Sleep

A hypothesized model of spindle organization of thalamic and hippocampal spike dynamics (Figure 1) suggests that sparsity operates in spindles as an essential component of thalamic activity that could be contributing to flexibility in learning (Varela & Wilson, 2020). We asked the question of whether sparse spindle-like (10Hz non-rhythmic) or 10Hz rhythmic activity in thalamic cells of the reuniens nucleus influence cognitive flexibility during learning after sleep. By comparing the two stimulation protocols (“nonrhythmic” and “rhythmic”), we tested if disrupting the characteristic sparsity reveals any changes in flexibility during learning after sleep. Results showed that sleep accompanied 10Hz rhythmic optogenetic stimulation of thalamic nucleus reuniens impaired rule-switching (or set-shifting) performance and disrupted the sleep enhancing rule-switch associated increase in vicarious trial and error (VTE), which we used as a metric for deliberation. We found that rule-switching was associated with a subsequent increase in VTE, as were incorrect choices, and when rats subsequently made correct choices. Instead, stimulating against the endogenous thalamocortical spindle oscillation (i.e. sleep accompanied 10Hz rhythmic optogenetic stimulation) resulted in a significant disruption in post-sleep performance and VTE during, but not prior to, rule-switching. Lastly, optogenetic 10Hz stimulation of the thalamic nucleus reuniens did not affect sleeping or waking behavior during the sleep box session but it did show a clear though nonsignificant increase in waking head velocities; thus, changes in cognitive flexibility and VTE cannot be explained by any changes in sleep itself, but rather due to the after-effects the specific patterns of 10Hz optogenetic stimulation in thalamic nucleus reuniens applied during sleep had on cognition.

Proper regulation of sleep and metabolism are critical to the survival of all organisms. In humans, dysregulation of sleep is linked to metabolic syndrome, including hypertension, hyperglycemia and hyperlipidemia. However, the mechanisms regulating interactions between sleep and metabolism are poorly understood. Although the fruit fly, Drosophila melanogaster, bears little anatomical resemblance to humans, it shares similar genetics essential in understanding normal development and disease in humans. From humans to flies, many disease-related genes and pathways are highly conserved, rendering the fruit fly ideal to understanding the interactions between sleep and metabolism. Therefore, using the fruit fly provides a framework for understanding how genes function between sleep and metabolism. During starvation, both humans and rats reduce their sleep. Similarly, previous studies have shown that fruit flies also suppress sleep to forage for food, further showing that sleep and metabolism are intricately tied to one another and that they are highly conserved across species. To further explore the interactions between sleep and metabolism, I have conducted multiple genetic screens to identify novel regulators of sleep-metabolism interactions. These experiments led to the identification of the mRNA binding protein translin (trsn) as being required for starvation-induced sleep suppression. A second screen that targeted metabolic genes from a genome-wide association study identified the ion channel accessory protein uncoordinated 79 (unc79) as a critical regulator of both sleep duration and starvation resistance. The genes function in different regions of the brain and suggest complex neural circuitry is likely to underlie regulation of sleep metabolism interactions. Taken together, a mechanistic understanding of how different genes function to regulate sleep in flies will further our understanding of how sleep and metabolism is regulated in humans.

Sparse thalamocortical cell population synchronicity during sleep spindle oscillations has been hypothesized to promote the integration of hippocampal memory information into associated neocortical representations 1. We asked the question of whether sparse or rhythmic activity in thalamocortical cells of the reuniens nucleus influence memory consolidation and cognitive flexibility during learning after sleep. For this study, I designed a novel attentional set-shifting task and incorporated optogenetics with closed-loop stimulation in sleeping rats to investigate the effects of sparse (nonrhythmic) or rhythmic spindle-like (~10Hz) activity in thalamic cells of the reuniens nucleus on learning and cognitive flexibility. We show that, as predicted, post-sleep setshifting performance improved after sleep with non-rhythmic optogenetic stimulation in the thalamic nucleus reuniens relative to rhythmic optogenetic stimulation. While both non-rhythmic and rhythmic optogenetic stimulation led to an increase in perseverative errors, only non-rhythmic optogenetic stimulation showed effects of learning from errors, which correlated with sleep, and which ultimately had a net benefit in set-shifting performance compared to rhythmic optogenetic stimulation and the control group.

