一氧化氮在新生大鼠离体延髓脑片标本上对节律性呼吸的调节作用
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摘要
目的 1)应用新生大鼠离体延髓脑片标本，记录与之相连的舌下神经呼吸节律性放电(respiratory rhythmical discharge activity，RRDA)，在灌流液中分别给予不同浓度的NO(nitric oxide，NO)供体硝普钠(sodium nitropmsside，SNP)，NO合成前体L-精氨酸(L-Arginine，L-Arg)以及神经元型一氧化氮合酶(neuronal nitric oxide synthase，nNOS)特异性抑制剂7-nitro indazole (7-NI)，观察其对RRDA的影响，从而探讨NO对节律性呼吸的产生和调节中的作用；2)在新生大鼠离体延髓脑片标本上，同步记录舌下神经根和面神经后核内侧区(the medial area of retrofacialis nucleus，mNRF)中的吸气神经元的放电活动，分别给以NO的合成前体、供体以及nNOS的特异性抑制剂，进一步探讨NO对节律性呼吸产生影响的可能机制。
方法 选用新生SD大鼠(0～3d)，雌雄不拘。1)参照并改良Suzue的方法制作离体延髓脑片标本，主要包含面神经后核内侧区，前包钦格氏复合体(Pre-B(?)tzinger Complex)、腹侧呼吸组以及背侧呼吸组的一部分，保留舌下神经根。用玻璃吸附电极记录舌下神经放电作为呼吸活动的指标，主要的呼吸参数包括呼吸周期(respiratory cycle，RC)、吸气时程(inspiratory time，TI)、呼气时程(expiratory time，TE)以及放电的积分幅度(integral amplitude，IA)，通过灌流液给药，观察不同浓度的不同药物对神经根放电活动的影响；2)在面神经后核内侧区(mNRF)同步记录吸气神经元单位的放电活动，灌流给予上述不同药物药物，观察其对吸气神经元放电的影响。
结果 1在记录神经根的实验部分：1)灌流给予不同浓度的NO合成前体L-Arg(从500μmol/L到2mmol/L)，与给药前对照相比，舌下神
经根的各项放电指标均未出现显著性变化;2)灌流给予不同浓度的NO
供体sNP(浓度100脚。UL~2~1几)，不论给予上述任何浓度的SNP，
舌下神经根放电指标与对照相比都未出现明显变化;3)灌流给予不同浓
度的nNOs的特异性抑制剂7一I(500娜ol/L~2~ol几)后，Rc、TE
无显著性变化，而随着给药时间的逐渐延长，TI和IA均同时出现逐渐
的下降的趋势:500娜。比7一I实验组，正常MKS液灌流时TI为1.39
士0.055、I^为1387.00士65.16林V·ms，给药后第smin时Tl为1.01士
0 .055、I^为1274.00士55.82”V·ms(p<0.05)，第10min时TI为0.89士
0.055、IA为1218.00士55.34衅·ms(p<0 .05);lmmo比的7一I实验组，
正常对照组Tl为1.01士0.185、IA为1642.83士226.90四.ms，给药后
Slnin，Tl为0.87士 0.145、认为1528.43士189.02四·ms(P<0 .05)，10rliln
时，Tl为0 .37士0.125、IA为1278.83士180.92四.ms(P<0 .05);Znnno讥
7一I实验组，正常对照的TI为1.17士0.065、IA为1356.00士65.16四·ms，
给药后smin时，Tl为0.87士0.055、IA为1190.00士57.82衅.ms(P<0 .05)，
给药10min时，Tl为0.32士0.035、IA为966.00士58.34四.ms(’I’<0 .05)。
7一I的浓度和这种抑制作用还存在量效关系，10min时，给予500洲mo比
Tl下降约36%，IA下降12%，Inuno比时TI下降63%，IA下降22%，
2~泥时Tl下降73%，IA下降28%;4)单纯给予相应浓度的7一I
溶剂二甲亚矾(d如ethyl sulfoxide，DMSO)后，舌下神经根放电活动
无显著性变化。
2同步记录舌下神经根和mNRF区吸气神经元放电活动的实
验:l)给药前，正常MKS灌流时，吸气神经元放电的平均频率为
25.39士5.04次HZ，放电时程为0.5811士0.02975;2)先后分别给予L一A飞、
SNP之后，吸气神经元放电的平均频率分别为26.79士5.llHz和
26.43土5.02Hz，放电时程分别为0.5733士0.02535和0.5628士0.02655，与
对照相比平均频率和放电时程并无显著性变化(卜0.05);3)给予nNOS
特异性抑制剂7一I之后，吸气神经元的放电时程为0.1894士0.02475，平
均频率为17.58士4.78HZ，和对照相比均显著性下降(P<0 .05)，分别下
降67.4%和30.8%:4)灌流给予上述三种试剂后，TE均未发生明显变
化。
结论1.NO对节律性呼吸中吸气活动的抑制作用参与了吸气中止
机制;2.NO对呼吸强度和呼吸频率均有调节作用，但对前者的作用更
为重要;3.在NO对基本节律性呼吸的调节过程中，主要由神经元合成
并释放的内源性NO就可能满足其对节律性呼吸的调节功能的需要，而
外源性NO，则可能由于对其需要量不大，或由于其半衰期过短，所以
在呼吸调节中的作用不大;4.NO对mNRF中吸气神经元的放电时程和
放电平均频率有明显的抑制作用，提示NO对吸气中止机制的影响，可
能是通过对mN盯的吸气神经元的作用而实现的。
Nitric oxide is involved in the modulation of the basic rhythmical respiration in vitro brainstem slice from neonatal
rat
Object
1. This experiment was expected to test whether NO (nitric oxide) exerted significant effects on basic rhythmical respiration.
