Journal: The Journal of Neuroscience

Article Title: Extracellular pH and Neuronal Depolarization Serve as Dynamic Switches to Rapidly Mobilize trkA to the Membrane of Adult Sensory Neurons

doi: 10.1523/JNEUROSCI.4408-12.2013

Figure Lengend Snippet: Increased mobilization of trkA to neuronal membrane enhances response to NGF challenge. A, Fluorescence photomicrographs depicting relative levels of activated/phosphorylated trkA (phospho-trkA) detected under permeabilizing conditions in representative adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media (in the presence of anti-BDNF) for 30 min, followed by a 15 min challenge with pH-specific medium + anti-BDNF alone or with anti-BDNF + 50 ng/ml NGF. Scale bar, 10 μm. Scatter plots illustrate the relationship between phospho-trkA-like labeling intensity (y-axis; normalized to the mean signal intensity from the control pH group) and perikaryal diameter (x-axis) for all sensory neurons analyzed from three separate experiments exposed to control (pH 7.4, B) or acidic (pH 6.5, C) media as above and then challenged with 50 ng/ml NGF (open circles) or not (filled circles). D, Summary bar graph depicting relative levels of activated trkA (phospho-trkA; normalized to pH 7.4 control) in representative adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media for 30 min and then challenged with 50 ng/ml NGF and as normalized to the mean signal intensity from the control pH group. E, Fluorescence photomicrographs of sensory neurons exposed to identical conditions as in A and processed to detect activated/phosphorylated p38MAPK Note: A significant increase is observed in the levels of phospho-trkA detected in sensory neurons in response to acidosis with a parallel response observed for phospho-p38MAPK (E) (one-way ANOVA with post hoc Tukey's; ***p < 0.001; n = 131–210 neurons analyzed per experimental condition). Scale bar, 20 μm.

Article Snippet: Primary antibodies included a rabbit polyclonal anti-trkA (1:2000; Alexis Biochemicals), a rabbit polyclonal anti-phospho-p38MAPK (1:50; Cell Signaling Technology), and a rabbit polyclonal anti-phospho-trkA (1:100; Neuromics) diluted in blocking solution, and incubated at room temperature (trkA) or 4°C (phospho-trkA and phospho p38MAPK) for 24 h. The anti-trkA antibody recognizes the extracellular fragment of the trkA protein at amino acids 1–416, the phospho-trkA antibody recognizes the intracellular phosphorylated Y490 residue, while the phospho-p38MAPK recognizes p38MAPK phosphorylated at T180 and Y182 residues.

Techniques: Fluorescence, Labeling