The effects of target skeletal muscle cells on dorsal root ganglion neuronal outgrowth and migration in vitro

Abstract

Targets of neuronal innervations play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. During development, neurons extend axons to their targets, and then their survival become dependent on the trophic substances secreted by their target cells. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The interdependence of sensory neurons and skeletal muscle (SKM) cells during both embryonic development and the maintenance of the mature functional state has not been fully understood. In the present study, neuromuscular cocultures of organotypic dorsal root ganglion (DRG) explants and dissociate SKM cells were established. Using this culture system, the morphological relationship between DRG neurons and SKM cells, neurites growth and neuronal migration were investigated. The migrating neurons were determined by fluorescent labeling of microtubule-associated protein-2 (MAP-2) and neurofilament 200 (NF-200) or growth-associated protein 43 (GAP-43). The expression of NF-200 and GAP-43 and their mRNAs was evaluated by Western blot assay and real time-PCR analysis. The results reveal that DRG explants showed more dense neurites outgrowth in neuromuscular cocultures as compared with that in the culture of DRG explants alone. The number of total migrating neurons (the MAP-2-expressing neurons) and the percentage NF-200-immunoreactive (IR) and GAP-43-IR neurons increased significantly in the presence of SKM cells. The levels of NF-200 and GAP-43 and their mRNAs increased significantly in neuromuscular cocultures as compared with that in the culture of DRG explants alone. These results suggested that target SKM cells play an important role in regulating neuronal protein synthesis, promoting neuritis outgrowth and neuronal migration of DRG explants in vitro. These results not only provide new clues for a better understanding of the association of SKM cells with DRG sensory neurons during development, they may also have implications for axonal regeneration after nerve injury.

Figure 1. SEM photomicrographs of the neuromuscular coculture (A–F) and DRG explants culture alone (G–I).

Panel A: DRG explants send numerous large radial projections (thin arrows) to the peripheral area in neuromuscular coculture. Many neurons (thick arrows) migrated from DRG explants to the peripheral area. Panel B: The enlargement of the box in Panel A. Panel C: The axons form a dense lace-like network (thin white arrows) with crossing patterns on the surface of single layer SKM cells (thick black arrow) in neuromuscular coculture. The single migrating neurons (thick white arrows) scattered in the space of the network and send axons (thin black arrows) joining the network. Panel D: The axons cross (thin white arrows) on the surface of a single SKM cell (thick black arrow). Panel E: The endings of the axons enlarge and terminate on the surface of a single SKM cell (thick black arrow) to form NMJ-like structures (thin white arrows). Panel F: The enlargement of the box in Panel E. Panel G: DRG explants sends radial projections (thin arrows) to peripheral area in DRG explants culture. A few neurons (thick arrows) migrated from DRG explants to the peripheral area. Panel H: The enlargement of the box in Panel G. Panel I: The axons form a sparse lace-like network (thin white arrows) with crossing patterns in the peripheral area in DRG explants culture. The single migrating neuron (thick white arrow) sends axons (thin black arrow) joining the network. Scale bar = 50 µm in Panel A, G; Scale bar = 25 µm in Panel B, H; Scale bar = 10 µm in Panel C; Scale bar = 5 µm in Panel D, E, I; Scale bar = 2.5 µm in Panel F.

Figure 2. Double fluorescent labeling of MAP-2 (for neurons) and muscle actin (for muscle cells).

Panel A : MAP-2 for DRG neurons; Panel B: muscle actin for SKM cells; Panel C: overlay of Panel A and B. The migrating neurons send axons cross over (thick arrow) and terminate on (thin arrow) the surface of SKM cells. Scale bar = 50 µm.

Figure 3. Nerve fiber bundles extended from DRG explants.

Panel A, B: The example images to show how to quantify nerve fiber bundles. Nerve fiber bundles extended from DRG explants as far as 200 µm from the edge of a quarter of each DRG explants was counted in each sample. Panel A is neuromuscular coculture (thick arrows show SKM cells). Panel B is DRG explant culture. Panel C: The number of nerve fiber bundles extended from DRG explants. The number of nerve fiber bundles increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 10 different samples). *P<0.001. Scale bar = 40 µm.

Figure 4. The example images to show how to count cells.

The full visual field showed in the circle in which neurons were counted in one sample. The neurons in the box were showed in figure 6. Panel A is the total neurons (MAP-2-IR neurons). Panel B is NF-200-IR neurons. Panel C is the overlay of Panel A and B.

Figure 5. Total migrating neurons from DRG explants.

Total number of migrating neurons from DRG explants increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 38 different samples). *P<0.001.

Figure 6. Double fluorescent labeling of MAP-2 and NF-200.

Panel A: neuromuscular coculture (A1: MAP-2; A2: NF-200; A3: overlay of A1 and A2). Panel B: DRG explant culture (B1: MAP-2; B2: NF-200; B3: overlay of B1 and B2). Panel C: The percentage of migrating NF-200-IR neurons. The percentage of NF-200-IR neurons increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 20 different samples), Scale bar = 50 µm. *P<0.05.

Figure 7. Double fluorescent labeling of MAP-2 and GAP-43.

Panel A: neuromuscular coculture (A1: MAP-2; A2: GAP-43; A3: overlay of A1 and A2). Panel B: DRG explant culture (B1: MAP-2; B2: GAP-43; B3: overlay of B1 and B2). Panel C: The percentage of migrating GAP-43-IR neurons. The percentage of GAP-43-IR neurons increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 18 different samples), Scale bar = 50 µm. *P<0.001.

Figure 8. The mRNA levels of NF-200 and GAP-43.

The mRNA levels of NF-200 and GAP-43 increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 6). *P<0.01, **P<0.001.

Figure 9. The protein levels of NF-200.

The protein levels of NF-200 increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 6). *P<0.001.

Figure 10. The protein levels of GAP-43.

The protein levels of GAP-43 increased in neuromuscular coculture as compared with that in DRG explants culture alone. Bar graphs with error bars represent mean ± SEM (n = 6). *P<0.001.