【作者】 陈咏华；

【导师】 孙凤艳； 王继江；

【作者基本信息】 复旦大学 ， 神经生物学， 2008， 博士

【摘要】 5-羟色胺（serotonin or 5-hydroxytryptamine,5-HT）是一种小分子吲哚胺化合物,广泛分布于外周组织和中枢神经系统中,作为血管活性物质或神经递质在痛觉调制、睡眠、内分泌功能、心血管功能、呼吸功能以及精神活动等多方面发挥重要作用。脑内5-HT能神经元胞体主要集中在低位脑干中线附近的中缝核群（中缝苍白核,中缝隐核,中缝大核）,通过中缝核群发出的纤维下行支配脊髓,上行支配前脑和脑干的广大区域,同时还可通过脑脊液到达其他脑区发挥广泛作用。近年来大量在体动物实验研究表明5-HT参与心血管与呼吸活动的反射性调节,并有多种5-HT受体亚型（5-HT₁,5-HT₂,5-HT₃,5-HT₄,5-HT₇）参与。在中枢阻断5-HT_1A受体或5-HT₇受体可以减弱心肺反射引起的心动过缓;在孤束核（nucleus tractus solitarius,NTS）,不同5-HT₂受体亚型有不同作用,其中5-HT_2B受体起兴奋作用而5-HT_2C起抑制作用。但是具体的神经反射通路及作用机制目前还不甚清楚。以往的研究主要采用解剖学和在体电生理研究的方法,采用膜片钳技术在突触水平研究5-HT对心肺迷走神经节前神经元的调节作用有可能在突触水平阐明5-HT对心血管与呼吸功能反射性调节的部分机制。以往对心肺迷走神经节前神经元的膜片钳研究主要关注的是心迷走节前神经元[cardiac vagal（parasympathetic）preganglionic neurons,CVPNs],其中枢定位及突触支配和调制的研究已经取得了一定的成果,而对肺迷走神经节前神经元突触支配及调制的研究则开展得很少。因此,本论文的研究目的分为三部分:一,研究5-HT对CVPNs突触支配的调节作用。二,研究呼吸道副交感节前神经元[airway-related parasympathetic（vagal）preganglionic neurons,APPNs]在中枢的解剖定位及电生理特性。三,研究5-HT对APPNs突触支配的调节作用。本实验以2-6天SD大鼠为研究对象,先用荧光染料逆行标记目的神经元,在离体延髓脑片上采用全细胞膜片钳模式对神经元进行研究。结果和结论如下:一、5-HT_1A/7受体激动剂8-OH-DPAT显著抑制CVPNs的自发性GABA能和甘氨酸能抑制性突触后电流（spontaneous inhibitory postsynaptic currents,sIPSCs）的频率和幅度,显著抑制刺激NTS引起的GABA能突触后电流,而对微小的GABA能和甘氨酸能抑制性突触后电流（miniature inhibitory postsynapticcurrents,mIPSCs）、外源性GABA和外源性甘氨酸引起的突触后电流及自发的谷氨酸能兴奋性突触后电流（spontaneous excitatory postsynaptic currents,sEPSCs）均没有明显作用。这些结果提示5-HT_1A/7受体激动剂通过突触前机制抑制GABA能和甘氨酸能突触传入间接兴奋CVPNs。这可能是5-HT参与心迷走神经反射调控的机制之一。二、通过喉返神经标记的APPNs均位于疑核致密部,而通过气管壁标记的APPNs位于疑核致密部或疑核腹侧/腹外侧。位于疑核致密部的APPNs主要接受抑制性GABA能和甘氨酸能突触传入,其中部分神经元这些抑制性突触传入在吸气期间增加,而在另外部分神经元减少;位于疑核腹侧/腹外侧的APPNs除了接受抑制性GABA能和甘氨酸能突触传入外,还接受兴奋性谷氨酸能突触传入,部分神经元在吸气时抑制性突触传入增加从而被抑制,而另外部分神经元在吸气时兴奋性突触传入增加从而被兴奋。吸气时抑制性突触传入增加提示这些神经元在吸气期被抑制,在功能上可能是与呼气或后吸气相关的,而吸气时抑制性突触传入减少或兴奋性突触传入增加提示这些神经元在吸气期兴奋,在功能上可能是与吸气相关的。这些结果表明支配呼吸道不同节段的副交感节前神经元在中枢的定位及功能是分离的。此外,我们采用消除细胞跨膜电化学梯度的方法,在单个吸气性呼吸道副交感节前神经元（inspiratory airway-related parasympathetic preganglionic neurons,I-APPNs）上成功记录到了电突触电流（gap junction currents,GJCs）,并且这些GJCs与呼吸活动是密切相关的。这个结果提示电突触存在于APPNs中,并且可能在呼吸中枢神经元电活动的同步性中起重要作用。这种记录方法可用于快速探测具有自发活动的神经元网络中的GJCs。关于5-HT对APPNs突触支配的调控作用这一部分实验还处于研究阶段,需要在接下来的时间里继续探索。更多还原

