Subtypes of Bladder-Innervating Sensory Neurons revealed by Immuno-Fluorescence In Situ Hybridization

Sensory neurons that innervate the urinary bladder are critical for triggering voiding and mediating pain perception under pathological conditions. Despite functional and immunohistochemical evidence indicating the existence of several bladder afferent populations, the subtypes of dorsal root ganglion (DRG) neurons that innervate this organ are poorly defined and specific markers to study them have not been described. Here, we combined retrograde tracing with the cholera toxin {beta} subunit and immunofluorescence in situ hybridization with markers defined by transcriptomic analysis of C and A{delta} afferents to classify the lumbosacral DRG neurons that innervate the urinary bladder. Four lumbosacral (L6-S2) bladder-innervating DRG neuron populations were identified: three Calca+ peptidergic populations that express Adra2a/Th, Th/Ntrk2, or Ntrk2, and one non-peptidergic population that expresses Ntrk2. Among the whole population of peptidergic lumbosacral sensory neurons, Adra2a+/Th+, Th+/Ntrk2+ and Ntrk2+ neurons were rare. Thus, our studies identified multiple populations of bladder-innervating DRG neurons, revealing unappreciated diversity among the sensory neuron subtypes present at the lumbosacral level that innervate visceral and somatic tissues.