延髓后角脑啡肽超微结构定位和非突触部位释放——脑啡肽突触前抑制痛觉传递的形态学基础

阿片肽在后角镇痛的作用机理,被认为是通过突触前抑制一级传入纤维P物质释放的结果,然而始终未获得形态学的证实。鉴于一级传入纤维存在大量阿片受体的事实,曾提出阿片肽突触前抑制可能是通过非突触的轴-轴作用。为了验证这一设想,本文用免疫组化方法,详细观察了大鼠延髓后角浅层亮氨酸脑啡肽(L-ENK)轴突终末的突触结构和胞吐释放。电镜观察显示,延髓后角ENK终末可分为两类,第一类终末除了含圆形小清亮囊泡外,还有较多的大颗粒小泡(一般7个以上),主要分布于Ⅰ层,很少看到此类终末形成突触;第二类终末,一般含较多圆形清亮小泡和少量大颗粒小泡(一般不超过3个),它们分布于Ⅰ层和Ⅱ层,此类终末主要形成轴-树突触和少量的轴-体突触。只见到一例轴-轴突触,其突触后成分为未标记的R型终末,此外还见到ENK阳性树突成为中央终末的突触后成分。在去传入神经条件下,上述各类终末皆可见到ENK阳性大颗粒小泡的胞吐形成,它们皆位于非突触区,而在突触部位可见到清亮小泡胞吐像,上述结果提示后角ENK非突触部位释放可能是哭触后抑制一级传入纤维P物质释放的形态学基础。     

大鼠延髓后角神经降压肽(NT)的亚细胞定位和胞吐释放

神经降压肽(NT)广泛分布于哺乳动物的中枢神经系统,具有明显的镇痛作用,为了探索其镇痛机理的形态学基础,本文应用电镜免疫组化技术,对大鼠延髓后角NT的超微结构和胞吐释放进行了研究。超微结构显示延髓后角浅层NT轴突终末形态多样,大小不一,含有圆形或多形性清亮小泡及数量不等的大颗粒小泡,它们主要与未标记的树突形成轴-树突触,其突触后成分有的还含有少量清亮小泡。NT免疫反应阳性树突可分为两类:一类主要含微管;另一类主要含大颗粒小泡,有的尚可见少量清亮小泡。这两类NT树突可成为未标记的含圆形小泡终末、多形性小泡终末以及突触小球中央轴突终末的突触后成分,提示后角浅层NT神经元可接受不同种类轴突终末(包括一级伤害性传入纤维)的传入(?)动,然后可能再通过一个抑制性中间神经元,抑制痛觉的传递。本文还观察到有少量NT终末内的大颗粒小泡靠近突触活性区处,而更多见它们沿非突触部位轴膜分布,并与其融合,形成胞吐。本文认为NT既可在突触活性区处又可能在非突触部位释放。     

大颗粒小泡非突触部位的胞吐——论神经肽释放的另一种形式

本文在以前的工作基础上,进一步用电镜及免疫细胞化学方法,研究了大颗粒小泡非突触部位胞吐作用。实验结果表明,切除大鼠刚髭部皮肤1—24小时之后,术侧延髓后角浅层大颗粒小泡胞吐比对照侧明显增多(P<0.01),术后3—9天复又下降(近似对照动物),术后14—15天又急剧上升(P<0.01)。这些胞吐大部分出现于延髓后角浅层四种轴突终末的非突触部位,少最也发生于树突及轴突中。从术后第6天开始,术侧P物质明显减弱,而甲硫-脑腓肽略有增强。研究结果提示;1)后角浅层胞吐增多,P物质下降及脑腓肽增高,反映了中枢内不同神经元对去传入神经的功能调整作用;2)大颗粒小泡在非突触部位释放神经肽,弥散地作用于远距离的受体,可能起着神经调制物的作用。     

