CTBPro: A Next-Generation Cholera Toxin Subunit B-Based Neuroanatomical Tracer With Superior Brightness, Stability,
Xinghua Quan1,2, Yude Lou1,2, Huiqi Xie1,2
1Laboratory of Neurovascular Biology, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.
Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|April 22, 2026
Summary
Researchers developed CTBPro, a brighter, more stable neural tracer by fusing Cholera Toxin Subunit B (CTB) with a fluorescent protein. This advanced tracer enables high-fidelity mapping of neural circuits across multiple administration routes.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Mapping neural circuits is crucial for understanding brain function.
- Conventional tracers like Cholera Toxin Subunit B (CTB) conjugates have limitations in brightness and stability.
- Suboptimal brightness hinders high-fidelity morphological reconstruction of neural pathways.
Purpose of the Study:
- To engineer a next-generation CTB-based tracer with enhanced brightness and stability.
- To overcome the limitations of existing CTB tracers for improved neural circuit tracing.
- To develop a versatile and genetically encodable tool for high-resolution neuronal tracing.
Main Methods:
- Genetically fusing CTB to the ultra-stable fluorescent protein mBaojin to create CTBPro.
- In vitro assessment of CTBPro's molar brightness compared to conventional CTB-Alexa conjugates.
- In vivo tracing studies using CTBPro across four administration routes: intraparenchymal, renal, cerebrospinal fluid (CSF), and intravenous.
- Packaging CTBPro into an AAV-BI30 serotype vector for endothelial-specific expression.
Main Results:
- CTBPro demonstrated a nine-fold enhancement in molar brightness compared to conventional CTB-Alexa conjugates.
- Superior in vivo tracing was achieved with high-fidelity labeling of fine somatic and axonal details.
- CTBPro enabled successful neural tracing via intraparenchymal, renal, CSF, and intravenous administration.
- Genetically encoded CTBPro in an AAV vector recapitulated intravenous injection labeling patterns.
Conclusions:
- CTBPro significantly overcomes the fundamental limitations of traditional CTB-based tracers.
- Its enhanced brightness, versatile administration, and stability make it a powerful tool for high-resolution neuronal circuit tracing.
- CTBPro offers an extensible platform for advanced neuroscience research and diagnostics.
