient. Further, spatially confining stimulation to dendritic projections resulted in whole-cell calcium activity, revealing a structural dependence of stimulation. Targeting dendrites elicited calcium activity in dendritic spines that is qualitatively distinct from somatic activity. However, the induced calcium activity did not drive cytoskeletal remodeling, distinguishing photothermal-evoked calcium activity from traditional spine calcium signaling. These results demonstrate that cortical neuronal responses to INS depend on the spatiotemporal thermal gradient and the neuronal structures exposed, providing a framework for designing INS devices around the mechanisms and net physiologic effect they intend to recruit.