In the thoracic spinal cord neither

In the thoracic spinal cord, neither glacial acetic challenge nor HBO2 had an effect on nNOS expression or expression of the phosphorylated β3 subunit of the GABAA receptor. This result was not unexpected due to the preponderance of nociceptive signaling in the lumbar versus thoracic spinal cord. In addition, the acetic acid test is not necessarily a valid model of visceral pain due to specificity problems with the model; for example, non-analgesics have been found to have antinociceptive activity according to the model, and the abdominal constrictions include the muscles of the abdominal wall rather than just the organs of the viscera (Langford and Mogil, 2011, Ness, 1999). Despite this criticism, many laboratories use the acetic acid test as a model of visceral pain because it has been a very powerful predictive tool and has a unique ability to detect weaker antinociceptive drugs (Langford and Mogil, 2011). Indeed, some labs use the acetic acid test as an assay of chemical nociception rather than a model of visceral pain (Lariviere et al., 2002, Spindola et al., 2012). Our study found that an injection of glacial acetic acid into the peritoneal cavity had no effect on protein expression in the spinal cord. In other pain models such as the formalin and capsaicin models, nNOS expression has been shown to increase in response to noxious insult in the spinal cord (Shi et al., 2005, Wu et al., 2001).
Conclusions We found evidence that the GABAA receptor is involved in HBO2-induced antinociception because HBO2-induced acute antinociception was antagonized in a dose-dependent manner by the selective competitive GABAA antagonist SR 95531. Preventing GABA reuptake using the GAT inhibitor nipecotic acid increased the antinociceptive response to an 11-min treatment of HBO2. The GABAB antagonist CGP 35348 failed to antagonize HBO2-induced antinociception at the doses used. HBO2 did restore expression of the phosphorylated β3 subunit of the GABAA receptor in the presence of acetic acid. It is possible that the antinociceptive effects of HBO2 are caused by preventing or restoring activity of GABAergic inhibitory interneurons in the lumbar region of the spinal cord. We were unable to conclusively link this effect to NO system because HBO2 failed to restore nNOS expression in the presence of acetic acid or by itself.
Experimental procedure
Acknowledgements This research was supported by NIH Grant AT-007222, the Allen I. White Distinguished Professorship from Washington State University, the Chico Hyperbaric Center (Chico, California), and the Chinese Scholarship Council (Joint Doctoral Program).
Introduction Neurosteroids such as pregnanolone (1, Chart 1), allopregnanolone (2) and tetrahydrodeoxycorticosterone (THDOC, 3) are potent positive allosteric modulators of GABA type-A receptors (GABAAR), producing sedative, anxiolytic and anticonvulsant effects [[1], [2], [3], [4], [5]]. The synthetic allopregnanolone derivative alphaxalone (4) acts as a general anesthetic [6], but was withdrawn from clinical use due to anaphylactoid side effects associated with its ethoxylated castor oil formulation [7]. Currently, alphaxalone is used in veterinary medicine [8] and there is strong interest in restoring the clinical use of this and structurally related drugs [9,10]. More broadly, analogs of endogenous neurosteroids are of interest as potential drugs for treatment of epilepsy, anxiety, mood disorders and depression, and as probes for interrogation of GABAAR pharmacology [11]. Pharmacological studies show the GABAAR binding site(s) for neuroactive steroids differ from those of GABA and benzodiazepines, which bind to homologous sites at subunit interfaces in the extracellular domain (ECD), or of other intravenous general anesthetics including propofol, barbiturates, and etomidate, which bind to homologous sites at subunit interfaces in the extracellular third of the transmembrane domain (TMD) [4,[12], [13], [14]]. In recent crystal structures of chimeric homo–pentameric receptors containing GABAAR α subunit TMDs, pregnanolone and THDOC bound to a subunit interface site, whereas the inhibitory steroid pregnenolone sulfate (PS) bound to a distinct intrasubunit site [15,16]. Both the latter sites are near the cytoplasmic end of the TMD and in close proximity to a residue previously photolabeled by 6-azipregnanolone (5) in homomeric β3 GABAARs [17].