大鼠喉运动神经元的呼吸功能和非呼吸功能的中枢调控

发布时间：2018-08-01 11:09

【摘要】：目的：喉化学反射,由液体误吸入喉诱导,产生显著的呼吸抑制。喉上神经电刺激能诱导呼吸暂停反射,抑制中枢吸气活动,激发呼气性喉运动神经元放电活动。但是喉上神经电刺激能诱导呼吸暂停反射产生呼吸暂停的机制并不清楚。我们目的是的是研究由喉上神经刺激诱发呼吸暂停反射的神经调控路径。方法：喉上神经电刺激(20Hz,0.2ms)诱导呼吸暂停反射,继而微量注射GABAA受体激动剂isoguvacine (10 mM,20-40 nl)到同侧包钦格复合体和对侧的孤束核。经过60mmin的注射后恢复期,仅微量注射isoguvacine到对侧孤束核。喉返神经活动记录用来监测呼气性喉运动神经元的簇发放电活动。微量注射isoguvacine两分钟后,记录喉返神经和膈神经的电信号来评价isoguvacine的作用效果。结果：喉上神经刺激(20 Hz)诱发呼吸暂停反射,膈神经放电减低到基线的12士9%。微量注射isoguvacine到同侧的包钦格复合体后,呼吸暂停反射明显减弱,然后微量注射isoguvacine到对侧的孤束核,喉上神经刺激诱发的呼吸暂停反射完全被取消。但是,喉上神经刺激诱导的呼气性喉运动神经元的簇发放电活动没有受到影响。仅微量注射isoguvacine到对侧的孤束核,呼吸暂停反射几乎没有影响。的包钦格复合体和对侧的孤束核后,呼吸暂停反射几乎被完全取消,但是呼气性喉运动神经元的簇发放电活动没有受到影响。结果提示从孤束核到双侧的疑核尾段和包钦格复合体的神经投射可能分别介导呼气性喉运动神经元的簇发放电活动和呼吸暂停反射。目的：研究证明很多运动神经元和呼吸神经元接受酪氨酸羟化酶类免疫阳性递质的传递,但是位于疑核尾段的喉运动神经元是否接受该递质的投射至今并不清楚。本实验目的是1)酪氨酸羟化酶免疫阳性的神经终端是否和呼气性喉运动神经元形成密切接触结构；如果结果是阳性,2)研究大鼠喉运动神经元接受的儿茶酚胺类神经递质的来源。方法：本实验中,通过结合细胞内标记和免疫组织化学的方法,我们主要研究SD大鼠的呼气性喉运动神经元是否接收酪氨酸羟化酶免疫阳性神经递质的传递。呼气性喉运动神经元的辨识依据它的吸气后相放电活动特点和对喉返神经刺激产生的逆反射。进一步的神经示踪实验,通过注射霍乱毒素B亚单位到尾段疑核。逆向标记的神经元和酪氨酸羟化酶免疫阳性结构通过荧光双标记的方法来显示。结果：所有的呼气性喉运动神经元上均发现酪氨酸羟化酶免疫阳性的神经终端形成密切接触结构,其平均数目是每个神经元上约18±5(n=7,mean±SD)个密切接触的结构。大多数的密切接触的结构常在远端树突上被发现,近端树突上的数目较少,但是在胞体和轴突上,没有酪氨酸羟化酶免疫阳性的密切接触结构发现。通过将霍乱毒素B亚单位注射到尾段疑核,进行进一步的示踪方法来研究投射到喉运动神经元的儿茶酚胺类神经递质的来源。结果发现,注射部位同侧的孤束核和延髓最后区内有大量的逆向标记的儿茶酚胺类神经元,并且密度最高的位置出现在闩部尾向0.2-0.4 mm。结论：我们首先证实了呼气性喉运动神经元与酪氨酸羟化酶免疫阳性神经终端形成密切接触结构,表明儿茶酚胺类神经递质可能对呼气性喉运动神经元的活动起到一定作用。逆向示踪法研究揭示了大量的逆向标记的儿茶酚胺类神经元,其在孤束核的定位和喉上神经的输入信息投射到孤束核的水平相同,表明孤束核内位于闩部水平的儿茶酚胺类神经元可能对喉上神经介导的气道保护性反射起到重要作用。目的：位于疑核尾段的喉运动神经元,通过控制喉内肌的运动来表达不同的喉功能,包括呼吸,发声,和气道的保护性反射,例如咳嗽反射,喷嚏和吞咽反射。喉运动神经元接受来自不同脑部核团的不同神经化学物质的传递,不同神经化学物质可能对喉运动神经元有不同的作用和影响,从而引起不同的喉功能。