2. To investigate the role of NO in the basic rhythmical respiration generation and modulation by exploring the effects of substrate N donor of NO and inhibitor of nNOS (neuronal nitric oxide synthase) on the discharge activity of inspiratory neurons in mNRF
Methods:
Experiments were performed on in vitro brainstem slice preparations isolated from neonatal rats (0-3 d). These preparations include the medial region of the nucleus retrofacialis (mNRF); a part of pre-Botinger Complex, ventral respiratory group (VRG) and dorsal respiratory group (DRG).
l)Respiratory-related burst activities were recorded from hypoglossal nerve rootlets before and during perfusion of the slice preparation with L-Arg (L-Arginine), SNP(sodium nhroprusside) or 7-NI (7-nitro indazole, an inhibitor of neuronal nitric oxide synthase).
2)As monitoring the respiratory-related burst activities of hypoglossal nerve rootlets, we recorded the discharge of inspiratory neurons in the mNRF by inserting the glass microeletrode into the mNRF of the slice. These inspiratory neurons were determined according to the temporal relationship between the neuronal discharge and the hypoglossal nerve activity. Drugs
were administered by bath application and their effects on the inspiratory
neuronal activity were investigated.
Results:
1. Results from experiments on recording respiratory-related burst activities of hypoglossal nerve rootlets. 1) After perfused with different concentrations of L-Arg (500@mol/L~2mmol/L) and SNP (100@mol/L~2mmol/L) , there was no significant change in RRDA; 2) 7-NI, an inhibitor of nNOS, had no effect on the TE, RC,but it decreased the integral amplitude of burst (IA) and inspiratory time (TI), and this kind of inhibition is dose-dependent and time-dependent: during the control perfusion, TI(inspiratory time) is 1.39 ± 0.05s, IA (integral amplitude) is 1387.00 + 65.16@V.ms , at 5min after application of 500@mol/L 7-NI, TI is 1.01±0.05s, IA is 1274.00 ±55.82@Vms (P<0.05), and at lOmin, TI is 0.89±0.05s, IA is 1218.00+55.34uV'ms (P<0.05) . In 1mmol/L 7-NI group, before given the drug, TI is 1.01 ± 0.18s, IA is 1642.83±226.90@V.ms; at 5min after given the drug, TI is 0.87±0.14s, IA is 1528.43±189.02@V.ms (P<0.05); at lOmin, TI is 0.37 + 0.12s, IA is 1278.83+ 180.92uV
-ms (PO.
05); In 2mmol/L 7-NI group, TI is 1.17+0.06s, IA is 1356.00+65.16uV-ms when perfusion with MKS; at 5min, TI is 0.87±0.05, IA is 1190.00±57.82uV-ms (PO.05); at lOmin, TI is 0.32 +0.03s, IA is 966.00+ 58.34uV'ms (PO.05). As shown above, at lOmin after addition of different concentrations of 7-NI, TI decreased 36%, IA decrease 12% when the concentration of 7-NI is 500umol/L; when the concentration of 7-NI is Immol/L, TI decrease 63%, IA decrease 22%; at 2mmol/L, TI decrease 73 %, IA decrease 28 %. 3) After only given the same concentration of DMSO (dimethyl sulfoxide, DMSO) as dissolving the 7-NI, there is no significant difference between the MKS group and experimental group.
2. Result from experiments on recording of discharge of inspiratory neurons in the mNRF: 1) In the MKS group, TI is 0.5811 ± 0.0297s , Fn (average neuronal discharge frequency) is 25.39 ± 5.041Hz; 2) Afer
given L-Arg, SNP respectively, Fn is 26.79 ± 5.11 Hz and 26.43 ± 5.02Hz , TI is 0.5733 ± 0.0253s and 0.5628 ± 0.0265s , there is no significant change compared with the control group (P>0.05) ; 3) After application of 7-NI, TI is 0.1894 ± 0.0247s, Fn is 17.58±4.783Hz, they decrease 67.4% and 30.8% respectively.
