Optical imaging of cerebrospinal fluid via AAV-mediated secretory fluorescent protein

Neurons and glial cells are surrounded by interstitial fluid (ISF), which primarily originates from cerebrospinal fluid (CSF). The fluid dynamics of ISF and CSF have been examined by cisterna magna injection of fluid tracers such as fluorescent dextran or contrast agents for magnetic resonance imaging. While this approach has unveiled major CSF flow dynamics in the recent decade, cisterna magna injection introduces transient intracranial pressure changes that can lead to experimental variability. In this study, we attempted to label the CSF/ISF via endogenously expressed secretory fluorescent protein. Adeno-associated viral vectors (AAVs) were made to express albumin-fused or the secretion peptide IgK leader-fused bright green fluorescent protein, mNeonGreen, (Alb-mNG or IgKL-mNG, respectively) in neurons and astrocytes. The AAVs were injected into the cortex, striatum, and hippocampus on one hemisphere of the mouse brain. The contralateral cortex (i.e., non-injected side) was imaged by two-photon microscopy in anesthetized mice a month after AAV injection. Both Alb-mNG and IgKL-mNG signals were observed in the perivascular space and extracellular space. Additionally, sparse axonal labeling was observed for Alb-mNG, which is corroborated by cytosolic Alb-mNG expression in a subset of neurons at AAV injection sites. These results appeals the utility of contralaterally expressed secretory protein as a CSF/ISF label for longitudinal studies including various brain states. Moreover, the interhemispheric transport of secretory fluorescent protein elucidates an underappreciated fluid pathway whose turnover dynamics shall be studied further.