The Effects Of 5-HT_2A Receptors Via ROS On The MNRF And Respiratory Pacemaker Neurons

Our research have demonstrated that the medial area of nucleus retrofacialis(mNRF) is the site of respiratory rhythmogenesis. It is vital to respiratoryrhythmogenesis. Smith conformed that the pre-B(o|¨)tzinger complex (PBC) was the siteof respiratory rhythmogenesis in 1991.mNRF and PBC are located in the medullarylateral region of abdomen. The anatomical position of them has difference, but itoverlapped. The site of basic respiratory rhythmogenesis has been ascertained in themedullary head lateral region of abdomen.There are two hypothesis for themechanism of respiratory rhythmogenesis, that is pacemaker neuron hypothesis andneurons network hypothesis. Pacemaker neuron hypothesis is supported by majorityof the present findings. We had found Expiratory-Inspiratory phase spanning (E-I PS)neuron characteristic of pacemaker on mNRF in vivo and in vitro in neonate mice.The type of E-I PS neurons begin to discharge before inspiration, burst to initiationinspiration by constant frequency, continue and terminate simultaneously withdischarge of inspiration. The type of E-I PS neurons may be pacemaker neurons.Foreign scholars identified pacemaker neuron as cadmium-insensitive and cadmium-sensitive pacemaker. Cadmium-insensitive pacemakers are characterized bybursting that persist in the presence of Cadmium, dependent on persistent andtransient sodium current (I_NaP and I_NaT) and blocked by TTX. Cadmium-sensitivepacemakers are characterized by bursting that is blocked in the presence of Cadmium,dependent on Ca²⁺-activated nonspecific cationic current (I_CAN) and blocked byflufenamic acid (FFA).5-Hydroxytryptamine (5-HT) now appears that there are at 15 receptor subtypesthat belongs to seven receptors:5-HT_{1(A,B,D,E,F,like)},5-HT_2(A,B,C),5-HT₃,5-HT₄,5-HT_5(A,B),5-HT₆ and 5-HT₇．Among the 5-HT receptors,5-HT₂ receptors are ofsignificant clinical interest because of their potential involvement in mediating manyof the central and peripheral physiological functions of serotonin. There are threereceptor subtypes within the 5-HT2 classes: 5-HT_2A, 5-HT_2B, and 5-HT_2C.5-HT_2Asubtype is functionally the most important, the others having a much more limiteddistribution and functional role. 5-HT_2A receptors have been found in many parts ofthe CNS including the cerebral cortex, basal ganglia, hippocampus, thalamus,cerebellum, and hypothalamus. However, it is most highly enriched in the cerebralcortex. An ERK activation pathway had been confirmed in rat renal mesangial cellsas follows: 5-HT_2A receptor→G(q) protein→phospholipase C→diacylglycerol→protein kinase C (PKC)→NAD(P)H oxidase→reactive oxygen species (ROS, i.e.,H2O2 and superoxide)→mitogen-activated extracellular signal-regulated kinase→extracellular signal-regulated kinase (ERK). ROS are kown to be toxic and harmfulmolecules in pathophysiological domain, that can lead to various degenerativediseases. However, low quantity of ROS has been shown lately to be beneficial to thecell and appears to mediate the physiological functions of growth factors andcrtokines via redox signaling. Aside its harmful effect, it has physiological functionsas a small molecular messenger. Effects of PKC, ROS and ERK have been proved relation to regulation sodium channel in rat ventricular myocardium and renalmesangial cells.Ectogenic 5-HT_2A receptror agonists have excitory effect onrespiratory activity. Activation of 5-HT_2A receptors with 4-iodo-2,5-dimethoxyamph--etamine (DOI) increased the frequency of respiratory activity. Blockade ofendogenously activated 5-HT_2A receptors decreased the frequency, amplitude, andregularity of respiratory population activity. At the cellular level, blockade of 5-HT_2Areceptors reduced the action potential discharge in all examined respiratory neurons,which was associated with a reduction in the fast and the persistent sodium current.Continuous application of 5-HT_2A-receptor antagonists differentially affectedpacemaker neurons. Pacemaker