大鼠三叉神经脊束间质核内接受面口部躯体伤害性信息的calbindinD-28k神经元向孤束核的投射

为探讨三叉神经脊束间质核内的 calbindin D-2 8k神经元是否接受并传递面口部躯体伤害性信息到孤束核 ,本研究应用荧光金逆行束路追踪结合 FOS和 calbindin D-2 8k免疫荧光组织化学技术 ,观察了三叉神经脊束间质核的 calbindin D-2 8k和FOS双重免疫反应阳性的神经元向孤束核的投射。向右侧孤束核内注射荧光金并向右侧上、下唇皮下注射福尔马林 ,发现荧光金逆标细胞和 FOS免疫反应阳性细胞主要分布于注射侧的三叉神经脊束间质核的背侧边缘旁核和三叉旁核 ;大量的 calbindinD-2 8k免疫阳性细胞分布于双侧三叉神经脊束间质核内。此处的大部分荧光金逆标细胞 (约 74.4% )呈 calbindin D-2 8k免疫反应阳性。在此二者的双重阳性细胞中 ,又有一部分 (约 41.0 % )为同时呈 FOS免疫反应阳性的三重阳性神经元。结果提示 ,三叉神经脊束间质核内接受面口部躯体伤害性信息的 calbindin D-2 8k神经元可直接投射至孤束核 ,calbindin D-2 8k神经元可能在躯体伤害性信息经三叉神经脊束间质核向孤束核的传递过程中发挥重要作用     

面口部三叉神经躯体性初级传入和内脏性初级传入三叉神经脊束间质核汇聚的电镜研究

本教研室既往研究曾发现三叉神经脊束间质核不仅接受支配面、口部皮肤和粘膜的下牙槽神经、舌神经和眶下神经等三叉神经范围的初级传入，还接受舌咽、迷走神经的内脏初级传入。继之又证明，投射于三叉神经脊束间质核的舌神经眶下神经和舌咽、迷走神经的初级传入终末都主要分布在相当于闩水平的三叉神经脊束间质核的梭形、柱状膨大部分，且两者的终末分布区存在着明显的重叠。因此设想，此两种传入终末可能汇聚于三叉神经脊束间质核内的神经元并与之形成突触联系。为了证实此点，本研究将HRP和神经毒ricin分别注入于舌咽、迷走神经和眶下神经，在电镜下对它们的终末分布状态和联系进行了探索，发现有多数的此两类终末与树突分别构成突触连结；还发现一些标记的和溃变的终末共同汇聚于同一间质核神经元，其数量分别占标记末总数的1．43％和变性终末总数的6．57％。本文首次在形态学上证明直接来自三叉神经躯体性和来自Ⅸ、X脑神经内脏初级传入终末汇聚于间质核神经元形成双重突触连结。推率，来源不同的外周躯体性和内脏性传入信息可能 藉此汇聚而互相作用实现功能上的整合。     

面口部三叉神经躯体性初级传入和内脏性初级传入在三叉神经脊束间质核汇聚的电镜研究

本教研室既往研究曾发现三叉神经脊束间质核不仅接受支配面、口部皮肤和粘膜的下牙槽神经、舌神经和眶下神经等三叉神经范围的初级传入 ,还接受舌咽、迷走神经的内脏初级传入。继之又证明 ,投射于三叉神经脊束间质核的舌神经、眶下神经和舌咽、迷走神经的初级传入终末都主要分布在相当于闩水平的三叉神经脊束间质核的梭形、柱状膨大部分 ,且两者的终末分布区存在着明显的重叠。因此设想 ,此两种传入终末可能汇聚于三叉神经脊束间质核内的神经元并与之形成突触联系。为了证实此点 ,本研究将 HRP和神经毒 ricin分别注入于舌咽、迷走神经和眶下神经 ,在电镜下对它们的终末分布状态和联系进行了探索。发现有多数的此两类终末与树突分别构成突触连结 ;还发现一些标记的和溃变的终末共同汇聚于同一间质核神经元 ,其数量分别占标记终末总数的 1.43%和变性终末总数的 6 .5 7%。本文首次在形态学上证明直接来自三叉神经躯体性和来自 、 脑神经内脏性初级传入终末汇聚于间质核神经元形成双重突触连结。推论 ,来源不同的外周躯体性和内脏性传入信息可能藉此汇聚而互相作用实现功能上的整合     

