Ultrastructural subtypes of glutamate-immunoreactive terminals on rat trigeminal motoneurones and their relationships with GABA-immunoreactive terminals

 Electron-microscopic immunolabelling methods were used to study the relationships between glutamate-immunoreactive and γ-aminobutyric acid (GABA)-immunoreactive synapses on trigeminal motoneurones labelled by the retrograde transport of horseradish peroxidase. Serial sections were cut through the motor nucleus, alternate sections were incubated with antibodies to glutamate and GABA, and the immunopositive nerve terminal profiles were recognized using a quantitative, postembedding immunogold method. Boutons exhibiting high levels of glutamate immunoreactivity and GABA-immunoreactive boutons both formed axo-dendritic and axo-somatic synaptic contacts on labelled motoneurones. Boutons strongly immunopositive for glutamate were not immunopositive for GABA, and vice versa. Strongly glutamate immunoreactive boutons received axo-axonic synaptic contacts but did not form such contacts, while GABA-immunoreactive boutons formed axo-axonic synapses but did not receive them. The presynaptic elements at all axo-axonic synapses on to glutamate-immunoreactive boutons sampled were GABA-immunopositive. These data provide ultrastructural evidence in support of the roles of glutamate and GABA as transmitters at synapses on trigeminal motoneurones, and for presynaptic control of transmission at glutamatergic synapses by GABA acting at receptors at axo-axonic synapses. The vast majority (more than 90%) of strongly glutamate immunoreactive boutons contained spherical synaptic vesicles, in contrast to GABA-immunoreactive boutons, which contained pleomorphic vesicles. Most of the glutamate-immunoreactive boutons (67%) formed asymmetrical synaptic active zones, many of which (47% of total) were associated with subsynaptic dense ”Taxi” bodies (T-terminals), while a smaller population of boutons (21%) formed symmetrical synapses, and a few (11%) made synapses associated with subsynaptic cisternae (C-terminals). The heterogeneity of active zone ultrastructure of boutons identified as being glutamatergic on the basis of their high levels of immunolabelling is discussed in relation to possible differences in co-transmitters released, origins of the synaptic input or post-synaptic receptor subtypes activated. Received: 13 May 1996 / Accepted: 9 September 1996     

大鼠中脑导水管周围灰质腹外侧区5-羟色胺样、P物质样和亮氨酸-脑啡肽样免疫反应阳性亚微结构的电镜观察

本文用免疫电镜技术研究了大鼠中脑导水管周围灰质腹外侧区内5-HT样、SP样和L-ENK样的免疫反应阳性亚微结构。5-HT样免疫反应阳性的胞体较多,常见5-HT样阳性树突与阴性轴突终末形成多为非对称性的轴-树突触;偶见阳性轴突终末与阴性树突以及阴性轴突终末与阳性胞体分别构成轴-树和轴-体突触.SP样阳性胞体数目较少,可见少量含多形性小泡的阴性轴突终末与之形成轴-体突触;由阴性轴突终末与阳性树突所形成的轴-树突触最常见;阳性轴突终末与阴性胞体和阳性树突分别构成轴-体突触和轴-树突触。L-ENK样阳性胞体数目也较少,L-ENK样阳性树突与阴性轴突终末所形成的轴-树突触最多见,可见L-ENK样阳性胞体与阴性轴突终末构成轴-体突触;偶见阳性轴突终末与阴性树突形成轴-树突触。上述各种突触均主要含圆形小泡,有时有少量扁平小泡、椭圆形小泡和颗粒囊泡。     

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。     

Primary afferent terminals in the nucleus of the solitary tract of the frog: An electron microscopic study

In the frog solitarius nucleus, primary afferent terminals of the facial and glossopharyngeal-vagal nerves were identified with cobalt labelling and electron microscopy. The labelled terminals were grouped in two main categories, one with small (1-2 micron) and pale terminals, and another with large (3-5 micron) and dark terminals. The small terminals greatly outnumbered the large ones. In addition many terminals intermediate in size and staining reactions were found. All kinds of labelled boutons contained medium-size clear synaptic vesicles, among which dense-core vesicles of the smaller type frequently occurred. The labelled primary afferent terminals established axo-dendritic contacts of the asymmetric type. Close to these contact sites they were themselves very frequently contacted by a profile interpreted as presynaptic in relation to them. Such profiles contained spherical, pleomorphic (including dense-core) or flattened vesicles; a fourth kind was interpreted as presynaptic dendrites. It is concluded that viscerosensory fibres, as opposed to somatosensory fibres, predominantly generate small and lightly stained terminals. It is likely that the effect of synaptic transmission at the solitarius tract terminals is modulated in a very versatile manner by the various presynaptic profiles converging on these terminals.     

