Using synaptic connections to dissect neural microcircuitry

Abstract

There are a number of methods used to study synapses through selective labeling and control. However, these techniques are often cytotoxic, difficult to implement, or lack the selectivity required to most effectively study microcircuits. We aim to create two novel techniques for studying how the brain is wired – a method for transsynaptic genetic labeling and a method for selectively destroying synapses. For synaptic labeling we are using the C. elegans orthologs for the Notch-Delta signaling system, Glp-1 and Lag-2. Glp-1 was modified by replacing the intracellular portion of the protein with cre recombinase, a site directed DNA recombination enzyme. It is hypothesized that when a cell expressing the chimeric Glp1-Cre protein comes in close contact with a cell expressing Lag-2, cre recombinase will be released allowing for the selective expression of a fluorescent molecule based on cell proximity. In vitro testing via confocal microscopy indicates that the labeling mechanism performs as expected, labeling cells based on contact in HEK 293T cells.To selectively destroy synapses, we have developed a chimeric protein, CadPlexin, that will activate repulsion signals through homophilic binding between two cells connected by a synapse. CadPlexin is composed of the extracellular domain of fruit fly DE-Cadherin and the transmembrane and intracellular domains of mouse plexin-B2. Cadherins bind to one another homophillically, acting to bind cells with one another. Plexins, when dimerized and activated by their usual ligand, Semaphorin, will cause a downstream signaling cascade that induces cell-cell repulsion. We hypothesize that, when two cells expressing CadPlexin are in close contact (i.e. at a synapse), CadPlexin will selectively destroy the synapse, as the extracellular domains from DE-Cadherin will bind together and mimic semaphorin binding and clustering required to activate plexin signaling. Preliminary results from in vitro experiments suggest that CadPlexin performs as expected, seen through stress fibers in the actin cytoskeleton of Cos7 cells transfected with CadPlexin – a characteristic cytoskeletal change induced through Plexin signaling. Together, these techniques will allow us to label synaptically connected neurons and destroy synapses with surgical precision. Funding Sources: Murchison Summer Research Fellowship, Trinity University Start-Up Funds, McNair Scholars Program, the Brain and Behavior Research Foundation NARSAD Young Investigator Award, Mary E. Groff Foundation, Women in STEM Program. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.