5-HT_1A Receptors Are Involved In The Modulation Of Rhythmical Respiration And Discharge Activities Of Respiratory Neurons In The Medulla Oblongata Slice Preparation Of Neonatal Rats

ObjectSerotonin(5-hydroxytryptamine; 5-HT) is well known to be a key modulator ofrespiration in the central nervous system(CNS), 1) The present study was carried outto determine the role of 5-HT_1A receptors in the generation and modulation of basicrespiration rhythm; 2) Experiments were performed to determine whether 5-HT_1Areceptors could modulate the discharge activities of biphasic expiratory neurons(BE-neurons) and inspiratory neurons (I-neurons).MethodsIn this study, Newborn SD rats(0-3days) brainstem slices were made accordingto the method of Suzue, et al. These preparations included the medial region of thenucleus retrofacialis(mNRF) with the hypoglossal nerve rootlets retained. Respiratoryrhythmical discharge activity(RRDA) of BE-neurons/I-neurons in mNRF andactivities of the hypoglossal nerve(Ⅻnerve) were simultaneously recorded by usingextracellular microelectrodes and suction electrode, respectively. The effects of5-HT_1A receptors on the respiratory rhythm were investigated by application of5-HT_1A receptors specific agonist (+/-)-8-hydroxy-2-(di-N-propylamino)tetralinhydrobromide(8-OH-DPAT) and its specific antagonist [4-iodo-N-[2-[4-(methoxyph enyl)-1-piperazinyl]ethyl]-N-2-pyridynyl-benzamide hydrochloride](PMPPI) inthe perfusion solution. 1) Ten medulla oblongata slice preparations were divided intotwo groups. In groupⅠ, 5-HT_1A receptor specific agonist 8-OH-DPAT (20μmol/L)was added into the perfusing solution for 10 min first, after washing out, the 5-HT_1Aantagonist PMPPI (10μmol/L) was applied to the perfusing solution for I0 min. IngroupⅡ, after application of 8-OH-DPAT for 10 min, additional PMPPI was addedinto the perfusing solution for 10 min. The discharges of the rootlets of hypoglossalnerve were recorded. RRDA were recorded from hypoglossal nerve rootlets beforeand during perfusion of the slice preparation with 5-HT_1A receptor agonist8-OH-DPAT and its specific antagonist PMPPI into a modified Kreb's perfusionsolution (MKS); 2) 20 isolated neonatal rat brainstem slices were divided into 4groups: groupⅠ～Ⅳ: 8-OH-DPAT groups in which preparation was perfusedcontinuously for 10 min with different concentrations of 8-OH-DPAT(1,5,10,20μmol/L). RRDA were recorded before and 1, 3, 5 min after 8-OH-DPAT perfusion. 3)RRDA of the hypoglossal nerve rootlets were recorded by using suction electrodes. Inorder to record respiratory neurons, glass microelectrodes fixed on amicro-manipulator were inserted into the ventral respiratory group (VRG) in medullawith a step pace(10μm every time) till a spontaneous respiratory unit, and thedischarge activities of BE-neurons or 1-neurons in mNRF discharge were obtained.These biphasic expiratory neurons were characterized by the discharge activityrestricted in the late expiratory phase and the early expiratory phase of the followingcycle as well as inhibition in the inspiratory phase. The inspiratory neurons wasidentified by their on-going activities that were correlated with RRDA of thehypoglossal nerve. Drugs were administered by bath application and their effects onthe neuronal activities were investigated. Ten medulla oblongata slice preparationswere divided into two groups. In groupⅠ, 5-HT_1A receptor specific agonist8-OH-DPAT (20μmol/L) was added into the perfusing solution for 5 min first, afterwashing out, the 5-HT_1A antagonist PMPPI (10μmol/L) was applied to the perfusingsolution for 5 min. In groupⅡ, after application of 8-OH-DPAT for 5 min, additional PMPPI was added into the perfusing solution for 5 min.Results1. The discharges of hypoglossal nerve rootlets were recorded: Controlexperiments were performed without drug application.1) Bath application of 20μmol/L 8-OH-DPAT for 1 min 8-OH-DPAT extendedRC by 30.11% (t=2.709, P=0.027) and TE by 32.24% (t=2.676, P=0.028), at thesame time, reduced the integral amplitude(IA) by 