A GABA immunocytochemical study of rat motor thalamus: light and electron microscopic observations.

A light and electron microscopic study of GABA-immunoreactive neurons and profiles in the ventroanterior-ventrolateral and ventromedial nuclei of rat dorsal thalamus was conducted using antiserum raised against GABA. Less than 1% of the neurons in these motor-related nuclei exhibited GABA immunoreactivity, confirming previous reports that these nuclei are largely devoid of interneurons. Immunoreactive neurons in the ventral anterior-ventral lateral complex and ventromedial nucleus were bipolar or multipolar in shape, and tended to be smaller than non-immunoreactive neurons. GABA immunoreactivity in the neuropil consisted of labeled axon terminals and myelinated and unmyelinated axons, and was lower in the ventral anterior-ventral lateral complex and ventromedial nucleus than in neighboring thalamic nuclei. The density of neuropil immunolabeling was slightly higher in ventral anterior-ventral lateral complex than in ventromedial nucleus. GABA-immunoreactive axon terminals, collectively termed MP boutons for their medium size and pleomorphic vesicles (and corresponding to "F" profiles of some previous studies of thalamic ultrastructure), formed symmetric synapses and puncta adhaerentia contacts predominantly with large and medium-diameter (i.e. proximal) non-immunoreactive dendrites. Approximately 12 and 18% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, were GABA-immunopositive. Many of these immunoreactive profiles probably arose from GABAergic neurons in the thalamic reticular nucleus, substantia nigra pars reticulata and entopeduncular nucleus. Two types of non-immunoreactive axon terminals were distinguished based on differences in morphology and synaptic termination sites. Boutons with small ovoid profiles and round vesicles that formed prominent asymmetric synapses onto small-diameter dendrites were observed. Mitochondria were rarely observed within these boutons, which arose from thin unmyelinated axons. These boutons composed approximately 82 and 74% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, and were considered to arise predominantly from neurons in the cerebral cortex. In contrast, boutons with large terminals that contained round or plemorphic vesicles and formed multiple asymmetric synapses predominantly with large-diameter dendrites were also observed. Puncta adhaerentia contacts were also common. Mitochondria were numerous within large boutons with round vesicles, which arose from myelinated axons. Many of the large boutons were likely to have originated from neurons in the cerebellar nuclei. Approximately 6% of the boutons in the ventral anterior-ventral lateral complex and 8% in ventromedial nucleus were of the large type.(ABSTRACT TRUNCATED AT 400 WORDS)     

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。     

大鼠纹状体边缘区的突触特征

本文在电镜下对大鼠纹状体边缘区的突触特征进行了研究。边缘区中可见四种类型的突触:轴—体,轴—树,轴棘及轴—轴。在胞体、树突或树突棘上均可见有对称性及非对称性突触。多数对称性突触含有多形性小泡,而非对称性突触主要含圆形小泡。我们还在边缘区中首次观察到少量轴—轴—棘突触。边缘区中这些突触特征和纹状体其余部位有区别。     

Neuronal and synaptic organization of the centromedian nucleus of the monkey thalamus: a quantitative ultrastructural study, with tract tracing and immunohistochemical observations

Summary The ultrastructure of the centromedian nucleus of the monkey thalamus was analysed qualitatively and quantitatively and projection neurons, local circuit neurons, and synaptic bouton populations identified. Projection neurons were mostly medium-sized, with oval-fusiform or polygonal perikarya, few primary dendrites, and frequent somatic spines; local circuit neurons were smaller. Four basic types of synaptic boutons were distinguished: (1) Small- to medium-sized boutons containing round vesicles (SR) and forming asymmetric contacts, identified as corticothalamic terminals. (2) Heterogeneous medium-sized boutons with asymmetric contacts and round vesicles, similar to the so-called large round (LR) boutons, which were in part of cortical origin. (3) Heterogeneous GAD-positive small- to medium-sized boutons, containing pleomorphic vesicles and forming symmetric contacts (F1 type), which included pallidothalamic terminals. (4) Presynaptic profiles represented by GAD-positive vesicle-containing dendrites of local circuit neurons. Complex synaptic arrangements, serial synapses and triads with LR and SR boutons engaging all parts of projection neuron dendrites and somata, were seen consistently, whereas classical glomeruli were infrequent. LR and SR boutons also established synapses on dendrites of local circuit neurons. F1 boutons established synapses on projection neuron somata, dendrites and initial axon segments. Compared to other previously studied motor-related thalamic nuclei, differences in synaptic coverage between proximal and distal projection neuron dendrites were less pronounced, and the density of synapses formed by local circuit dendrites on projection neuron dendrites was lower. Thus, compared to other thalamic nuclei, the overlap of different inputs was higher on monkey centromedian cells, and centromedian inhibitory circuits displayed a different organization.     