P物质,酪氨酸羟化酶和5-羟色胺免疫阳性神经终端投射到到喉运动神经元已经在光镜水平下得到研究,但是它们在疑核尾段的分布比较还不明确。而且,这些神经化学物质的免疫阳性神经终端与喉运动神经元在超微结构水平的关系的研究,也是欠缺的。而这种超微结构水平的研究是用来证实神经化学突触结构和关系的必要方法。因此,我们研究的目的是(1)评价和比较P物质,酪氨酸羟化酶和5-羟色胺免疫阳性神经终端在疑核尾段的分布；(2)通过电生理细胞内记录,免疫组织化学和电镜学,研究P物质免疫阳性神经突触终端与喉运动神经元的超微结构关系。方法：我们通过应用多重免疫荧光法和共聚焦显微镜学方法来同时评估P物质,酪氨酸羟化酶和5-羟色胺免疫阳性神经终端在疑核尾段的分布。疑核尾段运动神经元被胆碱乙酰转移酶的免疫反应阳性标记和辨识。突触体素是一种突触前结构的蛋白。突触体素在疑核尾段内的表达代表了总的神经突触终端的数量。运用Image J软件共定位分析后,突触体素阳性和P物质,酪氨酸羟化酶或者5-羟色胺免疫阳性共同表达的区域代表了P物质,酪氨酸羟化酶或者5-羟色胺的神经突触终端区域。P物质,酪氨酸羟化酶或者5-羟色胺的神经突触终端占总神经突触终端区域的比例,被用来评价和比较它们在疑核尾段的突触终端的分布。在比较P物质,酪氨酸羟化酶和5-羟色胺的神经突触在疑核尾段的分布的基础上,我们进一步研究了P物质免疫阳性神经终端与喉运动神经元的超微结构关系。在一例实验中,一个吸气性喉运动神经元通过细胞内记录,颈迷走神经刺激和它的疑核尾段的定位被辨识和确认。然后注射神经生物素(biotinamide,1.5%)入这个吸气性喉运动神经元。被生物胞素注射的神经元和P物质免疫阳性结构,通过电镜包埋前免疫组化染色法,同时显示出来。包含有喉运动神经元和P物质免疫阳性结构的超薄切片染色并在电镜下观察和分析。结果：我们研究发现,P物质,酪氨酸羟化酶或者5-羟色胺的神经突触终端占疑核尾段总神经突触终端的强度比例,均不超过10%,三者合计不超过15%。和酪氨酸羟化酶或者5-羟色胺比较, P物质神经突触终端在疑核尾段有相对较高的强度比例。在超微结构水平,53.3%(114/206)的突触膨大终端与该吸气性喉运动神经元的树突形成不对称突触结构,22.3%(46/206)形成对称突触结构,其他的膨大终端与神经元形成接触,但是没有清晰的特殊突触结构。在这206个神经终端中,16%(33/206)是P物质免疫阳性神经终端并与神经元形成突触结构。29个形成不对称突触结构,4个形成对称突触结构。少数几个P物质免疫阳性神经终端与神经元胞体形成突触结构,但是在神经元轴突上没有发现P物质神经终端形成突触结构。非免疫反应性的神经终端也在神经元树突棘上形成突触结构,一些具有突触下特殊结构。在吸气性喉运动神经元所在的组织切片上,还可以观察到几个具有大胞体(直径约为30-40μm)的,疑核非免疫阳性神经元。它们含有大的胞核和明显的核仁。结论：首先,我们的结果第一次证实了,在大鼠疑核尾段,P物质,酪氨酸羟化酶和5-羟色胺的神经突触终端仅占突触体素免疫阳性的总的神经突触终端的少数,这间接表明了P物质,酪氨酸羟化酶和5-羟色胺在喉运动神经元的功能上仅起到适中的调控作用。第二,我们证实了P物质免疫阳性神经终端在喉运动神经元上形成突触结构。在一例实验中,一个吸气性神经元,位于疑核尾段,颈迷走神经刺激后受到激活,标记并进行超微结构分析。该标记的神经元接受大量的对称和不对称的突触传递。共计发现33个P物质的神经终端在吸气性喉运动神经元的树突上形成突触结构。其中,大多数(29)为不对称性突触,少数(4)为对称性突触,表明表达P物质的神经元可能直接提高和增强吸气性喉运动神经元的兴奋性,主要通过近端树突上的化学突触传递。