Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

Abstract

Both neurotrophins (NTs) and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT), calcitonin-gene related peptide (CGRP), neurofilament 200 (NF-200), and microtubule associated protein 2 (MAP-2) in dorsal root ganglion (DRG) sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG) neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF) or neurotrophin-3 (NT-3) on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

Figure 1

Inverted phase contrast microscopic photomicrographs of the living cells in neuromuscular coculture at six days of culture age. DRG neuronal cell body (thin arrows) sends axons across or terminating on SKM cells (thick arrows). Panel A: neuromuscular coculture with NGF treatment; Panel B: neuromuscular coculture with NT-3 treatment; Panel C: neuromuscular coculture without NT treatment. Scale bar = 50 µm

Figure 2

PPT mRNA expression at different culture conditions. Normalized levels of PPT mRNA were higher in NGF treated cocultures than control. NT-3 treatment increased PPT mRNA levels. Bar graphs with error bars represent mean ± SD (n = 12). *p < 0.01

Figure 3

CGRP mRNA expression at different culture conditions. Normalized levels of CGRP mRNA were higher in NGF treated cocultures than control. NT-3 treatment increased CGRP mRNA levels. Bar graphs with error bars represent mean ± SD (n = 12). *p < 0.05, **p < 0.01

Figure 4

NF-200 mRNA expression at different culture conditions. Normalized levels of NF-200 mRNA were higher in NGF treated cocultures than control. NT-3 treatment increased NF-200 mRNA levels. Bar graphs with error bars represent mean ± SD (n = 12). *p < 0.05, **p < 0.001

Figure 5

MAP-2 mRNA expression at different culture conditions. Normalized levels of MAP-2 mRNA were not changed significantly in NGF or NT-3 treated cocultures than control. Bar graphs with error bars represent mean ± SD (n = 6)