与眶下神经相关的初级传入纤维终末的溃变、非突触部位胞吐及突触联系

切断眶下神经的各组大鼠存活2～30天后分别杀死,于其三叉神经尾侧脊束核胶状质亚核内观察了一级传入纤维轴突终末的溃变过程.非突触部位胞吐及突触联系.结果发现:(1)眶下神经的跨节溃变、以突触小泡聚集、融合、空泡形成为主要特征,无微丝增生现象:(2)部分溃变终末内的线粒体明显肿胀变暗.呈球形改变:(3)大致密核心小泡的溃变时间远滞后于突触小泡.两者并不同步进行;(4)轴突终未在溃变过程中,其内的大致密核心小泡仍然进行非突触部位胞吐;(5)溃变纤维终末于胶状质内分别形成轴一树、轴一体、轴一轴三种类型的突触、并参与了突触复合体的形成.     

针刺镇痛中大致密核心小泡非突触部位的胞吐及胞吐过程的研究

本文首次采用形态与机能相结合的方法研究了大致密核心小泡(LDV)非突触部位的胞吐和胞吐的动态过程。电镜下观察了在针刺镇痛中三叉神经尾侧脊束核胶状质亚核内出现的各阶段的胞吐影像。据此认为,胞吐是按下列过程连续进行的:1.LDV趋近并伸出一细颈与质膜接触、融合,融合后形成只有一层单位膜的隔,进而隔部分地开放,小的通道形成。2.通道进一步扩大,此时LDV是典型的“Ω”断面像,递质开始排入细胞间隙。3.随通道的进一步扩大,LDV内腔完全开放于细胞间隙,成为间隙的一部分。4.LDV的膜并入终末质膜,仅呈弧形弯曲,其凹侧仍留有少量致密物质,随胞吐物质的弥散,弯曲部分的质膜展平,胞吐的痕迹消失。在针刺镇痛过程中,实验组动物出现的非突触部位胞吐影像比对照组明显增多,两组间的差异非常显著(P<0.01),从而有力地说明与痛觉相关的神经元主要是通过非突触部位胞吐LDV内的递质而参与镇痛过程的。本研究为进一步揭示针刺镇痛机理提供了形态学依据,同时也有力地支持了LDV非突触部位胞吐可能是神经肽释放的主要方式的学说。     

针刺过程中有衣小泡的形成、形态特征及其数量变化的研究

针刺大鼠“人中”、“四白”穴使之产生明显镇痛效果后,再将针刺时间分别延长至1、2、4、6、8、10、12h,到预定时间立即将动物灌流杀死取材,采用定量电镜方法观察计数了三叉神经尾侧脊束核胶状质亚核内各种有衣小泡的数量、形态以及在不同针刺时间内数量的变化。结果发现:大单壁有衣小泡的形成与针刺时间无明显正比例关系;而双壁有衣小泡则显然与之不同,它不仅形态多样,数目也随针刺时间而改变。本文将观察到的双壁有衣小泡归纳为以下五种类型:1、尚未与相邻两终末质膜脱离的孤立存在者。2、游离于终末内孤立存在者。3、与终末质膜相连且融合而成簇存在者;4、游离于终末内融合成簇者;5、树突棘凸入另一轴突或树突内并与之共同形成的不典型的双壁有衣小泡簇。本研究还发现,在针刺过程中双壁有衣小泡在1～8h内的形成与时间成正比,即1h时开始增多,4h显著增多,8h达高峰;以后开始下降,10h已恢复到4h的水平,到12h恢复到针刺前的状态。对照组仅见少数孤立的双壁有衣小泡,其形成基本上不随时间而变化。本文认为双壁有衣小泡的形成,是较大单壁有衣小泡的形成更为有效地继大致密核心小泡非突触部位胞吐之后膜再循环的一条新途径。     

