GENERATION AND TRANSMISSION OF RESPIRATORY OSCILLATIONS IN MEDULLARY SLICES - ROLE OF EXCITATORY AMINO-ACIDS
GENERATION AND TRANSMISSION OF RESPIRATORY OSCILLATIONS IN MEDULLARY SLICES - ROLE OF EXCITATORY AMINO-ACIDS
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DOI：
10.1152/jn.1993.70.4.1497
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发表时间：
1993-10-01
影响因子：
2.5
通讯作者：
FELDMAN, JL
中科院分区：
文献类型：
--
作者：
FUNK, GD;SMITH, JC;FELDMAN, JL
1.在体外制备的新生大鼠延髓切片上，观察了兴奋性氨基酸(EAA)受体参与呼吸节律的产生和吸气驱动向舌下运动神经元的传递。脑片在XII神经上产生有节奏的吸气活动。通过分析EAA受体拮抗剂浸浴或延髓腹外侧区(中央呼吸组)呼吸神经元主柱局部微量注射EAA受体拮抗剂后呼吸网络活动的扰动，确定EaS在节律产生中的作用，特别是在Botzinger前复合体(Pre-Botzinger Complex)。将EAA受体拮抗剂微量注射到XII运动核团或延髓腹外侧区驱动传递回路中间神经元的部位，通过记录XII神经放电或运动神经元突触输入的扰动来研究EaS参与驱动传递到XII运动神经元的作用。用6-氰基-7-硝基-2，3-二酮(CNQX)阻断非N-甲基-D-天冬氨酸(Non-NMDA)受体，可逆地降低XII神经爆发频率和幅度，呈浓度依赖关系，完全阻断浓度为4微米的呼吸运动输出。用D，L AP-4激活2-氨基-4-膦酸丁酸(AP-4)敏感受体，可使XII神经爆发幅度降低30%，但不改变爆发频率。用(+)-5-甲基-10，11-二氢-5H-二苯并[a，d]环庚烯-5，10-亚胺马来酸(MK-801)阻断NMDA受体通道，不影响运动输出的频率和幅度。二氢海人酸浴对脑片EAA摄取的抑制作用可逆地增加XII运动放电的频率和幅度。通过单侧微量注射CNQX，在延髓腹外侧区呼吸神经元主柱的多个位置阻断非NMDA受体，包括前BotC，可引起剂量依赖性的双侧XII神经爆发幅度的降低，而呼吸振荡的频率没有明显的扰动。阻断Pre-BotC中位于波幅改变部位腹侧的非NMDA受体可导致频率降低，并最终阻断双侧呼吸网络振荡。单侧运动核内微量注射CNQX，阻断非NMDA受体，可使同侧XII神经放电幅度呈剂量依赖性降低，但不影响呼吸振荡的频率。对侧XII神经爆发波幅的扰动明显较小。XII运动核内的NMDA通道阻断不影响吸气爆发波幅，而激活AP-4受体则导致波幅降低30%。XII运动神经元突触电流和电位的全细胞记录表明，呼吸调制突触输入的XII运动神经元接受150+/-80pA(平均+/-SD；n=26)的节律性吸气驱动电流，电位为11+/-6 mV(n=25)。外源性应用CNQX可逆性阻断90-95%的节律性突触输入。局部应用D，L AP4使吸气调制突触电流降低21+/-6%，而应用MK-801不影响吸气驱动电流或电位。用河豚毒素阻断动作电位依赖性突触传递后，局部应用L-谷氨酸产生内向电流，该电流为对照的5%，但不受D，L AP-4的影响。局部应用非N-甲基-D-天冬氨酸受体激动剂[奎斯奎特(Quis)、(R，S)-α-氨基-3-羟基5-甲基-4-异恶唑-4-丙酸氢溴酸盐(AMPA)和海人酸(KAIN)]和N-甲基-D-天冬氨酸受体激动剂可引起膜去极化或内向电流。局部应用CNQX可有效地竞争性阻断Quis、AMPA和Kain的突触后活动。MK-801.7不可逆地阻断运动神经元对N-甲基-天冬氨酸的反应。我们的结果表明：1)体外新生大鼠延髓脑片呼吸节律的产生依赖于内源性EAA作用于BotC前C区的非NMDA受体；2)延髓前BotC区神经元之间相互兴奋性的、非NMDA受体介导的突触相互作用有助于维持呼吸节律；3)XII运动神经元具有NMDA和非NMDA(AMPA和KAIN)受体；以及4)主要作用于非NMDA受体的EAA样物质介导吸气驱动向XII运动神经元的传递。
1. The involvement of excitatory amino acid (EAA) receptors in the generation of respiratory rhythm and transmission of inspiratory drive to hypoglossal (XII) motoneurons was examined in an in vitro neonatal rat medullary slice preparation. Slices generated rhythmic inspiratory activity in XII nerves. The role of EAAs in rhythm generation was determined by analyzing perturbations of respiratory network activity after bath application of EAA receptor antagonists or local microinjection of antagonists into the main column of respiratory neurons in the ventrolateral medulla (central respiratory group), particularly in the pre-Botzinger complex (pre-BotC). The involvement of EAAs in drive transmission to XII motoneurons was examined by recording perturbations in XII nerve discharge or motoneuron synaptic inputs after microinjection of EAA receptor antagonists into either the XII motor nuclei or sites in the ventrolateral medulla containing interneurons of the drive transmission circuit.2. Block of non-N-methyl-D-aspartate (non-NMDA) receptors by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly reduced XII nerve burst frequency and amplitude in a concentration-dependent manner, completely blocking respiratory motor output at concentrations >4 muM. Activation of 2-amino-4-phosphonobutyric acid (AP-4)-sensitive receptors