A Chemically Tailorable Multifunctional Protein Platform for Glutamatergic Neuron Targeting in Mouse Brain

Abstract Targeting glutamatergic neurons is critical for understanding brain functions and treating neurological disorders linked to glutamate dysregulation. However, existing methods face challenges including low specificity, slow kinetics, poor blood‐brain barrier (BBB) penetration, and reliance on transgenic pathways. Here, we report the cyanobacterial ferredoxin‐dependent glutamate synthase (Fd‐GOGAT cb ) as a non‐transgenic, multifunctional platform for rapid and selective targeting of glutamatergic neurons in vivo. We demonstrate the high affinity of Fd‐GOGAT cb to glutamatergic neuron membranes that allows for fixation‐free labeling within 1–2 h, and its self‐deliverable BBB‐crossing capability via systemic administration in living mice. When conjugated with functional motifs (e.g., fluorophores or photosensitizers), Fd‐GOGAT cb enables in vivo glutamatergic neuron‐targeted imaging and neuromodulation, displaying region‐specific enrichment in glutamate‐dependent brain circuits and light‐responsive neuron inhibition through intravenous delivery of photodynamic conjugates. This platform addresses longstanding barriers for direct neuron targeting and manipulation in vivo, proving its adaptability for versatile applications by chemical functionalization and potential transformative value in glutamate‐centered fundamental research and clinical translation.