【作者】 潘秉兴；

【导师】 吴中海；

【作者基本信息】 第一军医大学 ， 神经生理学， 2001， 硕士

【摘要】 目的 1）应用新生大鼠离体延髓脑片标本，同步记录舌下神经根和呼吸神经元单位的放电活动，探查不同类型的呼吸神经元在新生大鼠延髓脑片上的分布情况；并对照与面神经后核内侧区（the medialarea of retrofacialis nucleus，mNRF）的结构关系，以进一步明确mNRF在离体脑片标本基本呼吸节律发生中的作用；2）观察非N-甲基-D-天门冬氨酸（N-methyl-D-asparate，NMDA）类兴奋性氨基酸受体相应的激动剂和拮抗剂对呼吸相关神经元放电活动的影响，进一步探讨其在基本呼吸节律产生和调节中的作用。 方法 选用新生SD大鼠（0-3d），雌雄不拘。参照并改良Suzue的方法制作离体延髓脑片标本，记录舌下神经放电作为呼吸活动的指标，并在延髓腹外侧区同步记录呼吸神经元单位的放电活动。通过灌流液给药，观察不同药物对神经元放电活动的影响。 结果 1）在实验中，共记录到429个具有自发节律性放电的神经元单位，其中呼吸相关神经元326个，非呼吸相关神经元103个；2）根据呼吸神经元单位放电和舌下神经电活动之间的时相关系，可将呼吸神经元分为吸气神经元（245/326）和呼气神经元（81/326）两大类。吸气神经元可进一步分为前吸气神经元（Pre-inspiratory neuron，Pre-I，4/245）、早吸气神经元（Early-inspiratory neuron，E-I，125/245）和晚吸气神经元（Late-inspiratory neuron，L-I，116/245），并且大部分的E-I和L-I呈现出多单位的放电形式。Pre-I在舌下神经吸气性放电前数百毫秒开始放电，一直持续到吸气相结束；E-I在吸气早期以频率递减型的形式发放；而L-I则在吸气相中期开始放电，频率递增至吸气相结束时达最高峰；3）呼气神经元可分为全呼气神经元（Throughout expiratory neuron，T-E，24/81）、双相呼气神经元（Biphasic explratory neuron，B－E，22／81）和晚呼气神经n（Late explratory neuron， L＊，35／81）；T＊在舌下神经放电静息期（呼气相）开始时突然发放， 以相对恒定的频率发放至呼气相结束；B工在呼气相晚期开始放电， 但在吸气相时则停止发放，而吸气相结束后又开始放电并持续一段时 间：LE在呼气相晚期开始发放，以比较缓慢而恒定的频率持续到呼 气相结束；4）各类神经元都沿疑核呈混杂柱状分布，在解剖位置上 与mN’RF有较大重叠。在头尾方向，吸气神经元较呼气神经元略偏 尾侧，约 18石％的吸气神经元分布在闩部尾侧，而在相应的位置上， 仅有3％的呼气神经元；在水平方向和腹背方向，两者的分布则无显 著性差异；5）吸／呼气神经元的各亚型神经元在不同方向上的分布均 无显著性差异：6）正常对照时，双相呼气神经元放电的峰频率是39厂5 土5．43次沾，以非N’MDA受体激动剂红藻氨酸（Kainic acid，KA）灌 流5分钟后，峰频率上升到55＊ 土10．17次沾；同时，神经元的放电 形式也发生了明显的变化，全程的放电频率显著提高；再以相应的抬 抗齐 6－氰基－7－硝基睦恶咐土卫四（trans－l－ammo－cycloPlntane－trans4， 3＊carboxylic acid，DNQX）灌流5分钟后，峰频率则下降至39．56土 7．45次沾；全程放电的频率也明显下降；7）正常对照灌流时，吸气 神经元放电的峰频率为40刀1土1＊0次／s，以非NMDA受体激动剂KA 灌流5分钟后，峰频率上升到50厂0士1．43次／S，但KA对吸气神经 元放电形式的影响只表现为使中期放电的频率出现显著的提高，而早 期和晚期放电的频率则没有明显的改变；以相应的抬抗剂 DNQX灌 流 5分钟后，峰频率下降至 41．41土 0石2次七，中期放电的频率也出 现明显的下降。 结论 以上结果提示，在新生大鼠延髓脑片标本上：1）呼吸神 经元存在多种类型的放电形式，各种神经元在脑片上呈混杂分布；2） 呼吸神经元的分布范围与面神经后核内侧区（mN’RF）有着较大的重 叠，提示mNRF可能在离体脑片标本基本呼吸节律的产生过程中同 样发挥重要的作用；3）与在体动物相似，在离体延髓脑片的腹侧区， 同样可以探查到一类始终在吸气前开始放电并持续到吸气相结束的呼 吸神经元（Pre．I），提示哺乳动物的基本呼吸节律可能是由此类神经 元所发起的：4）非NM＊A受体在呼吸节律的形成和调节中发挥着 3重要的作用，但它对吸气神经元和双相呼气神经元的放电活动有着不同的影响。更多还原