Conlusion
1. NO may take part in the IOS (inspiratory off-switching mechanism because of its regulation to the inspiration.
2. NO can regulate the amplitude and frequency of respiration, but the regulation on amplitude of respiratory bursts of NO is more powerf
方法 选用新生SD大鼠(0～3d)，雌雄不拘。1)参照并改良Suzue的方法制作离体延髓脑片标本，主要包含面神经后核内侧区，前包钦格氏复合体(Pre-B(?)tzinger Complex)、腹侧呼吸组以及背侧呼吸组的一部分，保留舌下神经根。用玻璃吸附电极记录舌下神经放电作为呼吸活动的指标，主要的呼吸参数包括呼吸周期(respiratory cycle，RC)、吸气时程(inspiratory time，TI)、呼气时程(expiratory time，TE)以及放电的积分幅度(integral amplitude，IA)，通过灌流液给药，观察不同浓度的不同药物对神经根放电活动的影响；2)在面神经后核内侧区(mNRF)同步记录吸气神经元单位的放电活动，灌流给予上述不同药物药物，观察其对吸气神经元放电的影响。
结果 1在记录神经根的实验部分：1)灌流给予不同浓度的NO合成前体L-Arg(从500μmol/L到2mmol/L)，与给药前对照相比，舌下神
经根的各项放电指标均未出现显著性变化;2)灌流给予不同浓度的NO
供体sNP(浓度100脚。UL~2~1几)，不论给予上述任何浓度的SNP，
舌下神经根放电指标与对照相比都未出现明显变化;3)灌流给予不同浓
度的nNOs的特异性抑制剂7一I(500娜ol/L~2~ol几)后，Rc、TE
无显著性变化，而随着给药时间的逐渐延长，TI和IA均同时出现逐渐
的下降的趋势:500娜。比7一I实验组，正常MKS液灌流时TI为1.39
士0.055、I^为1387.00士65.16林V·ms，给药后第smin时Tl为1.01士
0 .055、I^为1274.00士55.82”V·ms(p<0.05)，第10min时TI为0.89士
0.055、IA为1218.00士55.34衅·ms(p<0 .05);lmmo比的7一I实验组，
正常对照组Tl为1.01士0.185、IA为1642.83士226.90四.ms，给药后
Slnin，Tl为0.87士 0.145、认为1528.43士189.02四·ms(P<0 .05)，10rliln
时，Tl为0 .37士0.125、IA为1278.83士180.92四.ms(P<0 .05);Znnno讥
7一I实验组，正常对照的TI为1.17士0.065、IA为1356.00士65.16四·ms，
给药后smin时，Tl为0.87士0.055、IA为1190.00士57.82衅.ms(P<0 .05)，
给药10min时，Tl为0.32士0.035、IA为966.00士58.34四.ms(’I’<0 .05)。
7一I的浓度和这种抑制作用还存在量效关系，10min时，给予500洲mo比
Tl下降约36%，IA下降12%，Inuno比时TI下降63%，IA下降22%，
2~泥时Tl下降73%，IA下降28%;4)单纯给予相应浓度的7一I
溶剂二甲亚矾(d如ethyl sulfoxide，DMSO)后，舌下神经根放电活动
无显著性变化。
2同步记录舌下神经根和mNRF区吸气神经元放电活动的实
验:l)给药前，正常MKS灌流时，吸气神经元放电的平均频率为
25.39士5.04次HZ，放电时程为0.5811士0.02975;2)先后分别给予L一A飞、
SNP之后，吸气神经元放电的平均频率分别为26.79士5.llHz和
26.43土5.02Hz，放电时程分别为0.5733士0.02535和0.5628士0.02655，与
对照相比平均频率和放电时程并无显著性变化(卜0.05);3)给予nNOS
特异性抑制剂7一I之后，吸气神经元的放电时程为0.1894士0.02475，平
均频率为17.58士4.78HZ，和对照相比均显著性下降(P<0 .05)，分别下
降67.4%和30.8%:4)灌流给予上述三种试剂后，TE均未发生明显变
化。
结论1.NO对节律性呼吸中吸气活动的抑制作用参与了吸气中止
机制;2.NO对呼吸强度和呼吸频率均有调节作用，但对前者的作用更
为重要;3.在NO对基本节律性呼吸的调节过程中，主要由神经元合成
并释放的内源性NO就可能满足其对节律性呼吸的调节功能的需要，而
外源性NO，则可能由于对其需要量不大，或由于其半衰期过短，所以
在呼吸调节中的作用不大;4.NO对mNRF中吸气神经元的放电时程和
放电平均频率有明显的抑制作用，提示NO对吸气中止机制的影响，可
能是通过对mN盯的吸气神经元的作用而实现的。
Nitric oxide is involved in the modulation of the basic rhythmical respiration in vitro brainstem slice from neonatal
rat
Object
1. This experiment was expected to test whether NO (nitric oxide) exerted significant effects on basic rhythmical respiration.
2. To investigate the role of NO in the basic rhythmical respiration generation and modulation by exploring the effects of substrate N donor of NO and inhibitor of nNOS (neuronal nitric oxide synthase) on the discharge activity of inspiratory neurons in mNRF
Methods:
Experiments were performed on in vitro brainstem slice preparations isolated from neonatal rats (0-3 d). These preparations include the medial region of the nucleus retrofacialis (mNRF); a part of pre-Botinger Complex, ventral respiratory group (VRG) and dorsal respiratory group (DRG).
l)Respiratory-related burst activities were recorded from hypoglossal nerve rootlets before and during perfusion of the slice preparation with L-Arg (L-Arginine), SNP(sodium nhroprusside) or 7-NI (7-nitro indazole, an inhibitor of neuronal nitric oxide synthase).
2)As monitoring the respiratory-related burst activities of hypoglossal nerve rootlets, we recorded the discharge of inspiratory neurons in the mNRF by inserting the glass microeletrode into the mNRF of the slice. These inspiratory neurons were determined according to the temporal relationship between the neuronal discharge and the hypoglossal nerve activity. Drugs
were administered by bath application and their effects on the inspiratory
neuronal activity were investigated.
