The endogenous hydrogen sulfide producing enzyme cystathionine-β synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of visceral hypersensitivity, a characteristic pathophysiological feature of irritable bowel syndrome (IBS), remains elusive. Recent studies suggest a role for hydrogen sulfide (H₂S) in pain signaling but this has not been well studied in visceral models of hyperalgesia. We therefore determined the role for the endogenous H₂S producing enzyme cystathionine-β-synthetase (CBS) in a validated rat model of IBS-like chronic visceral hyperalgesia (CVH). CVH was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were performed at 8–10 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labeled by injection of DiI (1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall.</p> <p>Results</p> <p>In rat DRG, CBS-immunoreactivity was observed in approximately 85% of predominantly small- and medium-sized neurons. Colon specific DRG neurons revealed by retrograde labeling DiI were all CBS-positive. CBS-positive colon neurons co-expressed TRPV1 or P2X3 receptors. Western blotting analysis showed that CBS expression was significantly increased in colon DRGs 8 weeks after neonatal AA-treatment. Furthermore, the CBS inhibitor hydroxylamine markedly attenuated the abdominal withdrawal reflex scores in response to colorectal distention in rats with CVH. By contrast, the H₂S donor NaHS significantly enhanced the frequency of action potentials of colon specific DRG neurons evoked by 2 times rheobase electrical stimulation.</p> <p>Conclusion</p> <p>Our results suggest that upregulation of CBS expression in colonic DRG neurons and H₂S signaling may play an important role in developing CVH, thus identifying a specific neurobiological target for the treatment of CVH in functional bowel syndromes.</p

Nanomolar oxytocin synergizes with weak electrical afferent stimulation to activate the locomotor CPG of the rat spinal cord in vitro.

Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM-1 \u3bcM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits

Expression of GABA\u3csub\u3eB\u3c/sub\u3e Receptors in Magnocellular Neurosecretory Cells of Male, Virgin Female and Lactating Rats

GABA is one of the key neurotransmitters that regulate the firing activity of neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. In the present study, we used immunohistochemical techniques to study the distribution and subcellular localisation of metabotropic GABAB receptors in magnocellular neurones in the SON and PVN. Robust GABAB receptor immunoreactivity (GABABR; both subunit 1 and subunit 2 of the heterodimer), was observed in the SON and PVN. At the light microcope level, GABABR immonoreactivity displayed a clustered pattern localised both intracytoplasmically and at the plasma membrane. Densitometry analysis indicated that GABABR immunoreactivity was significantly more intense in vasopressin cells than in oxytocin cells, both in male, virgin female and lactating rats, and was denser in males than in virgin females. Light and electron microscope studies indicated that cytoplasmic GABABR was localised in various organelles, including the Golgi, early endosomes and lysosomes, suggesting the cycling of the receptor within the endocytic and trafficking pathways. Some smaller clusters at the level of the cell plasma membrane were apposed to glutamic acid decarboxylase 67 immunoreactive boutons, and appeared to be colocalised with gephyrin, a constituent protein of the postsynaptic density at inhibitory synapses. The presence of GABABR immunoreactivity at synaptic and extrasynaptic sites was supported by electron microscopy. These results provide anatomical evidence for the expression of postsynaptic GABAB receptors in magnocellular neurosecretory cells