Role Of Glycine In The Modulation Of Rhythmical Respiration And Discharge Activities Of Respiratory Neurons In The Medulla Oblongata Slice Preparation Of Neonatal Rats

ObjectGlycine(Gly)is an important inhibitory neurotransmitter in the central nervous system(CNS),it is a key modulator of respiration.1) The present study was carried out to determine the role of Gly in the generation and modulation of basic respiratory rhythm;2)Experiments were performed to determine whether Gly could modulate the discharge activities of inspiratory neurons(I-neurons).MethodsIn this study,newborn SD rats(0-3 days) brainstem slices were made according to the method of Suzue,et al.These preparations included the medial region of the nucleus retrofacialis(mNRF) with the hypoglossal nerve rootlets retained. Respiratory rhythmical discharge activity(RRDA) of I-neurons in mNRF and activities of the hypoglossal nerve(Ⅻnerve) were simultaneously recorded by using extracellular microelectrodes and suction electrode,respectively.The effect of Gly on the respiratory rhythm was investigated by application of Gly,Gly receptor specific antagonist STR in the perfusion solution.1) Eighteen medulla oblongata slice preparations were divided into three groups.In groupⅠandⅡ,Gly(10μmol/L),Gly receptor specific antagonist STR(1μmol/L) was added into the perfusion solution for 20 min separately.RRDA were recorded from hypoglossal nerve rootlets before and during perfusion of the slice preparation with Gly and Gly receptor specific antagonist STR into the modified Kreb's perfusion solution(MKS).In groupⅢ, after application of Gly for 20 min,additional STR was added into the perfusion for another 20 min.The discharges of the rootlets of hypoglossal nerve were recorded.2) RRDA of the hypoglossal nerve rootlets were recorded by using suction electrodes.In order to record respiratory neurons,glass microelectrodes fixed on a micro-manipulator were inserted into the ventral respiratory group(VRG) in medulla with a step pace(10μm every time)till a spontaneous respiratory unit,and the discharge activities of I-neurons in mNRF discharge were obtained.The inspiratory neurons were identified by their on-going activities that were correlated with RRDA of the hypoglossal nerve.Drugs were administered by bath application and their effects on the neuronal activities were investigated.Five medulla oblongata slice preparations were used in this group.Gly(10μmol/L) was added into the perfusing solution for 20 min first,after washing out,the Gly receptor specific antagonist STR (1μmol/L) was applied to the perfusing solution for 20 min.Results1.The discharges of hypoglossal nerve rootlets were recorded:Control experiments were performed without drug application.1) Bath application of 10μmol/L Gly for 20 min,Gly reduced inspiratory time (TI,t=4.741 P=0.005)and integral amplitude(IA,t=2.995 P=0.030),but the changes of respiratory cycle(RC)and expiratory time(TE)were statistically nonsignificant (RC,t=0.775 P=0.473;TE,t=0.683 P=0.525) compared with the control.In 10 min, Gly reduced TI by 21.46%and IA by 13.74%.After application of 1μmol/L STR, STR decreased RC and TE(RC,t=4.048,P=0.010;TE,t=4.082,P=0.010),but the changes of inspiratory time(TI) and integral amplitude(IA) were statistically nonsignificant(TI,t=0.283 P=0.789;IA,t=1.558 P=0.180) compared with the control. In 10 min,STR reduced RC by 5.93%and TE by 6.22%. 2) After application of Gly,TI and IA were shortened but the changes of TE and RC were statistically nonsignificant compared with the control.These effects could be reversed by additional administration of STR.TI and IA were increased by 16.90 %(P=0.022 vs Gly) and 13.07%(P=0.038 vs Gly).(TI,F=6.131 P=0.019;IA, F=6.935 P=0.018;TE,F=1.677 P=0.250;RC,F=1.613 P=0.259).2.The rhythmical discharges of respiratory neurons and activities of the hypoglossal nerve rootlets were simultaneously recorded:Effects of Gly and STR on the discharge activity of I-neurons:10μmol/L Gly shortened IA by 22.63% (P=0.027) and TI by 22.74%(P=0.005),but TE,RC and the peak discharge frequency(PFn) were statistically nonsignificant(TE,P=0.068;RC,P=0.085;PFn, P=0.202).After washout of Gly,application of 1μmol/L STR for 20 min changed the discharge activity of I-neurons with a 5.97%(P=0.017) decrease in RC and a 5.68% (P=0.021)decrease in TE.STR have no significant effects on TI,IA and PFn(TI, P=0.095;IA,P=0.07;PFn,P=0.452).ConlusionThe results indicated that in neonatal rat brainstem slice in vivo:1) Gly plays an important role in the modulation of the respiratory rhythmical discharge activity in the medullary slice preparation of neonatal rats.2) Gly acts predominantly via Gly receptor.Gly receptor can short IA and TI.3)Gly can inhibit inspiratory neurons,and short TI and IA.4)Gly may inhibit the dischange activities of inspiratory neurons,and then inhibits the respiratory rhythmical discharge activity.