Technology Development for the Functional and Structural Analysis of the Brain

Abstract

Tool development for neuroscience has allowed us to study brain function and structure with great detail. However, as all technologies have limitations, we are yet far from fully deciphering brain dynamics and its relationship to its anatomical structure. In this thesis, I describe the development of two technologies: genetically encoded potassium indicators for functional studies of the brain, and an approach for constructing a partial connectome of the zebrafish brain. In functional studies, methods to study cell dynamics have relied on the use of electrodes or dyes. Genetically encoded indicators have gained popularity because they are non-invasive and targetable. We focus on potassium as it’s proposed its homeostasis in the brain is supported by glia, which modulates neuron activity, and potassium’s dysregulation has been observed in several neurological diseases. I present the design and characterization of a family of genetically encoded potassium indicators using a potassium binding protein (Kbp) and the mNeonGreen fluorescent protein, named KRaIONs. These indicators were developed by using structure-guided mutagenesis of Kbp’s potassium binding site and by identifying alternative potassium binding proteins from metagenomic databases. In structural studies, efforts to map small organism’s brains have relied on electron microscopy or cell-labeling, which are limited in protein detection, cell-labeling diversity, or imaging resolution. Using the zebrafish model, we propose a strategy for constructing a partial connectome of the fish larvae brain by optimizing a version of Brainbow, a method that stochastically labels cells. My work focuses on identifying endogenous markers to detect cell and synapse location and type, which would complement the cell labels. To detect all proposed cell labels and endogenous markers in the zebrafish brain, I describe a protocol that uses DNA-conjugated antibodies to enable multiplex imaging at high resolution, with the use of expansion microscopy. Taken together, the work presented in this thesis introduces a method to readout potassium dynamics and my contributions to the zebrafish partial connectome project.