Sleep is required for normal physiological function in all organisms. In particular, sleep is hypothesized to have a critical role in brain development. However, systems have not effectively studied sleep during the earliest periods of neural development, when neurons are first being born.
The fruit fly, Drosophila melanogaster, exhibits sleep behaviors. Studies show that behavioral characteristics of sleep are shared between mammals and flies. While this model is a powerful tool, neurogenesis is almost complete in the adult brain. The Kayser Lab has expanded upon classic fly work to devise methods of studying fly larvae. The creation of the LarvaLodge, a device that allows for the study of activity of individual larvae, has allowed us to study sleep during early stages of brain development and determine that larvae meet the criteria for sleep.
The goal of this work is to identify specific neurons in 2nd instar larvae that play a role in sleep and wake control. These experiments are a part of a larger plan to map and describe sleep/wake circuits in an immature nervous system, helping us understand how neural control of sleep/wake changes throughout the lifespan.
For this screen, I crossed female UAS-TrpA1 virgins and males from a specific GAL4. In neurons with GAL4 and the UAS, the activator protein Gal4 can bind to the UAS, expressing TrpA1, which encodes for a thermosensitive cation channel. By increasing the temperature to 30ºC, I activated TrpA1, which depolarizes every neuron containing GAL4 and TrpA1.
Next, I loaded the larvae of this cross into a LarvaLodge and used MATLAB to compare the sleep patterns of GAL4 flies with that of control flies containing the UAS but no GAL4. Specifically, I looked at the average number of bouts, average bout length, average activity, and total sleep time for each group of larvae in the experiments.
Finally, I selected GAL4 lines in which larvae displayed deviations in total sleep, but not in average activity. This removed flies with possible locomotive defects that could be mistaken for abnormal sleep patterns. To visualize the neurons where these interesting GAL4s are active, I expressed UAS-GFP labeled 2nd instar larval brains with a GFP antibody and imaged them using a confocal microscope.
My contribution to this project has been a wonderful opportunity to become immersed in a lab setting and truly understand what it means to conduct research. I started out my freshman year as a pre-medical student determined to get an M.D. and become a doctor as fast as possible. However, after experiencing the joys of research and feeling the excitement of getting interesting data, I have realized that I should make more room for research in my future. I am now convinced that an M.D./Ph.D. program may be more suited for me. Making connections between various studies and building upon the findings of others evokes a kind of exhilaration that is unique to conducting research.