(370) Intramuscular versus intravesical retrograde labeling techniques identify anatomically and electrophysiologically distinct groups of mouse bladder lumbosacral primary afferent neurons

Abstract

Four functionally distinct classes of pelvic nerve mechanosensitive bladder afferents have been characterized and described as urothelial, muscular/mucosal, muscular, and serosal, but the actual anatomical location of their respective terminal endings is unknown. We utilized two strategies to retrogradely label lumbosacral (LS) bladder primary afferent neurons: intramuscular (IMus) consisted of microinjection of tracer into the bladder parenchyma (the current gold standard), and intravesical (IVes) consisted of transurethral infusion of tracer into the bladder lumen. IMus and IVes techniques labeled two distinct populations of bladder neurons that could be differentiated on the basis of labeling in the dorsal root ganglia (DRG), dye distribution within the bladder, and electrophysiological properties reflecting neuronal excitability. IMus- and IVes-labeled neurons accounted for 91.5% (378.3 ± 32.3) and 8% (33.0 ± 26.0) of all labeled neurons, respectively (p<0.01), with only 2.0 ± 1.2 neurons labeled by both techniques (total of 1240 labeled cells from 3 mice). IMus labeling resulted in distribution of tracer throughout the bladder wall but not in the urothelium; in contrast, only the urothelium and in some cases the adjacent submucosa was labeled by the IVes method. Whole mount patch-clamp recordings of labeled neurons in LS DRG revealed significant differences between IMus- and IVes-labeled neurons, with IVes neurons exhibiting lower rheobase and membrane capacitance, higher input resistance, and lower total K+ current density, suggesting increased excitability of IVes afferents. This study is the first to demonstrate that IVes labeling is a minimally invasive approach for retrograde labeling of bladder afferent neurons, and to selectively identify urothelial versus muscular bladder afferent soma in the DRG. An important consideration is whether the current findings change the interpretation of published studies that only used IMus labeling techniques.