The Roles And Mechanism Of Hydrogen Sulfide In The Regulation Of Rat Central Neuronal Excitability And Epilepsy And Feeding Behaviors

Part Ⅰ Hydrogen sulfide promotes the surface insertion of AMPARs in rat hippocampal CA1regionAim:Hydrogen sulfide （H2S） has been widely accepted as a gas neurotransmitter/neuromodulator to regulate synaptic transmission. Our recent study has proved that H2S is a regulator of neuronal excitability and enhances a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor （AMPAR） function. However, the precise mechanism underlying this phenomenon was yet unresolved.Methods:Herein, we set out to determine the mechanisms by western blot analysis, patch clamp and biotin switch assay. The expression and phosphorylation of AMPARs, and the activation of PKA, PKC, CaMKII and ERK1/2were examined by western blot analysis. The currents of AMPARs were recorded by patch clamp recording. The sulf-sulfhydration levels were detected by biotin switch assay.Results:We observed that bath application of H2S donor NaHS （50and100μM） rapidly promoted surface insertion of AMPAR GluAl subunit in the hippocampal slices and increased AMPAR-mediated excitatory postsynaptic currents. This effect can be abolished by dithiothreitol （DTT）, indicating that a sulfhydration-dependent mechanism may be involved. Notably, NaHS did not increase the sulfhydration of AMPAR subunits, but it significantly increased the phosphorylation of GluAl at Serine-831and Serine-845sites. Postsynaptic signal pathways that control GluAl phosphorylation, such as protein kinase A （PKA）, protein kinase C （PKC） and calcium-calmodulin-dependent protein kinases Ⅱ （CaMKII）, were activated by NaHS and this activation can be occluded by DTT. Furthermore, we observed that H2S increased S-sulfhydration of protein phosphatase type2A （PP2A） in the hippocampus, which may be responsible for the activation of postsynaptic signal pathways.Conclusion:These data suggest that H2S promotes surface insertion of AMPARs in the hippocampus via phosphorylation of GluAl at Serine-831/Serine-845sites, which depends on a S-sulfhydration-mediated activation of postsynaptic signal pathways. Part Ⅱ Hydrogen sulfide increases the cortical neuronal excitability and aggravates seizure-like events in ratsAim:Epileptic seizures are well-known neurological complications following stroke, occurring in3% of patients. However, the intrinsic correlation of seizures with stroke remains largely unknown. Hydrogen sulfide is a gas transmitter that may mediate cerebral ischemic injury. But the role of H2S in seizures has not been understood yet. We examined the effect of H2S on seizure-like events （SLEs） and the underlying mechanisms.Methods:Pentylenetetrazole （PTZ） and pilocarpine-induced rat epileptic seizure models were tested. Low-Mg2+/high-K+and4-aminopyridine （4-AP）-induced epileptic seizure models were examined using patch-clamp recordings in rat brain slices. The functions of voltage-gated sodium channels （VGSC）, AMPAR and NMDAR were also investigated with the treatment of hydrogen sulfide. Results:It was found that NaHS aggravated both PTZ （PTZ30:control,0.75±0.26, NaHS,2.37±0.57; PTZ75:control,4.12±0.23, NaHS,5.5±0.27） and pilocarpine-induced SLEs （30:control,2.25±0.16, NaHS,3.1±0.38;60:control,3.75±0.41, NaHS,4.75±0.42） in rats, while both low-Mg2+/high-K+（Control,70±19s; NaHS,173±32s） and4-AP-induced SLEs （Control,29±8s; NaHS,42±13s） were also exacerbated by NaHS in brain slices, which may be due to its regulation on the functions of VGSC, NMDAR, and AMPAR. Furthermore, these effects were reversed by blocking VGSC, NMDAR, and AMPAR.Conclusion:These results suggest a pathological role of increased H2S level in SLEs in vivo and in vitro. Enzymes that control H₂S biosynthesis could be interesting targets for antiepileptic strategies in poststroke epilepsy treatment. Part III Hydrogen sulfide increases AMPK activation in ARC region and feeding behavior of ratsAim:Hydrogen sulfide is an endogenous gaseous molecule which plays important physiological and pathological roles in many tissues and organs including the brain. Hypothalamal arcuate nucleus （ARC）, is a critical modulation centre for energy metabolism and feeding behavior. Hypothalamal AMPK is a critical gauge molecule in the maintainace of energy status and controlling of energy metabolism. But the role of hydrogen sulfide in the hypothalamus has not been investigated.Methods:We carried out experiments with behavioral testing, intracerebroventricular （i.c.v） cannulation and injection, primary culture of ARC neurons, western blot analysis, biotin switch assay, calcium imaging assay, gas inhalation experiments, immunocytochemical and immunofluorescent methods, to explore the efficacy and mechanism of H2S on the feeding consumption of rats.Results:Here we show that hydrogen sulfide increased rat food intake and c-Fos expression in ARC neurons, enhanced the sulfur-sulfydryl level, the intracellular free Ca2+concentration [Ca2+]i and AMPK activation in ARC. Then, blocking the sulfur-sulfydrylation effect of hydrogen sulfide by DTT abolished the orexigenic effect of H2S, the increased AMPK activation and intracellular Ca2+level in ARC neurons. What is more, fasting also increased the sulfur-sulfydryl level in ARC, and blocking fasting-induced sulfur-sulfydrylation in ARC with DTT partially attenuated the enhanced AMPK activity and increased food intake.Conclusion:These results suggest that gaseous molecule H2S, which is also an endogenous metabolite of proteins and amino acids, promotes appetite via its S-sulfydrylation effect in the hypothalamal ARC region, and this function also partially mediates fasting-induced feeding increase and AMPK activation in ARC.