[Abstract Title]. - Society for Neuroscience

Poster
269. Pain: Visceral Pain II
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 269.12/GG27
Topic: D.08.n. Visceral pain
Support: NS 19112
Title: Identification and characterization of anterogradely traced extrinsic primary afferents of
the mouse colon
Authors: *P. R. BRUMOVSKY, J. LA, G. F. GEBHART;
Ctr. for Pain Res., Univ. of Pittsburgh, Pittsburgh, PA
Abstract: Nerve fibers innervating the mammalian colon are divided into extrinsic (sensory and
autonomic axons) and intrinsic (enteric neurons producing intracolonic axons). Interestingly,
most published anatomical data on both systems in the colon is based on whole mount
preparations, where only the tangential distribution of nerve fibers is shown. The main objective
of the present study is thus to reveal the distribution and phenotype of extrinsic primary afferent
fibers through the different layers of the mouse colon. Distal colon and innervating pelvic nerves
of C57Bl/6 mice were dissected out and placed in a chamber with circulating oxygenated Krebs
solution at 34 °C. The colon was opened longitudinally and pinned flat to the bottom of the
chamber. The pelvic nerves, passed through a gate into a second chamber with mineral oil, were
split into 10-12 bundles and explored electrophysiologically to certify the presence of
mechanosensitive fibers. This was done by mechanical stimulation of the colon using a 1g von
Frey filament. Subsequently, the nerve bundles were exposed for 20-24 h to 5-10% biotinamide
(BTA), used as an anterograde tracer. During this time, the Krebs solution was exchanged with
oxygenated DMEM-12 containing fetal bovine serum, calcium chloride and nifedipine.
Afterwards, the colon was transversely cryo-sectioned in the longitudinal axis and processed for
immunohistochemistry. Traced fibers were detected by incubation with Streptavidin-Alexa Fluor
(488), and their phenotype as primary afferents evaluated using calcitonin gene-related peptide
(CGRP). We found that extrinsic nerves penetrating the colon extend both longitudinally and
transversely. Moreover, some traced fibers were observed in the mucosal layer, supporting
previous electrophysiological studies suggesting the presence of mucosal and muscular-mucosal
afferents in the colonic wall. A proportion of the traced nerves expressed CGRP, although BTA-
or CGRP-only fibers were also observed. The present data contribute to the anatomical
description of nerve fibers of extrinsic origin in the colon, and will be useful for future
correlations with the neurophysiological events occurring in the colonic wall. Supported by NS
19112.
Disclosures: P.R. Brumovsky, None; J. La, None; G.F. Gebhart, None.