A Super‐Resolution Approach for Astrocyte‐Specific Molecular Imaging Reveals the Nanoscale Distribution of Monoacylglycerol Lipase, the Metabolic Node Between Endocannabinoid and Prostaglandin Signaling

Astrocytes play essential roles in brain function and disorders. Yet, compared to neurons, our knowledge of the physiological and pathological signaling mechanisms in astrocytes remains limited. As a major challenge, the ultrathin (~10-100 nm) processes of astrocytes render high-throughput quantitative molecular imaging within well-defined cellular contexts very difficult. Here, we introduce a single-molecule localization microscopy-based methodology that achieves unprecedented resolution of the intricate astrocytic arbor in intact brain circuits. Postnatal tagging of the plasma membrane by electroporation in mice resulted in selective and sparse labeling of hippocampal astrocytes and enabled the complete visualization of individual astrocytes with nanoscale precision by using STochastic Optical Reconstruction Microscopy (STORM). We also developed high-yield and easy-to-implement approaches to segment, measure, analyze, and visualize nanoscale molecular information within astrocytic compartments. As a proof-of-concept, we could readily differentiate between synaptic and astrocytic proteins by using dual-color STORM super-resolution imaging. Moreover, we identified cell-type-specific differences in the distribution of monoacylglycerol lipase (MAGL), an enzyme regulating synaptic plasticity in neurons and coupling endocannabinoid signaling to prostaglandin signaling in astrocytes. Our findings demonstrate the feasibility of nanoscale molecular measurements within ultrathin astrocytic processes. Moreover, the results provide insights into the synapse-independent nanoscale arrangement of the astrocytic MAGL pool that controls neuroinflammatory processes.