舌咽、迷走神经初级传入与三叉神经初级传入在三叉神经脊束间质核的汇聚

三叉神经脊束间质核是位于三叉神经脊束内的一些灰质团块，三叉神经的一些分支和舌咽、迷走神经均有初级传入纤维投射于此核．为了更确切地认识这些传入投射在此核内的分布范围及其相互关系，本文在光学显微镜下详细观察了Nissl引染色和未染色的透明大鼠脑切片，利用计算机图像分析技术首先重塑此核的立体构形，继之又重塑了经HRP跨节追踪方法显示的三叉神经分支的舌神经、眼下神经以及舌咽、速走神经初级传入终末向此核内投射的立体分布状况．结果显示三叉神经脊束间质核在吻尾方向上整体呈连续且中间膨大的桂形佳状结构；投射于此核的各神经的终末量多少、分布区域虽然都不尽相同，但三叉神经躯体初级传入和Ⅸ、X脑神经内脏初级传人终末在此梭形结构中段膨大部呈明显的重叠分布。本文结果为内脏传入和面目部躯体使人信息在三叉神经脊柬问质核神经元的汇聚提供了形态学根据．     

舌咽,迷走神经初级传入与三叉神经初级传入在三叉神经脊束？…

三叉神经脊束间质核是位于三叉神经脊束内的一些灰质团块，三叉神经的一睦分支和舌咽，迷走神经均有初级传入纤维投射于此核，为了更确切地认识这些传入投射在此核内的分布范围及其相互关系，本文在光学显微镜下详细观察了Ｎｉｓｓｌ染色和未染色的透明大鼠脑切片，利用计算机图像分析技术首先重塑此核的立体构形，继之又重塑了经ＨＲＰ跨节追踪方法显示的三叉神经分支的舌神经，眶下神经以及舌咽，迷走神经初级传入终末向此核内投射     

大鼠三叉神经脊束间质核内接受内脏伤害性信息的含CB神经元向孤束核的投射

目的 探讨大鼠三叉神经脊束间质核(INV)内接受内脏伤害性信息的含calbindin D-28K(CB)神经元与孤束核(NTS)的投射联系。方法 用福尔马林刺激上消化道，应用荧光金(FG)逆行束路追踪结合Fos和CB的免疫荧光组织化学三重标记法。结果 INV的背侧边缘旁核(PaMd)和三叉旁核(PaV)内可见到大量FG逆标细胞，以注射FG的同侧为主。大部分FG逆标细胞(约71.2％)为CB免疫反应阳性。部分FG和CB双标记神经元(约31．5％)同时呈Fos免疫反应阳性的三重标记。结论 INV内部分接受内脏伤害性刺激的CB神经元可直接投射至NTS，含CB的神经元在内脏伤害性信息经INV向NTS的传导通路中，可能发挥重要作用.     

大鼠三叉神经节神经元向三叉神经脊束核尾侧亚核和孤束核的分支投射

为研究三叉神经节神经元向三叉神经脊束核尾侧亚核（ＶＣ）和孤束核（ＮＴＳ）的分支投射，用荧光素逆行追踪双标记方法，Ｆａｓｔｂｌｕｅ（ＦＢ）和Ｎｕｃｌｅａｒｙｅｌｏｗ（ＮＹ）分别注射入三叉神经脊束核尾侧亚核和孤束核后，荧光显微镜（３６０ｎｍ激发波长）下可见三叉神经节内的多种逆行标记神经元。ＦＢ显示明亮的蓝色标记胞浆，而ＮＹ标记则为黄绿色的圆形胞核。虽大多数神经元为单标记，但部分也呈黄色胞核，蓝色胞浆的双标记细胞。双标记细胞多为直径３０μｍ以下的中、小型，鲜见大型细胞。分支投射神经元可能扩散传递口、舌伤害性痛觉信息并在ＶＣ和ＮＴＳ内与其他来源的内脏、躯体感觉传入信息相互影响。     

