Identifying Neural Pathways of Stress and Fear with Retrograde Viral Tracing and Single Cell RNA Sequencing

Thesis (Ph.D.)--University of Washington, 2022The brain is made up of millions of neurons, often making thousands of connections each. Interrogating individual neurons activated by stressors is the equivalent of finding a needle in a haystack. Here we describe experimental and computational methods for tracing neural connections and identifying neurons involved in the stress-response pathway of corticotropin releasing hormone (CRH) signaling. We perform single cell RNA sequencing on neurons upstream of CHR neurons (CRHNs) and show incredible diversity and co-expression of signaling molecules. Subsequent studies sort cells to enrich for activated neurons upstream of CHRNs in response to restraint stress. This analysis reveals known neuron subtypes in activated CHRNs and reports new genes of interest in CHRN signaling in response to restraint stress. These studies provide a framework for mapping neural connections upstream of targeted cell populations, and the identification of activated neurons through computational analysis