Results:
1. Results from experiments on recording respiratory-related burst activities of hypoglossal nerve rootlets. 1) After perfused with different concentrations of L-Arg (500@mol/L~2mmol/L) and SNP (100@mol/L~2mmol/L) , there was no significant change in RRDA; 2) 7-NI, an inhibitor of nNOS, had no effect on the TE, RC,but it decreased the integral amplitude of burst (IA) and inspiratory time (TI), and this kind of inhibition is dose-dependent and time-dependent: during the control perfusion, TI(inspiratory time) is 1.39 ± 0.05s, IA (integral amplitude) is 1387.00 + 65.16@V.ms , at 5min after application of 500@mol/L 7-NI, TI is 1.01±0.05s, IA is 1274.00 ±55.82@Vms (P<0.05), and at lOmin, TI is 0.89±0.05s, IA is 1218.00+55.34uV'ms (P<0.05) . In 1mmol/L 7-NI group, before given the drug, TI is 1.01 ± 0.18s, IA is 1642.83±226.90@V.ms; at 5min after given the drug, TI is 0.87±0.14s, IA is 1528.43±189.02@V.ms (P<0.05); at lOmin, TI is 0.37 + 0.12s, IA is 1278.83+ 180.92uV
-ms (PO.
05); In 2mmol/L 7-NI group, TI is 1.17+0.06s, IA is 1356.00+65.16uV-ms when perfusion with MKS; at 5min, TI is 0.87±0.05, IA is 1190.00±57.82uV-ms (PO.05); at lOmin, TI is 0.32 +0.03s, IA is 966.00+ 58.34uV'ms (PO.05). As shown above, at lOmin after addition of different concentrations of 7-NI, TI decreased 36%, IA decrease 12% when the concentration of 7-NI is 500umol/L; when the concentration of 7-NI is Immol/L, TI decrease 63%, IA decrease 22%; at 2mmol/L, TI decrease 73 %, IA decrease 28 %. 3) After only given the same concentration of DMSO (dimethyl sulfoxide, DMSO) as dissolving the 7-NI, there is no significant difference between the MKS group and experimental group.
2. Result from experiments on recording of discharge of inspiratory neurons in the mNRF: 1) In the MKS group, TI is 0.5811 ± 0.0297s , Fn (average neuronal discharge frequency) is 25.39 ± 5.041Hz; 2) Afer
given L-Arg, SNP respectively, Fn is 26.79 ± 5.11 Hz and 26.43 ± 5.02Hz , TI is 0.5733 ± 0.0253s and 0.5628 ± 0.0265s , there is no significant change compared with the control group (P>0.05) ; 3) After application of 7-NI, TI is 0.1894 ± 0.0247s, Fn is 17.58±4.783Hz, they decrease 67.4% and 30.8% respectively.
Conlusion
1. NO may take part in the IOS (inspiratory off-switching mechanism because of its regulation to the inspiration.
2. NO can regulate the amplitude and frequency of respiration, but the regulation on amplitude of respiratory bursts of NO is more powerf
引文
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22. Berger AJ. Recent advances in respiratory neurobiology using in vitro methods. Am. J. Physiol. 1990, 259(3): L24～L29.
23．胡德辉，吴中海，高扬．切割延髓、酸碱度及温度对新生大鼠离体延髓——脊髓标本呼吸节律性放电的影响．中国应用生理学杂志 2001年01期：25～28
24．吴中海，胡德辉，高扬．新生大鼠离体延髓脊髓标本的呼吸节律性放电及微切割延髓对其放电的影响．第一军医大学学报，1998，18：260～262．
25．高扬，胡德辉，吴中海．新生大鼠延髓面神经后核内侧区微量注射GABA 和 BIC 对呼吸节律的影响．第一军医大学学报，1999：197～200．
26. The medullary respiratory network in the rat. J Phusiol 1991, 435:631～644
27．潘秉兴，吴中海，王宁黔，史玥，李自强．吸气神经元在新生大鼠延髓脑片标本上的分类及分布。第一军医大学学报，2001，21(8)：565～567
28．王宁黔，吴中海 大鼠面神经后核内侧区与脑干呼吸相关区间纤维联系。第一军医大学学报，2002，19(5)：417～420
29. Dawson VL, Dawson TM. Nitric oxide in neuronal degeneration. Proc Soc Exp Biol Med 1996; 211(1): 33～40
30. Snyder SH, et al. Biological roles of nitric oxide. Sci Am,1992,266(5):28
31. Palmer RMJ, et al. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 1987,327:524
32. Ignarro LJ. et al. Endothelium-derived nitric oxide: actions and properties. J FASEB, 1989,3:31
33. Wolin MS, et al. Guanylate cyclase from bovine lung: A kinetic analysis of the regulation of the purified soluble enzyme by protoporphyrin Ⅸ, heme, and nitrosylheme. J Biol Chem, 1982, 257:13312
34. Radomskl MW, et al. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet, 1987, 2:1057
35. Garthwaite J, Garthwaite G, Palmer RM, Moncada S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur J Pharmacol 1989; 172(4-5):413～6
36. Meulemans A. Diffusion coefficients and half-lives of nitric oxide and N-nitroso-L-arginine in rat cortex. Neurosci Lett, 1994, 171: 89～93
37. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature, 1988, 336:385～88
38．李国君．中枢神经系统中一氧化氮和一氧化氮合酶研究进展．国外医学卫生分册 1999，26(2)：65-69
39. Nathan C, Xie Q W. Regulation of biosynthesis of nitric oxide. J Biol Chem, 1994, 19:13725～13728
40. Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder SH. Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci U. S. A,1994, 91:4214～4218
41． Forstermann U, Gorsky LD, Pollock JS, Schmidt HH, Heller M, Murad F. Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain. Biochem.Biophys.Res.Commun.1990,168(2) : 727-732
42． Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U.S.A, 1991, 88(7) :2811-2814
43． Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci U S A 1991; 88(17) :7797-801．
44． Bredt DS, Glatt C E, Hwang P M, Fotuhi M, Dawson TM, Snyder S H. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron, 1991,7(4) :615-624
45． Volgin D, Volgina A., Seredenko M. Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats. Acta Neurobiol Exp (Warsz.). 2000,60(2) : 175-186．
46． Garthwaite J, Southam E, Anderton M. A kainate receptor linked to nitric oxide synthesis from arginine. J Neurochem. 1989, 53(6) :1952-1954．
47． Wood PL, Emmett MR, Rao TS, Cler J, Mick S, Iyengar S. Inhibition of nitric oxide synthase blocks N-methyl-D-aspartate-, quisqualate-,kainate-,harmaline-,and pentylenetetrazole-dependent increases in cerebellar cyclic GMP in vivo. J Neurochem. 1990,55(1 ):346-8
48． Okada D. Two pathways of cyclic GMP production through glutamate receptor-mediated nitric oxide synthesis. J Neurochem. 1992, 59:1203-1210
49． Southam E, East SJ, Garthwaite J. Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. J Neurochem, 1991, 56:2072-2081
50． East SJ, Garthvvaite J. NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide
pathway. Neurosci Lett.l991,123(1) :17-19
51． Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U.S.A, 1990, 87:682-685
52． Knowles RG, Palacios M, Palmer RM, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U.S.A, 1989, 89:5159-5162
53． Ling L, Karius DR, Fiscus RR, Speck DF. Endogenous nitric oxide is required for an integrative respiratory function in the cat brain, J Neurophysiol 1992,68: 1910-2
54． Gozal D, Gozal E, Torres JE , Gozal YM, Nuckton TU, Hornbu PJ, Nitric oxide modulates ventilatory responses to hypoxia in the developing rat, Am J Respir Crit Care Med, 1997,155:1755-62
55． Gozal D, Torres JE, Gozal YM, Littwin SM. Effect of nitric oxide synthase inhibition on cardiorespiratory responses in the conscious rat, J Appl Physiol, 1996,2068-77
56． Miller MJ, and SM. Tenney. Hyperoxic-hyperventilation in carotid-deafferented cats., Respir. Physiol, 1975, 23: 23-30
57． Gautier H., M. Bonora, and J. H. Gaudy. Ventilatory response of the . conscious or anesthetized cat to oxygen breathing., Respir. Physiol., 1986 65: 181-196
58． David Gozal, et al , potentiation of hypoxic ventilatory response by hyperoxia in the conscious rat: putative of nitric oxide,J Appl Physiol, July 1998, 85(1) : 129-132
59． Suzue TM. Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the newnatal rat. J Physiol. 1984, 354: 173-183．
60． Armand LB, Denavit-Sanbie M and Champagnant J. Central control of breathing in mammals: neuronal circuitry, membrane properties and neurotransmitters. Physiol. Rev. 1995; 75(1) : 1-45．
61． Berger AJ. Properties of medullary respiratory neurons. Federation
Pro.1981,40:2378-2383．
62． Feldman JL. Interaction between brainstem respiratory neurons. Federation Pro.1981,40:2384-2388．
63． Merrill EG. Where are the real respiratory neurons? Federation Pro. 1981,40:2389-2394．
64． Ballantyne BD and Richter DW. Post-synaptic inhibition of bulbar inspiratory neurones in the cat. J Physiol. 1984, 348:67-68．
65． Feldman JL. Neurogenesis of respiratory rhythm and pattern: emerging concepts. Am. J. Physiol.1990,259: R879-R886．
66． Ogilvie MD. A network modei of respiratory rhythmogenesis. Am. J. Physiol. 1992,263: R962-R975．
67． Huang CC et al. Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal nevirons. J Physiol. 2001, 523(3) :731-748．
68． Niermann H, et al. A novel role of vasopressin in the brain: modulation of activity dependant water flux in the neocortex. J Neurosci. 2001,21: 3045-3051．
69． Terman GW et al. Mu opiates inhibit long-term potentiation induction in the spinal cord slice. J Neurophysiol. 2001,85: 485-94．
70． Lu YM, et al. Endogenous Zn(2+) is required for the induction of LTP at rat hippocampal mossy fiber CA3 synapses. Synapse. 2000, 38: 187-97．