【Abstract】 sABSTRACT?Study on distribution of respiratory neurons in the in vitro brainstem slice from neonatal rat and mechanisms of the primaryrespiratory rhythm generationObject1. To illustrate the distribution of different subtypes of respiratory neurons in the brainstem slices isolated from neonatal rats by simultaneous recording of respiratory neurons discharge and hypoglossal nerve activity; 2. To test the hypothesis that the medial area of nucleus retrofacialis (mNRF) may be the kernel site for primary respiratory rhythmogenesis by comparing the anatomical location of respiratory neurons with the mNRF. 3. To investigate the roles of non-NMDA receptors in the respiratory rhythm generation and modulation by exploring the effects of non-NMDA receptors agonist and antagonist on the activities of respiratory neurons.Methods:The present studies were performed on the in vitro brainstem slice isolated from neonatal rats. The respiratory activity was monitored by suction electrode applied on the central end of hypoglossal nerve and the respiratory neuronal discharges were recorded extracellularly by inserting the glass microelectrode into the ventral part of the slices. The respiratory neurons were classified into several subtypes with reference to the temporal relationship between the neuronal discharge and the hypoglossal nerve activity. The distributional status of different types of respiratory neurons were constructed by their distances from the midline, obex and the ventral surface of medullary slices. Drugs were administered by bath application and their effects on the respiratory neuronal activity were investigated.Results:51.In the present study, a total of 429 neuronal units with spontaneousdischarge were recorded. Out of them there were 326 respiratory neuronsand l03 non-respiratory neurons;2. The respiratory neurons were grossly classified into inspiratory neuronsand expiratory neurons with the reference to the temporal relationshipbetween the neuronal discharge and hyPoglossa1 nerve activity Moreovef,the inspiratory neurons were further classified into pre-insPiratory neuronsand early inspiratory neurons as we1l as late inspiratory neurons based ontheir temporal discharge pattems.3. On the other hand, the exPiratory neurons were then classified into threesubtyPes listed as throughout expiratory neurons, biphasic expiratoryneurons and late expiratory neurons.4. A1l the respiratory neurons were distributed and imrningled as a coluninalong the nucleus ambiguus. The inspiratory neurons, howevef, werelocated a little more caudal1y in comparison with the relatively rostrallocation of the expiratory neurons.5. SubtyPes of inspiratory or expiratory neurons showed no conspicuousdifference in the spatial distribution.6. During the control perfusion, the peak frequency of biphasic expiratoryneuronal discharge was 39.75 l 5.43Hz. After bathing aPPlication of KAfor 5 minutes, an agonist of non-N’MDA recePtors, the peak frequencywere increased to 55. l4 t l0.l7Hz and the whole discharge pattems wereelevated. These effects were idriited by administration of DNQX.7.After aPplication of KA, the peak discharge frequency of inspiratoryneurons were increased from 4l.0l l l.l0 Hz to 50.70 t l.43 Hz.MeanWile, the discharge pattems in the middle inspiratory phase wereelevated. In contrast, the discharge pattems in the early and late phaseexhibited no conspicuous changes. The KA excitatory effects could bereversed by ensuing aPplication of DNQX.Conclusions:These results indicated that in the l’n vitrO brainstem slice from6neonatal rats: 1) There were several subtypes of respiratory neurons with different discharge patterns and they were mingled as a column along the nucleus ambiguus; 2) The anatomical localization of respiratory neurons overlapped the structure of mNRF, which suggested the important role of mNRF in the primary respiratory rhythm generation in mammals; 3) Similar to the in vivo situation, Pr更多还原