家兔脊髓腰骶部中间外侧核区的超微结构——神经纤维网和突触

本实验用家兔7只,取腰髓2～4和骶髓2～4节中间外侧核区,做超薄切片,电镜观察。此区的神经纤维网内含树突、轴突、轴突终末、终端树突、突触和突触球。胶质细胞的突起穿行其间。树突散在,形态和大小多变。轴突则常成束分布。突触连接以轴树和轴体突轴为多见,偶见轴轴突触。多数突触单独存在,部分形成以树突或轴突为中心的突触球。突触内的突触小泡有清亮的圆形、椭圆形、扁平形和不规则形,还有相当多见的大致密核心小泡和少数有衣小泡。依终末囊内突触小泡的形态和突触前后膜的对称与不对称,所见突触可分为三类:1.圆形小泡不对称型;2.扁平小泡对称型;3.其它中间类型。     

大鼠三叉神经脊束核尾侧亚核胶状质非小球突触成分

用透射电镜观察了大鼠三叉神经脊束核尾侧亚核胶状质神经毡非小球的突触成分。非小球的突触大部分为轴树突触,此外还见到轴轴、树树及树轴突触。它们的轴突终末成分,按所含小泡的形状,区分为圆形小泡终末、扁平小泡终末、多形小泡终末及大颗粒小泡终末。圆形小泡终末根据小泡的大小又有大圆形小泡终末及小圆形小泡终末。本文还讨论了突触分类及各种轴突终末的机能意义。     

大鼠脊髓 V 层轴突终末的亚显微结构

本文用透射电镜观察大鼠颈段脊髓Ⅴ层正常神经毡的轴突终末及其突触联系。Ⅴ层的轴突终末中含清亮圆形小泡的终末(R)和含扁平小泡的终末(F),约各占40%;含大颗粒小泡的终末(LG)约占16%;突触小球中央终末(C)约占3%。以上这些终未中约有3%含神经微丝,0.9%的终末胞浆电子密度较低,小泡部分缺如或集聚。Ⅴ层神经毡与Ⅱ层比较,F 含量多,R 和 LG 含量少;Ⅴ层终末形成的突触形式以轴树突触为主,少量形成轴轴突触,与树突近端以及与神经元胞体形成的突触,明显多于Ⅱ层。     

三叉神经尾侧脊束核内突触的亚显微结构

在电镜下观察家兔和小白鼠三叉神经尾侧脊束核,于突触前膜上可见突触活性点;在突触前,后膜上有致密物质堆积,尤以突触后膜显著,偶尔在后膜下方的胞浆面见有突触下致密小体。在突触裂之间可见突触间丝。突触小泡有圆形和椭圆形、透明与颗粒小泡之分。尾侧脊束核内有大量轴突终末与树突。树突又分Ⅰ型(无突触小泡)和Ⅱ型(有少量突触小泡)。该核内的突触类型多数为轴-树突触,其次为轴-体突触和轴-轴突触,还见有树-树突触;按照突触小泡形状区分该核突触,则有 S 型、F 型、S-F 型与 F-S 型。突触丝球出现率为20%。本文讨论了突触类型、突触丝球与突触小泡的机能意义。     

Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: a possible mechanism for neuropeptide release

It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.     

肽类激素分泌机制的研究进展

肽类激素贮存在大型致密核芯分泌颗粒内,通过胞吐分泌,分泌颗粒被膜与细胞膜融合成单层,释放出全部内容物,或通过接触-分离的方式,多次部分释放。对新发现的孔体或融合孔的研究,揭示了分泌颗粒停泊、融合并释放出内容物的机制。排放到细胞外的囊泡内容物的消散速度,取决于融合孔的状态和囊泡内容物的化学性质。由于在细胞外存在完整的膜包分泌颗粒.因此,可能还存在着连同颗粒被膜的整体释放方式。     

Protein-protein interactions in neurotransmitter release

The arrival of a nerve impulse at a nerve terminal leads to the opening of voltage-gated Ca(2+) channels and a rapid influx of Ca(2+). The increase in Ca(2+) concentration at the active zone from the basal level of 100-200 mM triggers the fusion of docked synaptic vesicles, resulting in neurotransmitter release. A large number of proteins have been identified at nerve terminals and a cascade of protein-protein interactions has been suggested to be involved in the cycling of synaptic vesicle states. Functional studies in last half decade on synaptic-terminal proteins, including Ca(2+) channels, have revealed that the SNARE core complex, consisting of synaptobrevin VAMP, a synaptic vesicle-associated protein, syntaxin and SNAP-25, synaptic membrane-associated proteins, acts as the membrane fusion machinery and that proteins interacting with the SNARE complex play essential roles in synaptic vesicle exocytosis by regulating assembly and disassembly of the SNARE complex.     