activity was eliminated in cadmium-insensitivepacemaker neurons. In cadmium-sensitive pacemaker neurons, the frequency ofpacemaker activity was unaffected. The effect was blocked by activating PKC.Endogenously activated 5-HT_2A receptors are required for maintaining fictiverespiratory activity in the brainstem slice by modulating sodium conductances via aPKC pathway. We presume that activation 5-HT_2A receptors in respiratory centercoincide with an ERK activation pathway. It sustains and adjusts respiratory rhythmvia ROS.In this study, we explore the effect of 5-HT_2A-receptors coupled with superoxideanion (O₂^-) on respiratory regulation signal transductionin passageway in mNRF.Simultaneous recording of the hypoglossal nerve (â…«n) respiratory rhythmicdischarge (RRDA) with suction electrode and the respiratory neuronal discharge wereperformed with whole cell patch in the mNRF on the brainstem slice in vitro. Observethe electrophysiological characteristic of pacemaker, non-pacemaker,cadmium-sensitive pacemaker and cadmium-insensitive pacemaker neurons.Research the effect of DOI, Ketanserin, t-Butyl hydroperoxide (tBHP) andα-lipoicacid (α-LA) on respiratory rhythm and cadmium-insensitive pacemaker neuronsin mNRF. Demonstrate that activated 5-HT_2A receptors sustain and adjust respiratoryrhythmical in mNRF by modulating sodium conductances via ROS.1. Measurement of extracellular O₂^- after activated O₂^- in mNRF.Absorbance (A) of O₂^- producted by 5-HT (0.1, 0.2, 0.5, 1, 2, 5μmol/L) hadsignificant difference in mNRF (F =13.01, P =0.000). Absorbance (A) of O₂^-producted by 5-HT achieved peak at 1μmol/L and didn't insignificantly increasealong with density increased. Absorbance (A) of O₂^- producted by DOI (1, 2, 5, 10,20, 30, 40μmol/L) had significant difference in mNRF (F =16.032 , P=0.000). Absorbance (A) of O₂^- producted by DOI achieved peak at 20μmol/L anddidn't insignificantly increase along with density increased.5-HT and DOI can significantly increase absorbance (P<0.01) and they had nodifference (t=1.277,P=0.249). Ketanserin can decrease absorbance, but has nosignificant difference(t=1.638, P=0.153, n=4). Absorbance generated by 5-HT andDOI can be significantly blocked by antioxidantα-LA (α-LA+5-HT vs control,t=1.690, P=0.142, n=4 andα-LA+DOI vs control,t=1.553, P=0.171, n=4).We concluded that activated 5-HT_2A-receptors can significantly generate O₂^- inmNRF. It suggested that 5-HT_2A-receptors regulated respiratory correlated with ROS.Whther 5-HT_2A-receptors regulated respiratory via ROS ? We demonstrated withoxidants tBHP andα-LA by whole-cell patch technique in mNRF.2,Effects of DOI, ketanserine, tBHP andα-LA on respiratory cycle (RC) in mNRF atdifferent timeEffective density of DOI, ketanserine, tBHP andα-LA were (μmol/L) 20, 40,500 and 250 according to records, but the effective time was uncertain. So weobserved the effects of DOI, ketanserine, tBHP andα-LA on RC in mNRF atdifferent time.Observe the effect of DOI, ketanserine, tBHP andα-LA on RC by recording RRDA ofâ…«n in mNRF at different time point (min 0, 2, 5, 10, 15, 20, 30 and 60).We found that the effect of DOI, ketanserine, tBHP andα-LA on RC had significantdeviation by repeated measured data ANOVA test (P<0.05).LSD test and profileplot of DOI, ketanserine, tBHP andα-LA at different time factor indicated that thepeak time for shorting and prolonging RC of the four reagents were all 15 min.3. Synaptic isolation, identification, heterogeneity and membarne properties of mNRFrespiratory pacemaker neurons.Respiratory rhythmic discharge activity (RRDA) was recorded extracellularlyfrom hypoglosive nerve with suction electrode contain silver wire by means ofvacuum aspiration. These fictive inspiratory bursts were used to identify individualinspiratory neurons that were then recorded intracellularly, preferentially in themNRF. Simultaneous discharged with RRDA was inspiratory neuron. To moreconclusively identify pacemaker, we applied antagonists to block fast synaptictransmission, since the defining