17.08%, however, the changes of IAwere statistically nonsignificant. At 3 min, 8-OH-DPAT increased RC by 59.23%(t=3.670, P=0.006) and prolonged TE by 63.44% (t=3.670, P=0.006) as well asreduced IA by 24.82 % (t=3.406, P=0.009), but the changes of TI were statisticallynonsignificant. What's more, the effects of 8-OH-DPAT on the respiration rhythmcould be partially reversed by additional application of PMPPI. Bath application ofPMPPI 10μmol/L for 1 min shortened TI (t=3.641, P=0.007), Bath application of 10μmol/L PMPPI for 3 min induced a significant decrease in RC by 26.08%(t'=3.468, P=0.014), TE by 25.99% (t'=3.236, P=0.021) and TI by 26.14%(t=3.532, P=0.008), whereas no significant change on IA were observed.2) After application of 8-OH-DPAT for 3 min, RC and TE were prolonged withobvious decreases in IA, compared with the control. These effects were partiallyreversed by additional administration of PMPPI. RC and TE were decreased by 23.51%(t=2.802, P=0.023) and 23.92% (t=2.793, P=0.023), IA was increased by 11.58%(t=2.453, P=0.040).3) There were statistically significant in RC among different timepoints(F=181.219, P＜0.001). RC increased after 8-OH-DPAT given in eachconcentration, minimum before administration and maximum at 5 min afteradministration. There were also statistically significant among differentconcentrations(F=61.675, P＜0.001). At each time point after administration, RC wasminimum in 1μmol/L and maximum in 20μmol/L. There was crossover effectbetween time and concentration(F=22.940, P＜0.001). There were statistically significant in IA among different time points(F=20.949, P＜0.001). In 10μmol/L and20μmol/L, IA decreased significantly after 8-OH-DPAT given(F=5.050, P=0.017;F=51.389, P=0.001, respectively). There were also statistically significant amongdifferent concentrations(F=41.027, P＜0.001). At each time point after administration,IA was maximum in 1μmol/L and minimum in 20μmol/L. There was crossovereffect between time and concentration (F=3.483, P=0.002).2. The rhythmical discharges of respiratory neurons and activities of thehypoglossal nerve rootlets were simultaneously recorded: 1) Effects of 8-OH-DPATand PMPPI on the discharge activity of BE-neurons: Application of 20μmol/L8-OH-DPAT for 3 min changed the discharge activity of BE-neurons with a 63.09%(t=4.763, P=0.001) increase in RC and a 69.64% (t=4.647, P=0.002) increase inTE, but decreased IA by 23.63% (t'=2.802, P=0.011). At the same time, the peakdischarge frequency (PFn) was changed from (36.09±4.25) times/s to (19.65±3.98)times/s, 8-OH-DPAT decreased PFn by 45.55% (t=6.315, P＜0.001). The changes ofTI were statistically nonsignificant. However, after washout of 8-OH-DPAT,application of 10μmol/L PMPPI for 3 min decreased RC by 26.33% (t=2.475,P=0.039) and TE by 26.76% (t=2.353, P=0.046). The changes of TI, PFn and IAwere statistically nonsignificant. 2) Effects of 8-OH-DPAT and PMPPI on thedischarge activity of I-neurons: 20μmol/L 8-OH-DPAT extended RC by 78.17%(t=4.775, P=0.001) and TE by 83.01% (t=4.780, P=0.001), but depressed IA by21.78% (t=5.148, P=0.001). What's more, the PFn was changed from (37.40±4.37)times/s to (20.96±3.64) times/s, 8-OH-DPAT decreased PFn by 43.96% (t=6.455,P＜0.001). 8-OH-DPAT had no significantly effect on TI. After washout of8-OH-DPAT, application of 10μmol/L PMPPI for 3 min changed the dischargeactivity of I-neurons with a 26.35% (t=2.359, P=0.046) decrease in RC and a27.01%(t=2.334, P=0.048) decrease in TE. PMPPI had no significantly effect on TI,IA and PFn. ConlusionThe results indicated that in neonatal rat brainstem slice in vivo: 1) 5-HT_1Areceptors play an important role in the modulation of the respiratory rhythmicaldischarge activity in the medullary slice preparation of neonatal rats. 2) 5-HT_1Areceptors are involved in the phase-switching between expiration and inspiration byaffecting BE-neurons. 3) 5-HT_1A receptors are possibly involved in the modulation ofrhythmical respiration through the inhibitory synaptic input from I-neurons.