Convergence of bladder and colon sensory innervation occurs at the primary afferent level

Abstract

Dichotomizing afferents are individual dorsal root ganglion (DRG) neurons that innervate two distinct structures thereby providing a form of afferent convergence that may be involved in pelvic organ cross-sensitization. To determine the distribution of dichotomizing afferents supplying the distal colon and bladder of the Sprague-Dawley rat and the C57Bl/6 mouse, we performed concurrent retrograde labeling of urinary bladder and distal colon afferents using cholera toxin subunit B (CTB) fluorescent conjugates. Animals were perfused 4-5 days after sub-serosal organ injections, and the T10-S2 DRG were removed, sectioned, and analyzed using confocal microscopy. In the rat, CTB-positive afferents retrogradely labeled from the bladder were nearly three times more numerous than those labeled from the distal colon, while in the mouse, each organ was equally represented. In both species, the majority of colon and bladder afferents projected from lumbosacral (LS) ganglia and secondarily from thoracolumbar (TL) ganglia. In the rat, 17% of the total CTB-positive neurons were retrogradely labeled from both organs with 11% localized in TL, 6% in LS, and 0.8% in thoracic (TH) ganglia. In the mouse, 21% of the total CTB-positive neurons were dually-labeled with 12% localized in LS, 4% in TH, and 4% in TL ganglia. These findings support the existence of dichotomizing pelvic afferents, which provide a pre-existing neuronal substrate for possible immediate and maintained pelvic organ cross-sensitization and ultimately may play a role in the overlap of pelvic pain disorders.

Figure 1

Retrogradely labeled cell bodies in the rat L1 DRG 5 days following urinary bladder and distal colon injections of Alexa Fluor 488- and 647-conjugated cholera toxin B (CTB), respectively, as visualized under confocal microscopy. CTB-positive bladder afferents appear green (A), CTB-positive distal colon afferents appear red (B), and dually-labeled cells appear yellow (C). (Bar represents 20 um)

Figure 2

Retrogradely labeled cell bodies in the mouse L6 DRG 5 days following urinary bladder and distal colon injections of Alexa Fluor 488- and 647-conjugated cholera toxin B (CTB), respectively, as visualized under confocal microscopy. CTB-positive bladder afferents appear green (A), CTB-positive distal colon afferents appear red (B), and dually-labeled cells appear yellow (C). (Bar represents 20 um)

Figure 3

Bar graphs illustrating the number (A) and percentage (B) of bladder-specific, distal colon-specific, and dually-labeled cells across all DRG levels for both the rat and mouse.

Figure 4

Bar graphs illustrating the percentage of bladder-specific, distal colon-specific, and dually-labeled cells with respect to all labeled cells (T10-S1) and stratified by TH, TL, and LS DRG levels for the rat (A) and mouse (B).

Figure 5

Pie graphs illustrating the percentage of TH, TL, and LS bladder-specific, distal colon-specific, and dually-labeled cells with respect to the total number of labeled cells at each respective DRG level for the rat (A) and mouse (B).

Figure 6

Bar graphs illustrating the number of bladder-specific, distal colon-specific, and dually-labeled cells in control and bladder denervated rats with respect to all labeled cells (T10-S1) (A), TL afferents (B), and LS afferents (C).