with D,L AP-4 reduced XII nerve burst amplitude by 30% but did not alter burst frequency. Block of NMDA receptor channels by bath application of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) did not perturb the frequency or amplitude of motor output. Inhibition of EAA uptake in the slices by bath application of dihydrokainic acid reversibly increased the frequency and amplitude of XII motor discharge.3. Block of non-NMDA receptors at multiple sites along the main column of respiratory neurons in the ventrolateral medulla, including the pre-BotC, by unilateral microinjection of CNQX produced a dose-dependent, bilateral reduction in XII nerve burst amplitude without substantial perturbations of the frequency of respiratory oscillations. Block of non-NMDA receptors within the pre-BotC at sites ventral to amplitude altering sites produced a reduction in frequency and ultimately bilateral block of respiratory network oscillations.4. Non-NMDA receptor block within the XII motor nucleus by unilateral microinjection of CNQX produced a dose-dependent reduction in ipsilateral XII nerve discharge amplitude without perturbing the frequency of respiratory oscillations. Perturbations of contralateral XII nerve burst amplitude were significantly smaller. NMDA channel block within the XII motor nucleus did not affect inspiratory burst amplitude, whereas activation of AP-4 receptors caused a 30% reduction in amplitude.5. Whole-cell recordings of XII motoneuron synaptic currents and potentials indicated XII motoneurons with respiratory-modulated synaptic inputs receive rhythmic inspiratory drive currents of 150 +/- 80 pA (mean +/- SD; n = 26) and potentials of 11 +/- 6 mV (n = 25). Exogenous application of CNQX reversibly blocked 90-95% of the rhythmic synaptic inputs. Local application of D,L AP4 reduced inspiratory-modulated synaptic currents by 21 +/- 6%, whereas MK-801 application did not affect inspiratory drive currents or potentials.6. After blocking action potential-dependent synaptic transmission with tetrodotoxin, local application of L-Glutamate produced an inward current that was reduced to 5% of control by CNQX but was unaffected by D,L AP-4. Local application of non-NMDA receptor agonists [quisqualate (Quis), (R,S)-alpha-amino-3-hydroxy 5 methyl isoxazole-4-propionic acid hydrobromide (AMPA), and kainate (Kain)] and NMDA receptor agonists caused membrane depolarization or inward current. The postsynaptic actions of Quis, AMPA, and Kain were potently and competitively blocked by local application of CNQX. The motoneuron responses to NMDA were irreversibly blocked by MK-801.7. Our results indicate that: 1) respiratory rhythm generation in the in vitro neonatal rat medullary slice is dependent on endogenously released EAAs acting at non-NMDA receptors within the pre-BotC; 2) mutually excitatory, non-NMDA receptor-mediated synaptic interactions between pre-BotC neuron populations on each side of the medulla contribute to maintenance of respiratory rhythm; 3) XII motoneurons possess NMDA and non-NMDA (AMPA and Kain) receptors; and 4) an EAA-like substance acting primarily at non-NMDA receptors mediates transmission of inspiratory drive to XII motoneurons.