The Effects Of Hydrogen Sulfide On The Electrical Activity Of Oxytocin Neurons And The Underlying Cellular Mechanisms

Background and purpose Oxytocin（OT）has a wide range of biological effects and is mainly used to promote uterine contractions during labor and postpartum hemostasis in clinical applications.OT is mainly synthesized by OT neurons in the supraoptic nucleus（SON）and paraventricular nucleus（PVN）of the hypothalamus.The function of OT neurons is regulated by various factors,such as excitatory and inhibitory synaptic activity,astrocyte plasticity,neuropeptide and ion concentrations,expression of intracellular signaling molecules,and the activity status of ion channels.Among them,the modulatory activity of the gaseous transmitter hydrogen sulfide（H₂S）on hypothalamic OT neurons has been rarely reported.Existing studies have demonstrated that hypothalamic H₂S production is mainly catalyzed by cystathione-β-synthase（CBS）,and CBS activity is primarily regulated by calmodulin kinase II（Ca MKII）.In the SON,both OT neurons and astrocytes express CBS,and inhibition of CBS reduces the basal firing rate of OT neurons and prevents the excitatory effects of OT on OT neurons.However,the source of H₂S in the SON and mechanisms underlying its action on OT neurons remains unclear.In the functional activity of the body,the synchronization of OT neuronal activity and the subsequent pulsatile OT secretion have important physiological and medical values,and the diffusible neurotransmitter and signaling molecule H₂S has a more important role in synchronizing neural activity than other types of neurotransmitters.Therefore,an in-depth study on the mechanism for H₂S production and its regulation of OT neuronal activity is important for understanding the activity pattern of OT secretion system and for developing OT clinical applications.Hence,this study observes the effect of astrocytic H₂S on OT neuronal activity and the mechanism underlying H₂S action,especially its effect on astrocyte plasticity.Methods In this study,5-6 weeks-old SD male rats（100-140 g）were used,and after decapitation and separation of brain tissue,the hypothalamus was sectioned into 300μm-thick coronal brain slices.Under artificial cerebrospinal fluid（ACSF）perfusion in vitro,brain slices were treated with OT,the CBS allosteric agonist SAM,the H₂S slow-releasing donor GYY4137,the CBS inhibitor aminooxyacetic acid（AOAA）,the Ca MK II-specific inhibitor KN-62,and the gliotoxin L-α-aminoadipic acid（L-AAA）,either alone or in combination,while whole-cell patch-clamp recording techniques were used to record spontaneous firing activity and excitatory postsynaptic currents（EPSCs）of OT neurons.After different drug treatments,expression of CBS,glial fibrillary acidic protein（GFAP）and Ca MK II protein in the SON were analyzed using Western blotting technique,and the spatial relationship between CBS and GFAP expression around OT neurons was analyzed using immunofluorescence technique.In addition,changes in the length of processes of primarily cultured hypothalamic astrocytes under different experimental conditions were measured.In analyzing the results,t-test and repeated-measures ANOVA in SPSS 26 software were applied for statistical analysis in this study.Results In hypothalamic slices,administration of physiological concentrations of OT significantly increased the firing rate,frequency and amplitude of EPSCs in OT neurons.Similar to the effect of exogenous OT,upregulation of H₂S concentration in tissues using SAM significantly increased the firing rate,frequency and amplitude of EPSCs in OT neurons.Similarly,GYY4137 also significantly increased the firing rate of OT neurons.AOAA,blocking H₂S production by inhibiting CBS,reduced the firing rate,frequency and amplitude of EPSCs in OT neurons,and also blocked the excitatory effects induced by OT.Application of KN-62,inhibiting Ca MKII activity,blocked the excitatory effects of OT.Application of L-AAA to disrupt astrocyte plasticity not only reduced the excitability of OT neurons,but also blocked the excitatory effects of SAM on OT neurons.The expression of GFAP within SON was significantly reduced after application of OT or GYY4137 incubated in brain slices in vitro.OT had no significant effect on the expression of CBS and Ca MK II.However,KN-62 preincubation blocked the effects of OT.Immunofluorescence and microscopy observations revealed that OT treatment of brain slices could significantly shorten the length of GFAP filaments and increase the expression of CBS in the ventral glial lamina,a region where astrocytic somata were aggregated.Finally,GYY4137 could time-dependently shorten the length of astrocytic processes in the primary cultures of hypothalamic astrocytes.Conclusions The auto-regulation of OT neuronal activity in the SON is associated with the activation of CBS and H₂S production in astrocytes.The production and action of H₂S depend on the activation of Ca MKII in astrocytes.H₂S can excite OT neurons by increasing the frequency and amplitude of EPSCs in OT neurons while causing retraction of astrocyte processes,which underlie the excitatory effect of OT on OT neurons.