Anatomical reappraisal of the relationship between vagal preganglionic motor neurons and the spleen in the male mouse

INTRODUCTION: The direct innervation of the spleen by the vagus nerve has been a subject of ongoing controversy for many years. Specifically, studies in laboratory rodents disagree as to whether vagal preganglionic motor neurons are directly connected to the spleen. METHODS: Employing several orthogonal anatomical approaches in the mouse, we reassessed the anatomical relationship between vagal preganglionic motor neurons and the spleen. RESULTS: First, using the Phox2b-Cre-Tomato mouse model, which allows for the permanent labeling of all vagal preganglionic motor neurons, vagal afferent neurons, and subsets of sympathetic neurons, we observed Tomato-positive axons within the spleen. A combination of double-immunohistochemistry and nerve cut studies revealed that intrasplenic Tomato-positive axons were of sympathetic rather than vagal origin. Second, anterograde tracing with AAV-GFP-labeled vagal preganglionic motor neurons revealed no direct innervation to the spleen. Third, through immunohistochemistry and quantitative mass spectrometry-based proteomics, the mouse spleen was found to be devoid of cocaine and amphetamine regulated transcripts, a neuropeptide of vagal origin. Fourth, retrograde tracing with intrasplenic FluoroGold did not label specifically vagal preganglionic motor neurons after intrasplenic administration in the mouse. Instead, we showed it is possible to map neurons connected to the spleen by examining the expression of Activating transcription factor 3 (Atf3) mRNA, a marker of axotomy, after splenectomy. Atf3 was induced in a subset of celiac ganglion neurons, but not in vagal preganglionic motor neurons after splenectomy. CONCLUSION: Our anatomical findings reaffirm that vagal preganglionic motor neurons do not innervate the spleen to a significant degree in the laboratory mouse.