Selective Silencing of Vertebrate Neurons: Strategies Using Invertebrate Ligand-Gated Ion Channels

Citation

Slimko, Eric Michael (2006) Selective Silencing of Vertebrate Neurons: Strategies Using Invertebrate Ligand-Gated Ion Channels. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/RYJJ-JW20. https://resolver.caltech.edu/CaltechETD:etd-05142008-004518

Abstract

Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of certain human neurological disorders and (2) provide insight into the roles of specific sets of neurons, both in the local circuit and in the behavior of the intact organism. This work focuses on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl α and β) in vertebrate neurons first in vitro using viral and tranfection techniques, and then finally in vivo using genetic techniques, to produce inhibition via a Cl- conductance when activated with IVM. We have considerably engineered these two genes by (1) re-coding the genes such that vertebrate-preferred codons are used throughout the sequences, (2) incorporating fluorescent tags within the proteins, and (3) finding a mutation to remove the undesirable glutamate sensitivity of the channel while retaining IVM efficacy. Expression of this new channel does not affect the normal spike activity of the target cell, yet the experimentor can effectively “shut-off” the cell with concentrations of as low as 5 nM IVM. Chapter 1 provides a broad overview of the many “selective silencing” approaches that experimenters have tried. In Chapter 2, the author describes the basic “GluCl/IVM” technique and initial experiments in cultured hippocampal neurons. Chapter 3 refines the technique by describing the strategy and mutation that allowed great reduction in the native glutamate response while maintaining the IVM response. Chapter 4 develops the final engineering of the channel: recoding the sequence for optimal expression and the introduction of fluorescent tags for identification. Finally, Chapters 5 and 6 discuss the successes and failures of in vivo work with what we now call the “GluCl/IVM method.”

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