【作者】 张长征；

【导师】 王建军；

【作者基本信息】 南京大学 ， 生物学， 2013， 博士

【摘要】 5-HT和组胺是中枢神经系统中两类重要的单胺能神经递质。长期以来,人们主要关注它们对非躯体性活动的调节,然而,起源于脑干中缝核(raphe nuclei)及延髓与脑桥网状结构(medullary and pontine reticular formations)的5-HT能神经纤维和起源于下丘脑结节乳头体核(tuberomammillary nucleus, TMN)的组胺能神经纤维也直接支配皮层下许多重要运动结构(如小脑、基底神经节、红核和前庭核团等)。这些纤维以曲张体(varicosity)的形式释放5-HT或组胺,主要通过相应的促代谢型受体与靶细胞发生联系并调节神经元的活动,进一步影响运动功能。除了大脑皮层、脑干和脊髓这三个运动控制的等级性组构之外,小脑(cerebellum)和基底神经节(basal ganglia)构成了皮层下参与运动控制的两个重要侧环。小脑是最大的皮层下运动结构,在动物的平衡控制、肌张力调节和随意运动控制中发挥重要作用,其损伤将导致共济失调等运动障碍。而基底神经节主要负责随意运动的发起,其损伤将导致帕金森病(Parkinson’s disease, PD)和亨廷顿病(Huntington’s disease, HD)等。但是,到目前为止,中枢5-HT能传入纤维和中枢组胺能传入纤维对经小脑和基底神经节介导的运动行为的作用仍不清楚,对其神经机制就更知之甚少。本论文采用实时定量RT-PCR、免疫荧光组织化学、电生理学以及行为学等方法,研究了5-HT和组胺分别对这两个皮层下运动结构神经元活动的作用,特别是二者对运动行为的调控。一、5-HT对经小脑顶核介导的运动行为的调控及其机制5-HT能传人纤维是继苔状纤维和爬行纤维之后小脑最大的传入系统,不仅直接支配皮层中的细胞,还直接支配核团中的神经元。另外,放射自显影、原位杂交、免疫组织化学和神经药理学等研究已经发现,包括5-HT2受体在内的多种5-HT受体亚型均在小脑内有分布。但是,激活5-HT2受体对小脑神经元的活动发挥何种效应,特别是对小脑运动行为产生什么样的影响,尚不清楚。在小脑神经元环路中,小脑核团是小脑内信息处理的最终结构而并非仅仅是接受来自小脑皮层Purkinje细胞(小脑皮层主神经元)传出的中继站。事实上小脑核团整合来自小脑皮层、苔状纤维和爬行纤维的相关运动信息,以及包括5-HT能传入在内的第三类传入系统传来的复杂调节信息,与绒球小结叶一起,构成小脑的最终输出。在小脑核团中,顶核(fastigial nucleus, FN)是系统发生上最古老的核团,主要负责躯干和肢体近端肌肉的精确调节。由此,本研究重点探讨5-HT2受体家族在小脑顶核神经元活动中的作用,以及在经顶核介导的小脑运动行为中的功能意义。实时RT-PCR和免疫组织化学实验显示,在5-HT2受体家族中,只有5-HT2A受体在小脑顶核中有表达和分布；通过离体脑片的细胞外电生理记录发现,所记录到的大多数神经元(92/116,79.3%)对5-HT的刺激表现出单一的兴奋性效应,且这种兴奋性效应具浓度依赖性特点。重要的是,我们发现低钙高镁人工脑脊液不能阻断5-HT对顶核神经元所诱发的兴奋性效应(n=9),提示5-HT引起顶核神经的兴奋性反应主要是突触后效应。进一步地,我们还发现,5-HT2A受体的激动剂TCB-2(n=42)能够模拟5-HT对顶核神经元产生的兴奋性效应,且5-HT2A受体的阻断剂M100907(n=32)不仅能阻断TCB-2,而且也能阻断5-HT对顶核神经元引起的兴奋性效应,表明突触后5-HT2A受体在5-HT对顶核神经元的兴奋性效应中发挥重要作用。接下来,我们通过向小脑顶核微量注射的方法来评估5-HT及其它5-HT能药物对顶核介导的运动行为影响,结果发现,向双侧小脑顶核微量注射5-HT能药物虽然不能影响大鼠在开场活动中的基础运动行为(n=12),但注射5-HT后,大鼠在rota-rod走步机和平衡木的运动成绩显著提高(n=11),同时在足印实验中发现大鼠行进时两足间距缩小(n=12)。向小脑顶核微量注射5-HT2A受体的激动剂TCB-2(n=12或n=13)可模拟5-HT对运动行为的这种易化效应,而微量注射5-HT2A受体的阻断剂M100907(n=12)可对抗核团内源性5-HT传入而显著降低大鼠的运动平衡与协调能力,并且使得大鼠行进时的步宽增大。这些结果强烈提示5-HT或5-HT能神经传入可以通过调节小脑顶核神经元的活动来增强大鼠的运动能力,在这个过程中5-HT2A受体发挥了重要作用。此外,向小脑顶核微量注射5-HT(n=12)并不影响大鼠的步长和肌张力,表明小脑顶核内5-HT或5-HT能传入精确调节躯干肌或近端肌的活动,而非远端肌。因此,我们推测小脑5-HT能传入纤维可以通过对核团神经元活动的调节,影响小脑的最终输出,而实现对小脑介导的运动平衡和协调能力的调控。综上所述,中枢5-HT能神经系统能够通过5-HT2A受体直接调节小脑顶核神经元的电活动,并进而调控经小脑顶核介导的运动行为。二、组胺兴奋大鼠底丘脑核神经元并改善帕金森病模型大鼠的运动能力底丘脑核(subthalamic nucleus, STN)是基底神经节环路中唯一的兴奋性谷氨酸能核团,它接受苍白球及大脑运动皮层的传入并发出广泛的纤维投射到基底神经节的其它核团,由此,STN被认为在基底神经节的运动整合功能中起着驱动及控制作用。许多研究表明在PD患者及PD模型动物中STN神经元的自发放电频率升高并且出现不规则的p同步化震荡,由此,现在临床上STN一直被认为是深部脑刺激治疗PD的重要靶点。放射自显影、免疫组化和原位杂交实验发现地松鼠下丘脑结节乳头体核有到STN直接的组胺能投射,并且在人类及豚鼠的STN中分别发现有组胺H1和H2受体。另有研究发现PD病人脑内组胺能纤维密度增高,基底神经节(壳核、黑质网状部、苍白球)及血液内组胺的浓度显著升高。然而,组胺对STN神经元活动的影响以及中枢组胺能神经系统在PD发生和发展中的作用的研究相对较少,本研究旨在探讨组胺对正常及PD模型大鼠经STN介导的运动行为的影响及其作用的神经机制。行为学实验发现,向STN微量注射组胺能药物显著改善阿普吗啡诱导PD模型大鼠的旋转行为(30min内的旋转圈数)。与生理盐水组(234.1±12.5,n=12)相比,组胺组(125.7±7.6,n=12；P<0.01)、H2受体激动剂dimaprit组(174.9±8.5,n=12；P<0.01)和高钾组(176.9±10.4,n=12；P<0.01)PD模型大鼠的旋转圈数显著减少；H1受体激动剂2-PyEA组(228.5±12.9,n=12；P>0.05)、H1受体阻断剂mepyramine组(243.1±15.3,n=12；P>0.05)、H4受体激动剂VUF8430组(240.2±13.3,n=12；P>0.05)和H4受体阻断剂JNJ7777120组(236.6±12.3,n=12；P>0.05)PD模型大鼠的旋转圈数没有显著性变化；H2受体阻断剂ranitidine组(337.6±13.7,,n=12；P<0.01)和HCN通道的阻断剂ZD7288组(334.9±18.7,n=12;P<0.01)PD模型大鼠的旋转圈数显著增加。这些结果表明组胺通过作用STN改善PD模型大鼠的运动能力,并且组胺的这种改善作用是通过H2受体及其所耦联的下游HCN通道所介导的。进一步地,我们在粘附移除和足印实验中也证实组胺能够通过STN中H2受体作用而改善PD模型大鼠的运动发起和运动协调能力。细胞外电生理记录发现,组胺通过H2受体突触后兴奋STN神经元,这与我们用RT-PCR和免疫组织化学方法发现STN中只有H2受体表达与分布的结果相一致。此外,我们通过全细胞膜片钳记录的方法发现组胺在STN神经上所诱发的兴奋性反应是通过H2受体所耦联的HCN通道介导的。同时,在PD模型大鼠离体脑片的细胞外记录中,我们发现组胺不仅能够提高STN神经元的自发放电频率而且能够显著降低STN神经元自发放电的峰-峰间期的变异系数(n=30),而高钾仅提高STN神经元的自发放电频率(n=16)。考虑到组胺也可以兴奋正常大鼠的STN神经元,随后我们通过rota-rod和平衡木测试,证实组胺可以通过作用于STN神经元上组胺H2受体而提高正常大鼠的运动平衡和运动协调能力。综合上述,我们的结果表明：组胺通过激活STN神经元上组胺H2受体,并通过与之相耦联的HCN通道,改善PD模型大鼠的运动能力。结合已有的组胺兴奋黑质、纹状体、苍白球等基底神经节神经元的研究结果,我们认为中枢组胺能神经纤维可能通过直接的平行投射兴奋基底神经节直接通路和间接通路的各核团而调节运动的发起和执行,同时基底神经节中组胺H2受体和HCN通道可能成为临床治疗PD疾病的靶点。更多还原