第三,我们研究了包含吸气性喉运动神经元的疑核尾段内的大胞体神经元的总体超微结构和突触学。在这些神经元上发现大量的对称和不对称的突触结构。目的：在呼吸运动以及其他条件下和其他活动中,例如低碳酸血症和睡眠,喉内肌接受不同条件的调控。以往的解剖学和药理学研究表明,在疑核水平的乙酰胆碱起到调控喉运动神经元的活动的作用。疑核松散结构含有吸气性和呼气性喉运动神经元。本实验目的是研究疑核松散结构内的喉运动神经元接受胆碱能输入信息的解剖学特点。方法：我们采用体内细胞内记录,染色剂细胞内注射,和免疫组织化学的方法来研究喉运动神经元和胆碱能免疫阳性神经终端之间的解剖学关系。突触体素是突触的标志蛋白,因此突触体素免疫反应阳性的神经终端可视为神经突触终端。我们采用共聚焦显微镜的免疫荧光学方法和共定位分析,即胆碱能免疫阳性神经终端与突触体素的免疫反应性共定位的分析,进一步研究和评估了与喉运动神经元有“密切接触”关系的胆碱能免疫阳性神经终端中,同时又呈现突触体素免疫阳性的比例。结果：结果证实了呼气性喉运动神经元与囊泡乙酰胆碱转运体免疫阳性神经终端之间存在“密切接触”。共有12个呼气性喉运动神经元被细胞内记录识别和被神经生物素注射而标记。其中,呼气性喉运动神经元上发现较多的囊泡乙酰胆碱转运体免疫阳性神经终端形成“密切接触”(mean±SD,32±9;n=8)。与远端树突相比,喉呼气性运动神经元的细胞体和近端树突有更多的“密切接触”(two-way ANOVA,P0.0001).共聚焦显微镜荧光学方法证实了约90%的“密切接触”结构中囊泡乙酰胆碱转运体免疫阳性神经终端与突触体素存在共定位结构(突触终端=45,呼气性喉运动神经元=4)。结论：本实验的创新点在于,在体实验功能性识别的呼气性喉运动神经元,与囊泡乙酰胆碱转运体免疫阳性神经终端存在“密切接触”。与远端树突相比较,在细胞体和近段树突发现较多的“密切接触”。通过细胞内记录,共聚焦显微镜学和突触标志蛋白的使用,我们还评价了“密切接触”是否代表突触结构。大多数囊泡乙酰胆碱转运体免疫阳性神经终端与喉运动神经元之间的“密切接触”,呈现突触体素免疫阳性。总之,我们的实验发现为胆碱能递质输入对喉运动神经元活动存在潜在的重要作用提供了解剖学证据。但是喉运动神经元上胆碱能受体的表达,以及所接受的胆碱能递质输入的来源,需要进一步的研究。
[Abstract]:Objective: the laryngeal chemical reflex is induced by the larynx induced by the liquid inhalation of the larynx. The electrical stimulation of the superior laryngeal nerve can induce apnea reflex, inhibit the activity of the central nervous system and stimulate the discharge activity of the exhalation laryngeal motor neurons. However, the mechanism of the electrical stimulation of the superior laryngeal nerve can induce the respiratory suspension reflex to produce apnea. The aim is to study the neural regulation pathway of apnea reflex induced by stimulation of superior laryngeal nerve.
