Mapping spinal cord and brain targets of colonic extrinsic nerves in the mouse

Aim: Identify the central circuits into which extrinsic nerves from the distal colon convey nociceptive signalling in the mouse. Methods: Male and female adult mice underwent retrograde tracing from the distal colon using cholera toxin subunit b (CTb). CTb labelled sensory neurons were quantified within CLARITY cleared dorsal root ganglia (DRG, T9-S2) and nodose ganglia (N=6) after four days. CTb labelled projections were identified in spinal and medulla sections (N=5) after seven days. Anterograde trans-neuronal tracing with herpes simplex virus 1 expressing EGFP (HSV-H129-EGFP) was performed from the distal colon wall. Neuronal expression of EGFP was identified within spinal and brain sections after 72-120 hours (N=4/time). To identify brain regions engaged by colonic nociceptive stimuli the number of neurons activated by noxious in vivo colorectal distension (N=5) was compared to that evoked by innocuous (N=5) and no distension (N=5). Immunolabelling for phosphorylated MAP kinase (pERK) was used to identify activated neurons.
Results: Colonic CTb-labelled neurons were present in the DRG, peaking in L1 and S1, and nodose ganglia. CTb-labelled projections were observed in the spinal cord dorsal horn, spanning T11-L1 (in lamina I/ V and in lateral tracts ofLissauer) and L6-S2 (in lamina I, dorsal grey commissure and lateral tracts into the sacral parasympathetic nuclei (SPN)), and within the medulla (nucleus tractus solitarius (NTS) and area postrema). CTb-labelled cell bodies were present in the intermediolateral nuclei (IML), SPN and dorsal motor vagal nuclei (DMV). 72-96 hours after colonic injection, EGFP-expressing neurons were observed in the spinal cord dorsal horn spanning T11-L1 (lamina I, IML and around the central canal) and L6-S2 (laminae I-V, dorsal grey commissure and SPN) and within the caudal and rostral ventrolateral medulla (VLM) and the rostral ventromedial medulla (RVM). After 120 hours, EGFP-labelling was present in the DMV, NTS, hindbrain locus coeruleus (LC), subcoeruleus, Barrington’s nucleus, lateral parabrachial nucleus (lPbN) and pontine reticular A7. Labelling was also evident in the hypothalamic paraventricular nucleus, periaqueductal gray (PAG), thalamic nuclei and somatosensory motor cortex. Of the brain regions in which EGFPneurons were observed, noxious CRD evoked a significant increase in neuronal activation
relative to innocuous distension in the VLM, RVM, LC, lPbN, PAG and medial thalamic nuclei.
Conclusion: These data localised colonic input within the spinal cord dorsal horn and medulla dorsal motor vagal complex and the subsequent brain circuits into which colonic nociceptive stimuli is conveyed.