Sleep is a complex phenomenon that has been the subject of research for decades. Nearly all animals sleep more early in life, suggesting an important role for sleep in development. While the Drosophila brain contains significantly less neurons than mammals, 60% of its genome is shared with humans. Its simplicity and similarity makes the fruit fly an ideal model organism for sleep research. Our goal is to define a neuronal wake promoting system. Thus far, we have found that octopamine (OA), the fly analog of mammalian norepinephrine, is essential for wake promotion in larvae. My research this summer used intersectional genetic approaches to identify the octopaminergic cell clusters relevant for sleep-wake balance.
In past research, we discovered when expressing thermos-sensitive sodium channels (NaChBac) in octopaminergic neurons, wake behaviors increase. However, when repressing NaChBac expression in those that additionally express tsh, that wake-promotion is lost. Over the summer I raised flies that specifically expressed green fluorescent protein (GFP) in octopaminergic cells to map their location throughout the larval central brain (CB) and ventral nerve chord (VNC). Next, I introduced a transgene that prevented GFP expression in teashirt (tsh) expressing neurons, the same subgroup that removed the wake-promoting effect of NaChBac activation. I dissected the whole brain of at least 10 adults and larvae of each group, immunostained with anti-GFP and visualized them with confocal microscopy. The immunostaining helped the cells become more visible under the confocal microscope when imaging. Once the brains were imaged, I counted every fluorescent cell and analyzed them. Since the GFP was membrane-bound rather than nuclear, it was difficult to distinguish the exact position of every single neuron. Consequently, I was forced to analyze the clusters of octopaminergic neurons rather than individual ones.
Upon analyzing the cell counts, we were able to conclude that tsh expressing octopaminergic neurons are found in both the CB and VNC. In adults, the clusters most affected by tsh suppression were localized in the ventral CB, as well as the middle and posterior areas of the VNC. In larva, however, tsh suppression was most apparent in the anterior and middle part of the VNC and the sVM region of the brain. Our results imply that the neuron clusters we identified play an important role in maintaining proper sleep-wake balance during Drosophila larval development.
Spending my summer at the Kayser Lab has taught me more than simple lab skills. I was thrown in the deep end and forced to learn how to work more independently. I learned how to plan my experiments and pace myself. Furthermore, working with Dr. Kayser reinforced my interest in pursuing a graduate degree after college and a career in academia.