【Abstract】 5-HT and histamine are two important monoaminergic neurotransmitters in central nervous system. It has long been focused on their non-somatic modulatory functions, however, the serotonergic fibers, derived from the raphe nuclei as well as the medullary/pontine reticular formations, and the histaminergic fibers originated from tuberomammillary nucleus of the hypothalamus, also directly innervate some subcortical motor structures, such as cerebellum, basal ganglia, red nucleus and vestibular nuclei. These fibers release5-HT or histamine through varicosities, which modulates neuronal activities mainly via the correspondingly metabotropic receptors and subsequently influences animal’s motor abilities, such as motor balance and coordination.In addition to the cerebral cortex, brainstem, and spinal cord in the three motor control hierarchies, the cerebellum and the basal ganglia constitute two important subcortical side loops responsible for the motor control. The cerebellum, the largest subcortical brain region, holds a key position in the control of balance, equilibrium, muscle tone, and the coordination of voluntary movement. Cerebellar damage could cause a variety of motor dysfunctions, such as cerebellar ataxia. Basal ganglia principally accounts for the initiation of voluntary movement, and its disorders could lead to a series of movement disorders, including Parkinson’s disease (PD) and Huntington’s disease (HD).However, the role and the underlying mechanism of central serotonergic/histaminergic inputs on cerebellum-/STN-mediated motor behaviors are still largely unknown. In this study, by using RT-PCR, immunofluorescence, electrophysiological, and behavioral methods, the effects of5-HT/histamine on cerebellar/STN neuronal activities, and especially, their motor behavioral influences were investigated.1. Modulation of5-HT on cerebellar FN-mediated motor behavior and the underlying mechanismThe serotonergic fibers are the largest afferent population except the mossy fibers and climbing fibers, innervating not only the cerebellar cortex but also the cerebellar nuclei. Correspondingly, radioligand binding, in situ hybridization, neuropharmacological and immunohistochemical studies have revealed an existence of several subtypes of5-HT receptors including5-HT2receptors in the cerebellum. However, effect mediated by the activation of5-HT2receptors on cerebellar neurons, particularly on cerebellar motor behaviors, still remains enigmatic.In the cerebellar circuitry, the cerebellar nuclei are the final integrative processing unit rather than a relay station just receiving projections from Purkinje cells (the principal neurons) in the cerebellar cortex. The cerebellar nuclei integrate information from the cerebellar cortex, signals from the mossy fibers and climbing fibers, as well as complicated modulatory messages from the third type of inputs including the serotonergic to make the ultimate outputs of the cerebellum together with the flocculonodular node. Among the cerebellar nuclei, the fastigial nucleus (FN) is the phylogenetically oldest nucleus, and holds a key position in fine-tuning body and proximal limb movements. Therefore, in the present study, we investigated role of5-HT2receptors