Proper function of precisely wired neural circuits depends on a close physical and functional relationship with an equally complex and overlapping vascular network. Vascular and perivascular cells are heterogeneous both within and across brain regions, and this heterogeneity is thought to underlie the functional specialization that caters to local neuronal circuitry demands. Moreover, emerging evidence shows that vascular cells actively influence the activity of neuronal ensembles, and reciprocally, neuronal activity controls the function and patterning of vascular networks. Understanding the molecular and structural heterogeneity underlying these unique local interactions is fundamental to understanding how non-neuronal cells contribute to the emergence of neural activity patterns underlying cognition and behavior. However, an inventory of vascular cell types within a defined brain region or within functionally defined brain circuits does not exist, and methods to identify or manipulate intercellular connections at the neuro-glio-vascular interface to determine their influence on circuit function are lacking. To date, the creation of a vascular cell inventory with regional specificity has proven challenging because it is difficult to obtain adequate vascular and perivascular cell yields for analysis. This is because vascular cells represent only 5% of brain cells4 , and of course we cannot pool across regions without losing regional specificity. To overcome this challenge, we developed a dissociation protocol to enrich for vascular cell survival in mice, which allows us to study small, defined brain regions. As a proof of principle, we focused on the median eminence (ME) and a size-matched region of somatosensory cortex, regions selected for their distinct functions and small, defined spatial structures (~0.05 x 0.2 x 1.2 mm3). Our preliminary single- cell RNA sequencing (scRNA-seq) data revealed differences in cell composition and gene expression among vascular and perivascular cells. Moreover, we identified ligand-receptor interactions underlie different neuro-glio- vascular interactions. We will capitalize on this work to generate the first comprehensive inventory of vascular and perivascular cells in these regions and two additional regions, the subfornical organ (SFO) and the hippocampus. We will identify cell type- and regional-specific markers and generate mouse Cre lines to target these cells, and we will generate a neuro-glio-vascular connectome database from which other researchers can rapidly access the spatial arrangement of cells in a brain region, with morphology, cell-cell contacts, and ultrastructure for all cell types identified in our inventory. Our tools will provide an invaluable resource to identify the contribution of non-neuronal cells to structural and functional heterogeneity of neural circuits across brain regions. In addition, the cell type- and regional-specific markers are likely to be useful in other mammalian species for viral delivery of Cre recombinase. Finally, given the importance of non-neuronal cells in neurological diseases, this work will provide a vast source of understudied molecular and cellular targets, which could change how we think about brain disease and identify new modes of treatment.
(public health relevance): Many symptoms of nervous system disease are preceded and perpetuated by impaired neuro-vascular interactions. The tools and database we generated and knowledge we learned will accelerate our understanding of neuro-vascular interactions and thus advance the prevention, diagnosis, and treatment of CNS diseases including Alzheimer's disease, stroke, cerebral hemorrhages, migraine, ALS, and MS. Moreover, our inventory in the first two regions included in my proposal will provide immediate benefit in identifing key molecular targets for manipulating the blood-brain barrier to facilitate CNS drug delivery.
