Tracking epithelial calcium dynamics and immune surveillance in the choroid plexus in vitro and in vivo

Abstract

Cerebrospinal fluid (CSF) is the ever-changing milieu of the brain, and its primary source is the choroid plexus (ChP). The epithelial surface is responsible for adjusting CSF composition by secretion of de novo proteins as well as selective transcytosis of blood-borne factors. Additionally, the ChP serves as a unique immunological niche, and there is evidence that it serves as a gateway for peripheral immune cells to enter the central nervous system (CNS). Despite its importance to brain development and function, the ChP-CSF field has lacked tools to address even basic questions of function, such as what signals is the ChP epithelium sensitive to, and how do ChP immune cells protect the brain from injury? Here we develop imaging tools to interrogate these functions in the adult lateral ventricle ChP in wholemount explants. After microdissecting the ChP, we used immunostaining to characterize the organization of vasculature and immune cells. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events with cellular resolution. Additionally, we noted increased sensitivity and secretory frequency following delivery of serotonergic agonists. We established and validated a protocol for implanting a chronic imaging window into the lateral ventricle to perform in vivo two-photon imaging in awake mice. We observed spontaneous subcellular calcium events and agonist-evoked apocrine secretion of cytoplasm into the CSF. Three-dimensional imaging of resident ChP immune cells revealed several commonalities with cortical microglia, namely: local surveillance, physiological housekeeping, and response to injuries. Immune cell imaging also revealed multiple subpopulations, distinguishable based on position, motility, mobility, and function. All experimental tools, particularly three-dimensional imaging, necessitated the development of custom analysis tools, including segmentation and registration, which are also presented here. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface, and serve as a foundation for future studies of ChP in coordinating brain development and maintaining brain health across the lifespan.