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Objective Neural tracing technique, as an advanced neuroanatomical approach, has been applied for the morphological study on the acupuncture for 30 years.The aim of this study is to investigate the correlation between acupoint and nervous system using a new generation of fluorescent neural tracer, cholera toxin subunit B conjugated with Alexa Fluor 488, 594 (CTB-Alexa488, 594).It will help us to further understand the neuroanatomical characteristic of acupoints.Methods Micro-injection with CTB-Alexa488 or CTB-Alexa 594 was carried out in the aeupoints of "Chengshan", "Jinggu"and "Dazhong" in the adult male SD rats.After 3 surviving days, the rats brain, spinal cord and dorsal root ganglia (DRGs) were dissected following perfusion with 4% paraformaldehyde, cut into sections and observed under fluorescent microscope equipped with a digital camera.The neurons labeled by CTB-Alexa 488 and CTB-Alexa 594 were recorded and counted.Results Under the fluorescent microscope system, the labeled neurons with CTB-Alexa 488 and CTB-Alexa 594 appeared in green and red respectively, and located ipsilaterally on the injection side.The sensory and motor neurons innervating each acupoint distributed on the corresponding DRGs and spinal cord.Conclusion The present fluorescent tracing technique may be quite useful for investigating the neuroanatomical basis associated with different acupoints.