property of pacemaker neurons is their ability togenerate voltage-dependent spontaneous bursts of action potentials in the absence ofsynaptic inputs. The antagonists called Cocktail were NMDA receptor 10μmol/LMK801, non-NMDA receptor CNQX 20μmol/L, glycine receptor 1μmol/LStrychnine and GABA_A receptor 20μmol/L bicuculline. Pacemakers continue to burstrhythmically and non-pacemakers cease to burst when fast synaptic transmission isblocked. Pacemaker neurons characteristically generate ectopic bursts in response toapplication of a depolarizing current. In the experiment, we recorded 362 respiratoryneurons, including 118 pacemakers and 244 non pacemakers.In Current-clamp, recording for five minutes at stabilization status of neuronafter seal and rupture of membrane . To take down the capacitance membrane (Cm)(PF), input resistance membrane (Rm) (MΩ) and leak inward current (Ileak)(pA).Results of Cm, Rm and Ipeak had insignifant deviation by independent t test (P>0.05) between pacemaker (n=6) and non-pacemaker (n=7) neurons.In the presence of the cocktail, pacemaker neurons can be classified into twotypes: Cd-sensitive and Cd-insensitive pacemakers, depending on whether or not200mol/L Cd2+ blocks their bursting. Both types have voltage-dependent burstbehavior, as confirmed by application of depolarizing and /or hyperpolarizingcurrent.Of the 118 intracellularly recorded inspiratory neurons, only 4 neurons(3.39%) had Cd-sensitive pacemaker properties in the experiment.Characteristic of discharge for pacemaker neurons was spontaneous discharge,generating ectopic bursts in response to application of a depolarizing current andvoltage-dependent burst behavior by application of depolarizing and /orhyperpolarizing current according to the above research. Pacemaker neurons weremajority of Cd-insensitive in the mNRF for neonant rats.4. Ascertain 5-HT_2A receptors maintaining and regulating respiratory rhythm viaROS in the respiratory center.â‘ Effects of agonist and antagonist of 5-HT_2A receptors on RRDA ofâ…«n andCd-insensitive pacemaker neurons.Observe the effects of DOI and ketaserine on RRDA ofâ…«n and Cd-insensitivepacemaker neurons in medullary slices of neonant S-D rat. The results indicated thatDOI had excitory effects and ketaserine had rejection capability. With continousperfusion of 20μmol/L DOI for 15min, RC) (s) was significantly decurtated (P<0.01) and peak value of discharge (μV) significantly increased(P<0.001). DOIsignificantly increased the burst duration (s), burst amplitude (mV) and spikefrequency (Hz) (P<0.05) of Cd-insensitive pacemaker neurons. With continousperfusion of 40μmol/L ketanserine for 15min, RC (s) was significantly prolonged (P< 0.001), peak value of discharge((μV) significantly increased (P<0.01). Significantly reduced the burst duration (s), burst amplitude (mV) and spikefrequency (Hz) of Cd-insensitive pacemaker neurons (P<0.05).â‘¡The effects of tBHP, an oxidant, on RRDA ofâ…«n and Cd-insensitive pacemakerneurons in mNRF.The effects of DOI should be mimicked by direct application of molecules,which generate ROS, if 5-HT_2A receptors maintaining and regulating respiratoryrhythm via ROS in the respiratory center. So we selected tBHP to do the followingexperiment.With continous perfusion of 500μmol/L tBHP for 15min, tBHP had excitoryeffects on RRDA ofâ…«n and Cd-insensitive pacemaker neurons similar to DOI inmedullary slices of neonant S-D rat. RC (s) was significantly decurtated from12.67±2.47 to 7.87±1.21(n=5,t=5.546, P =0.005), peak value of discharge (μV)increased from 345.10±21.39 to 385.17±21.39 (n=5; t=6.659,P=0.003). The burstduration (s), burst amplitude (mV) and spike frequency (Hz) were all significantlyincreased (P<0.05). Burst duration(s): 2.52±0.56 vs 2.71±0.58 (n=5;t=5.593,P=0.005), burst amplitude(mV): 58.68±4.96 vs 64.53±5.20 (n=5; t=5.034, P=0.007)and spike frequency(Hz): 2.67±0.84 vs 3.16±0.94 (n=5;t=4.377, P=0.012).