Neonatal Maternal Deprivation Sensitizes Sodium Channels By Cystathionine β-synthetase Activation In Colon Specific Sensory Neurons In Adult Rats

Background and Aims: Irritable bowel syndrome （IBS） is a commongastrointestinal disorder characterized by abdominal pain in association with alteredbowel movements. The underlying molecular mechanisms of visceral hypersensitivityremain elusive. Recent studies suggest a role for hydrogen sulfide （H₂S） in painsignaling but this has not been well studied in animal models of visceral hyperalgesia.This study was designed to examine the roles for voltage-gated sodium channels andCBS-H₂S signaling in a rat model of chronic visceral hyperalgesia induced by neonatalmaternal deprivation （NMD）. Methods: Neonatal male rats （PND2-15） were exposed toa3hour period of daily maternal separation with temperature maintained at³3°C.Colon-specific dorsal root ganglion （DRG） neurons were labeled with DiI and acutelydissociated for measuring excitability and sodium channel current under whole-cellpatch clamp configurations. The expression of NaV1.8was analyzed by western blot,Quantities Real time-PCR and immunofluorescence study. The endogenous H₂Sproducing enzyme CBS antagonist was injected intraperitoneally. Results: NMDsignificantly increased AWR scores, which lasted for⁶weeks when compared withcontrol rats. Administration of CBS antagonist significantly reduced AWR score ofNMD model rats, and reversed the visceral hypersensitivity in a dose dependent manner.NMD also produced hyperexcitability of colon DRG neurons. TTX-resistant but notTTX-sensitive sodium current density was greatly enhanced in colon DRG neurons inNMD rats. Compared with controls, activation curve showed a leftward shift in NMD rats while the inactivation curves did not differ significantly. NMD markedlyaccelerated the activation time （10⁹0%） of peak current amplitude without anychanges in inactivation time. Furthermore, NMD remarkably enhanced expression ofNaV1.8at protein level but not at mRNA level in colon DRGs. The expression ofNaV1.9was not altered after NMD. We showed that CBS was colocalized with NaV1.8in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-（Carboxymethyl）hydroxylamine hemi hydrochloride （AOAA）, an inhibitor of CBS, significantly reducedexpression of NaV1.8in NMD rats when compared with NS treatment. AOAA treatmentalso inhibited the neurons’ excitability，reduced TTX-R sodium current density andnormalized the Vhalfof activation curve in colon-specific DRG neurons in NMD rats.Compared with controls, activation curve showed a rightward shift while theinactivation curves did not differ significantly after injection of AOAA inhibitor.Furthermore, addition of NaHS, a donor of H₂S, greatly enhanced the excitability andthe TTX-R sodium current density of DRG neurons in age-matched healthy rats.Compared with controls, activation curve showed a leftward shift while the inactivationcurves did not differ significantly after addition of NaHS. The potentiation of TTX-Rcurrent by NaHS was reversed by pre-incubation of a PKA inhibitor, H-89. Conclusions:These data suggest that sensitization of sodium channel activity is most likely mediatedby upregulation of CBS expression of colon DRG neurons in NMD rats, thusidentifying a potential target for treatment for chronic visceral pain in patients with IBS.