三叉神经初级传入纤维与孤束核的联系——HRP跨越神经节追踪研究

本文用HRP跨节追踪技术在光镜下观察了猫的眶上神经、眶下神经、耳颞神经、颊神经、舌神经和下牙槽神经的初级传入纤维向孤束核的投射状况。另外,向舌咽、迷走和鼓索神经也注入HRP进行了观察和对比。结果表明,上述三叉神经各分支中只有舌神经和下牙槽神经向孤束核投射。舌神经的标记出现于孤束核的闩以上部分,以三叉神经吻侧亚核尾段水平的部位最为浓密,标记终末多分布于腹外侧亚核、中间亚核和内侧亚核。下牙槽神经只有少量标记终末呈斑块状局限分布于闩附近的孤束核间质亚核区,小部分投射于连合核。舌咽神经投射至孤束核全长,以内侧亚核区为最多。迷走神经的大量纤维主要投射于极间亚核平面的孤束核。鼓索神经大部投射至孤束核吻极段。本文对比分析了三叉神经初级传入与Ⅶ、Ⅸ、Ⅹ神经内脏传入在孤束核分布的相互关系,结合文献讨论了投射到孤束核的三叉初级传入神经的性质。     

投射于腰骶髓后连合核的传递伤害性刺激的初级传入纤维的来源探索—c-fos表达方法

本课题组的既往研究曾发现 ,膀胱的初级传入投射纤维中有一部分传递伤害性刺激 ,投射于腰骶髓的后连合核。本研究的目的是通过比较盆腔内脏和后肢躯体性结构的伤害性刺激诱导 FOS阳性反应在大鼠后连合核的表达状况 ,藉以确定投射于后连合核的传递痛信号的初级传入的来源。本实验分别向盆腔内脏的膀胱和直肠以及坐骨神经支配的小腿外侧皮肤及腓肠肌等四个不同部位注入 2 %福尔马林溶液 ,用免疫组织化学方法观察了腰骶段脊髓内 FOS的表达状况 ;并向此四部位注射生理盐水作为对照。结果证明 ,将 2 %福尔马林溶液注入膀胱或直肠内腔后可主要诱导 L6,S1 ,S2 节段后连合核出现大量 FOS阳性细胞核( 2 5～ 95个 /片 ) ;向小腿外侧皮肤或腓肠肌注射福尔马林溶液 ,则主要在背角浅层出现浓密的 FOS阳性细胞核而在后连合核仅发现极少量的 FOS阳性细胞核 ( <5个 /片 ) ;在不给予福尔马林刺激的对照组 ,后连合核内也出现极少量散在的 FOS阳性细胞核 ( <3个 /片 )即后连合核内出现极少量的传递躯体伤害性刺激的传入成分与不给予福尔马林刺激的对照组结果无明显差别 ,这种极少量的神经元在神经机能的传递上不应有何作用。因而 ,本研究结果提示 ,盆腔内脏来源的痛信号初级传入成分是后连合核内唯一的有机能意义的痛传入成     

外周伤害性刺激后颈髓和三叉神经脊束核尾侧亚核中向中缝背核投射神经元的 c-fos 表达

中缝背核是内源性镇痛系统中的重要核团之一,为了估价该核与外周伤害性信息的传递与调控之间的关系,文内采用荧光金逆行追踪与 FOS 免疫荧光标记相结合的方法进行研究,结果显示将荧光金注入中缝背核后,在三叉神经脊束核尾侧亚核和颈髓后角浅层中可观察到许多荧光金标记神经元的胞体,其中有一部分神经元在给予外周伤害性刺激后被激活而引起 c-fos 表达,该文进一步证明了来自脊髓和三叉神经脊束核尾侧亚核到中缝背核的直接投射至少有一部分与中缝背核的痛觉谓节机制有关。     

