Journal: Frontiers in Neuroscience
Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons
Figure Lengend Snippet: M 1 R antagonists, MT7 and pirenzepine, augment neurite outgrowth in primary sensory neurons and M 1 R overexpression inhibits neurite outgrowth. (A,B) Whiskers box (Tukey) showing total neurite outgrowth per neuron. Neurons were grown for 48 h in defined media containing LGF (A) or HGF (B) condition and treated with 100 nM MT7 or 1 μM pirenzepine (PZ), respectively. N = 1249 (LGF) and 1517 (HGF), respectively. P -values were calculated by one-way ANOVA followed by post hoc multiple comparison tests. Dunnett’s multiple comparisons test was used to compare the MT7 and PZ treatment groups with the control group and Sidak’s multiple comparisons test was used to compare between the MT7 and PZ treatment groups; ∗ indicates the p -value obtained by Sidak’s multiple comparisons test. (C,D) Binning of the entire data set presented in (A,B) . (E) Immunoblot showing GFP-tagged muscarinic receptors (M 1 R to M 5 R) and GFP expression in transfected adult rat DRG neurons. pEGFP-C1-(M 1 R-M 5 R) plasmids were transfected in to DRG neurons and the lysate was resolved in SDS page and subsequently immunoblotted with anti-M 1 R (bottom panel) and anti-GFP (top panel) antibodies. (F) Time lapse confocal images showing increasing internalization (white arrows) of the GFP-M 1 R following treatment with carbachol (10 μM). Scale bar: 10 μm. (G) Immunofluorescence images showing colocalization of 24h CCh treated GFP-M1R with endosomal marker Rab5. Scale bar: 10 μm. (H) Whiskers box (Tukey) showing total neurite outgrowth per neuron, N = 634 (GFP), and N = 553 (GFP-M 1 R), neurons, respectively. P -value was calculated by t -test (unpaired). (I) Immunofluorescence images showing β-tubulin III staining and corresponding neurite trace (red lines) images in GFP and GFP-M 1 R overexpressed neurons. The total neurite outgrowth measurement was performed in Cellomics ArrayScan HCS Reader using neuronal profiling software. Scale bar: 10 μm.
Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).
Techniques: Over Expression, Expressing, Transfection, SDS Page, Immunofluorescence, Marker, Staining, Software