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Two-photon Optogenetic Stimulation Of Cells.
Dhakal, Kamal Raj
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The controlled stimulation of cells, especially neurons, is of significant interest both for basic understanding of neuronal circuitry as well as clinical interventions. Existing electrode-based methods of stimulation are invasive and non-specific to highly localized regions or specific cell types. Recently, optical stimulation of targeted neurons expressing light-sensitive proteins (opsins) using visible light has emerged as a powerful technique in neuroscience. However, the use of these optogenetic tools as a minimally invasive technique for in-vivo applications is limited to the study/intervention of superficial regions of the brain since, due to absorption and scattering, a significant amount of the visible light is lost as depth of penetration increases. Therefore, the deep-brain (at depths > 1 mm) optogenetic stimulation of neurons in behaving animals is equally invasive as the electrical stimulation since it requires delivery of light in close proximity to the cells of interest. It has been recently shown that by non-linear interaction of light with opsin, invitro two-photon stimulation using near-infrared (NIR) light is possible. While these experiments used microscope objectives to stimulate cells in vitro, this thesis reports in vivo, in-depth fiber-optic two-photon optogenetic stimulation (FO-TPOS) of neurons in mouse models. In order to optimize the deep-brain stimulation strategy, two-photon activation efficacy at different near-infrared laser parameters (average power density, wavelength, exposure, etc.) were characterized. The significantly-enhanced depth stimulation efficiency of FO-TPOS as compared to conventional single-photon beam was demonstrated both by experiments and Monte Carlo simulation. Manipulation of depth neuronal circuitry in ventral tegmental area (VTA) of the midbrain was achieved using FO-TPOS and confirmed by immunohistochemistry. FO-TPOS as demonstrated in this report will lead to a better understanding of in- vivo neural circuitry, and may help in therapeutic modulation of brain activity because this technology permits precise (few micron) and less invasive anatomical delivery of stimulation with high spatial and temporal specificity.