Ultrastructure of substance P immunoreactive elements in the periaqueductal gray matter of the rat.

Substance P (SP) is a non-opioid peptide that generates a potent analgesia when injected into the periaqueductal gray matter (PAG). The aim of this study was to investigate the fine neuronal structures and synaptic circuits involved in SP action in rats by means of electron microscopy, using immunocytochemical (ICC) pre-embedding methods. A conventional ultrastructural study, carried out to interpret the ICC data correctly, shows small sized nerve cell bodies with a high nucleus-cytoplasmic ratio; absence of an extensive granular endoplasmic reticulum; and few axo-somatic contacts having symmetrical and asymmetrical junctions in equal proportions. The large neuropil is characterized by numerous thin unmyelinated axons and axo-dendritic synapses mainly showing pleomorphic vesicles and asymmetrical junctions. The ICC analysis showed moderately labeled nerve cell bodies with the same structural, synaptic, and dimensional features as the negative cells. In the neuropil SP immunoreactivity is shown by dendrites, synapses, and thin elements which are unidentifiable structurally. No SP terminals synapsing on SP nerve cell bodies were found and only occasional SP light labeled terminals synapsing on negative perikarya were seen. The SP boutons generally have pleomorphic vesicles and asymmetrical junctions. On the basis of these data a possible excitatory activity of PAG SP synapses could be hypothesized. This activity would take place on postsynaptic neurons generally at a dendritic level. Our ultrastructural findings give support to an excitatory role carried out by SP neurons of the PAG, as suggested by the role of PAG circuitry on spinal nociception.     

Parvalbumin-like immunostaining in the cat inferior colliculus. Light and electron microscopic investigation

The presence of the calcium-binding protein parvalbumin (PV) was studied in neuronal elements of the cat's inferior colliculus (IC) by means of light and electron microscopic immunocytochemistry. Immunostaining of PV was detected in all three main parts of the IC. Several subtypes of large neurons that differed in size and shape were immunostained, comprising approx. 15% of the total number of PV-containing neurons. Approx. half of the labeled neurons were medium sized. Two types of small neurons were found to be PV synthesizing, and comprised approx. 35% of the total PV-containing population. Ultrastructurally, many dendrites were heavily immunolabeled, and the reaction product was present in dendritic spines as well. Several types of synaptic boutons contained reaction product, and terminated on both labeled and unlabeled postsynaptic targets forming asymmetric and symmetric synapses. Approx. 70% of all PV-immunolabeled terminals contained round synaptic vesicles and formed asymmetric synapses. The majority of these boutons were of the "large round" type and corresponded to the terminals of cochlear nuclei. A lower number were of the "small round" type, and were probably corticotectal terminals. The remaining 30% of PV-containing terminals contained pleomorphic or elongated vesicles and formed symmetric synapses. These terminals corresponded with "P" and "F1" bouton types. Part of these boutons appeared to arise from nuclei of the lateral lemniscus and the superior olive, and a certain percentage likely represented endings of inhibitory interneurons.     

Synaptic organization of the nucleus raphe dorsalis of the cat

Summary This electron microscopic study describes the different types of synaptic terminals found in the nucleus raphe dorsalis of the adult cat. Serial section analysis was used extensively to confirm the nature of the synaptic contact established by the various classes of terminals.Five different classes of terminals are identified according to the shape and packing density of the synaptic vesicles and type of contact they establish. The most common class (RDI-type) contains densely packed, round, agranular synaptic vesicles and establishes asymmetrical synaptic contacts. A second class (RDII-type) also contains spherical synaptic vesicles, but establishes symmetrical synaptic contacts with dendrites of all sizes. Most of the terminals in these two classes contain a few dense-cored synaptic vesicles, but a small sub-group contains many dense-cored vesicles. A third, less frequent, class (RSI-type) contains sparsely packed spherical synaptic vesicles and the majority of these terminals have asymmetrical contacts. A fourth terminal class contains pleomorphic synaptic vesicles (P-type), contacts dendrites of all sizes, and usually establishes symmetrical synaptic contacts. Finally, boutons thought to be the vesicle-filled excrescences of dendrites (postsynaptic dendrites) are found and in some cases the dendritic origin of these profiles was confirmed by serial sectioning. Such boutons containing pleomorphic vesicles are presynaptic to other such dendrites as well as conventional dendrites, and are postsynapticto the other terminal types described.Somata within the nucleus exhibit somatic spines but receive few synaptic contacts. Most axo-somatic terminals contain either round or pleomorphic vesicles and have postsynaptic thickenings intermediate to the symmetric and asymmetric types.     

