To understand how the nervous system processes information, a map of the connections among neurons is essential. Viral transsynaptic transmission has gained popularity as a method for labeling neural circuits. In particular, the development of retrograde monosynaptic tracing vectors has enabled visualization of the pre-synaptic inputs onto defined sets of postsynaptic neurons. This system utilized the rabies virus (RABV), in which the glycoprotein gene in the virus was deleted, and re-supplied in trans. In order to build alternative, more flexible tracers, we made recombinant VSV genomes, first developing the use of vesicular stomatitis virus (VSV) for tracing neuronal connections. Viruses encoding several different fluorescent proteins were made, giving brilliantly labeled neurons, bright enough for live imaging and characterization of the detailed morphologies of cells. Expression was very rapid, facilitating identification of neurons both in vivo and in ex vivo applications. In addition, the use of an avian glycoprotein (ASLV-A) allowed specific targeting to cells expressing an avian glycoprotein receptor (TVA). This allowed monosynaptic tracing from defined starter cells. In order to alter the direction and cell type specificity of transmission, we then fitted VSV with a glycoprotein from one of multiple other viruses. Glycoproteins such as the rabies virus glycoprotein (RABV-G) endowed VSV with the ability to spread in a retrograde transsynaptic pattern, while the glycoproteins from viruses such as the lymphocytic choriomeningitis virus (LCMV) gave an anterograde pattern of transsynaptic spread. This anterograde or retrograde spread was observed in all species tested, and even for other non-VSV viruses, such as lentiviruses. We also developed transsynaptic tracing viruses which direct viral spread between defined cell types, instead of from a defined cell type to any upstream of downstream cell. In all, we developed an extensive transsynaptic tracing repertoire for tracing neuronal connections.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10336868 |
| Date | January 2012 |
| Creators | Beier, Kevin |
| Contributors | Cepko, Connie |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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