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Optogenetic Tools for In-Vitro Neurophysiology

Norman, Olivia Rose
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1408643520

Abstract Details

2014, Master of Science, University of Toledo, Bioengineering.
Since its development, electrophysiological methods have provided invaluable information regarding the electrical and biological nature of countless cell types. The ability to form a direct electrical connection with the cell of interest allows the experimenter to observe and measure membrane potential and ion channel gating behavior. While the field has and continues to become more advanced, certain limitations have proved difficult to overcome. In particular, the invasive nature of the method itself has seemed like an unavoidable vice. With the development of optogenetics, this issue has been resolved. This technology utilizes genetic engineering to deliver light-sensitive ion channels to desired cell populations both in-vitro and in-vivo. Once the ion channel is delivered and expressed, all that is required to activate it is the correct wavelength of light. In this way, cells of interest can be stimulated with spatiotemporal precision in a non-invasive manner. The focus of this study is to implement optogenetic tools into excitable and non-excitable cell lines while using electrophysiological techniques to quantify and characterize their electrical behavior under various conditions. Channelrhodopsin-2, an excitatory light-gated ion channel was delivered to HEK-293, MC3T3-E1, and primary dissociated hippocampal neurons via a constitutively active plasmid driven by the CMV promoter. Control and ChR2-positive cells were subjected to voltage and current clamp recordings as well as exposure to fluorescent light in an attempt to observe light-induced depolarization. To further explore the efficacy of optogenetic tools in-vitro, physical connections between transfected and control cells were exploited in an attempt to observe propagation of light-induced depolarization between adjacent cells. It was found that ChR2 was able to be successfully expressed and activated in both the HEK-293 and MC3T3-E1 cells. It appeared that the transfection process was highly toxic to the hippocampal neurons and thus did not permit the study of ChR2 functionality in this particular cell line. When the role of gap junctions in electrical signal propagation was explored, it was observed that upon illumination with the appropriate wavelength of light, gap junctions are able to pass depolarizing responses from a transfected cell to a control cell.
Scott Molitor, PhD (Committee Chair)
Ronald Fournier, PhD (Committee Member)
Patricia Relue, PhD (Committee Member)
Biology; Biomedical Engineering; Neurosciences
Optogenetics; Channelrhodopsin-2; Voltage Clamp; Current Clamp; HEK-293; MC3T3-E1; Gap Junctions

Recommended Citations

Citations

  • Norman, O. R. (2014). Optogenetic Tools for In-Vitro Neurophysiology [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1408643520

    APA Style (7th edition)

  • Norman, Olivia . Optogenetic Tools for In-Vitro Neurophysiology . 2014. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1408643520.

    MLA Style (8th edition)

  • Norman, Olivia . "Optogenetic Tools for In-Vitro Neurophysiology ." Master's thesis, University of Toledo, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1408643520

    Chicago Manual of Style (17th edition)

toledo1408643520
817
© 2014, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.