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

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.