与坐骨神经和盆神经对应的后根节中CGRP、SP、NOS样阳性神经元分布特点分析——荧光金逆标和荧光免疫组化结合的研究

为了探索大鼠坐骨神经（躯体性）和盆神经（内脏性）内与传递痛信号有关的初级传入神经元在后根节内的分布特点,本研究采用荧光金逆行追踪与免疫荧光组化技术相结合的方法,对ＣＧＲＰ能、ＳＰ 能和ＮＯＳ样神经元在相应的后根节内（坐骨神经,Ｌ４～Ｌ６ ;盆神经,Ｌ６～Ｓ１）的分布状况进行了分析。结果表明：（１）坐骨神经和盆神经初级传入神经元中有相当数量的ＣＧＲＰ和ＳＰ样阳性细胞,与这二者相比,ＮＯＳ样细胞数量稀少;（２）盆神经初级传入神经元中ＣＧＲＰ／ＦＧ、ＳＰ／ＦＧ、ＮＯＳ／ＦＧ 双标细胞的比率高于坐骨神经,而其前两种双标细胞与各该活性物质单标细胞的比率则低于坐骨神经;（３）三种物质与ＦＧ 的双标神经元以小型为主,少有中型细胞。因为既往的研究证明,分布有大量的ＣＧＲＰ、ＳＰ、ＮＯＳ样终末的骶髓后连合核（ＳＤＣＮ）接受盆腔脏器伤害性信息传入,并且ＣＧＲＰ、ＳＰ都以外周来源为主。故本文结果进一步核实了ＳＤＣＮ 区接受来自外周的ＣＧＲＰ、ＳＰ投射,且确为经盆神经传入的细纤维。     

Calcitonin gene-related peptide-immunoreactive nerve fibers in mandibular periosteum of rat: evidence for primary afferent origin

Peptidergic neurons may play a role in the local regulation of bone mineralization. The neuropeptide vasoactive intestinal peptide (VIP) increases bone resorption in vitro, while calcitonin gene-related peptide (CGRP) has been shown to inhibit bone resorption in vitro. We have previously reported that sympathetic nerves with VIP-immunoreactivity innervate bone and periosteum. In the present study we sought to determine if CGRP fibers, like VIP fibers, exist in periosteum and what their origin might be. In whole-mount preparations of mandibular periosteum from rat, CGRP- and VIP-immunoreactive (IR) nerve fibers were present as networks within the periosteum. In preparations using two-color immunofluorescence, most CGRP-IR fibers were also immunoreactive for substance P (SP). In rats in which the subperiosteal space subjacent to the mandibular molars was injected with Fast blue or Fluoro-gold, retrogradely labeled cells were seen in ipsilateral trigeminal ganglia, superior cervical ganglia, and nodose ganglia. Individual cells labeled with both CGRP immunoreactivity and retrograde tracer were seen only in the mandibular portion of the trigeminal ganglion. These data suggest that CGRP-IR nerve fibers in periosteum may be of primary afferent origin. Given the reported effects of CGRP on bone mineralization, the present results suggest that primary afferent nerves containing CGRP and SP, as well as sympathetic nerves containing VIP, may play a role in focal bone remodeling.     

