大鼠中缝大核内5-羟色胺样P物质样和亮氨酸脑啡肽样突触结构的免疫电镜观察

用免疫电镜法在大鼠中缝大核内观察到:(1) 5-羟色胺(5-HT)样阳性轴突终末与阴性胞体、阳性和阴性树突以及阴性轴突终末,分别形成轴-体突触、轴-树突触和轴-轴突触;阴性轴突终末与阳性胞体和阳性树突分别形成轴-体和轴-树突触;(2) P物质样(SP样)和亮氨酸脑啡肽(L-Enk)样阳性轴突终末与阳性和阴性的胞体和树突,以及阴性轴突终末与阳性胞体和树突分别形成轴-体突触和轴-树突触,L-Enk样阳性轴突终末之间形成轴-轴突触;(3) 上述5-HT、SP和L-Enk样结构所形成的突触中,阴性轴突终末与阳性树突所形成的轴-树突触最多见;(4) 上述阳性轴突终末内主要含透明圆形小泡。免疫反应产物为电子密度高的物质,主要沉积于膜性细胞器的表面、透明圆形小泡和部分颗粒囊泡内和小泡膜上。     

Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release

In Huntington disease (HD), polyglutamine expansion causes the disease protein huntingtin to aggregate and accumulate in the nucleus and cytoplasm. The cytoplasmic huntingtin aggregates are found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, we found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in [(3)H]glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function.     

Distribution of GABA immunoreactivity in the retino-recipient layer of the viper optic tectum. A light and electron microscope quantitative study

The distribution of GABA-immunoreactivity was investigated in the principal retino-recipient layer of the optic tectum in the snake Vipera aspis. This layer, the stratum griseum et fibrosum superficiale, contained an important proportion (approximately 50%) of small GABA-immunoreactive interneurons, characterized by a voluminous invaginated nucleus surrounded by a thin rim of cytoplasm poor in organelles and occasionally showing pleiomorphic synaptic vesicles, which could also be observed in some of the dendrites that contained synaptic vesicles. In the neuropile, the GABA-immunoreactive profiles containing synaptic vesicles could be subdivided into dendrites containing synaptic vesicles and axon terminals with pleiomorphic synaptic vesicles. The dendrites containing synaptic vesicles (23.4% of all profiles containing synaptic vesicles) were postsynaptic either to optic terminals (39.2%), GABA-immunoreactive axon terminals with pleiomorphic synaptic vesicles (48.2%) or to immunonegative (S₁) boutons with round synaptic vesicles (12.6%). These dendrites were presynaptic to GABA-immunoreactive (18%) neurons or immunonegative (82%) neurons. The axon terminals with pleiomorphic synaptic vesicles, which represented 47.4% of all profiles, were predominantly (99%) GABA-immunoreactive and four types could be distinguished according to cytological criteria. These axon terminals made synaptic contacts for the most part (78%) with immunonegative profiles, and more rarely (22%) with immunoreactive neurons. These data are compared to those previously obtained in the homologous structure of other vertebrate species, birds and mammals in particular.     

Ultrastructural study of cholecystokinin-like immunoreactive nerve terminals in the rat nucleus accumbens

The cholecystokinin (CCK)-like immunoreactive nerve terminals were studied in the caudal and medial parts of the rat nucleus accumbens (NA), using the indirect immunoperoxidase technique, at the electron microscopic level. In the labelled axon terminals the immunoprecipitate is localized inside large dense-cored vesicles which are occasionally present, and surrounds small and medium-sized, round, clear synaptic vesicles. The immunoreactive nerve terminals participate in synapses of both asymmetrical and symmetrical types containing mostly small synaptic vesicles. The asymmetrical synapses are much more numerous and mainly axo-spinous. The symmetrical synapses are less frequent and are axo-dendritic or axo-somatic.     

Ultrastructural visualization of glutamate and aspartate immunoreactivities in the rat dorsal horn, with special reference to the co-localization of glutamate, substance P and calcitonin-gene related peptide.

