Project 2: Periarterial CSF pumping: Dependence on state of brain activity

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Abstract_Project 2 Many lines of work show that glymphatic flow plays a key role in brain waste clearance during sleep. Yet, the mechanisms driving glymphatic flow and its regulation by the state of brain activity are poorly understood. Like the other projects in the U19 application, Project 2 will focus on understanding what drives periarterial CSF influx. We are basing the periarterial CSF pumping model on the well-documented mechanism of functional hyperemia, i.e., transient increases in blood flow and volume in response to neural activity. We postulate that CSF in parallel is pumped along the periarterial spaces to compensate for local or global changes in vascular volume. Thus, glymphatic fluid transport peaks during NREM sleep because coordinated neural activity drives larger changes in blood volume than in the awake state. Aim 1 will with high spatio-temporal resolution image the effect of whisker stimulation on Ca2+/cAMP signaling in the individual cell populations of the neurovascular unit on the diameters of penetrating arterioles, and on the velocity of CSF in periarterial spaces (as measured by particle tracking). We will determine the drivers of periarterial CSF pumping using optogenetic stimulation of neurons, astrocytes, or smooth muscle cells with simultaneous observations via 2-photon or macroscopic imaging in mice. Aim 2 will test the hypothesis that natural sleep states determine not only the activity pattern of neural circuits, but also the physical dimensions and the functional properties of the perivascular spaces, and thereby the efficacy of periarterial CSF pumping. Operationally, we will use the same paradigms proposed in Aim 1, including sensory whisker stimulations and cell-specific optogenetic activation, in awake and sleeping (NREM) mice. Aim 3 will establish whether periarterial CSF pumping predicts glymphatic clearance. We will take advantage of a novel clearance assay based on intracortical injection of a small fluorescent tracer (DB53, BBB-impermeable) that accumulates in blood after clearance from brain, so that the signal in blood reflects total DB53 brain efflux, independent of the clearance routes. We will systematically manipulate periarterial CSF pumping and determine its effect on DB53 clearance after intracortical delivery. Overall, Project 2 will contribute to the U19 proposal by testing the hypothesis that neural circuit activity determines glymphatic solute clearance by controlling the structural and physical properties of the neurovascular unit and thereby the efficacy of periarterial CSF pumping in a brain state dependent mechanism. Project 2 depends critically on the fluid dynamic modelling in Project 1, while Project 3 will provide key information on which neuronal populations control neurovascular coupling during sleep. Projects 2 and 3 will together serve as an important source of cellular information for the neuroimaging studies in Project 4, bridging cellular mechanisms and consequences for solute clearance with the multiscale dynamics observed in the human brain.