视网膜节细胞投射到视交叉上核AVP神经元的研究——假狂犬病毒跨神经元追踪结合胶体金免疫电镜双重标记法

用９ 只成年ＳＤ 大鼠向单侧眼球玻璃体内注入假狂犬病毒３～４ μｌ,存活７２ ｈ 或９６ ｈ 后灌注、取材、Ｅｐｏｎ ８１２ 包埋,行免疫电镜胶体金标记。结果：（１）两个时间组视交叉上核内精氨酸血管加压素神经元被胶体金标记;（２）假狂犬病毒感染的视交叉上核神经元以含病毒颗粒为特征;（３）视交叉上核内少量神经元既含病毒颗粒又被精氨酸血管加压素胶体金标记。本研究结果首次证实视网膜节细胞与视交叉上核内的少数精氨酸血管加压素神经元之间有直接的突触联系,提示此类精氨酸血管加压素神经元可能直接接受来自视网膜的光信息。     

视网膜节细胞与视交叉上核AVP神经元联系的研究Ⅱ.假狂犬病毒顺行追踪与免疫荧光双重标记

为进一步揭示视网膜节细胞与视交叉上校精氨酸血管加压素神经元间有无直接的联系，本文将假狂犬病毒注入大鼠眼球内通过顺行追踪结合免疫荧光双重标记法观察到：（1）视交叉上核神经元被病毒感染的时间始于病毒注入后56h，并随存活时间的延长而增多；（2）呈绿色荧光的病毒感染神经元见于双侧视交叉上核，注射对侧优于同侧，主要位于视交叉上核的腹外侧部和嘴侧份，个别散在于二者之外;（3）视交叉上核内个别病毒感染的神经元可同时呈精氨酸血管加压素的红色荧光，此类双标神经元系病毒由视网膜节细胞顺行运输并跨突触传给视交叉上核的精氨酸血管加压素神经元所致。表明视网膜节细胞与视交叉上核的精氨酸血管加压素神经元间有直接的突触性联系。这一结果为视交叉上核节律机制和精氨酸血管加压素神经元的机能调控提供了进一步研究的线索。     

视网膜节细胞投射到视交叉上核AVP神经元的研究—假狂犬病毒跨 …

用９只成年ＳＤ大鼠向单侧眼球玻璃体内注入假狂犬病毒３－４μｌ,存活７２ｈ或９６ｈ后灌注,取材,Ｅｐｏｎ８１２包埋,行免疫电镜胶体金标记。结果：（１）两个时间组视交叉上核内精氨酸血管加压素神经元被胶体金标记;（２）假狂犬病毒感染的视交叉上核神经元以含病毒颗粒为特征;（３）视交叉上核内少量神经元既含病毒颗粒又被精氨酸血管加压素胶体金标记。     

大鼠前连合核神经元对渗透压和血管紧张素Ⅱ刺激的神经性反应——脑片电生理研究

在大鼠下丘脑离体脑片上,用电生理细胞外记录法研究了前连合核神经元的放电频率及其波形。实验观察到的前连合核自发放电神经元有不规则或规则的连续性放电活动,少数神经元有短阵性放电或不放电。这些神经元中的绝大部分可因脑片浸浴液中给予血管紧张素Ⅱ而被兴奋(放电频率增加),约1/3—1/2神经元对高渗透压刺激呈抑制性反应(放电频率减少),也有少数神经元可因高渗透压刺激而兴奋或无反应。前连合核的这些电生理特性大致与视上核、室旁核相似,也有一些与视上核、室旁核不同之处。对记录后的脑片进行组织化学及免疫组织化学染色的结果表明,记录部位在前连合核内,该部位有许多催产素样免疫阳性社经元。上述研究结果提示:前连合核的大细胞神经分泌神经元在水平衡的调节中起重要作用。     

大鼠下丘脑催产素及加压素样神经元与脑啡肽样传入纤维末梢的关系

用免疫组织化学ABC双标记法,显示大鼠下丘脑各大细胞神经分泌核团中催产素样免疫反应(OT-li)、加压素样免疫反应(VP-li)神经元、亮-脑啡肽样免疫反应(L-ENK-li)和甲-脑啡肽样免疫反应(M-ENK-li)纤维末梢,并对不同神经分泌核团内脑啡肽样传入纤维末梢与OT-li及VP-li神经元的关系加以分析。结果发现,L-ENK-li及M-ENK-li纤维末梢在下丘脑各神经分泌核团中,与OT-li及VP-li神经元均有一定程度的接触关系。L-ENK-li与M-ENK-li纤维末梢在第三脑室周、前连合核、背内侧和背外侧副核、穹窿前和后核的OT-li及VP-li神经元周围最密集,在视上核、室旁核次之,在血管周细胞群内的OT-li及VP-li神经元周围密度较低。这些神经分泌性核团内L-ENK-li纤维末梢,均较M-ENK-li纤维末梢密集。表明脑啡肽能传入纤维末梢,与视上核、室旁核及各神经分泌副核(除血管周细胞群外)中的OT-li及VP-li神经元,均有不同程度的接触关系。因此,脑啡肽能传入纤维末梢可能在大细胞神经分泌系统OT-li及VP-li神经元分泌活动的调节中,起较广泛的重要作用。     