Immunohistochemical study of neuropeptides in vagal and glossopharyngeal afferent neurons in the rat

The presence and distribution of multiple neuropeptides in vagal and glossopharyngeal afferent ganglia of the rat were studied using immunohistochemistry. Substance P-, calcitonin-gene related peptide-, cholecystokinin-, neurokinin A-, vasoactive intestinal polypeptide-, and somatostatin-immunoreactive neurons were detected in each visceral afferent ganglion. Neurotensin-immunoreactive cells were not observed. In the nodose ganglion (inferior ganglion of the vagus nerve) occasional immunoreactive cells were scattered throughout the main (caudal) portion of the ganglion with small clusters of cells seen in the rostral portion. The pattern of distribution of the various peptides in the nodose ganglion was similar, with the exception of vasoactive intestinal polypeptide-immunoreactive neurons which exhibited a more caudal distribution. The relative numbers of immunoreactive cells varied, with the greatest numbers being immunoreactive for substance P or vasoactive intestinal polypeptide, and the lowest numbers being immunoreactive for neurokinin A and somatostatin. A build-up of immunoreactivity for each of the peptides, except somatostatin and neurotensin, was detected in vagal nerve fibers of colchicine-injected ganglia. Numerous peptide-immunoreactive cells were also found in the petrosal (inferior ganglion of the glossopharyngeal nerve) and jugular (superior ganglion of the vagus nerve) ganglia. No specific intraganglionic distribution was noted although the relative numbers of cells which were immunoreactive for the different peptides varied considerably. Substance P and calcitonin-gene related peptide were found in large numbers of cells, cholecystokinin was seen in moderate numbers of cells, and neurokinin A, vasoactive intestinal polypeptide and somatostatin were seen in fewer cells. These data provide evidence for the presence and non-uniform distribution of multiple peptide neurotransmitters in vagal and glossopharyngeal afferent neurons. In general, relatively greater numbers of immunoreactive cells were located in the rostral compared with caudal nodose ganglion, and in the petrosal and jugular ganglia compared with the nodose ganglion. Thus, multiple neuropeptides may be involved as afferent neurotransmitters in the reflexes mediated by vagal and glossopharyngeal sensory nerves.     

Characterization of sheep (Ovis aries) palatine tonsil innervation

Palatine tonsils (PTs), together with ileal Peyer's patches, rank among the first colonization sites for infectious prions. After replicating in these lymphoid tissues, prions undertake the process of “neuroinvasion,” which is likely mediated by the peripheral nerves connecting lymphoid tissues to the central nervous system (CNS). To study the connections between the tonsils and the CNS, we injected fluorescent tracers into the PTs of lambs; the highest number of Fast Blue (FB)–labeled neurons was found in cranial cervical ganglia (CCG), whereas a progressively decreasing number of cells were detected in proximal glossopharyngeal, proximal vagal, trigeminal, pterygopalatine, and cervicothoracic ganglia. Immunohistochemistry was carried out on tonsil and ganglia cryosections. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and calcium-binding protein S100 (S100), was observed in the fibers around and within PT lymphoid nodules. In the trigeminal, proximal glossopharyngeal and vagal ganglia the retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR. In all ganglia some retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR co-localization. It is worth noting that only 66±19% and 75±13% of retrogradely-labeled neurons in CCG showed TH- and DBH-IR, respectively. The present results allow us to attribute PT innervation mainly to the sympathetic component and to the glossopharyngeal, vagal and trigeminal cranial nerves. Furthermore, these data also provide a plausible anatomic route through which infectious agents, such as prions, may access the CNS, i.e. by traveling along several cranial and sympathetic nerves, as well as by migration via glial cells.     

