Viral-mediated fluorescent labeling of hyaluronan reveals extracellular matrix dynamics in the mouse brain in vivo

The extracellular matrix (ECM) of the brain is primarily composed of the glycan polymer hyaluronan (HA), a core scaffold that nucleates proteoglycans forming a self-assembled matrix that acts as structural framework and signaling hub. Since most of the neural matrix is composed of sugars, development of genetically encoded tags has been limited. Therefore, although several staining protocols exist for ECM in fixed tissue, there are no reliable matrix labels for live imaging. Here we report a viral-mediated fluorescent probe that binds to HA and labels the mouse brain ECM. The vector encodes the HA binding domain from neurocan fused to GFP and an externalization tag (AAV-Ncan-GFP), enabling transduced cells to secrete the fluorescent hyalectan into the extracellular space, thereby labeling HA. We demonstrate stable probe expression in organotypic brain slices, as well as in vivo in the mouse cortex, where it labels both perineuronal nets and interstitial matrix. We validate HA labeling through colocalization with HABP and sensitivity to hyaluronidase, and confirm the probes extracellular localization by shadow imaging. As a proof of concept, we combine AAV-Ncan-GFP with dendritic spine imaging ex vivo and calcium transient imaging in vivo, providing a real-time map of local ECM alongside neural function. The probe enables time-lapse imaging of ECM dynamics in live mice, facilitating longitudinal studies across a wide range of timescales, from minutes to days. The results establish AAV-Ncan-GFP as a valuable tool for real-time observation of brain ECM and a promising resource to explore ECM dynamics and brain function in vivo.