Methods: 20Hz (0.2ms) induced apnea reflex, and then microinjection of GABAA receptor agonist isoguvacine (10 mM, 20-40 NL) to the ipsilateral paratagal complex and contralateral nucleus of solitary tract. After 60mmin injection, only microinjection of isoguvacine to the contralateral nucleus of the solitary tract was used to monitor the recurrent laryngeal nerve activity. After two minutes of microinjection of isoguvacine, the electrical signals of the recurrent laryngeal nerve and phrenic nerve were recorded to evaluate the effect of isoguvacine.
Results: the apnea reflex was induced by the stimulation of the superior laryngeal nerve (20 Hz), and the phrenic nerve discharge decreased to the baseline of 12 isoguvacine to the same side of the pachtchin complex. The apnea reflex was markedly weakened, and then isoguvacine was injected into the contralateral nucleus of the solitary tract, and the apnea reflex induced by the upper laryngeal nerve stimulation was completely cancelled. However, the cluster discharge activity of the exhaled laryngeal motor neurons induced by the stimulation of the superior laryngeal nerve was not affected. Only microinjection of isoguvacine to the contralateral nucleus of the solitary tract had little effect on the apnea reflex. After the package Qinge complex and the contralateral nucleus of the solitary tract, the apnea was almost completely cancelled, but exhaled larynx movement The results suggest that the neural projections from the nucleus of the nucleus to the bilateral nucleus of the nucleus and the Bao Qin lattice may mediate the cluster discharge and apnea of the exhaled laryngeal motoneurons, respectively.
Objective: to demonstrate that many motor neurons and respiratory neurons accept the transfer of tyrosine hydroxylase - like immuno - positive transmitters, but it is not clear whether the projection of the laryngeal motoneurons located in the nucleus of the nucleus of the nucleus is not clear. The purpose of this experiment is 1) whether the tyrosine hydroxylase immunoreactive nerve terminal and exhalation larynx are used. If the results are positive, 2) Study the source of catecholamine neurotransmitters received by rat laryngeal motor neurons.
Methods: in this experiment, we mainly study whether the expiratory laryngeal neurons in SD rats receive the transmission of tyrosine hydroxylase immunoreactive neurotransmitters by combining the intracellular labeling and immunohistochemistry. The identification of exhaled laryngeal motoneurons is based on the characteristics of its discharge activity in the post inhalation phase of the larynx and on the recurrent laryngeal nerve spines. A further neural tracer experiment, through the injection of cholera toxin B subunit to the caudal nucleus. Reverse labeled neurons and tyrosine hydroxylase immunoreactive structures are displayed by a fluorescence double labeling method.
Results: the close contact structure of tyrosine hydroxylase immunoreactive nerve terminals was found on all exhaled laryngeal motoneurons, with the average number of close contact structures about 18 + 5 (n=7, mean + SD) on each neuron. Most of the close contact structures were often found on the distal dendrites and the number of the proximal dendrites. There is less, but there is no close contact structure of tyrosine hydroxylase immunoreactivity on the cell body and axon. The source of catecholamine neurotransmitters projecting into the laryngeal motoneurons is studied by injecting the cholera toxin B subunit into the caudal nucleus, and the results show that the isolated bundle of the injection site is on the same side. A large number of retrogradely labeled catecholamine neurons were found in the nucleus and medulla oblongata, and the highest density was found in the caudal part of the latch 0.2-0.4 mm.