Sleep is a complex behavioral state with ramifications on multiple levels of homeostasis including bodily function, neural activity, and molecular signaling. Sleep is conserved across evolution, though significant variations in sleep duration, architecture, and behavior are found across phyla. Decoding neural processing underlying behavior, including sleep, is a fundamental aim in neuroscience, and understanding how such behavior has evolved remains largely unknown. The encompassing goal of this dissertation is to elucidate the genetic and neuronal factors at play in the evolution of sleep loss in the blind Mexican tetra, Astyanax mexicanus. To this end, the work found within will explore peripheral sensory systems regulating distinct mechanisms of sleep loss, demonstrate how evolved changes in specific hypothalamic circuits drive sleep reductions, apply computational techniques to understand whole-brain evolution, and finally, will show how the generation of transgenic tools in a novel model system can be harnessed to assist functional experimental paradigms in relation to evolution and behavior.

Dysregulation of sleep and metabolism has enormous health consequences. Sleep
loss is linked to increased appetite and insulin insensitivity, and epidemiological studies
link chronic sleep deprivation to obesity-related disorders. Interactions between sleep and
metabolism involve the integration of signalling from brain regions regulating sleep,
feeding, and metabolism, as well as communication between the brain and peripheral
organs. In this series of studies, using the fruit fly as a model organism, we investigated
how feeding information is processed to regulate sleep, and how peripheral tissues
regulate sleep through the modulation of energy stores.
In order to address these questions, we performed a large RNAi screen to identify
novel genetic regulators of sleep and metabolism. We found that, the mRNA/DNA
binding protein, Translin (trsn), is necessary for the acute modulation of sleep in
accordance with feeding state. Flies mutant for trsn or selective knockdown of trsn in
Leucokinin (Lk) neurons abolishes starvation-induced sleep suppression. In addition, genetic silencing of Lk neurons or a mutation in the Lk locus also disrupts the integration
between sleep and metabolism, suggesting that Lk neurons are active during starvation.
We confirmed this hypothesis by measuring baseline activity during fed and starved
states. We found that LHLK neurons, which have axonal projections to sleep and
metabolic centers of the brain, are more active during starvation. These findings suggest
that LHLK neurons are modulated in accordance with feeding state to regulate sleep.
Finally, to address how peripheral tissues regulate sleep, we performed an RNAi
screen, selectively knocking down genes in the fat body. We found that knockdown of
Phosphoribosylformylglycinamidine synthase (Ade2), a highly conserved gene involved
the biosynthesis of purines, regulates sleep and energy stores. Flies heterozygous for two
Ade2 mutations are short sleepers and this effect is partially rescued by restoring Ade2 to
the fly fat body. These findings suggest Ade2 functions within the fat body to promote
both sleep and energy storage, providing a functional link between these processes.
Together, the experimental evidence presented here provides an initial model for how the
peripheral tissues communicate to the brain to modulate sleep in accordance with
metabolic state.

Accumulating evidence points to a fundamental connection between sleep and feeding behavior. However, the temporal, genetic, and neuronal architecture that defines these relationships is poorly understood. Drosophila are amenable to high-throughput studies and offer numerous genetic tools which have advanced our understanding of the mechanistic relationships between these behaviors. However, certain features of the sleep-feeding axis have remained elusive, largely due to the separate measurement of sleep and feeding. Here, I develop a system which simultaneously measures sleep and feeding in individual animals by employing high resolution machine vision tracking and micro-controller interface functionality. Using this system, I show that food consumption drives a transient rise in sleep, which depends on food quality, quantity, and timing of a meal. The leucokinin system mediates these effects, particularly in response to protein ingestion. We further use the system to examine sleep homeostasis and demonstrate sleep dependence on energy expenditure and fat-brain communication. Collectively, these findings provide novel insight into the fundamental connections between sleep and feeding behavior.