【Abstract】 5-hydroxytryptamine（serotonin,5-HT）,an indolamine that is widely distributed in the peripheral tissue and central nervous system,is involved in a large variety of physiological functions and pathogenesis including pain modulation,sleep,endocrine, cardiopulmonary function and psychiatric disorders such as depression,anxiety and aggressiveness.Anatomical studies have found that 5-HT containing neurons are mainly within the raphe nuclei（pallidus,obscurus,magnus）.Serotonergic neurons project to the whole brain and synapse upon many kind of neurons.In recent years, many in vivo electrophysiological studies have shown that 5-HT plays an important role in mediating the reflex control of cardiopulmonary functions,which involves many receptor subtypes including 5-HT₁,5-HT₂,5-HT₃,5-HT₄,5-HT₇.Blocking 5-HT_1A or 5-HT₇.receptors attenuated bradycardias evoked by stimulating baroreceptor and cardiopulmonary afferents.In the nucleus tractus solitarius（NTS）, 5-HT₂ receptors had variable effects;5-HT_2B receptors excite and 5-HT_2C receptors inhibit.However,little is known about the neural pathway and the synaptic mechanisms.Using patch-clamp techniques to study the effect of 5-HT on the synaptic inputs of cardiopulmonary vagal preganglionic neurons is important to elucidate the reflex control of cardiopulmonary functions at the synaptic level.Previous studies have made much progress about the central distribution and the synaptic control of cardiac vagal preganglionic neurons（CVPNs）.With respect to pulmonary vagal preganglionic neurons,the study is scarce.So,our aim is to investigate:（1） the effect of 5-HT on the synaptic inputs of CVPNs.（2） the central distribution and electrophysiological characteristics of airway-related parasympathetic preganglionic neurons（APPNs）.（3）the effect of 5-HT on the synaptic inputs of APPNs.The CVPNs were retrogradely labeled by injecting rhodamine into the fat pads of the heart or into the cardiac sac of 2-6-day-old Sprague-Dawley rats and the APPNs were retrogradely labeled by injecting rhodamine into the recurrent laryngeal nerve （RLN） or tracheal wall.The CVPNs and APPNs were identified by the presence of fluorescence in brainstem slices and studied using whole cell patch-clamp recording technique.Following are the results and conclusions:1.5-HT_1A/7 receptor agonist 8-OH-DPAT significantly inhibited the spontaneous inhibitory postsynaptic currents（sIPSCs） of GABAergic and glycinergic inputs and also caused significantly amplitude decrease of the GABAergic currents evoked by stimulation of NTS,whereas it had no effect on the miniature inhibitory postsynaptic currents（mIPSCs） of GABAergic and glycinergic inputs.8-OH-DPAT had no effect on the currents evoked by exogenous GABA and glycine and had no effect on the glutamatergic spontaneous excitatory postsynaptic currents（sEPSCs）.These findings suggested that 5-HT_1A/7 receptor agonist might excite CVPNs through inhibition of their GABAergic and glycinergic inputs.This conclusion at least in part revealed the synaptic mechanisms involved in the 5-HT mediated reflex control of cardiac vagal tone.2.Tracer application directly to the RLN only labeled the putative APPNs within the compact portion of the nucleus ambiguous（cNA）,while tracer injection into the tracheal wall labeled the putative APPNs both in cNA and in the area ventro/ventrolateral to cNA（vNA）.The putative APPNs within cNA receive mainly inhibitory synaptic inputs,which in some neurons showed an inspiratory-related augmentation and in others showed an inspiratory-related attenuation.The putative APPNs in vNA received both excitatory and inhibitory synaptic inputs,and central inspiratory activity excited some of them via augmentation of their excitatory inputs and inhibited others via augmentation of their inhibitory inputs.The APPNs inhibited during inspiration might be expiration-related or postinspiratory-related motoneurons and others excited during inspiration might be inspiratory-related motoneurons. Theses results provide evidence that APPNs controlling different segments of the airway might be dissociated in the ventrolateral medulla both anatomically and in functional control.In addition,we recorded the gap junction currents（GJCs） in single inspiratory APPN（I-APPN） by elimination of the transmembrane electrochemical gradients in voltage patch-clamp recording.The GJCs were rhythmically activated by central inspiration activity.These findings indicated that gap junctions exist in the APPNs and might play an important role in the synchronization of the central respiratory neurons. This method may be used as a fast way to detect GJCs within spontaneously active neuronal network.The study about the effect of 5-HT on the synaptic inputs of APPNs is still on the way.更多还原