71． Bonde C et al. GDNF and neuroblastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures. Neuroreport. 2000,11: 4069-73．
72． Xiang Z and Bergold PJ. Synaptic depression and neuronal loss in transiently acidic hyppocampal slice cultures. Brain Res. 2000, 88: 77-87．
73． Richter DW et al. Calcium currents and calcium dependant potassiium currents in mammalian medullary respiratory neurons. J Physiol. 1993,470:23-33．
74． 吴中海，徐小元，张枫桐．面神经后核内侧区微量注射GABA对 呼吸节律的影响．第一军医大学学报．1995，15(1) ：21-23．
75． Smith JC et al. Neural mechanism generating respiratory pattern in mammalian brainstem-spinal cord in vitro: Ⅰ . Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 1990, 64: 1149-1169．
76． Hamada O, Garcia-Rill E and Skinner RD. Respiration in vitro: Ⅰ . Spontaneous activity. Somatosen. Motor Res. 1992, 9: 313-326．
77． Onimaru H. Studies of the respiratory center using isolated brainstem-spinal cord preparations. Neurosci. Res. 1995,21:183-190．
78． Rekling JC and Feldman JL. Pre-Botzinger Complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu Rev. Physiol. 1998, 60:385-405．
79． Ballanyi K, Onimaru H and Homma I. Respiratory nerwork function in the isolated brainstem-spinal cord of newborn rats. Prog. Neurobiol. 1999, 59: 583-634．
80． Haji A, Takeda R, Okazaki M. Neuropharmacology of control of respiratory rhythm and pattern in mature mammals. Pharmacol Ther 2000 Jun; 86(3) : 277-304
81． Liu Y Y. Ju G. Wong Riley.M-T, Distribution and colocalization of neurotransmitters and receptors in the pre-Botzinger complex of rats. J Appl Physiol 2001; 91(3) : 1387-95．
82． Volgin D. Volgina A. Seredenko M, Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats. Acta Neurobiol Exp (Warsz) 2000; 60(2) : 175-86．
83． Haxhiu MA, Chang CH, Dreshaj IA, Erokwu B, Prabhakar NR, Cherniack NS. Nitric oxide and ventilatory response to hypoxia. Respir Physiol, 1995,101:257-266
84． Pyatin V F, Miroshnichenko I V. Effects of nitric oxide on respiratory activity in bulbospinal preparation from rat fetus. Bull Exp Biol Med 2001; 132(2) : 723-6．
85． Pyatin V F, Miroshnichenko I V. Kul'chitskii. Role of NO-ergic
mechanism in regulation of rhythmogenesis in respiratory center in bulbospinal preparation from newbom rat. Bull Exp Biol Med 2001, 132(2) : 719-22
86． Ogawa H, Mizusawa A, Kikuchi Y, Hida W, Miki H, Shirato K. Nitric oxide as a retrograde messenger in the nucleus tractus solitarius of rats during hypoxia. J Physiol 1995,486(Pt2) : 495-504．
2. Lumsden T. Obervation on the respiratory centers. J Physiol. 1923,57:354～367
3. Lumsden T. The regulation of respiration. J Physiol. 1923,58:81～91
4. Bianchii AL. Localization et etude des neurons respiratoires bulbaires. Mise en jeu antidromique par stimulation spinale ou vagale. J Physiol (Paris). 1971,63:5～40
5. Ezure K. Synaptic connections between medullary resiratory neurons and considerations on the genesis of respiratory rhythm. Prog Neurobiol. 1990, 35: 429～450.
6. Kala MP. Anatomical organization of central respiratory neurons. Ann Rev Physiol. 1981,43:105～120.
7. Smith JC, et al. Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science. 1991,254:726～729.
8. Onimaru H, Arata A and Homma I. Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat. Brain Res. 1988. 445:314～324
9. Onimaru H, Arata A, Homma I. Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro. Exp Brain Res. 1989, 76:530～536
10． 吴中海，张枫桐，李有仁．家兔延髓区域阻滞对呼吸的影响。广东生理学通报．1986，3(2)：60～61．
11． 张枫桐，吴中海，李有仁．阻滞面神经后核内侧区对呼吸节律的影响．第一军医大学学报．1989，9(2)：87～93
12. Zhang FT, Wu ZH and Li YR. Effect of blocking media area of nucleus retrofacialis on respiratory rhythm. Respi. Physiol. 1991, 85:73～81.
13． 吴中海，张枫桐，李有仁．阻滞面神经后核内侧区对节律性呼吸的影响．第一军医大学学报，1994，14(1)：9～11．
14．吴中海，张枫桐，李有仁．毁损面神经后核内侧区神经元胞体对呼吸节律的影响．第一军医大学学报．1992，12(3)：223～227．
15．吴中海，张枫桐，李有仁．家兔延髓区域对呼吸的影响．生理学报．1988，40(3)：250～257．
16．吴中海，张枫桐，徐小元．延髓面神经后核内侧区呼吸相关神经元的形式．生理学报．1997，49(4)：389～394．
17．吴中海，张枫桐，徐小元．家兔面神经后核内侧区在呼吸节律起源中的作用．生理学报．1990，42(4)：68～75
18. Dobbins EG and Feldman JL. Brainstem network controlling descending drive to phrenic motoneurons in rat. J Comp. Neurol. 1994, 347: 64～86.
19. Suzue TM and Tamai S. Electrophysiology of reflexes in an isolated brainstem-spinal cord preparation of the newborn rat. Biomed Res. 1983, 4:611～614.
