CTBPro: A Next‐Generation Cholera Toxin Subunit B‐Based Neuroanatomical Tracer With Superior Brightness, Stability, and Sensitivity for Enhanced Neural Circuit Mapping

ABSTRACT Mapping complex neural circuits demands bright and stable tracers, yet conventional Cholera Toxin Subunit B (CTB) conjugates exhibit suboptimal brightness, which limits high‐fidelity morphological reconstruction. Here, we engineer CTBPro, a next‐generation CTB‐based tracer, by genetically fusing CTB to the ultra‐stable fluorescent protein mBaoJin. In vitro, CTBPro exhibits an estimated nine‐fold enhancement in peak fluorescence output per mole of tracer compared to conventional CTB‐Alexa488 conjugates. This enhanced brightness translates to superior in vivo tracing across four distinct administration routes—intraparenchymal, peripheral (renal), cerebrospinal fluid (CSF), and intravenous—enabling high‐fidelity labeling of fine somatic and axonal details. Critically, because CTBPro is an entirely protein‐based fusion, it can be genetically encoded. We leverage this property by packaging CTBPro into an AAV‐BI30 serotype vector, which enables endothelial‐specific expression that recapitulates the distinctive labeling pattern of intravenous injection. Taken together, CTBPro overcomes the fundamental limitations of CTB‐based tracers. Its superior brightness, versatile administration, and multimodal stability establish it as a powerful and extensible tool for high‐resolution neuronal circuit tracing.