Cerebrospinal fluid (CSF) drains into venous blood and into meningeal lymphatic vessels. In this application, we will test the overarching hypothesis that there exists a third CSF exit route via skull channels into the cranial bone marrow. Three years ago, we described the existence of sub-millimeter channels connecting skull marrow cavities with the dural vasculature in mice and humans, and that leukocytes produced in the cranial bone marrow migrate towards the inflamed central nervous system (CNS) through these channels. Investigating the skull channels further, we recently made the surprising observation of CNS — skull marrow crosstalk in the opposite direction: fluorescent CSF tracers injected into the cisterna magna migrate through skull channels in a perivascular fashion and accumulate in specific skull marrow areas distinct from regions labeled by intravenous blood pool markers. We here propose to systematically study CSF egress through skull channels in the steady state and in bacterial meningitis, testing the hypothesis that the skull bone marrow is a privileged site of hematopoiesis that constantly surveys brain health via sampling the CSF for danger signals. We will begin with a rigorous assessment of CSF skull channel outflow in healthy mice, establishing its route, size constraints, and the marrow microenvironment receiving CSF. Motivated by our preliminary data showing bacterial migration through skull channels into the cranial bone marrow, we will then test the relevance of CSF skull egress in mice with bacterial meningitis induced by Streptococcus pneumoniae, clinically the most prevalent strain. The work in mice with meningitis will reveal how skull channel CSF drainage changes in inflammatory CNS conditions. This Co-PI application unites three different labs, creating a unique set of complimentary expertise in neuroscience, intravital imaging, immunology and hematology. We propose to leverage custom-developed tools such as advanced imaging of the hematopoietic niche, image-guided single cell sequencing, non-invasive imaging for phenotyping of infection and inflammation, in combination with the fields' gold standard assays. The program is based on bold preliminary data, will clarify the importance of previously unknown CSF signaling to the skull marrow, and potentially advance knowledge in a setting of clear clinical need, bacterial meningitis.