Antisera raised against the fixation products of L-glutamate and L-aspartate were used, singly or in combination, to study the ultrastructural localization of the amino acids in the rat dorsal horn, with post-embedding immunogold techniques. Immunostaining for each of the amino acids was also combined with immunolocalization of GABA, an important inhibitory neurotransmitter in the spinal cord, or synaptophysin, a synaptic vesicle glycoprotein. In addition, we examined the localization of glutamate immunoreactivity in relation to that of calcitonin-gene related peptide and substance P, two neuropeptides present in high concentrations in the dorsal horn. Glutamate- and aspartate-immunoreactive neuronal cell bodies, dendrites, axons and terminals were apparent in the first three laminae of the dorsal horn. In somatic and dendritic profiles, the immunolabel was present over the general cytoplasm and mitochondria; in the terminals, it was found over small, agranular vesicles, mitochondria and, at times, synaptic densities. Quantitative estimation indicated that the colloidal gold density in the glutamate-immunoreactive terminals was five-fold more than in any other neuronal profile. Both glutamate- and aspartate-immunopositive terminals made asymmetric synaptic contacts onto unlabelled dendrites; glutamate-positive terminals often formed the core of type I and II glomeruli. After double labelling of the same sections, glutamate and aspartate immunoreactivities consistently occurred in different axonal and terminal profiles. In these preparations, it was clearly seen that glutamate-immunoreactive terminals were far more numerous than (more than 10-fold) those immunoreactive for aspartate. Double labelling for glutamate or aspartate and GABA also revealed distinct staining of different terminals. Simultaneous immunolocalization of each of the amino acids and synaptophysin showed the amino acid and glycoprotein immunoreactivities co-localized in small, agranular vesicles in immunoreactive terminals. Finally, triple labelling of the same sections for glutamate, calcitonin gene-related peptide and substance P revealed that glutamate was often co-localized with either of the two neuropeptides in the same axonal boutons; terminals that showed simultaneous labelling for glutamate, calcitonin gene-related peptide and substance P were also noted. In all cases, the glutamate immunoreactivity was restricted to small, clear vesicles whereas the neuropeptide immunoreactivities were present in larger, dense-cored vesicles. Our observations demonstrate that there is an abundant glutamate immunoreactivity in the superficial layers of the rat dorsal horn, localized in neuronal profiles distinct from those containing aspartate or GABA.(ABSTRACT TRUNCATED AT 400 WORDS)     

降钙素基因相关肽、甘丙肽免疫反应在大鼠臂旁外侧核分布的超微结构特征分析

应用免疫电镜包埋前染色技术对降钙素基因相关肽、甘丙肽免疫反应在大鼠臂旁外侧核分布的超微结构特征进行了研究。在臂旁外侧核腹外侧区中,常见降钙素基因相关肽阳性轴突终末与阴性树突及树突棘形成轴-树或轴-棘突触,大多为非对称型,少数为对称型;也可见阳性树突或阳性胞体与阴性轴突终末形成的轴-树或轴-体突触。阳性轴突终末含清亮小泡和致密核心小泡。臂旁外侧孩腹外侧部中甘丙肽阳性结构大多为阳性轴突终末,部分为阳性树突及阳性胞体。阳性树突常呈丛状分布于血管附近,阳性轴突常与阴性树突,阳性树突常与阴性轴突形成轴-树突触,大多为非对称型。臂旁外侧核腹外侧亚核的甘丙肽阳性结构分布基本与前者相似,但轴-树突触大多为对称型,而且一些阳性轴突与血管基膜紧贴。     

Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures.

The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P-immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P-containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase-immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase-immunoreactive dendrites. These findings reveal that two types of transmitter-specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase.     

