Abstract

The mechanisms by which the energetically intensive brain maintains tissue homeostasis remains a fundamental mystery. We propose to test the hypothesis that a newly discovered brain lymphatic endothelial cell (BLEC) is involved in solute clearance from the brain during periods of high neuronal activity. Understanding how this novel lymphatic-related cell population participates in brain clearance will provide insight into how dysfunctions in this system may contribute to diseases of ageing. Our programme of study will firstly carry out molecular, immunohistochemical and ultrastructural studies to determine whether BLECs are present in mammalian, including human, meninges. This work will build on our preliminary data and firmly establish the evolutionary conservation of BLECs. To gain functional insight into BLECs, we will use the experimentally tractable, optically transparent zebrafish larvae, in which BLECs can be non-invasively 4D imaged during behavior using confocal and 2-photon microscopy. To manipulate neuronal activity, we will use both natural visual stimuli and 24-hour sleep-wake cycles, as well as pathological states, including sleep deprivation and seizure. Following behavioral manipulation, we will use 4D imaging to assess morphological changes and combine cell sorting and RNA sequencing to determine molecular changes in BLECs during behavior. We will also functionally assess macromolecule uptake by BLECs using dye injection and photobleaching techniques. Finally, to assess whether BLEC function is required for normal behavior, we will either laser or genetically ablate BLECs and quantify changes in the recovery of larval zebrafish locomotor behavior following sleep deprivation or seizure.