大鼠视交叉上核至视上核的纤维联系—免疫组化双重标记法研究

为了观察视交叉上核（SCN）的传出纤维，本实验采用兔抗VIP及AVP血清作为一抗对大鼠下丘脑SCN与视上核（SON）进行免疫组化双重染色研究，结果观察到在SCN尾段，位于腹侧份的VIP样神经纤维走向SON，而在SON的腹侧份的VIP样神经纤维走向SON，而在SON的腹侧份及背侧份可见VIP样纤维分布，并明显可见该纤维包绕AVP样神经元周围,由此本推测在SCN至SON之间可能存在直接的纤维联系，它可能是体内血浆和神经垂体的加压素（VP），催化素（OT）水平呈现24小时节律的又一原因。     

加压素、催产素神经元在大鼠丘脑下部的分布——免疫组化PAP技术

本文应用免疫组化 PAP 技术观察了正常与经秋水仙素处理的大鼠丘脑下部及其附近区域加压素、催产素神经元的分布及形态特征。加压素、催产素神经元分布在丘脑下部的前连合核,室旁核,视上核,视前区、室周区,副视上核,环状核,穹窿周围区域,和丘脑下部外侧区。另外在丘脑下部以外的区域如终纹床核,丘脑靠近内囊膝处,苍白球腹内侧区,室间孔前壁腹侧份,也有少数这两种神经元的分布。经秋水仙素处理的大鼠,在前连合核和视前区出现较多淡染的加压素神经元。分布在室旁核、室周区的加压素、催产素神经元的突起多伸向第三脑室室管膜下神经毡,有的伸入室管膜上皮内,甚至伸达第三脑室,少数催产素、加压素神经元似居于室管膜上皮细胞之间。     

用免疫组织化学方法显示电生理记录部位神经元的化学性质——离体脑片法研究

500μm厚的冠状脑片取自新鲜大鼠下丘脑前部。在人工孵育条件下进行电生理观察,看到前连合核和第三脑室前部室周区部分神经元的电活动可被人工脑脊液中加入血管紧张素Ⅱ所兴奋,被提高人工脑脊液中的Ca~(++)浓度所抑制。这些电生理反应与大细胞内分泌神经元的电生理特性一致。记录结束时,记录部位用记录电极中的染料标记,脑片浸泡固定4—8h,恒冷箱切片,用抗催产素和抗加压素抗体孵育,ABC反应。结果在记录电极尖端所在部位看到数量不等的催产素或加压素免疫阳性神经元。上述电生理和免疫组化结果表明,研记录到的部分神经元可能含催产素或加压素免疫反应物质。本文报告了用免疫组织化学方法显示离体电生理标本,特别是记录部位神经元的免疫化学特性的实验结果。     

视网膜节细胞与视交叉上核AVP神经元联系的研究：Ⅱ.假狂犬病？…

为进一步揭示视网膜节细胞与视交叉上核精氨酸血管加压素神经元间有无直接的联系，本文将假狂犬病毒注入大鼠眼球内通过顺行追踪结合免疫荧光参重标记法观察到：（１）视交叉上核神经元被病毒感染的时间始于病毒注入后５６ｈ，并随存活时间的延长而增多；（２）呈绿色荧光的病毒感染神经元见于双侧视交叉上核，注射对双侧优于同侧，主要位于视交叉上核的腹外侧部和嘴侧份，个别散在于二者之外；（３）视交叉上核内个别病毒感染的神经     

针刺“足三里”穴下丘脑血管加压素(Vp)样细胞的形态学变化——免疫组织化学法

采用免疫组化PAP技术,在电针大鼠“足三里”穴位后,观察丘脑下部Vp样神经元的变化。发现视上核与室旁核Vp样大神经元数目增多、胞体胀大,并伸出细长突起。室旁核少数串珠状神经纤维可插入第三脑室壁的细胞之间。在视交叉上核腹内侧Vp小神经元密集,而背外侧反应阳性神经元较少。视上核与室旁核间的神经元岛的细胞体积增大、轴索变长伸向视上区。该区的串珠状纤维集合成束达正中隆起的外层。以上实验结果提示,电针“足三里”可促进下丘脑有关核团Vp样物质的合成与分泌。     

大鼠视交叉上核内VIP、AVP及SOM样神经元的相互关系──免疫组化双重反应法研究

视交叉上核具有生物钟功能已为很多实验研究所证实，但其功能机制尚在探索之中。本实验采用双重免疫组织化学反应技术对大鼠视交叉上核内ＶＩＰ、ＡＶＰ及ＳＯＭ样三大神经元群之间的相互联系进行了观察．结果表明：（１）ＶＩＰ样扣结广泛分布于ＡＶＰ样神经元周围，数量最多、密度最大；而ＳＯＭ样扣结贴附于ＶＩＰ及ＡＶＰ样神经元的数量次之；（２）ＡＶＰ样扣结与ＶＩＰ样神经元之间，ＶＩＰ样扣结与ＳＯＭ样神经元之间也形成联系．上述发现为视交叉上核功能机制的研究提供了进一步的形态学依据．     