Conclusion: we first confirmed the close contact structure of the exhaled laryngeal motoneurons with the tyrosine hydroxylase immunoreactive nerve terminal, indicating that the catecholamine neurotransmitters may play a role in the activity of the exhaled laryngeal motoneurons. The location of the nucleus in the nucleus of the solitary tract and the input information of the superior laryngeal nerve projected to the same level of the nucleus of the solitary tract, indicating that the catecholamines at the latch level in the nucleus of the solitary tract may play an important role in the protective reflex of the upper laryngeal nerve.
Objective: the laryngeal motor neurons, located in the nucleus of the nucleus, express different laryngeal functions by controlling the movement of the intramuscular muscles, including respiratory, vocal, and protective reflex of the airway, such as coughing reflex, sneezing and swallowing reflex. Laryngeal motor neurons accept the transfer of different neurochemicals from different brain nuclei and different neurochemistry. Substances may have different effects and effects on laryngeal motor neurons, resulting in different laryngeal function.P substances. Tyrosine hydroxylase and 5- serotonin immunoreactive nerve terminals have been projected to the laryngeal motoneurons at the level of light microscopy, but their distribution in the nucleus of the nucleus is not clear. The study of the relationship between the immunoreactive nerve terminals and the laryngeal motoneurons at the ultrastructural level is also deficient. This ultrastructural level is a necessary method to confirm the synaptic structure and relationship of the neurochemical. Therefore, the purpose of our study is to evaluate and compare P, tyrosine hydroxylase and 5- hydroxyl. The distribution of serotonin immunoreactive nerve terminals in the caudal caudal segment; (2) the ultrastructural relationship between the immunoreactive synapse terminal of substance P and the laryngeal motoneuron was studied by electrophysiological cell recording, immunohistochemistry and electron microscopy.
Methods: We used multiple immunofluorescence and confocal microscopy to evaluate the distribution of P, tyrosine hydroxylase and 5- serotonin immunoreactive nerve terminals in the nucleus caudal. The nucleus caudal motoneurons were labeled and identified by the immunoreactivity of choline acetyltransferase. Synaptophysin is a synapse. The expression of synaptophysin in the nucleus of the nucleus of the nucleus represents the number of the total synaptic terminals. After the Image J Software Co localization analysis, the regions of synaptosomal positive and substance P, tyrosine hydroxylase, or 5- HT are represented by P matter, tyrosine hydroxylase or 5- hydroxytryptamine. The synaptic terminal region.P substance, tyrosine hydroxylase or 5- serotonin synaptic terminal accounts for the proportion of the total synaptic terminal region, which is used to evaluate and compare the distribution of synaptic terminals in the nucleus caudal. Based on the comparison of the distribution of P, tyrosine hydroxylase and 5- hydroxytryptamine in the caudal segment of the nucleus of the nucleus of the nucleus, We further studied the ultrastructural relationship between the P substance immunoreactive nerve terminal and the laryngeal motoneuron. In an experiment, an inspiratory laryngeal motor neuron was identified and confirmed through intracellular recording, the cervical vagus nerve stimulation and its localization of the nucleus caudal. Then, the nerve biotin (biotinamide, 1.5%) was injected into this inhalation. Sexual laryngeal motoneurons. Immunoreactive structures of neurons and substance P injected with biotin were detected by electron microscopy before embedding immuno histochemical staining, and were displayed at the same time. Ultrathin sections of the immune positive structures of laryngeal motoneurons and substance P were stained and observed and analyzed under electron microscopy.
