This week I’ve been diving in a little more into doing some actual research myself. Nothing breakthrough mind you, just some simple experiment to sort of understand the world around and inside of me a little better. My partner and I are looking into how the optic nerve develops inside of zebra fish and how its development may be affected by developing the fish in total darkness. We are trying to stain 1-2 day old zebra fish with Dye-I by simply poking the dye into the retina with any sort of small sharp object we can find. We’ll then separate the groups of stained fish into those that develop in a small flask with normal exposure to light (about 10-15 hours a day of UV light) and those that develop in a tin-foil-wrapped flask with no UV exposure in which feeding will be done under red light.
Every few days, I hope to take some of the fish out and look at how their brains are developing using a fluorescent microscope that allows me to see how the dye is traveling. With any luck, I’ll see a decently clear pattern of paths of nerves from the retina to the lateral geniculate. I’m not sure where it leads from there as the zebra fish has mainly the midbrain rather than our large forebrain with a thalamus and cerebral cortex. More research on my fish is definitely needed before I can do any real analyzing of the staining technique, but my real problem right now is just getting some fish stained!
Using a pin head dipped in dye, I had been trying to poke the retinas of these fish. Once the retina ruptures, almost any amount of dye should stain the retina and lead to further staining of the optic nerve as development continues (from what I’ve read, usually the rods and cones develop about 4 days at the earliest). I ran into many problems with this of course and have been fishing (oh so hilarious, I know) for new solutions ever since.
The first problem I had is that the head of the pin is about the size of the fish’s eye itself, so trying to rupture the eye with it is like trying to shove a baseball bat through my own. Of course actually rupturing the retina with this gigantic tool will only happen if my mounted fish would stop moving every time I get close to it, which is the second problem. So far, I’ve created new tools by heating TLC spotters over a flame in the organic chemistry lab and then pulling it in half so that the glass is pulled into a tiny pipette with a head just smaller than the fish’s retina. This has been successful after coupling it with a technique for knocking out my fish to keep them still. I burst a small bubble of anesthetic (0.5 MESAB) over the water that I’m using to mount my fish before injecting the fish in augar onto the slide. This does a great job in keeping the fish still so I can do my dirty work of staining its eye, but sometimes the concentration of anesthetic is too strong and the fish will die. All in all, with much more research and some patience this may turn out to be a fun little project. If you have any ideas as to where I should be looking for research and anything else I could be doing with this experiment, let me know.