Vasoactive intestinal polypeptide and the nervous system: Immunohistochemical evidence for localization in central and peripheral neurons,particularly intracortical neurons of the cerebral cortex

Immunohistochemical studies on rat brain revealed large numbers of vasoactive intestinal polypeptide (VIP)-positive nerve cell bodies all over the limbic cortex and neocortex. Large numbers of nerve terminal plexus' were found within all layers of the limbic cortex, but mainly within layers II–IV of the neocortex. Thus, association neurons probably exist in the cerebral cortex which store VIP or VIP-like peptides, making possible a role for such peptides in higher nervous processes. A neuroendocrine function may also be postulated in view of the presence of VIP-positive terminals in the central amygdaloid nucleus, suprachiasmatic nucleus, medial preoptic nucleus and the anterior hypothalamic nucleus.     

Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macaca fuscata)

Summary The hypothalamic oxytocin and vasopressin neurons of the monkey, Macaca fuscata, were demonstrated in Golgi-like images by a modified immunoperoxidase method. The magnocellular oxytocin and vasopressin neurons were distributed mainly in the supraoptic and paraventricular nuclei. In addition to these main nucleic, both types of magnocellular neurons were found in the accessory supraoptic nucleus, the periventricular and perifornical areas, the nucleus of the stria terminalis, the lateral hypothalamic area, and the pars interna of the globus pallidus. Magnocellular oxytocin neurons were seen immediately ventral to the anterior commissure, and parvocellular vasopressin neurons were localized in the medial portion of the suprachiasmatic nucleus. The preferential distribution of the oxytocin and vasopressin neurons was recognized not only in the supraoptic and paraventricular nuclei, but also in other areas. In all areas observed, the cytological difference between the oxytocin and vasopressin neurons could be identified. The area, of the perikarya of the vasopressin neurons was determined to be larger than that of the oxytocin neurons. Most of the axons of the oxytocin neurons issued from the perikarya, while the axons of the vasopressin neurons originated in most cases from the thick proximal dendrites. These results show that the oxytocin and vasopressin neurons are distributed in areas much broader than has hitherto been assumed, and that these two peptidergic neurons can be definitely differentiated morphologically as well as functionally.Supported by grants (No. 56440022, 56770037) from the Ministry of Education, Science, and Culture, Japan     

血管活性肠肽、加压素和生长抑素免疫阳性细胞和纤维在大鼠交叉上核的分布

用免疫组化技术观察了正常和秋水仙素预处理后的血管活性肠肽、加压素和生长抑素免疫反应阳性胞体和纤维在ＳＤ大鼠交叉上核的分布。（１）血管活性肠肽：在交叉上核嘴侧部的腹外侧部有少量阳性胞体和纤维分布；在交叉上核中部，大量阳性胞体集中在腹外侧亚核，相当多的阳性纤维分布于整个核区；在交叉上核尾侧部可见少量阳性胞体和中等密度的阳性纤维。（２）加压素：在交叉上核嘴侧部和尾侧部可见中等密度的阳性胞体和纤维，以核的背内侧为多，大量阳性胞体和纤维见于交叉上核中部的背内侧亚核。（３）生长抑素：阳世胞体较少，散布于交叉上核的背内侧亚核，以核的中部为多，中等密度的阳性纤维遍及交叉上核全长。（４）双标显示血管活性肠肽、加压素和生长抑素存在于交叉上核的不同神经元之中，没有共存。     

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.     

Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus

The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not know to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111-122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G. P. McGregor et al. (1984) Neuroscience 13, 207-216; S. A. S. Shehab and M. E. Atkinson (1984) J. Anat. 139, 725; S. A. S. Shehab and M. E. Atkinson (1986) Expl Brain Res. 62, 422-430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. (Neuroendocrinology 44, 338-346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurotensin expressing neurons developed earlier than vasoactive intestinal polypeptide and vasopressin expressing neurons in the human suprachiasmatic nucleus

Development of neurotensin (NT), vasoactive intestinal polypeptide (VIP), vasopressin (AVP) and neuropeptide-Y (NPY) expressing neurons was investigated in the human fetal suprachiasmatic nucleus of nine subjects ranging from 20-40 weeks of gestation using immunocytochemistry and morphometry. Results obtained showed that NT expressing neurons developed earlier than VIP, AVP and NPY expressing neurons. Consistent with results obtained from animal studies, we also found VIP expressing neurons were born earlier than AVP expressing neurons. Whether the NT expressing neurons play a role in generating circadian rhythms in the early life of humans needs to be further investigated.