Friday, August 01, 2008

Stressed Induced Analgesia (SIA) and OFQ

This is to highlight excellent work done by our friends at SRI and AfaSci.

Our Orphanin FQ ab was used in experiments by the team. Here's the latest publication highlighting their work:

Xinmin Xie, Jonathan P. Wisor, Junko Hara, Tara L. Crowder1, Robin LeWinter, Taline V. Khroyan, Akihiro Yamanaka, Sabrina Diano, Tamas L. Horvath, Takeshi Sakurai, Lawrence Toll and Thomas S. Kilduff. Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia. J. Clin. Invest. 118(7): 2471-2481 (2008). doi:10.1172/JCI35115. Copyright © 2008, The American Society for Clinical Investigation.

...Brain sections (10 μm) were treated with 0.3% H2O2 to quench endogenous peroxidases and then incubated overnight in primary anti-N/OFQ (1:5,000; RA10106, anti-FGGFTGARKSARKLANQ, Neuromics) and anti-orexin-B (1:5,000; sc-8071, Santa Cruz Biotechnology Inc.) antisera at 4°C with agitation. Sections were incubated in blocking buffer for 1 hour, followed by a 2-hour incubation at room temperature in secondary antisera (Alexa Fluor 546 donkey anti-goat [1:750] and 488 donkey anti-rabbit [1:500]; Molecular Probes, Invitrogen). As a negative control, additional sections were treated similarly, but the primary antibody was omitted. Preadsorption with the N/OFQ peptide FGGFTGARKSARKLANQ was used as a positive control and blocked all specific staining as also found by others...

N/OFQ-containing fibers innervate Hcrt neurons, and N/OFQ inhibits Hcrt neuronal activity. (A) Left panel shows confocal image of N/OFQ-immunoreactive fibers in the vicinity of, and in putative contact with, Hcrt-immunoreactive neurons in the PLH of WT mice. N/OFQ (green) fibers are in close proximity to Hcrt-immunoreactive (red) cells. The arrow indicates the N/OFQ innervation of an Hcrt cell body. Middle panel shows light micrograph of a light brown immunolabeled Hcrt neuron contacted by a dark black bouton (arrow) representing immunolabeling for N/OFQ. Right panel shows electron micrograph taken from ultrathin sections of the same labeled terminal and dendrite shown in the light micrograph in the middle panel. Black arrow indicates the N/OFQ-immunolabeled axon terminal in synaptic contact (red arrowhead) with the dendrite of the Hcrt cell. Scale bars: 10 μm (left and middle panels); 1 μm (right panel). (B) Under current clamp, bath application of N/OFQ (1 μM) hyperpolarized Hcrt neurons, decreased input resistance, and blocked spontaneous firing of action potentials. The resting potential of this cell was –54 mV and was manually adjusted to –60 mV with DC current. Membrane resistance was monitored using hyperpolarizing current pulses (–0.3 nA, 800 ms) delivered every 5 seconds throughout the experiment. (C) Under voltage-clamp mode at a Vh of –60 mV, N/OFQ (1 μM) induced an outward current (–53 pA) in an Hcrt neuron. Notice that the frequency but not the amplitude of the miniature synaptic currents (inward currents) recorded in the presence of TTX (0.5 μM) was also reduced.

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