Fine structure of GABA-labeled axonal endings in the inferior colliculus of the cat: immunocytochemistry on deplasticized ultrathin sections.

Antisera to GABA conjugates and postembedding techniques were used to identify GABA-containing axonal endings at the electron microscopic level in the inferior colliculus. Over 90% of the GABA-labeled axonal endings had a similar morphology. They contained pleomorphic synaptic vesicles and made symmetrical synapses. The exceptional endings contained round vesicles and made symmetrical synaptic contacts or had pleomorphic vesicles with asymmetrical contacts. The majority of GABA-labeled axonal endings synapsed on dendrites; however, a few labeled axosomatic synapses were also found. Potential sources for these GABAergic synapses are neurons intrinsic to the inferior colliculus or from the dorsal nucleus of the lateral lemniscus. These findings suggest a basic similarity for most GABAergic endings in the inferior colliculus despite their possible origin from different cell types.     

Light and electron microscopical studies of the normal nuclei ventralis lateralis and ventralis anterior thalami in the cat

Summary A light and electron microscopical investigation of the nucleus ventralis lateralis (VL) and nucleus ventralis anterior (VA) of the cat's thalamus was made. Light microscopical examination of Golgi impregnated material revealed the existence of two types of cells based on differences in their dendritic arborization and branching of the axon. One of the cells is considered to be the thalamocortical relay cell, whereas the other is tentatively considered to be a Golgi type II neuron. Electron microscopical investigations of the two nuclei revealed the existence of a high number of profiles containing pleomorphic vesicles, and which have been identified as dendrites. Based on correlation with the Golgi material as well as on cytological features of the parent cell bodies, the dendrites containing vesicles are believed to belong to Golgi type II neurons. In addition to the vesicle-filled dendritic profiles, five different types of boutons have been identified. Two of these boutons, type LR and type SR, contain ovoid vesicles and establish asymmetrical synaptic contacts with dendrites of both types of neurons. Type F1, F2 and F3 boutons contain pleomorphic vesicles, but can be distinguished from dendritic profiles containing pleomorphic vesicles. Type F2 and F3 boutons establish symmetrical contacts with dendrites of both thalamocortical relay cells and Golgi type II neurons. Type F1 boutons establish symmetrical synaptic contact with the proximal dendrites or soma of the thalamocortical relay neurons only.Dendrites of both thalamocortical relay cells and Golgi type II neurons, as well as type LR, SR, F2 and F3 boutons, are engaged in glomeruli. Dendro-dendritic synapses between Golgi type II dendrites and relay cell dendrites are frequently seen, whereas no evidence of axo-axonic synapses have been found.Differences and similarities in the ultrastructural organization of VL and VA are described in some detail.Some of the observations reported in this and the following two papers were presented at the 6th Symposium of the International Society for Research in Stereoencephalotomy, Tokyo, October 12th & 13th, 1973.     

SP,CGRP,ENK样阳性纤维终末在猫骶髓后连合核内的分布特征及超微结构

本实验用免疫组化电镜技术对骶髓后连合核中SP祥、CGRP样、L-ENK样阳性终末进行了观察,结果表明:SP样阳性终末主要含圆形清亮小泡,间有少量颗粒囊泡,主要与中、小树突形成不对称型轴-树突触(93%);还可见到不对称型轴-体突触(5%);也可见到少量的轴-轴突触(2%),SP样阳性终末为突触后成分。CGRP样阳性终末以含圆形清亮小泡为主,有的终末内混有颗粒囊泡。大多数终末(89%)与树突构成轴-树突触,但以远侧树突为主;也有少数(6%)的CGRP样阳性终末与胞体形成轴-体突触;还观察到由阴性终末与CGRP样阳性终末构成的轴-轴突触。L-ENK样阳性终末以含圆形清亮小泡为主,有时可见散在的颗粒囊泡,多与中、小树突形成不对型轴-树突触(92%);也观察到轴-体突触(5%)和轴-轴突触(3%)。     

大鼠下丘脑室旁核的突触结构

在PVN神经网中,最多见的是轴—树突触,其次是轴—体突触,偶尔可见树—树突触。根据所含的突触小泡不同,可把轴突末稍分为三类:Ⅰ、含有大量的清亮小泡,混有一些小的致密核心小泡。Ⅱ、含有大量圆的清亮小泡,混有一些大的致密核心小泡。Ⅲ、单纯含有大量圆及椭圆的清亮小泡,大小不等。多种不同性质的轴突末稍共同参与PVN的神经内分泌整合过程。     

