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William Stoy
BME PhD Thesis Defense
Date: Monday, December 3rd, 2018
Time: 1:00 PM
Location: EBB Children's Healthcare Seminar Room
Thesis advisor:
Craig Forest, PhD (Georgia Tech, Mechanical Engineering, Biomedical Engineering)
Thesis Committee:
Garrett Stanley, PhD (Georgia Tech, Biomedical Engineering)
Todd Sulchek, PhD (Georgia Tech, Biomedical Engineering)
Jun Ueda, PhD (Georgia Tech, Mechanical Engineering)
Albert Lee, PhD (Janelia Research Campus, HHMI)
Title: Automated single-cell electroporation and subcortical whole-cell recording in vivo
Abstract:
Whole-cell patch clamping is uniquely suited to investigations of cell type and function in the living brain because of its stable intracellular access; however, the success rate of whole-cell patch clamping trials in the mouse thalamus, 3 mm deep in the brain, is only 1% compared to >70% in vitro.
In order to successfully apply this technique to the creation of an in vivo cell-types database or to the study of high-value preparations, such as those that are virally modified or behaviorally trained, the success rate of individual trials of whole-cell patch clamp in the living brain must be drastically improved.
The two main sources of failure of a patch clamp trial stem from pipette contamination and tissue movement. The formation of a tight, high resistance seal between the pipette tip and the membrane is crucial for the isolation and longevity of the recording, and it can be prevented or disrupted by contamination or tissue movement. In Aim 1) I demonstrate a tool to provide automated, multimodal (electrical, physiological, and genetic) access to single cells in the living brain by recording with a lower resistance seal (cell-attached patch clamping) and electroporating fluorescent protein plasmids into the recorded cell. This effectively bypasses the requirement for a high resistance seal but does not provide access to subthreshold electrical activity in the recorded cell.
In Aim 2) I demonstrate that the primary source of pipette contamination is the penetration of blood vessels while moving the pipette to the region of interest and deploy an algorithm to move laterally around blood vessels, effectively dodging them. I demonstrate that this step significantly improves the success rate of whole-cell recordings in deep brain structures. Finally, in Aim 3) I demonstrate that precise positioning of the pipette tip with respect to the membrane is crucial to the formation the high resistance gigaseal and deploy a method to compensate for respiratory and cardiac-induced brain motion by feedforward axial position compensation. I demonstrate significantly higher success rates of whole-cell recordings in the cortex and thalamus of the mouse.
Together, these techniques represent automated, multimodal access to tissue throughout the living brain with high success rate, a crucial requirement for the study of cell types and sensory neuroscience.
