Dense lace-like network (thin white arrows) with crossing patterns on the

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 GW-0742 custom synthesis 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 mm in Panel A, G; Scale bar = 25 mm in Panel B, H; Scale bar = 10 mm in Panel C; Scale bar = 5 mm in Panel D, E, I; Scale bar = 2.5 mm in Panel F. doi:10.1371/journal.pone.0052849.gFurthermore, the levels of NF-200 and GAP-43 and their mRNAs also 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 inFigure 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 mm. doi:10.1371/journal.pone.0052849.gthe regulation of neuronal protein synthesis, promoting neurites outgrowth and neuronal migration of DRG explants in vitro. MAP-2 is a cytoskeletal protein. It plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons [37]. MAP-2 has been tentatively implicated in neuronal outgrowth and polarity of neuronal cells [15]. It has been shown that MAP-2 is specifically expressed in neuronally differentiated cells [16]. MAP-2 is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and it appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites [38]. It has been suggested that MAP-2 can interact with cytoskeletal components and might be critically involved in neurites initiation [39]. Within the neuronal cell, MAP-2 proteins are known to interact with btubulin, neurofilaments (NFs) and actins, and contribute to dendrite outgrowth and maintenance of neuronal cytoarchitecture [40?1]. In the present study, MAP-2 was used as a neuronal marker to detect neurons in different culture conditions. The migrating MAP-2-IR neurons increased significantly in neuroTarget SKM on Neuronal Migration from DRGFigure 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 mm from the edge of a quarter of each DRG explants was counted in each sample. Panel A is neuromuscular PD 168393 site coculture (thi.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 mm in Panel A, G; Scale bar = 25 mm in Panel B, H; Scale bar = 10 mm in Panel C; Scale bar = 5 mm in Panel D, E, I; Scale bar = 2.5 mm in Panel F. doi:10.1371/journal.pone.0052849.gFurthermore, the levels of NF-200 and GAP-43 and their mRNAs also 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 inFigure 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 mm. doi:10.1371/journal.pone.0052849.gthe regulation of neuronal protein synthesis, promoting neurites outgrowth and neuronal migration of DRG explants in vitro. MAP-2 is a cytoskeletal protein. It plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons [37]. MAP-2 has been tentatively implicated in neuronal outgrowth and polarity of neuronal cells [15]. It has been shown that MAP-2 is specifically expressed in neuronally differentiated cells [16]. MAP-2 is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and it appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites [38]. It has been suggested that MAP-2 can interact with cytoskeletal components and might be critically involved in neurites initiation [39]. Within the neuronal cell, MAP-2 proteins are known to interact with btubulin, neurofilaments (NFs) and actins, and contribute to dendrite outgrowth and maintenance of neuronal cytoarchitecture [40?1]. In the present study, MAP-2 was used as a neuronal marker to detect neurons in different culture conditions. The migrating MAP-2-IR neurons increased significantly in neuroTarget SKM on Neuronal Migration from DRGFigure 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 mm from the edge of a quarter of each DRG explants was counted in each sample. Panel A is neuromuscular coculture (thi.