in neuronal activity of the cerebellar FN and in FN-mediated motor behaviors.Real-time RT-PCR and immunostaining results demonstrated that only5-HT2A receptors rather than5-HT2B or5-HT2C receptors in5-HT2receptor subfamily were expressed and localized in the cerebellar FN. From in vitro extracellular recordings, we found that the majority of the recorded FN neurons (92/116,79.3%) responded to5-HT simulation with a unique excitatory effect in a concentration-dependent manner. Importantly, low-Ca2+/high-Mg2+medium did not block the5-HT-evoked concentration-dependent excitations on FN neurons(n=9), indicating that the excitatory effect of5-HT on FN neurons is primarily postsynaptic. Furthermore, selective5-HT2A receptor agonist TCB-2mimicked the5-HT-induced excitations (n=42), and selective5-HT2A receptor antagonist M100907significantly blocked the excitatory responses induced not only by TCB-2but also by5-HT (n=32), suggesting postsynaptic5-HT2A receptors contribute greatly to the excitatory effect of5-HT on rat FN neurons.Next, we microinjected serotonergic reagents to bilateral cerebellar FNs to evaluate the effect of5-HT/serotonergic inputs on FN mediated motor behaviors. Although microinjection of serotonergic compounds into cerebellar FNs did not influence the overground locomotion in an open field (n=12), microinjection of5-HT into FNs remarkably promoted motor performances on rota-rod and balance beam (n=11), and narrowed stride width in footprint tests (n=12). This facilitation effect of5-HT was mimicked by5-HT2A receptor agonist TCB-2(n=12or n=13). Moreover, microinjection of5-HT2A receptor antagonist M100907to block endogenous serotonergic inputs in FNs significantly attenuated motor balance and coordination and enlarged stride width (n=12). These results strongly suggest that5-HT/serotonergic inputs improve motor behaviors through modulation of FNs, to which5-HT2A receptors are greatly contributed. Furthermore, microinjection of5-HT into FNs did not affect the stride length, nor did it influence grip strength, demonstrating that5-HT/serotonergic inputs in FNs may precisely modulate trunk and proximal musculature rather than distal muscles. Thus, we speculate that serotonergic afferent inputs may actively regulate final cerebellar outputs by modulation of cerebellar nuclear neurons, and subsequently influence the cerebellum-mediated ongoing motor balance and coordination.These results demonstrate that central serotonergic system could influence crebellar FN neuronal activities via5-HT2A receptors, and consequently participate in modulation of cerebellum-mediated motor control.2. Histamine excites rat subthalamic nucleus neurons and restores motor behavior in animal models of Parkinson’s diseaseThe subthalamic nucleus (STN) is the only excitatory glutamatergic nucleus in the basal ganglia motor circuit, and its widespread projections to other basal ganglia regions lead it to be viewed as a ’driving force’ or ’control structure’ in the integrative function of the basal ganglia circuitry. Numerous studies show that in PD patients and PD animal models the spontaneous firing rate of STN neurons increased with irregular β-synchronized oscillatory. Thus, the STN has long been clinically regarded as an important target in deep brain stimulation for PD.A series of autoradiography, immunohistochemistry and in situ hybridization studies showed that the STN of squirrels received the hypothalamic histaminergic innervations, and histamine