功能确定后的HRP细胞内注射标记的三叉神经中脑核神经元的电镜研究

在猫的Ｐｒｏｂｓｔ束纤维内记录并注射ＨＲＰ，确定出支配咬肌肌梭的三叉神经中脑核神经元，对其进行了连续切片电镜观察．发现约有１０个不明来源的终扣与被标记的中脑核神经元密切接触，其中４个与之形成明确的突触．突触有两种类型：对称性含混合透明小泡（圆形、椭圆形和扁平小泡）者和非对称性含圆形透明小泡者．另外后者也含有为数很少、散在分布的小圆形致密芯颗粒小泡．在其它与标记胞体密切接触的终扣内，也可见到不规则分布的致密芯颗粒小泡．本文对上述所见的可能的功能机制进行了讨论。     

Nigrothalamic pathway in the cat demonstrated by autoradiography and electron microscopy

Summary Light microscopic autoradiography and electron microscopy were used to trace the nigrothalamic projections and to study the sites of termination of this pathway in the cat. Injections of tritiated amino acids or electrolytic lesions were placed in the substantia nigra pars reticularis (SNr). An accumulation of radioactivity was found in the ventral medial nucleus and in the ventromedial part of the ventral anterior nucleus. At the ultrastructural level degenerating medium size synaptic boutons and medium size myelinated fibers were observed in these nuclei. The boutons contained clear pleomorphic vesicles and formed symmetrical type synaptic contacts with regular type dendrites and vesicle-containing dendrites.The present findings indicate that the ventral medial nucleus is the principal site of termination of nigrothalamic projections in the cat.     

Ultrastructure of substance P immunoreactive elements in the periaqueductal gray matter of the rat

Substance P (SP) is a non-opioid peptide that generates a potent, analgesia when injected into the periaqueductal gray matter (PAG)The aim of this study was to investigate the fine neuronal structures and synaptic circuits involved in SP action in rats by means of electron microscopy, using immunocytochemical (ICC) pre-embedding methods. A conventional ultrastructural study, carried out to interpret the ICC data correctly, shows small sized nerve cell bodies with a high nucleus–cytoplasmic ratio; absence of an extensive granular endoplasmic reticulum; and few axo-somatic contacts having symmetrical and asymmetrical junctions in equal proportions. The large neuropil is characterized by numerous thin unmyelinated axons and axodendritic synapses mainly showing pleomorphic vesicles and asymmetrical junctions. The ICC analysis showed moderately labeled nerve cell bodies with the same structural, synaptic, and dimensional features as the negative cells. In the neuropil SP immunoreactivity is shown by dendrites, synapses, and thin elements which are unidentifiable structurally. No SP terminals synapsing on SP nerve cell bodies were found and only occasional SP light labeled terminals synapsing on negative perikarya were seen. The SP boutons generally have pleomorphic vesicles and asymmetrical junctions. On the basis of these data a possible excitatory activity of PAG SP synapses could be hypothesized. This activity would take place on postsynaptic neurons generally at a dendritic level. Our ultrastructural findings give support to an excitatory role carried out by SP neurons of the PAG, as suggested by the role of PAG circuitry on spinal nociception.     

Characterization of input synapses on intracellularly stained neurons in hippocampal slices: an HRP/EM study

Summary This study describes the fine structure of input synapses on identified neurons in slices of the guinea pig hippocampus. For morphological identification, granule cells of the fascia dentata and pyramidal neurons of regio inferior of the hippocampus were impaled and intracellularly stained with horse-radish peroxidase (HRP). Input synapses on the HRP-stained neurons were identified in the electron microscope by the location of the synapses in inner or outer zones of the dentate molecular layer, as in the case of the synaptic contacts on injected granule cells, or by unique fine structural characteristics, as in the case of the giant mossy fiber boutons on CA3 pyramidal cells. As in tissue fixed in situ by transcardial perfusion, a large number of terminals arising from the different afferents in inner and outer zones of the dentate molecular layer were well preserved and formed synaptic contacts with small spines, large complex spines, and dendritic shafts of the HRP-filled granule cells. Mossy fiber synapses on the stained CA3 neurons were densely filled with clear vesicles, contained a few dense-core vesicles, and formed synaptic contacts with large spines or excrescences. Occasionally electrondense degenerating boutons were also found impinging on the stained dendrites and spines. The significance of the present findings for electrophysiological and pharmacological studies on brain slices is discussed.     

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。