[Abstract Title]. - Society for Neuroscience

Poster
269. Pain: Visceral Pain II
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 269.14/GG29
Topic: D.08.n. Visceral pain
Support: NIH Grant NS19912
NIH Grant NS35790
Title: The biochemical properties of cultured mouse splanchnic and pelvic nerve dorsal root
ganglion neurons change over time
Authors: *D. R. ROBINSON, J.-H. LA, G. F. GEBHART;
Ctr. for Pain Res., Univ. of Pittsburgh, Pittsburgh, PA
Abstract: Functional studies of sensory neurons can require the use of cultured dorsal root
ganglia (DRG) cells. Previous studies have suggested that the phenotype of these cells change in
a culture time dependent manner. Here we report changes in Calcitonin Gene-Related Peptide
(CGRP)-like immunoreactivity and the binding of Isolectin B4 (IB4) in DRG somata with
specific emphasis on those innervating the colon.
DRG neurons innervating the descending colon were retrogradely labeled in adult male C57
BL/6 mice using Fast Blue. Four weeks later, splanchnic (TL; T8 - L1) and pelvic (LS; L6 - S1)
DRG neurons were dissected and cultured. Neurons were enzymatically treated, triturated, and
plated onto coverslips that were fixed 2, 6, 9, 12, 24, 48, and 72 hours (TL) or 2, 12, 24, and 72
hours (LS) later. Fixed coverslips were preblocked and sequentially incubated with primary
antibody, Cy3-conjugated secondary antibody, and AlexaFluor 488-conjuagated IB4. Analysis
was performed with NIH image analysis software, ImageJ.
23% of all DRG neurons were CGRP-like immunoreactive (LI) 2 hours after plating. This
increased to approximately 34% after 12 - 24 hours, then decreased to 25%. The number of
CGRP-LI cells in the colonic DRG population was greater: 68% after 2 hours, 91% after 24
hours. IB4 binding was relatively unchanged over time in the whole population of cells
(approximately 26%) but colonic DRG neurons showed a decrease from 49% after 2 hours to
approximately 34% after 12 hours. The relative number of CGRP-LI cells in TL and LS
populations was similar but more LS neurons bound IB4. A similar trend was seen for those cells
that were both CGRP-LI and bound IB4. After 9 hours, nearly all (90%) of IB4 binding colonic
DRG neurons were also CGRP-LI, compared to only 14% of unlabeled cells.
In conclusion, time- and marker-dependent changes were seen in cultured DRG neurons that
varied among specific populations (e.g., colonic afferents). When considering the colonic DRG