H1receptors and H2receptors were present in the STN of human and guinea pigs. Other studies demonstrated that the density of histaminergic fibers in the brain of PD patients and the concentration of histamine in their basal ganglia and the blood were significantly higher. However, research about effect of histamine on STN neuron activities and role of the central histaminergic nervous system in PD is relatively rare. Thus, in this study, the effect and the underlying mechanism of histamine on STN-mediated motor performances of normal and Parkinsonian rats were investigated. Behavioral tests demonstrated that microinjection of histamergic reagents to STN ipslateral to6-OHDA lesion considerably influenced apomorphine-induced rotation in PD rat models. Compared with the saline treatment (234.1±12.5in30min, n-12), Parkinsonian rats microinjected with histamine (125.7±7.6, n=12; P<0.01), H2receptor agonist dimaprit (174.9±8.5, n=12; P<0.01) and high K+(176.9±10.4, n=12; P<0.01) significantly reduced the rotation; Parkinsonian rats microinjected with H1receptor agonist2-pyridylethylamine (228.5±12.9, n=12; P>0.05), H1receptor antagonist mepyramine (243.1±15.3, n=12; P>0.05), H4receptor agonist VUF8430(240.2±13.3, n=12; P>0.05) and H4receptor antagonist JNJ7777120(236.6±12.3, n=12; P>0.05) had no significant effects on rotation; while Parkinsonian rats microinjected with H2receptor antagonist ranitidine (337.6±13.7, n=12; P<0.01) and HCN channel antagonist ZD7288(334.9±18.7, n=12;P <0.01) significant increased the rotation. These results indicated that histaminergic activities on STN improved the motor behaviors of PD rat models, which was mediated by H2receptors and their downstream HCN channels. Furthermore, we found that histamine improved Parkinsonian rat’s motor initiation and motor coordination through H2receptor in adhesive removal and footprint tests.Extracellular recording demonstrated that histamine postsynaptically excited STN neurons via H2receptor, which is consistent with our subsequent immunofluorescence and RT-PCR results that only H2recptor was found in STN. Moreover, using whole-cell patch clamp recordings, we found that histamine-elicited excitation on STN neuron was mediated through HCN channels coupled to H2receptors. Furthermore, our extracellular recordings showed that histamine not only increased the firing rate in STN neurons, but also markedly reduced the coefficient of variation in interspike interval (n=30), while high K+only showed an increase in the firing rate (n=16). Considering that histamine also excites STN neurons in normal rats, we subsequently used rota-rod and balance beam tests to evaluate histaminergic effects on the STN-mediated motor behaviors, and the results confirmed histamine promoted STN-mediated motor balance and motor coordination via H2receptor in normal rats. These results demonstrate that histamine restores motor performances of PD rat models by activation of H2receptors and the HCN channels. Considering histamine also excites the substantia nigra, neostriatum and globus pallidus, we speculate that the central histaminergic system may actively modulate the balance between direct and indirect pathways in the basal ganglia through its direct parallel innervations of those structures as well as the STN, and consequently regulate motor initiation and execution. Presumably, histamine H2receptor and the HCN channel in the basal ganglia is a potential target for clinical treatment of PD.更多还原