20. Funk, G. D., Smith, J. C., Feldman J. L., 1993. Generation and transmission of respiratory oscillations in medullary slices: roles of excitatory amino acids. J. Neurophysiol. 70, 1497～1515.
21. Si, X., Shao, M., Feldman, J. L., 1997. Respiratory rhythm generation and synaptic inhibition of expiratory neurons in Pre-Btzinger complex: Differential roles of glycinergic and GABAergic neural transmission. J. Neurophysiol. 77, 1853～1860.
22. Berger AJ. Recent advances in respiratory neurobiology using in vitro methods. Am. J. Physiol. 1990, 259(3): L24～L29.
23．胡德辉，吴中海，高扬．切割延髓、酸碱度及温度对新生大鼠离体延髓——脊髓标本呼吸节律性放电的影响．中国应用生理学杂志 2001年01期：25～28
24．吴中海，胡德辉，高扬．新生大鼠离体延髓脊髓标本的呼吸节律性放电及微切割延髓对其放电的影响．第一军医大学学报，1998，18：260～262．
25．高扬，胡德辉，吴中海．新生大鼠延髓面神经后核内侧区微量注射GABA 和 BIC 对呼吸节律的影响．第一军医大学学报，1999：197～200．
26. The medullary respiratory network in the rat. J Phusiol 1991, 435:631～644
27．潘秉兴，吴中海，王宁黔，史玥，李自强．吸气神经元在新生大鼠延髓脑片标本上的分类及分布。第一军医大学学报，2001，21(8)：565～567
28．王宁黔，吴中海 大鼠面神经后核内侧区与脑干呼吸相关区间纤维联系。第一军医大学学报，2002，19(5)：417～420
29. Dawson VL, Dawson TM. Nitric oxide in neuronal degeneration. Proc Soc Exp Biol Med 1996; 211(1): 33～40
30. Snyder SH, et al. Biological roles of nitric oxide. Sci Am,1992,266(5):28
31. Palmer RMJ, et al. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 1987,327:524
32. Ignarro LJ. et al. Endothelium-derived nitric oxide: actions and properties. J FASEB, 1989,3:31
33. Wolin MS, et al. Guanylate cyclase from bovine lung: A kinetic analysis of the regulation of the purified soluble enzyme by protoporphyrin Ⅸ, heme, and nitrosylheme. J Biol Chem, 1982, 257:13312
34. Radomskl MW, et al. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet, 1987, 2:1057
35. Garthwaite J, Garthwaite G, Palmer RM, Moncada S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur J Pharmacol 1989; 172(4-5):413～6
36. Meulemans A. Diffusion coefficients and half-lives of nitric oxide and N-nitroso-L-arginine in rat cortex. Neurosci Lett, 1994, 171: 89～93
37. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature, 1988, 336:385～88
38．李国君．中枢神经系统中一氧化氮和一氧化氮合酶研究进展．国外医学卫生分册 1999，26(2)：65-69
39. Nathan C, Xie Q W. Regulation of biosynthesis of nitric oxide. J Biol Chem, 1994, 19:13725～13728
40. Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder SH. Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci U. S. A,1994, 91:4214～4218
41． Forstermann U, Gorsky LD, Pollock JS, Schmidt HH, Heller M, Murad F. Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain. Biochem.Biophys.Res.Commun.1990,168(2) : 727-732
42． Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U.S.A, 1991, 88(7) :2811-2814
43． Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci U S A 1991; 88(17) :7797-801．
44． Bredt DS, Glatt C E, Hwang P M, Fotuhi M, Dawson TM, Snyder S H. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron, 1991,7(4) :615-624
45． Volgin D, Volgina A., Seredenko M. Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats. Acta Neurobiol Exp (Warsz.). 2000,60(2) : 175-186．
46． Garthwaite J, Southam E, Anderton M. A kainate receptor linked to nitric oxide synthesis from arginine. J Neurochem. 1989, 53(6) :1952-1954．
47． Wood PL, Emmett MR, Rao TS, Cler J, Mick S, Iyengar S. Inhibition of nitric oxide synthase blocks N-methyl-D-aspartate-, quisqualate-,kainate-,harmaline-,and pentylenetetrazole-dependent increases in cerebellar cyclic GMP in vivo. J Neurochem. 1990,55(1 ):346-8
48． Okada D. Two pathways of cyclic GMP production through glutamate receptor-mediated nitric oxide synthesis. J Neurochem. 1992, 59:1203-1210
49． Southam E, East SJ, Garthwaite J. Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. J Neurochem, 1991, 56:2072-2081
50． East SJ, Garthvvaite J. NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide
pathway. Neurosci Lett.l991,123(1) :17-19
51． Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U.S.A, 1990, 87:682-685
52． Knowles RG, Palacios M, Palmer RM, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U.S.A, 1989, 89:5159-5162
53． Ling L, Karius DR, Fiscus RR, Speck DF. Endogenous nitric oxide is required for an integrative respiratory function in the cat brain, J Neurophysiol 1992,68: 1910-2
54． Gozal D, Gozal E, Torres JE , Gozal YM, Nuckton TU, Hornbu PJ, Nitric oxide modulates ventilatory responses to hypoxia in the developing rat, Am J Respir Crit Care Med, 1997,155:1755-62
55． Gozal D, Torres JE, Gozal YM, Littwin SM. Effect of nitric oxide synthase inhibition on cardiorespiratory responses in the conscious rat, J Appl Physiol, 1996,2068-77
