The Bohórquez Lab focuses on the connection between the gut and the brain, especially how the brain perceives what the gut feels. Team members have performed a neurotracing using a monosynaptic rabies virus. The modified rabies virus they used could not move transynaptically because of a lack of its envelope glycoprotein rabG. They could permit the virus to jump a single synapse by expressing rabG in the target cell. By expressing rabG in PYY-expressing cells (often used as an indicator of being an enteroendocrine cell), they found that the virus could jump from PYY-expressing cells to efferent neighboring neurons. This strongly suggests a synaptic connection between enteroendocrine cells and the efferent neurons.
My project’s goal is to confirm this result using mammalian GFP reconstitution across synaptic partners (mGRASP). This technique uses constructs named pre-mGRASP and post-mGRASP. Pre-mGRASP uses a neurexin fragment to present half of a GFP fragment, while post-mGRASP uses neuroligin-1 to present its half of GFP. Normally, neurexin and neuroligin-1 connect across synapses, but in mGRASP, these two proteins reached across synapses to connect the halves of GFP. When connected, the protein fluoresces under the correct wavelength of light.
The aim of my project is to express one of these two constructs in cultured enteroendocrine cells and the other in cultured nodose neurons. The plan is to then co-culture the two groups of cells to see if there is GFP fluorescence. The constructs also express separate fluorescent proteins, mCerulean and dTomato. They are used to confirm that the cells are transfected with the constructs, even if there is not fluorescence from the reconstitution of GFP across a synapse.
