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Illuminating Neuronal-Astrocytic Pathways for Sleep homeostasis

Periodic Reporting for period 3 - iNAPS (Illuminating Neuronal-Astrocytic Pathways for Sleep homeostasis)

Período documentado: 2020-04-01 hasta 2021-09-30

ERC iNAPS 715933 aims to discern the mechanisms controlling sleep homeostasis. This means we wish to understand how the brain senses the need for sleep and drives appropriate sleep patterns. Most people know what sleep deprivation feels like, yet the impact of significant sleep loss on health, safety, and the economy is underappreciated. We believe if we can understand the brain circuits governing sleep deprivation and sleep drive, we can devise ways to augment them, enabling more restorative sleep and all the beneficial health, safety, and economic benefits that ensue.
The overall objectives are: 1) identify the cell types and brain regions involved in sleep homeostasis; 2) determine how these cells affect sleep drive; and 3) evaluate the role of astrocytes, a special type of brain cell, in sleep homeostasis.
We have tested genetic tools to identify the cell types involved in sleep homeostasis. We evaluated mouse lines that express a fluorescent marker only when a cell is active. This means we can find active groups of cells under different conditions of sleep need (e.g. sleep deprivation [SD], or rebound sleep after SD [RS]). We can use this tag to isolate the cell type and profile it. Nevertheless, we must ensure the sensitivity of the fluorescent tag is sufficient to mark enough brain cells in a given sleep state. One mouse line we tested did not express enough fluorescently tagged cells for us to pinpoint active cell groups. In contrast, another mouse line has more promising results (Fig 1). We are currently expanding these datasets to optimize the conditions. In parallel, we have successfully obtained fluorescent activity data from a distinct cell type in live brain sections, and are expanding this work to image these same cells in living mice. Additionally, we are building 3-dimensional maps of brain activity during SD and RS. This will help visualize the sleep drive network within the brain (Fig 1).

Once we have identified specific cell populations, we want to confirm the role of each group of cells, i.e. can we recapitulate aspects of sleep homeostasis by using specific tools that activate/inhibit those cells. For example, are certain cell types the engine driving sleep consolidation after sleep loss? We are currently optimising methodology required to target specific cell groups to assess their functionality.

Lastly, we are interested in how astrocytes – a specific type of brain cell – behave in sleep homeostasis. To do this we are using a mouse line that enables the expression of specific tags (e.g. fluorescent label or activity indicator) in astrocytes. We have observed marked morphological changes in astrocytes with sleep deprivation, and are exploring the functional consequences of this. We have also shown that astrocyte modulation affects the activity of cortical neurons of interest. Finally, we labeled astrocytes with a specific dye, facilitating the recording of their activity across sleep states.
• Successfully tagged SD- and RS-active cells in large numbers. We are now expanding this dataset and implementing cell sorting to purify and analyse which type of cells are active. We expect to identify new cell populations involved in sensing sleep need and promoting sleep responses

• Performed intact-brain mapping of cellular activity in SD and RS. We are now expanding this dataset and performing unbiased quantitative analysis to prioritise regions of interest. Combined with the results from the above point, we expect to establish which brain regions are involved in sensing sleep need. Concomitantly, we have performed intact-brain mapping of NOS1 cells across sleep states. Preliminary results indicate region-specific alterations in cell morphology.

• Imaged signaling dynamics of cortical cells in brain slices. We are expanding this dataset with sleep perturbations and have planned parallel in vivo recordings. We expect to confirm the dynamic modulation of NOS1 cell activity across sleep states.

• Preliminary analysis indicates alteration in astrocyte morphology with sleep deprivation. Further replication is required to determine if this effect is robust, reproducible and region-dependent.