Date of Award

5-2021

Document Type

Thesis campus only

Department

Chemistry

First Advisor

Gerard M.J. Beaudoin

Second Advisor

Christian Cooley

Abstract

Neuroscience is a rapidly expanding field that is outgrowing the currently available techniques. To help remedy this issue, we are developing new techniques that help to expand the molecular toolbox available to neuroscientists.

Cell culture work is typically a necessary first step before moving to an animal model. Many cell culture lines are inadequate representations for the cells they are trying to emulate. To help remedy this issue differentiation of HT22 cells, an immortalized hippocampal cell line, was tested to induce neuronal maturation and upregulate the expression of the neuron specific promoter CaMKII-α. This cell line has potential uses in studying CaMKII-α promoted genes prior to in vivo study.

Another technique is optogenetics. Optogenetics is the use of light as a stimulus to elicit a desired response. Previous research in our lab has used a blue-light gated cation channel, channelrhodopsin-2, for the selective activation of neurons in the context of understanding cocaine addiction. While useful, a single channelrhodopsin only provides understanding within a singular pathway. Usage of a second channelrhodopsin that responds to a different wavelength of light would allow for two pathways to be characterized both individually and together. Thus, a red-light gated channelrhodopsin, ChrimsonR, was thoroughly characterized and optimized for in vivo expression for optogenetic purposes. It was found that ChrimsonR can be effectively expressed in an in vivo model and displayed unique properties in comparison to other channelrhodopsins.

Lastly, current techniques exist to map various neural pathways and control synapses generally in the brain, but determining the information transferred between two areas of the brain has been extremely difficult. Utilizing adhesion proteins and neural repulsion signals, we developed a new chimeric protein, CadPlexin, to selectively destroy synapses between two brain regions without affecting additional outputs from those areas. This protein was first created and is currently being tested using in vitro cell culture models. Current results suggest that CadPlexin is expressed and properly trafficked to the cell membrane and the cells expressing the protein exhibit unique morphology, suggesting that the protein is potentially working as expected. Together, my thesis has laid the groundwork for sophisticated in vivo experiments vital for identifying necessary neural circuit components critical for encoding drug addiction.

Download

Share

COinS