Abstract

The development of optogenetic tools has greatly advanced the capability of researchers to understand the connection between neuronal activity and behavior. Optogenetics makes use of opsins, which are light activated ion-channels, ion pumps, or G-Protein coupled receptor (GPCR-based) proteins used to regulate cellular activity. Coupled with technological advances in genetics and optical physics/microscopy, optogenetics can be used to control specific populations of cells in a time-locked, spatially specific manner. The experiments possible with optogenetics depend largely on the capabilities and limitations of the opsin used; thus, there have been continuous efforts to design and employ new opsins with a variety of characteristics (such as speed, trafficking, and wavelength sensitivity) to allow for a greater variety of optogenetic manipulations. Some examples of this include finding solutions to increase the efficiency and efficacy of inhibitory opsins and the implementation of two-photon (2P) activated optogenetic tools; these two areas are the focus of this proposal. Ion-channel or Ion-pump opsins have been the most widely used for optogenetic inhibition, though they are restricted by off-target effects, poor performance at synaptic terminals, and require constant light to maintain their effect. Recently, there has been a shift in towards the development and use of GPCR-based opsins for inhibition, given they are more efficient synaptic terminal inhibition and have fewer off target effects. Opposed to ion-channel/pump direct hyperpolarization through the movement of Cl- or H+ ions, inhibitory GPCRs utilize Gi/o-protein effectors which lead to the activation G-Protein Coupled K+ channels (GIRKs) to hyperpolarize cells or inhibition of Voltage Gated Calcium Channels (VGCCs). The push to use GPCRs for optogenetic inhibition is recent, so there are just a few inhibitory GPCR opsins available. Lamprey parapinopsin, a reversibly activated inhibitory GPCR, is one example and the subject of this dissertation. To our knowledge, none of the GPCR-based inhibitory opsins have been characterized for 2P-use in neurons. 2P light has several advantages over single-photon excitation, allowing for greater depth penetration and excitation restrained to a single cellular-plane due to the lack of scattering. The off-target effects of ion channel inhibitory opsins along with the lack of GPCR-based opsin operational within multi-photon spectra excludes inhibitory optogenetics from many of the advantages of 2P microscopy, including multiplexing with other 2P sensitive tools. In this dissertation, we characterize LcPPO sensitivity to 2P-wavelengths by assessing LcPPO-GIRK coupling in a heterologous system and neurons. We then move on to understand how 2P-stimulated LcPPO (2P-LcPPO) affects somatic excitability in cultured neurons. The goal of this project is to demonstrate the potential LcPPO has for 2P optogenetic inhibition of neurons.

Committee Chair

Robert Gereau, IV

Committee Members

Bryan Copits; Alexxai Kravitz; Edward Han; Meaghan Creed

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-8-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-5465-6601

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