Correlation Between the Distribution of SP and CGRP Immunopositive Neurons in Dorsal Root Ganglia and the Afferent Sensation of Preputial Frenulum

The aim of this study was to explore the distribution of substance P (SP) and calcitonin gene‐related peptide (CGRP) immunoreactive nerve terminals in the penis prepuce and the preputial frenulum. The possible correlation between SP‐ and CGRP‐immunopositive neurons in dorsal root ganglia (DRG) and the afferent sensation of the penile preputial frenulum is also discussed. Immunohistochemistry showed SP‐ and CGRP‐positive nerve terminals in the epidermal basal layer of the prepuce and frenulum in adult human males. The majority of the nerve terminals presented as bundles of different lengths and a few as enlarged nodosities. The density of SP‐ and CGRP‐immunopositive nerve terminals in the preputial frenulum was significantly higher than those in the penis prepuce (P<0.01). Fluoro‐Gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in Sprague‐Dawley rats. SP and CGRP immunofluorescence labeling was employed to detect the distribution of SP‐ and CGRP‐immunoreactive neurons in DRG. FG retro‐labeled neurons were localized in L₆‐DRG and S₁‐DRG. All the FG/SP and FG/CGRP double‐labeled neurons were medium or small‐sized. One‐third of the FG‐labeled neurons were SP‐immunoreactive, and a half of them CGRP‐immunoreactive in L₆‐DRG and S₁‐DRG, respectively. The FG/SP/CGRP‐labeled neurons accounted for one fifth of the FG retro‐labeled neurons. Taken together, these data suggest that the SP‐ and CGRP‐immunopositive nerve fibers may participate in the transmission of afferent sensation in the preputial frenulum. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Pituitary adenylatecyclase-activating polypeptide-immunoreactive nerve fibers in the rat epiglottis and pharynx

The distribution of pituitary adenylatecyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers was studied in the rat epiglottis and pharynx. PACAP-IR nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant on the laryngeal side of the epiglottis and in the dorsal and lateral border region between naso-oral and laryngeal parts of the pharynx. PACAP-IR nerve fibers were also detected in taste buds within the epiglottis and pharynx. In addition, many PACAP-IR nerve fibers were found around acinar cells and blood vessels. The double immunofluorescence method demonstrated that distribution of PACAP-IR nerve fibers was similar to that in CGRP-IR nerve fibers in the epithelium and taste bud. However, distributions of PACAP-IR and CGRP-IR nerve fibers innervating mucous glands and blood vessels were different. The retrograde tracing method also demonstrated that PACAP and CGRP were co-expressed by vagal and glossopharyngeal sensory neurons innervating the pharynx. These findings suggest that PACAP-IR nerve fibers in the epithelium and taste bud of the epiglottis and pharynx which originate from the vagal and glossopharyngeal sensory ganglia include nociceptors and chemoreceptors. The origin of PACAP-IR nerve fibers which innervate mucous glands and blood vessels may be the autonomic ganglion.     

Occipital artery injections of 5-HT may directly activate the cell bodies of vagal and glossopharyngeal afferent cell bodies in the rat

The primary objective of this study was to determine whether circulating factors gain direct access to and affect the activity of vagal afferent cell bodies in the nodose ganglia and glossopharyngeal afferents cell bodies in the petrosal ganglia, of the rat. We found that the occipital and internal carotid arteries provided the sole blood supply to the nodose ganglia, and that i.v. injections of the tracer, Basic Blue 9, elicited strong cytoplasmic staining in vagal and glossopharyngeal afferent cell bodies that was prevented by prior ligation of the occipital but not the internal carotid arteries. We also found that occipital artery injections of 5-HT elicited pronounced dose-dependent reductions in heart rate and diastolic arterial blood pressure that were (1) virtually abolished after application of the local anesthetic, procaine, to the ipsilateral nodose and petrosal ganglia, (2) markedly attenuated after transection of the ipsilateral vagus between the nodose ganglion and brain and virtually abolished after subsequent transection of the ipsilateral glossopharyngeal nerve between the petrosal ganglion and the brain, (3) augmented after ipsilateral transection of the aortic depressor and carotid sinus nerves, and (4) augmented after transection of all ipsilateral glossopharyngeal and vagal afferent nerves except for vagal cardiopulmonary afferents. These findings suggest that blood-borne 5-HT in the occipital artery gains direct access to and activates the cell bodies of vagal cardiopulmonary afferents of the rat and glossopharyngeal afferents of undetermined modalities.