猫丘脑腹后内侧核内三叉丘系纤维终末的超微结构及其突触联系

本文采用顺行溃变法对猫丘脑腹后内侧核内发自三叉神经尾侧脊束核的三叉丘系纤维终末的超微结构及其突触联系进行了研究。在电灼损毁三叉神经尾侧脊束核四天后 ,在电镜下发现丘脑腹后内侧核内的三叉丘系终末存在三种溃变形式 ,即 :电子致密型溃变 ,电子透明型溃变及神经微丝型溃变 ,以电子致密型溃变终末最为常见。溃变的三叉丘系终末常较大 ,含有大量密集的圆形无颗粒小泡 ,在丘脑腹后内侧核内主要形成不对称型轴—树突触 ,多数溃变轴终末还参与形成以树突为中心的汇聚型突触复合体     

Ultrastructure of somatostatin-immunoreactive nerve terminals in laminae I and II of the rat trigeminal subnucleus caudalis.

The morphology and distribution of somatostatin-immunoreactive synaptic boutons was studied in the rat trigeminal subnucleus caudalis using pre-embedding electron microscopic techniques. Immunoreactive terminals were found in lamina I and throughout lamina II but were more concentrated in outer lamina II. All immunoreactive terminals contained many round or pleomorphic agranular small synaptic vesicles and some large dense-cored vesicles. Lamina I terminals were all simple dome-shaped and relatively small. They established one asymmetric or slightly asymmetric synapse over a dendritic spine or a small, medium or large dendritic shaft. The large dendrites are probably derived from Waldeyer neurons. Many lamina II immunoreactive terminals were also simple dome-shaped terminals and established asymmetric synaptic contacts with one postsynaptic structure, usually a dendritic spine or a small to medium-sized dendritic shaft. However, other lamina II immunoreactive terminals were larger and displayed more complex morphology and synaptology. Complex immunoreactive terminals had scalloped or smooth contours and made synaptic contacts with more than one postsynaptic profile. In outer lamina II they sometimes constituted the central terminals of typical glomerular synaptic complexes. We conclude that many of the immunoreactive simple terminals probably originate from intrinsic somatostatin-immunoreactive interneurons while some of the more complex ones and the central glomerular terminals are likely to originate from primary afferents. These results are consistent with our accompanying light microscopic study (Alvarez and Priestley, Neuroscience 38, 343-357, 1990) which indicates that somatostatin-immunoreactive primary afferents project preferentially to outer lamina II while the lamina I somatostatin-immunoreactive plexus is likely to originate largely from laminae I and II interneurons. In addition somatostatin-immunoreactive cell bodies were found in lamina II. The heaviest immunoreactivity in these cells was in the Golgi apparatus. Also some vesicles containing dendrites were immunostained, and these were most abundant in inner lamina II. Thus, in trigeminal subnucleus caudalis, somatostatin may be derived from primary afferent synaptic boutons, interneuron synaptic boutons and interneuron dendrites. However, each of these sites probably makes a proportionately different contribution to the total amount of somatostatin released in each lamina or sublamina.     

Innervation of thyrotropin-releasing hormone-containing neurons in the bullfrog pars intermedia

We studied immunocytochemical localization of thyrotropin-releasing (TRH) in the bullfrog pats intermedia. Many TRH-like immunoreactive terminals containing immunoreactive granular vesicles 80-150 nm in diameter (116 nm mean diameter) were found in the pars intermedia. Some TRH-like immunoreactive terminals were in synaptic contact with the intermedia cells. These findings support the hypothesis that TRH directly activates the intermedia cells via synapses to secrete alpha-melanocyte-stimulating hormone.     

