October 23rd, 2017
Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.
The overall goal of this viral transduction and time-lapse recording is to visualize movement of oligodendrocyte mitochondria in organotypic brain slices.This method can help answer key questions in the oligodendrocyte field.Such as how the oligodendrocytes and their organelles respond to changes in the local environment.The main advantage of this technique is that it gives detailed information about oligodendrocyte structure and organelle dynamics in a preparation where all the different brain cells are present and oligodendrocytes produce compact myelin.Transduce oligodendrocytes in slice cultures at day seven in vitro.First draw up 1.3 microliters of diluted AAV per slice.Making sure the outside of the pipette tip is dry, hold pipette above the cortex as close as possible without touching the slice with the pipette tip and slowly push solution out of the pipette.When the solution touches the slice, move the pipette tip over the cortex to spread the solution over the whole cortex area.Once all the slices in one dish have been transduced, return the dish to the incubator and then repeat the process on the other dishes.Examine the slices for expression of fluorescent markers and image the slices when the expression levels of the fluorescent markers are sufficient and the slices look healthy.Begin setting up the imaging bath by using modeling clay to attach syringe needles bent at a 45