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca²⁺ channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca²⁺ channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca²⁺ channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca²⁺ channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.     

Fine structure of the afferent synapse of the hair cells in the saccular macula of the goldfish, with special reference to the anastomosing tubules

Summary The fine structure of the afferent synapse has been studied in the hair cells of the goldfish saccular macula.A spherical dense body which is surrounded by synaptic vesicles is observed in association with the presynaptic membrane. An alternating, parallel arrangement of dense bars and of rows of synaptic vesicles is observed on the presynaptic membrane beneath the dense body. Each row consists of five to six immediately available synaptic vesicles, and five to six such rows of vesicles are observed per synapse.Sometimes anastomosing tubules are found around the dense body. The tubules are formed by direct infolding of the plasma membrane. Many coated vesicles are found at the periphery of the anastomosing tubules.A possible role of the anastomosing tubules in the turnover of the synaptic vesicle membrane is discussed.     

Exocytotic release of neurotransmitter substances from nerve endings in the taste buds of rat circumvallate papillae

Exocytotic release of neurotransmitters from nerve endings was demonstrated ultrastructurally in the taste buds of rat circumvallate papillae by stimulation of high K+ and Ca2+ Ringer perfusion and application of tannic acid-Ringer incubation (TARI) method. Omega-shaped images of large cored vesicles and small clear vesicles, indicating exocytotic release of their contents, were found only in the non-synaptic sites. Occasionally exocytosis occurred at sites facing other nerve fibers. Many coated pits were also seen, which presumably represent membrane retrieval at a later stage of exocytosis. It is likely that the taste buds receive more than one type of innervation.     

Tomosyn negatively regulates both synaptic transmitter and neuropeptide release at the C. elegans neuromuscular junction

The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans tomosyn gene, tom-1 . These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.     

Differential expression and subcellular localization of secretogranin II and synaptophysin during early development of mouse hypothalamic neurons in culture.

Mature neurons contain two distinct regulated secretory pathways, characterized electron microscopically by so-called large dense core vesicles and small synaptic vesicles, respectively. Each vesicle type is characterized by vesicle-specific proteins, such as the granins (chromogranins/secretogranins) for the matrix of large dense core vesicles and synaptophysin for the membrane of small synaptic vesicles. So far, no data exist on the biogenesis of these two distinct vesicle types during neuronal development. We have used secretogranin II and synaptophysin as markers for the biogenesis of these two vesicle types during the development of mouse hypothalamic neurons in culture, using immunocytochemistry and biochemical analyses. By immunofluorescence, we found that secretogranin II appears as early as synaptophysin, but in a subset of neurons only, and with different subcellular localizations. It was observed in cytoplasmic areas where little or no synaptophysin immunofluorescence was detected, such as lamellipodia, emerging neurites and growth cones. At later stages, the proportion of secretogranin II-containing varicosities remained steady whereas that of synaptophysin-containing varicosities increased dramatically. By quantitative analysis we found that the level of expression of synaptophysin increased several-fold during synaptogenesis whereas that of secretogranin II decreased. These data suggest that large dense core vesicles and small synaptic vesicles can be formed separately and expressed at different levels. They provide evidence for a differential biogenesis of these two distinct vesicle types.     

Localization by immunoelectron microscopy of spanning protein of triad junction in terminal cisternae/triad vesicles

Summary Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca²⁺ channel.Nomenclature SP spanning protein - SR sarcoplasmic reticulum - TC terminal cisternae - T-tubule transverse tubule - HMW high molecular weight - ELISA enzyme linked immunosorbent assay - PMSF phenylmethyl sulphonylfluoride - SDS sodium dodecyle sulphate