Immunoelectron microscopic observations of hypothalamic TRH-containing neurons in rats

Summary Immunoreactive TRH-containing neurons and their synaptic associations were studied electron microscopically in the paraventricular nucleus (PVN) and dorsomedial nucleus (DMH) of the rat hypothalamus. In propylthiouracil (PTU)-treated rats, the immunoreactive cell bodies in the PVN appeared to be activated, showing a hypertrophic perikaryon, well developed Golgi bodies and numerous secretory granules. No such alterations were evident in the TRH neurons in the DMH. These findings suggest that the PVN-TRH neurons are involved in the hypothalamic-hypophysial-thyroid axis. Further, it was shown that unlabeled nerve terminals containing small and large clear vesicles make synaptic contacts with the TRH perikarya in the PVN. Thus it is likely that PVN-TRH neurons are regulated both by thyroid hormones and by other neuronal signals. In the DMH, unlabeled nerve terminals containing small and large clear vesicles, and immunoreactive terminals form synapses with TRH neurons. Thus the DMH-TRH neurons may be under dual neuronal control. It was further noted that in the DMH and PVN, TRH nerve terminals make synaptic contacts with other unlabeled neurons. It is evident that TRH acts as a neurotransmitter or neuromodulator, although the origin of TRH terminals should be elucidated.     

Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal polypeptide have synaptic contacts with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat

An electron microscopic study showed by using a dual immunolabeling technique that in the suprachiasmatic nucleus of the rat, axon terminals immunoreactive for neuropeptide Y (NPY) made synaptic contacts upon neurons immunoreactive for vasoactive intestinal polypeptide (VIP). Diaminobenzidine (DAB)-labeled NPY axon terminals made synaptic contacts on silver-gold-labeled VIP perikarya and dendritic processes. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles labeled with DAB chromogen. At the synaptic portion, a symmetrical thickening of the pre- and post-synaptic membranes was evident.     

Leucine-enkephalin-like immunoreactive afferent fibers to pudendal motoneurons in the cat

Leucine-enkephalin-like immunoreactive (ENK-LI) afferent fibers to the feline homologue of the Onuf's nucleus (pudendal motoneurons) originate mainly from lamina X of the sacral cord. They make synaptic contacts chiefly upon dendrites of pudendal motoneurons; the synaptic terminals most often contain pleomorphic synaptic vesicles and occasionally round synaptic vesicles. Large-cored vesicles in the axon terminals within the Onuf's nucleus often showed ENK-LI.     

猫丘脑腹后内侧核的超微结构及突触联系

在电镜下对猫丘脑腹后内侧核内的超微结构及突触联系进行观察。该核内的轴突终末主要有３种类型：（１）含有圆形小泡的小轴突终末；（２）含有圆形小泡的大轴突终末；（３）含有扁平小泡的突终末。该核内的树突主要为不含突触小泡的Ⅰ型树突，此外还可见到少量含有突触小泡的Ⅱ型树突。     

Ultrastructure of the anterior ventral and anterior medial nuclei of the cat thalamus

Summary Electron microscopical studies of the thalamic AV-AM nuclei substantiated the presence of two main types of neurons, i.e. principal (or relay) cells and Golgi type II interneurons. Characteristic synaptic islands are found in abundance in the AV-AM. Four different types of synaptic terminals have been identified in these islands: RL-boutons = large axonal terminals with round synaptic vesicles; RS-boutons = small axonal terminals with round synaptic vesicles; F₁-boutons = small axonal profiles containing flattened synaptic vesicles, and F₂-profiles interpreted as presynaptic dendrite appendages, bearing pleomorphic vesicles, both belonging to the Golgi type II interneurons. — The synaptic relations were studied in normal preparations and after lesions in the mamillary body, limbic cortex and hippocampus. The specific afferents (RL-boutons) — originating from the medial mamillary nucleus — are presynaptic to both relay cell dendrites and presynaptic dendrite profiles of Golgi type II interneurons, which in turn are presynaptic to the same relay dendrites (synaptic triads). RS-boutons originate mainly from limbic cortex and hippocampus.     

