This study crossed my radar scope because the investigators referenced use of our phosphoERK1/2 for Western Blotting and Immunohistochemistry: Gisela Patrícia da Silva Borges , Juan Antonio Micó Segura , Fani Lourença Moreira Neto , Esther Berrocoso. Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons. DOI: http://dx.doi.org/10.1093/ijnp/pyv019 First published online: 25 February 2015
Conclusion: pain-induced anxiety is mediated by CRF neurotransmission in the LC through ERK1/2 signaling cascade.
Figure: a) Schematic representation of the anatomical pathways implicated. Briefly, the contralateral LC indirectly receives inputs from the inflamed paw (red dashed line; ascending pathways) and, subsequently, the information is sent to corticolimbic areas. Additionally, the LC sends direct projections to the spinal cord (blue straight line; descending pathways). b) Body weight of the control and MA rats. c) Body rectal temperature of control and MA rats. d) Mechanical hyperalgesia represented by a significant decrease in the paw withdrawal threshold of the ipsilateral paw of MA rats. e) Mechanical allodynia represented by a significant decrease in the force threshold of the ipsilateral paw of MA rats. Graph depicting the expression of pERK1/2 in the LC after intra-LC administration of the αCRF receptor antagonist, showing that the significant increase of pERK1/2 in MA4W animals was no longer observed when this antagonist was administered. g) Graph showing that the local administration of the αCRF antagonist had no significant effect on mechanical hyperalgesia in MA4W rats. h) Graph showing that local administration of h) Graph showing that local administration of the α-helical CRF antagonist had no significant effect on mechanical allodynia in the ipsilateral paw of MA rats. i) Graph showing that the time spent in the open arms decreased in MA4W rats receiving the vehicle alone but this effect was successfully reversed by administration of the αCRF antagonist. j) Graph showing that local administration of the α-helical CRF antagonist had no significant effect on the total distance traveled in the elevated zero maze. k) Graph showing that local administration of the α-helical CRF antagonist reversed the decrease in the number of entries into the open arms observed in MA4W rats receiving the vehicle alone. B=Baseline; LC=Locus Coeruleus; αCRF=antagonist of the corticotropin-releasing factor receptor I and II; W=Week; MA=Monoarthritis.
Western Blotting: The membranes were blocked with 5% Bovine Serum Albumin (BSA; Sigma, Spain) in TBST and probed overnight at 4 ºC with a rabbit anti-phospho-ERK1/2 (1:5,000; Neuromics). Immunohistochemistry: Brains were removed and processed for free-floating immunohistochemistry. One in five sequential transverse brain sections (30 µm) containing the PVN from each rat were washed, blocked and incubated with a rabbit antiserum against the phosphorylated ERK1 and ERK2 isoforms (pERK1/2; 1:1000; 48 hours at 4-8ºC: Neuromics, USA). Immunodetection was achieved with a biotinylated donkey anti-rabbit antiserum (1:500; 1 hour; Jackson ImmunoResearch, USA), followed by an ABC solution (1:200, 1 hour; ABC Elite kit, Vector Laboratories, UK) and a colorimetric reaction with 3,3-diaminobenzidine tetrahydrochloride (DAB; 10 min) in 0.05M Tris-HCl buffer containing 0.003% hydrogen peroxide (Cruz et al., 2005). Sections were then washed in PBS, mounted on gelatin-coated glass slides, cleared in xylene, cover-slipped with DPX and analyzed by light microscopy.
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