Zebra Finch Axons: Calcium Imaging and More!

Hello!

This is sadly my first blog post regarding actual lab work and data thus far. The delay has been caused by orienting myself with several new techniques, experiments, and culturing protocols that have been optimized this summer. Now that I am finally in the groove of things, I feel it necessary to share my experience!

If you may recall, my research focuses on axon degeneration in zebra finch and how we can better understand the mechanisms involved. There have been past studies with other subjects, but our lab is the first to observe degeneration (in particular, SARM’s role) in avian neurons. SARM is the engine of axon degeneration and induces the process to begin. Sarm1 is a major protein that induces swelling, fragmentation, AAD (acute axonal degeneration), retraction bulb formation, calpains, and calcium release in the axon, eventually leading to the destruction of said axon. If we can understand how this system works in zebra finch, we can target SARM, calcium channels, calpains and hopefully disrupt these pathways in an attempt to subsequently stop axon degeneration.

The first part of the summer has been focused on DRG (dorsal root ganglion) and retinal ganglion cell culturing techniques. Previously, we have struggled to get long, single axons stretching out across the plate so we can isolate and observe a particular axon. We assumed there might be problems with the substrate and adherence quality which leads to unstable axons that retract when cut (axotomized) or stress-induced to stimulate degeneration. Fortunately, we have made great strides on that front and have switched to creating explants of the DRG’s and retinal cells. For this process, we dissect the retinal tissue or remove the DRG’s from the embryonic spinal column and place the sample directly on to the well in our plate. We then add media to provide nutrition. With this technique, the axons will grow away from the explant on each well and provide an ideal subject to work on. We are still trying to determine the best methodology and substance for our substrate, but the best result we have determined is laminin-coated wells.

Another project I am involved in is calcium imaging in avian axons after stress. There is research that shows calcium release to be an integral part in axon degeneration. The idea is that there is a massive calcium wave before fragmentation which inevitably leads to the destruction of the axon distal to the cut. Calcium enters the mitochondria and just as in cell death, when there is large outpouring of calcium, the similar fate is simulated here. The cell believes it must kill itself, thus resulting in degeneration. There are multiple pathways involved in this process that can be interrupted on both the intracellular source (endoplasmic reticulum) and the target (mitochondria). I am working on how we can best inhibit these calcium channels to hopefully delay axon degeneration.

Currently, I am imaging calcium fluctuations within axotomized cells using the confocal microscope. I am treating the wells with a calcium indicator (Fluo-4), a chelator (EDTA), and a pH buffer (HEPES). The Fluo-4 emits green at the same frequency as typical GFP settings on the confocal (488 nm). The EDTA exists to remove calcium molecules from the extracellular space through chelation so that I can get a clearer image of intracellular activity. The pH buffer is necessary in timelapses for the time being because without HEPES, the pH of the media becomes too alkaline. In the future, I will use a live cell chamber to eliminate the need for HEPES. I have been running timelapses to observe calcium waves during axon degeneration and so far, the results have been promising. I have been able to see calcium spikes within the axon and cell body prior to, and during degeneration. I am continuing experimentation on this front in the following weeks.

The last project I am working on is creating an ex ovo experiment where I remove the zebra finch embryo from the egg and simulate that environment using a “hammock”. I will speak more to this in the upcoming posts but the overall idea is that in the future, I can inject a dye in the embryo’s vasculature using a micro needle (less than 2 microns wide)┬áthat will let us observe axon degeneration in a living organism. As you can imagine, this creates much more difficulty and is definitely a ways away from being credibly performed.

Thanks and I will see you next post!