大鼠前庭脊髓束终末与脊髓小脑束起源细胞间的突触联系

目的　探讨前庭脊髓束终末与脊髓小脑束起源细胞间的突触联系。方法　采用HRP逆行追踪和顺行溃变结合电镜技术 ,对大鼠的前庭脊髓束终末与脊髓小脑束起源细胞之间的突触联系进行了研究。结果　发现溃变的前庭脊髓束轴突终末与HRP标记的脊髓小脑束神经元胞体之间形成的突触数量较少 ,其中轴 -树型突触数量多于轴 -体型突触。突触前终末含有丰富的清亮型扁平突触小泡 ,其次为清亮型圆形小泡。前庭脊髓束轴突溃变终末与脊髓边缘细胞形成的突触数量多于Clark s柱细胞形成的突触 ,并形成一些以标记树突为中心的突触复合体。结论　本研究从形态上证实前庭脊髓束终末直接与脊髓小脑束起源细胞发生突触联系 ,也表明前庭脊髓束对脊髓小脑束起源细胞向小脑的信息传递具有直接调控使用     

GABA-containing neuronal processes in normal and cortically deafferented dorsal lateral geniculate nucleus of the cat: an immunogold and quantitative EM study

Summary The qualitative and quantitative synaptology of flat synaptic vesicle containing F type terminals was analysed in normal and in chronically cortically deafferented dorsal Lateral Geniculate Nucleus (dLGN) of the cat using an electron microscopic immunogold technique for visualization of GABA. A direct correlation was found between the density (number of gold particles) of GABA immunostaining and the number of synaptic vesicles in different F terminals. This suggested a quantitative relationship between transmitter content and the number of vesicles in the labeled terminals. Not only the number but also the size of synaptic vesicles was found to be different in the two main groups of F profiles, i.e. the axonal F1 and dendritic F2 terminals. Measurement of the size of synaptic vesicles in F1 axon terminals revealed two subpopulations of these endings: F1₁ and F1₂ terminals with vesicle diameters of 31.56 ± 0.08 nm and 33.73 ± 0.12 nm (P < 0.01; Chi² test) respectively. The size of synaptic vesicles in axonal F1₂ terminals was identical to that found in F2 dendritic profiles, suggesting that both processes belonged to the same, intrageniculate (interneuronal) cell population. F1₁ terminals, however, appeared to be axonal endings of extrageniculate (most probably of perigeniculate) neurons. Quantitative analysis of the two types of GABAergic axon terminals revealed the geniculocortical relay cells to be the main postsynaptic targets for F1₁ (extrageniculate) terminals while F1₂ axons terminated equally on both interneurons and relay cells. Following chronic decortication of the dLGN, the distribution pattern of both GABAergic axonal types had changed considerably. As a result of a severe loss in relay cells, more F1₁ and F1₂ axon terminals were found on GABA-containing interneuronal processes than on relay cells. An increase in the number of F1 axonal terminals per neuron was also revealed, (particularly on GABAergic interneurons), suggesting a compensatory reactive synaptogenesis by both F axonal types following decortication.     

猫丘脑中央外侧核内脊丘系终末与丘脑-皮质投射神经元之间的突触联系

本实验应用顺行溃变和HRP逆行追踪相结合的方法，首次在电镜水平对猫丘脑中央外侧核内脊丘系终末与丘脑-皮质投射神经元之间的突触联系进行了研究．在脊髓第4颈段刀切损毁一侧侧索和前索后，将HRP注射于同侧大脑前上薛氏回和中上薛氏回前端。在电镜下于损毁同侧中央外侧核内可见下列突触连结：（1）溃变的脊丘系轴突终末与标记树突形成的轴-树突触；（2）溃变的脊丘系轴突终末与非标记树突形成的轴-树突触，个别非标记树突含有突触小泡；（3）正常的轴突终末与HRP标记树突和胞体形成的轴-树突触和轮一体突触；（4）正常的两个轴突终末与HRP标记树突形成的轴-轴-树连续性突触；（5）非标记的含突触小泡的突触前树突与HRP标记树突形成的树-树突触。同时可见大量汇聚型突触复合体。本文首次报道在丘脑中央外侧核内，脊丘系终末与丘脑-皮质投射神经元之间存在着直接的突触联系。     

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

Summary Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.