Results: we found that the P substance, tyrosine hydroxylase or 5- serotonin terminals accounted for no more than 10% of the total synaptic terminals in the total nucleus of the nucleus caudate, and three were not more than 15%. and tyrosine hydroxylase or 5- hydroxytryptamine. The synaptic terminal of substance P had a relatively high intensity ratio in the nucleus of the nucleus. At the ultrastructural level, the synaptic expansion terminal of 53.3% (114/206) forms an asymmetric synaptic structure with the dendrites of the inhaled laryngeal motoneuron, and 22.3% (46/206) forms a symmetric synaptic structure. The other expansion terminals are in contact with the neurons, but there is no clear special synaptic structure. In these 206 nerve terminals, 16% (33/206) is P The substance immunoreactive nerve terminal and the synapse structure.29 formed an asymmetric synaptic structure with the neurons, and 4 formed symmetric synaptic structures. A few P substance immunoreactive terminals formed synaptic structures with the neuronal cell bodies, but there was no discovery of the synaptic structure of the P matter nerve terminal on the axon of the neuron. Non immune response was not found. The sexual nerve terminal also forms a synaptic structure on the dendritic spines, some with special structures under the synapse. On the tissue section of the inspiratory laryngeal motoneurons, several large cell bodies (about 30-40 m in diameter) are also observed, and the non immunoreactive neurons of the nucleus are suspected. They contain large nuclei and obvious nucleolus.
Conclusion: first of all, our results first confirmed that in the rat nucleus of the nucleus, substance P, tyrosine hydroxylase and 5- hydroxytryptamine are only a few of the synapse terminals of synaptopsin immunoreactive, which indirectly indicates the function of P, tyrosine hydroxylase and 5- HT in the function of laryngeal motoneurons. To moderate regulation. Second, we confirmed that substance P immunoreactive nerve terminals form synaptic structures on laryngeal motor neurons. In an experiment, an inspiratory neuron, located in the nucleus of the nucleus, was activated, marked and ultrastructural after stimulation of the vagus nerve of the neck. The labeled neurons accept a large number of symmetry. And asymmetric synaptic transmission. A total of 33 P substances were found to form synaptic structures on the dendrites of the inhaled laryngeal motoneurons. Most of them (29) were asymmetric synapses, and a few (4) were symmetric synapses, indicating that neurons expressing substance P may directly increase and enhance the excitability of the inhaled laryngeal motoneurons. Third, we studied the overall ultrastructure and synapse of large cell neurons in the caudal caudal segment of the inhaled laryngeal motoneurons, and found a large number of symmetrical and asymmetric synaptic structures on these neurons.
Objective: in breathing exercises and other conditions and other activities, such as hypocapnia and sleep, and the regulation of different conditions in the laryngeal muscles. Previous anatomical and pharmacological studies have shown that acetylcholine at the nuclear level plays a role in regulating the activity of the laryngeal motoneurons. The unconsolidated structure contains inhalation and exhalation. The aim of this study is to investigate the anatomical characteristics of cholinergic input to laryngeal motor neurons in the loose structure of nucleus suspected.
Methods: We used intracellular recording, intracellular injection of dyes, and immunohistochemical methods to study the anatomical relationship between laryngeal motor neurons and cholinergic immunoreactive nerve terminals. Synaptophysin is a marker protein of synapse, so the nerve terminal of synaptophysin positive immune response can be seen as a synapse terminal. We use the immunofluorescence method and co localization analysis of confocal microscopy, that is, the analysis of the co localization of the immunoreactivity of the cholinergic positive nerve terminal and the synaptophysin, and further studies and evaluates the cholinergic immunoreactive nerve terminal which has a "close contact" with the laryngeal motoneuron. The proportion of immunopositive to the tactis.
Results: the results confirmed that there was a "close contact" between the exhaled laryngeal motoneurons and the immunoreactive terminals of the vesicular acetylcholine transporter. A total of 12 exhaled laryngeal motoneurons were identified by intracellular recording and labeled by neurobiotin injection. Among them, more vesicle acetyl was found on the exhaled laryngeal motoneurons. Choline transporter immunoreactive nerve terminals formed "close contact" (mean + SD, 32 + 9; n=8), compared with the distal dendrites.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R338

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