â‘¢The effects ofα-LA, a antioxidants, on RRDA ofâ…«n and Cd-insensitivepacemaker neurons similar to ketanserineThe experiment indicated thatα-LA had rejection capability for RRDA ofâ…«nand Cd-insensitive pacemaker neurons similar to ketanserine in medullary slices ofneonant S-D rat. With continous perfusion of 250μmol/Lα-LA for 15min, RC (s)was significantly increased from 11.95±2.49 to 27.99±5.15 (n=4; t=11.993,P=0.001), peak value of discharge(μV) reduced from 352.13±24.68 to 303.59±26.51(n=4; t=13.064,P=0.001). The burst duration (s), burst amplitude (mV) and spikefrequency (Hz) were all significantly reduced (P < 0.05). Burst duration (s): 2.50±0.74 vs 2.27±0.73(n=4; t=4.540,P=0.020), burst amplitude(mV): 59.05±5.48 vs54.00±4.57 (n=4;t=9.374,P=0.003) and spike frequency(Hz): 2.65±0.76vs 2.19±0.52(n=4; t=3.878, P=0.030).â‘£The effects of DOI on RRDA ofâ…«n and Cd-insensitive pacemaker neurons wereblocked byα-LA.Antioxidants should attenuate the effect of DOI if 5-HT_2A receptors maintainingand regulating respiratory rhythm via ROS in the respiratory center. So we selectedα-LA to do the following experiment. Whether or notα-LA, an antioxidant, couldeliminate the effects of DOI on RRDA ofâ…«n and Cd-insensitive pacemaker neurons.With continous perfusion of 20μmol/L DOI and 250μmol/Lα-LA for 15min,α-LA could significantly eliminate the effects of DOI on RRDA ofâ…«n andCd-insensitive pacemaker neurons. RC (s): 11.92±2.73 vs 11.75±2.44 (n=4,t=0.742,P=0.512), peak value of discharge(μV): 336.14±29.86 vs 338.07±26.04 (n=4, t=0.670,P=0.551), Burst duration (s): 2.50±0.39 vs 2.48±0.45 (t=0.340, P=0.756, n=4), burstamplitude (mV): 58.11±5.23 vs 58.25±5.55 (t=0.350,P=0.750,n=4) and spikefrequency (Hz): 2.60±0.58 vs 2.63±0.63 (t=0.649, P=0.563, n=4).DOI had excitory effects on RRDA ofâ…«n and Cd-insensitive pacemakerneurons and the effects of oxidant was similar to DOI. Antioxidant could block theeffects of activated 5-HT_2A receptors. It indicated that activated 5-HT_2A receptorsmodulation respiratory via ROS, may be relation to sodium conductance.5. Inhibition of the respiratory rhythm produced by ketanserine is consistent with areduction of both transient and persistent sodium currentTo test the hypothesis that that ketanserine affects both the transient and thepersistent sodium current, we obtained recordings from 4 Cd-insensitive pacemakerneurons in the voltage-clamp configuration. Voltage steps from -80 to 20 mV andslow voltage ramps from -80 to 20 mV (90 mV/ sec) were applied to elicit transient and persistent sodium currents, respectively. I-V curve indicated that ketanserinecould inhibit the transient sodium current (F=33.759, t=0.000). The persistent sodiumcurrent elicited by Voltage ramp was inhibited by ketanserine. Both the persistent andtransient inward currents recording in the Cd-insensitive pacemaker neurons weresensitive to TTX. The experiment demonstrated that activation 5-HT_2A receptorsmodulated respiratory relation to sodium conductance.Conclusion:â‘ Activation of 5-HT_2A receptors can significantly generated O₂^-in mNRF. It suggested that 5-HT_2A-receptors regulated respiratory correlated with O₂^-.â‘¡Characteristic of discharge for pacemaker neurons was spontaneous discharge,generating ectopic bursts in response to application of a depolarizing current andvoltage-dependent burst behavior by application of depolarizing and /orhyperpolarizing current according to the above research. Pacemaker neurons weremajority of Cd-insensitive in the mNRF for neonant rats (1～3d).â‘¢There wereinsignificant difference on Cm, Rm and Ileak between pacemaker and non-pacemaerneurons.â‘£Activation of 5-HT_2A receptors in mNRF had excitory effects onrhythmic respiration.⑤tBHP had excitory effects on RRDA ofâ…«n and Cd-insensitivepacemaker neurons similar to DOI in mNRF.⑥α-LA could significantly eliminatethe effects of DOI on RRDA ofâ…«n and Cd-insensitive pacemaker neurons in mNRF.⑦5-HT_2A receptors modulated rhythmic respiration in mNRF relation to sodiumcurrents of Cd-insensitive pacemaker neurons.⑧Activation of 5-HT_2A receptorsmodulated rhythmic respiration via ROS relation to sodium currents.