Dissecting an asymmetric brain area implicated in sleep maintenance
Lead Research Organisation:
University College London
Department Name: Cell and Developmental Biology
Abstract
Just as most humans prefer to use either their right or left hand, asymmetries are built into the brains of most animals that are otherwise symmetrical. While we know some details about how these asymmetries arise during development, we are still discovering the functional importance of these asymmetries on thinking and behavior. One behavior for which little is known about the role of left-right asymmetry is sleep. Since sleep is essential for normal health and cognitive function across our lifespan, understanding the underlying organizing principles of sleep regulation is of great importance.
While studying sleep changes of zebrafish that have mutations associated with autism in humans, we discovered a new and unexpected brain asymmetry. Specifically, while normal zebrafish begin to lengthen the time they spend asleep in the evening before lights out, zebrafish lacking the autism risk gene called chd8 fail to do so. Using methods to visualize the brain activity of larval zebrafish, which are optically translucent in early stages, we found larvae that lack chd8 have high brain activity only on the right side of the midbrain during the evening. This led to the further discovery of a previously unreported physical asymmetry in same region of the brain. In this project, we now plan to investigate this new asymmetry by determining whether it follows the same developmental rules as other asymmetric brain regions, how this development may go wrong in zebrafish autism models, and whether and how this asymmetry is important for the regulation of sleep duration.
The zebrafish is an excellent system in which to study the links between brain asymmetries and sleep because of existing experimental tools to visualize and manipulate both properties. For example, there are zebrafish mutants and experimental manipulations that lead to altered brain asymmetries, such as animals with "double-left", "double-right" or "reversed" brain structures. We plan to test whether these manipulations also lead to changes in the asymmetry of this sleep regulating brain area, which will give us important clues about the extent to which this area follows known developmental rules for the creation of asymmetries. We will also use automated videotracking of zebrafish across the day-night cycle to see if changes in asymmetry affect sleep durations, which we can assess by measuring how long the zebrafish stay inactive in sleep states.
Using genetics, we will also label this midbrain asymmetry with fluorescent proteins to see how this area connects with the rest of the brain such as known sleep-regulating centers, to observe what molecules are selectively expressed in this region, and to understand how the formation of this area is disrupted in the short-sleeping animals that lack chd8. These experiments will tell us more about the structural and molecular properties of this asymmetry and will inform how the area may be regulating sleep.
Finally, we will test the role of this asymmetric area in sleep by taking direct control of the activity of this area. For example, we can selectively remove this area using laser ablation and ask whether this area is required for proper sleep durations. We will also use genetic interventions to put light- or drug-inducible proteins that can drive neuronal excitability specifically into this asymmetric area. This will give us direct control over the activity of this region, allowing us to test whether turning the area on or off is capable of altering zebrafish sleep.
Together these experiments will give us a new understanding of how brain asymmetries regulate sleep duration and knowledge about how this regulation might be altered in human disorders such as autism.
While studying sleep changes of zebrafish that have mutations associated with autism in humans, we discovered a new and unexpected brain asymmetry. Specifically, while normal zebrafish begin to lengthen the time they spend asleep in the evening before lights out, zebrafish lacking the autism risk gene called chd8 fail to do so. Using methods to visualize the brain activity of larval zebrafish, which are optically translucent in early stages, we found larvae that lack chd8 have high brain activity only on the right side of the midbrain during the evening. This led to the further discovery of a previously unreported physical asymmetry in same region of the brain. In this project, we now plan to investigate this new asymmetry by determining whether it follows the same developmental rules as other asymmetric brain regions, how this development may go wrong in zebrafish autism models, and whether and how this asymmetry is important for the regulation of sleep duration.
The zebrafish is an excellent system in which to study the links between brain asymmetries and sleep because of existing experimental tools to visualize and manipulate both properties. For example, there are zebrafish mutants and experimental manipulations that lead to altered brain asymmetries, such as animals with "double-left", "double-right" or "reversed" brain structures. We plan to test whether these manipulations also lead to changes in the asymmetry of this sleep regulating brain area, which will give us important clues about the extent to which this area follows known developmental rules for the creation of asymmetries. We will also use automated videotracking of zebrafish across the day-night cycle to see if changes in asymmetry affect sleep durations, which we can assess by measuring how long the zebrafish stay inactive in sleep states.
Using genetics, we will also label this midbrain asymmetry with fluorescent proteins to see how this area connects with the rest of the brain such as known sleep-regulating centers, to observe what molecules are selectively expressed in this region, and to understand how the formation of this area is disrupted in the short-sleeping animals that lack chd8. These experiments will tell us more about the structural and molecular properties of this asymmetry and will inform how the area may be regulating sleep.
Finally, we will test the role of this asymmetric area in sleep by taking direct control of the activity of this area. For example, we can selectively remove this area using laser ablation and ask whether this area is required for proper sleep durations. We will also use genetic interventions to put light- or drug-inducible proteins that can drive neuronal excitability specifically into this asymmetric area. This will give us direct control over the activity of this region, allowing us to test whether turning the area on or off is capable of altering zebrafish sleep.
Together these experiments will give us a new understanding of how brain asymmetries regulate sleep duration and knowledge about how this regulation might be altered in human disorders such as autism.
Technical Summary
Although left-right asymmetries are found in all animals with a bilateral body plan and are known to play critical roles in several types of behavior, the role of brain lateralization in the control of sleep has been relatively unexplored. Recently, we discovered a previously unknown functionally and molecularly asymmetric brain structure in the ventrolateral midbrain of larval zebrafish. Moreover, the activity of this area specifically in the evening is dysregulated in zebrafish harboring mutations in the autism risk gene, chd8, which also fail to lengthen their sleep bout durations in the evening. We will now test the hypotheses that this lateralized brain area regulates sleep bout architecture and that perturbations in the development of left-right asymmetry of this region in chd8 mutants are responsible for altered sleep times in the evening. Together this work will provide new insights into how brain asymmetries regulate the duration of sleep bouts.
Our program of study will first take advantage of genetic and other manipulations that alter left-right asymmetry of other zebrafish brain areas to establish whether the lateralisation of the putative sleep-regulating region develops following similar rules of symmetry-breaking. We will also test whether lateralization mutants show altered zebrafish sleep architecture using automated high-throughput videotracking. Next, we will examine whether chd8 mutants result in molecular or developmental changes to this asymmetric sleep-region, using a combination of transgenic-mediated imaging and circuit mapping, RNA sequencing of equivalent left and right areas, and marker analysis. Finally, we will test the importance of the asymmetric sleep-region in modulating sleep bout length by transgene-guided laser ablation and Cre-mediated opto- and chemogenetic manipulation of the cellular activity of this area in wild type and mutant animals at different times of day.
Our program of study will first take advantage of genetic and other manipulations that alter left-right asymmetry of other zebrafish brain areas to establish whether the lateralisation of the putative sleep-regulating region develops following similar rules of symmetry-breaking. We will also test whether lateralization mutants show altered zebrafish sleep architecture using automated high-throughput videotracking. Next, we will examine whether chd8 mutants result in molecular or developmental changes to this asymmetric sleep-region, using a combination of transgenic-mediated imaging and circuit mapping, RNA sequencing of equivalent left and right areas, and marker analysis. Finally, we will test the importance of the asymmetric sleep-region in modulating sleep bout length by transgene-guided laser ablation and Cre-mediated opto- and chemogenetic manipulation of the cellular activity of this area in wild type and mutant animals at different times of day.
