Document Type

Honors Thesis

Major

Biochemistry, Microbiology

Advisor(s)

Robert Wheeler, Marie Hayes

Committee Members

Edward Bernard, Margaret Killinger, Natalie Machamer

Graduation Year

May 2021

Publication Date

Spring 5-2021

Abstract

A known bidirectional relationship between intestinal microflora and the central nervous system, coined the gut-brain-axis, has stimulated work on the association between gut dysbiosis and inflammation, and sleep quality. Previous studies in the Hayes Lab have reported that a high fat (HF) diet was correlated with immobile phases, a marker of low motility during sleep found in some neurological disease and sleep duration[1]. Long sleep duration (>1 S.D. above norms for age) is associated with poor sleep quality or sleep fragmentation in participants who are overweight or obese according to body mass index values[2]. The current work utilizes a reverse translational model to propose that we can identify some of the molecular mechanisms underlying consumption of a HF diet and sleep fragmentation. The animal model for the current study was Danio rerio, or zebrafish. Within this Honors Thesis, an existing protocol for Danio rerio sleep analysis was modified in order to assess the correlation between inflammatory pathways induced via direct infection by the human fungal pathogen Candida vs. HF diet-induced gut dysbiosis on developed measures of sleep quality[3]. Sleep analysis was administered via a Noldus DanioVision behavioral tracking device and showed that, in 80% of replicate experimental trials (n=86), infected zebrafish larvae exhibited increased total sleep duration, sleep % and mean sleep bout length. In 60% of replicate experimental trials (n=68), infected zebrafish larvae exhibited a greater number of sleep bouts, a finding consistent with sleep fragmentation defined as the number of sleep bouts divided by the total sleep duration. Sleep analyses conducted for diet-controlled larvae showed that zebrafish larvae administered a high fat diet exhibited an increase in sleep bout number and larger fragmentation index, though this observation was not found to be trending or statistically significant.Zebrafish were euthanized and homogenized following sleep analysis in order to be used for single fish qPCR. In Candida infected larvae, gene expression of multifunctional pyrogenic and somnogenic cytokines TNF-ɑ and IL-6 were upregulated according to -ΔΔCt values which are calculated as the negative difference of the ΔCt of each individual treated larvae and the average ΔCt value of each control, non-treated larvae. Similarly, in HF diet-treated larvae, gene expression of IL-1β and IL-6 was also elevated. Positive gene fold expression was elevated for IL-6 in both infected and HF diet treated zebrafish. These findings suggest that a mutual inflammatory pathway, triggered via direct infection or diet-induced gut dysbiosis, may exist for IL-6 mediated sleep disruption

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