Structural tapering describes the branching and subsequent thinning of axons, decreasing in axon caliber or diameter. Recent evidence suggests that components of the axonal cytoskeleton may regulate axon caliber. This evidence specifically refers to the membrane periodic skeleton (MPS) of axons, which is composed of actin rings and myosin filaments that control the contractility of an axon, which is important for action potential conduction. The drug blebbistatin is a known myosin inhibitor. Therefore, we hypothesized that blebbistatin would cause the axons of zebrafish to relax and increase in caliber. To visualize the axons, we fluorescently labeled a subset of neurons green. We selected the fish that had a single Rohon-Beard neuron in the tail of the spine, and we treated half of the fish with blebbistatin and half of the fish with a control DMSO solution. Then, we used a confocal microscope to take images of the individual axons and measured the axon caliber using line scan analysis. Our preliminary data suggest that blebbistatin does increase axon caliber, leading us to conclude that myosin inhibition causes the MPS to relax, accounting for the structural tapering of RB neurons. While the data is promising, it is derived from a very small sample size. Thus, this experimental design should give us the necessary results to either support or reject our preliminary findings, but we will need to increase the number of fish to determine statistical significance.

Estradiol is known to inhibit calorie restriction-induced torpor in mice. The Medial Preoptic Area in the hypothalamus of the brain is a regulator of torpor in mice and known to be estrogen sensitive. In this experiment, we aimed to gauge the effects of estradiol in different brain regions through the expression of progesterone receptor (PR), an estrogen-responsive gene. Using ovariectomized mice, the subjects were split into four groups: those who received estradiol treatment directly to the MPA, those who received vehicle (placebo) treatment directly to the MPA, those who received systemic estradiol treatment, and those who received systemic vehicle treatment. We hypothesized that there will be a high concentration of progesterone receptors in the MPA, VMH of the mice who received estradiol treatment directly to the MPA, followed by the mice receiving systemic estradiol treatment, with little to none present in the mice who received vehicle treatments. We will evaluate these results through CellProfiler to measure the intensity of PR expression in each cell: the higher the PR intensity, the higher the response to estrogen. This evaluation will provide critical insight into the effectiveness of targeted estradiol delivery to the MPA. This will allow for more selective manipulations in the study of estradiol and its effect on calorie restriction-induced torpor in the brain, particularly in the MPA.

Sex differences in sleep amount are driven by multiple factors. The goal of this project is to determine the role of sex chromosomes and sex-linked genes in encoding these sex differences. Previous studies in mouse models have shown that hormones like estrogen and testosterone play a role in sex difference modulation. Despite the removal of hormones by gonadectomy, sex differences in sleep regulation remain. This suggests that sex differences in sleep are partially regulated by sex chromosomes. While there are many reports of sex differences in sleep amount, few studies have examined the role of sex chromosomes on sleep quality and sleep efficiency. In this study, we use a unique mouse model to examine the role of sex chromosomes on sleep fragmentation, which is a standard measure of sleep quality. The XY* mouse model allows us to investigate the specific effects of X chromosome dosage. We implanted gonadectomized XY* mice with EEG/EMG recording electrodes. We then measured sleep fragmentation over a 24-hour period. Our results show that X chromosome dosage has significant effects on a number of indices of sleep fragmentation which include phenotypes associated with REM sleep. Previous studies have shown that the presence of a Y chromosome has effects on sleep amount. The results of the current study suggest that the number of X chromosomes may influence sleep fragmentation and therefore sleep quality.

Researchers have been able to link the tau protein to several neurodegenerative diseases including Alzheimer’s disease (AD) and related disorder. Normal tau functions to stabilize microtubules in axons, but translational changes can result in the formation of aggregates that are pathological to neurons. Tau aggregation spreads in a prion-like manner, but its mechanism from cell-to-cell is not certain. Research suggests that extracellular vesicles may be responsible for the propagation of tau in these diseases. Exosomes are formed in multivesicular bodies (MVBs) within the cell and contain intraluminal vesicles (ILVs) that function to exocytose, or recycle nucleic acids, lipids, and proteins. ILVs are then released as exosomes into the extracellular space, and are taken up by neighboring cells. For this experiment, I separated exosomes from human brain samples from the hippocampus (HC), cerebellum (CBL), and occipital cortex (OC) using a rigorous ultracentrifugation process. Then, I used antibody-coupling techniques to isolate exosomes of neuronal and oligodendrocyte, cells that produce myelin in the brain. Tau seeding will be measured by other lab members. Trends in the extent of tau proliferation in different brain regions and cell types will allow researchers to understand the role exosomes play in pathological brain disorders, clearly differentiate the origin and movement of these diseases, and potentially develop treatments to disrupt the mechanism.