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 a calculated nine-fold enhancement in molar brightness compared to conventional CTB-Alexa 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.