Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: A . GDNF is increased in muscle after I/R compared to naïve animals (n=4 per group) as assessed by western blotting (WB). B . Representative image of GDNF distribution within myofibers in an injured forepaw muscle. DAPI was used to mark nuclei. Solid arrows: GDNF positive myofibers. Dashed arrows: GDNF negative myofiber. C . WB of GFRα1 in DRGs showed increased expression in the C7-T1 DRGs after I/R and in PenCON+I/R groups. Penα1 effectively prevented this upregulation (n=3 per group). D . Paw guarding is increased 1d after I/R and PenCON+I/R and this is partially prevented in Penα1+I/R animals. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation ( E ) and muscle squeezing ( F ) are decreased after I/R and PenCON+I/R. GFRα1 knockdown blocked these effects. G . Grip strength is decreased 1d after I/R and PenCON+I/R. This was partially reversed in Penα1+I/R animals. H . Only injured mice (I/R and PenCON+I/R) displayed a significant increase in MAP after forced exercise. The increase in MAP was not observed in Penα1+I/R mice. (D-H: n=12 per group. x p<0.5 vs Naïve, *p<0.5 vs Sham, **p<0.01 vs Penα1+I/R, ***p<0.001 vs Sham and Penα1+I/R, ****p<0.0001 vs Sham and Penα1+I/R, + p<0.01 vs Sham, ^ p<0.05 vs pre-exercise MAP; 1-way ANOVA with HSD post hoc test (A, C) or 2-way RM ANOVA with HSD post hoc test (D-H)).

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p<0.05 vs Naïve and Penα1+I/R, **p<0.01 vs Naïve and Penα1+I/R; 1-way ANOVA with HSD post hoc test). E. Representative functionally identified muscle afferent obtained during ex vivo recording intracellularly filled with neurobiotin (green) was found to be immunopositive for GFRα1 (red) and P2X5 (blue) and was responsive to both “low” and “high” metabolite mixtures.

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Ex Vivo

Journal: bioRxiv

Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

doi: 10.1101/627521

Figure Lengend Snippet: After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.

Article Snippet: Embedded DRGs were stored at −80°C, sections (15 µm) were cut on a cryostat and mounted on slides and processed for GDNF receptor GFRα1 (goat anti-GFRα1, 1:100; cat no. AF-560, R&D Systems, Minneapolis, MN, USA) and P2X5 (rabbit anti-P2X5, 1:100; cat no. APR005 Alomone Labs, Jerusalem, Israel).

Techniques: Muscles, Binding Assay, Over Expression