A novel anterograde neuronal tracing technique to selectively label spinal afferent nerve endings that encode noxious and innocuous stimuli in visceral organs

Background: One major weakness in our understanding of pain perception from visceral organs is the lack of knowledge of the location, morphology and neurochemistry of all the different types of spinal afferent nerve endings, which detect noxious and innocuous stimuli. This is because we lack techniques to selectively label only spinal afferents. Our aim was to develop an anterograde tracing technique that labels only spinal afferent nerve endings in visceral organs, without also labeling all other classes of extrinsic afferent and efferent nerves. Methods: Mice were anesthetized with isoflurane and dextran-biotin injected, via glass micropipettes (diameter 5 μm), into L6 and S1 dorsal root ganglia. Mice recovered for 7 days, were then euthanized and the colon removed. Key Results: Anterograde labeling revealed multiple unique classes of afferent endings that terminated within distinct anatomical layers of the colon and rectum. We characterized a particular class of intramuscular ending in the circular muscle (CM) layer of the colon that consists of multiple varicose axons that project circumferentially. Conclusions & Inferences: We demonstrate a technique for selective anterograde labeling of spinal afferent nerve endings in visceral organs. This approach facilitates selective visualization of the precise morphology and location of the different classes of spinal afferent endings, without visual interference caused by indiscriminant labeling of other classes of afferent and efferent nerve axons which also innervate internal organs. We have used this new technique to identify and describe the details of a particular class of intramuscular spinal afferent ending in the CM layer of mouse large intestine. The origin of the nerve endings of spinal afferent neurones which detect noxious stimuli from visceral organs remains elusive, due largely to the lack of the techniques that can selectively label only spinal afferents. In this study, we have developed, for the first time, a novel technique whereby dorsal root ganglia in mice can be injected with neuronal tracer in vivo, to selectively label spinal afferent nerve endings in visceral organs. We have used this technique to identify and characterise a specific class of intramuscular spinal afferent ending in the circular muscle layer.