Role Of Histamine H₁,H₂ Receptors In The Modulation Of Rhythmical Respiration And Discharge Activities Of Respiratory Neurons In The Medulla Oblongata Slice Preparation Of Neonatal Rats

ObjectHistamine (HA) is well known to be a key modulator of respiration in the central nervous system (CNS)1) The present study was carried out to determine the role of histamine H₁ and H₂ receptors in the generation and modulation of basic respiratory rhythm; 2) Experiments were performed to determine whether H₁ receptor 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 (XII nerve) were simultaneously recorded by using extracellular microelectrodes and suction electrode, respectively. The effects of HA receptors on the respiratory rhythm were investigated by application of HA, H₁ receptor specific antagonist pyrilamine and H₂ receptor specific antagonist cimetidine in the perfusion solution. 1) Six isolated neonatal rat brainstem slices were used in this group: The possible role of HA on RRDA was investigated by perfused different concentration of HA (0.5, 1, 5, 10μmol/L in the modified Kreb's solution (ACSF). RRDA of each concentrate was recored for 20 min. 2) Thirty medulla oblongata slice preparations were divided into five groups. In groupâ… ,â…¡andâ…¢, histamine (HA , 5μmol/L), H₁ receptor specific antagonist pyrilamine (10μmol/L ) and H₂ receptor specific antagonist cimetidine (5μmol/L) was added into the perfusion solution for 15 min separately. RRDA were recorded from hypoglossal nerve rootlets before and during perfusion of the slice preparation with HA, H₁ receptor specific antagonist pyrilamine and H₂ receptor specific antagonist cimetidine into the modified Kreb's perfusion solution (MKS).In groupâ…£, after application of HA for 15 min, additional pyrilamine was added into the perfusion for another 15 min. In groupâ…¤, after application of HA for 15 min, additional cimetidine was added into the perfusion for another 15 min. The discharges of the rootlets of hypoglossal nerve were recorded. 3) 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. HA (5μmol/L) was added into the perfusing solution for 15 min first, after washing out, the H₁ receptor specific antagonist pyrilamine (10μmol/L) was applied to the perfusing solution for 15 min.Results1. The discharges of hypoglossal nerve rootlets were recorded: Control experiments were performed without drug application.1 ) The possible role of HA on RRDA was investigated by perfusing different concentration of HA, 0.5, 1,5. 10μmol/L in the modified Kreb's solution (ACSF). HA increased RRDA at the range of concentration from 1 to 10μmol/L. HA could decrease RC and TE, but TI and IA were statistically nonsignificant (RC, F= 10.427 P=0.000; TE, F=10.534 P=0.000; TI, F=0.687 P=0.609; IA, F=0.432 P=0.784). In our experiment, we found 5μmol/L HA could decrease RC and TE most efifectively compared with control (RC, P=0.011; TE, P=0.011) . So we chose this concentrate as the used concentrate.2) Bath application of 5μmol/L HA for 15 min , HA reduced respiratory cycle(RC , F=5.887, P=0.000) and expiratory time(TE, F=16A95, P=0.000), but the changes of inspiratory time(TI) and integral amplitude(IA) were statistically nonsignificant (TI, F=0.333 P=0.801; IA, F=1.713 P= 0.207) compared with the control. In 5 min, HA reduced RC by 15.12% (P=0.014 vs control) and TE by 15.76% (P=0.012 vs control), however, TI and IA were statistically nonsignificant. The result of 10 min was almost the same with that of 5 min. After application of 10μmol/L pyrilamine, pyrilamine prolonged RC and TE (RC, F= 12.997, P=0.000; TE, F=12.874, P=0.000), but the changes of inspiratory time(TI) and integral amplitude(IA) were statistically nonsignificant(TI, F=0.333 P=0.801; IA, F=1.713 P= 0.207) compared with the control. In 5 min, pyrilamine prolonged RC by 18.13% (P=0.035 VS control) and TE by 18.95% (P=0.035 VS control). In 10 min, pyrilamine prolonged RC by 29.31% (P=0.009 vs control) and TE by 30.57% (P=0.009 vs control). But TI and IA were statistically nonsignificant (TI, F=0.674 P=0.581; IA, F=2.832 P=0.074). After application of 5μmol/L cimetidine, cimetidine had no effects on RRDA. (RC, F=0.819 P=0.503; TE, F=0.820 P=0.503; TI, F=1.517 P=0.238; IA, F=1.519 P= 0.250).3) After application of HA, RC and TE were shortened but the changes of TI and IA were statistically nonsignificant compared with the control. These effects could be reversed by additional administration of pyrilamine. RC and TE were decreased by 24.25%(P=0.005 vs HA) and 25.41 %(P=0.005 vs HA). (RC, F=7.289 P=0.003; TE, F=7.285 P=0.003 ;TI, F=0.594 P=0.628; IA, F=0.531 P= 0.668)4) After application of HA, RC and TE were shortened but the changes of TI and IA were statistically nonsignificant, compared with the control. After added additional administration of cimetidine, there were no significant changes in RRDA compared with application HA only. (RC, F=8.657 P=0.001; TE, F=8.575 P=0.001; TI, F=0.177 P=0.910; IA, F=0.605 P= 0.622)2. The rhythmical discharges of respiratory neurons and activities of the hypoglossal nerve rootlets were simultaneously recorded: Effects of HA and pyrilamine on the discharge activity of I-neurons: 5μmol/L HA shortened RC by 25.86% (P=0.038) and TE by 27.03% (P=0.037), but TI, IA and the peak discharge frequency (PFn) were statistically nonsignificant (TI, P=0.745; IA, P=0.288; PFn, P=0.874). After washout of HA, application of 10μmol/L pyrilamine for 10 min changed the discharge activity of I-neurons with a 21.46% (P=0.005 ) increase in RC and a 22.15% (P=0.005 ) increase in TE. Pyrilamine have no significant effects on TI, IA and PFn (TI, P=0.594; IA, P=0.448; PFn, P=0.149). ContusionThe results indicated that in neonatal rat brainstem slice in vivo: 1) HA plays an important role in the modulation of the respiratory rhythmical discharge activity in the medullary slice preparation of neonatal rats. 2) Histaminergic neuromodulation acts predominantly via H₁ receptors. H₁ receptors can short RC predominantly via TE, but had no significant effects on TI,IA and I-neurons. 3) H₂ receptor has no significant effects on RC,TE,TI and IA.