56． Miller MJ, and SM. Tenney. Hyperoxic-hyperventilation in carotid-deafferented cats., Respir. Physiol, 1975, 23: 23-30
57． Gautier H., M. Bonora, and J. H. Gaudy. Ventilatory response of the . conscious or anesthetized cat to oxygen breathing., Respir. Physiol., 1986 65: 181-196
58． David Gozal, et al , potentiation of hypoxic ventilatory response by hyperoxia in the conscious rat: putative of nitric oxide,J Appl Physiol, July 1998, 85(1) : 129-132
59． Suzue TM. Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the newnatal rat. J Physiol. 1984, 354: 173-183．
60． Armand LB, Denavit-Sanbie M and Champagnant J. Central control of breathing in mammals: neuronal circuitry, membrane properties and neurotransmitters. Physiol. Rev. 1995; 75(1) : 1-45．
61． Berger AJ. Properties of medullary respiratory neurons. Federation
Pro.1981,40:2378-2383．
62． Feldman JL. Interaction between brainstem respiratory neurons. Federation Pro.1981,40:2384-2388．
63． Merrill EG. Where are the real respiratory neurons? Federation Pro. 1981,40:2389-2394．
64． Ballantyne BD and Richter DW. Post-synaptic inhibition of bulbar inspiratory neurones in the cat. J Physiol. 1984, 348:67-68．
65． Feldman JL. Neurogenesis of respiratory rhythm and pattern: emerging concepts. Am. J. Physiol.1990,259: R879-R886．
66． Ogilvie MD. A network modei of respiratory rhythmogenesis. Am. J. Physiol. 1992,263: R962-R975．
67． Huang CC et al. Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal nevirons. J Physiol. 2001, 523(3) :731-748．
68． Niermann H, et al. A novel role of vasopressin in the brain: modulation of activity dependant water flux in the neocortex. J Neurosci. 2001,21: 3045-3051．
69． Terman GW et al. Mu opiates inhibit long-term potentiation induction in the spinal cord slice. J Neurophysiol. 2001,85: 485-94．
70． Lu YM, et al. Endogenous Zn(2+) is required for the induction of LTP at rat hippocampal mossy fiber CA3 synapses. Synapse. 2000, 38: 187-97．
71． Bonde C et al. GDNF and neuroblastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures. Neuroreport. 2000,11: 4069-73．
72． Xiang Z and Bergold PJ. Synaptic depression and neuronal loss in transiently acidic hyppocampal slice cultures. Brain Res. 2000, 88: 77-87．
73． Richter DW et al. Calcium currents and calcium dependant potassiium currents in mammalian medullary respiratory neurons. J Physiol. 1993,470:23-33．
74． 吴中海，徐小元，张枫桐．面神经后核内侧区微量注射GABA对 呼吸节律的影响．第一军医大学学报．1995，15(1) ：21-23．
75． Smith JC et al. Neural mechanism generating respiratory pattern in mammalian brainstem-spinal cord in vitro: Ⅰ . Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 1990, 64: 1149-1169．
76． Hamada O, Garcia-Rill E and Skinner RD. Respiration in vitro: Ⅰ . Spontaneous activity. Somatosen. Motor Res. 1992, 9: 313-326．
77． Onimaru H. Studies of the respiratory center using isolated brainstem-spinal cord preparations. Neurosci. Res. 1995,21:183-190．
78． Rekling JC and Feldman JL. Pre-Botzinger Complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu Rev. Physiol. 1998, 60:385-405．
79． Ballanyi K, Onimaru H and Homma I. Respiratory nerwork function in the isolated brainstem-spinal cord of newborn rats. Prog. Neurobiol. 1999, 59: 583-634．
80． Haji A, Takeda R, Okazaki M. Neuropharmacology of control of respiratory rhythm and pattern in mature mammals. Pharmacol Ther 2000 Jun; 86(3) : 277-304
81． Liu Y Y. Ju G. Wong Riley.M-T, Distribution and colocalization of neurotransmitters and receptors in the pre-Botzinger complex of rats. J Appl Physiol 2001; 91(3) : 1387-95．
82． Volgin D. Volgina A. Seredenko M, Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats. Acta Neurobiol Exp (Warsz) 2000; 60(2) : 175-86．
83． Haxhiu MA, Chang CH, Dreshaj IA, Erokwu B, Prabhakar NR, Cherniack NS. Nitric oxide and ventilatory response to hypoxia. Respir Physiol, 1995,101:257-266
84． Pyatin V F, Miroshnichenko I V. Effects of nitric oxide on respiratory activity in bulbospinal preparation from rat fetus. Bull Exp Biol Med 2001; 132(2) : 723-6．
85． Pyatin V F, Miroshnichenko I V. Kul'chitskii. Role of NO-ergic
mechanism in regulation of rhythmogenesis in respiratory center in bulbospinal preparation from newbom rat. Bull Exp Biol Med 2001, 132(2) : 719-22
86． Ogawa H, Mizusawa A, Kikuchi Y, Hida W, Miki H, Shirato K. Nitric oxide as a retrograde messenger in the nucleus tractus solitarius of rats during hypoxia. J Physiol 1995,486(Pt2) : 495-504．