Serial Ultrathin Sections Research Articles

Dendrodendritic and Axoaxonic Synapses in the Thalamic Reticular Nucleus of the Adult Rat

Currently, it is believed that cell-cell communications occur in the thalamic reticular nucleus (RT) during thalamocortical operations, but the anatomical substrate underlying these intrinsic interactions has not been characterized fully in the rat yet. To further our knowledge on this issue, we stained juxtacellularly rat RT neurons with biocytin or Neurobiotin and examined their intrinsic axon collaterals and "axon-like processes" at both light and electron microscopic levels. Of 111 tracer-filled RT cells for which the axon could be followed from its origin up to the thalamus, 12 displayed short-range, poorly ramifying varicose local axon collaterals, which remained undistinguishable from parent distal dendrites, raising the question as to whether their varicosities were presynaptic terminals. Correlated light and electron microscopic observations of the proximal part of these intrinsic varicose axonal segments revealed that their varicosities and intervaricose segments were, in fact, postsynaptic structures contacted by a large number of boutons that, for the most, formed asymmetric synapses and were nonimmunoreactive for GABA. Similarly, the so-called "axon-like processes" stemming from the soma or dendrites also were identified as postsynaptic structures. Two unexpected observations were made in the course of this analysis. First, the hillock and initial segment of some RT axons were found to receive asymmetric synaptic inputs from GABA-negative terminals. Second, examination of serial ultrathin sections of dendritic bundles cut in their longitudinal plane revealed the existence of several short symmetric dendrodendritic synapses and numerous puncta adhaerentia between component dendrites. In conclusion, dendrodendritic junctions might be a prominent anatomical substrate underlying interneuronal communications in the RT of the adult rat. Furthermore, excitatory axoaxonic synapses on the axon hillock, initial segment, and local axon collaterals might represent a powerful synaptic drive for synchronizing the firing of RT neurons. Future studies are essential to verify whether excitatory axoaxonic synapses with the axon hillock are a general feature in the RT.

Ultrastructure of the male reproductive organs in the interstitial annelid Sphaerosyllis hermaphrodita (”Polychaeta", Syllidae)

The reproductive organs of the simultaneous hermaphrodite Sphaerosyllis hermaphrodita (Syllidae, Exogoninae) were examined by TEM and reconstructed from ultrathin serial sections. Oocytes are produced in the 11–13th chaetigerous segments and then attached to the outer body surface. The male organs comprise a seminal vesicle, testes, sperm ducts and copulatory chaetae. The unpaired seminal vesicle is an uncompartmented cavity above the gut and within the chaetigerous segments 8–10. Its interior is lined with a layer of gland cells that degenerate as spermatogenesis in the vesicle proceeds. The testes are situated ventrolaterally, close to the seminal vesicle in the 9th chaetigerous segment. They contain cells at early stages of spermatogenesis, which are connected to one another by zonulae collares. The testes and seminal vesicle are enclosed in epithelia. Paired sperm ducts run ventrally from about the midline of the body under the seminal vesicle and into the parapodia of the 9th chaetigerous segment. There they open, together with the protonephridia of this segment, to the outside next to the stout copulatory chaeta. Each sperm duct consists of six cells, the luminal surface of which bears microvilli but no cilia. Only in animals with fully differentiated sperm does the small opening of the proximal duct cell in each duct give access to the seminal vesicle. The mode of sperm transfer is discussed.

Modification of dispersal patterns of branched photoreceptor axons and the evolution of neural superposition

Using 3D reconstructions of serial ultrathin sections and extended-focus pictures of Golgi-impregnated and cobalt-stained visual fibres, we studied the branched short photoreceptor axons found in ancestral dipterans and in the scorpionfly Panorpa. In the 'phantom' midge Chaoborus (Nematocera), each cartridge of the lamina neuropil is innervated by collaterals of 24 photoreceptor axons from 18 neighbouring and next-but-one ommatidia, in a regular pattern of asymmetrical neural pooling. Comparison of axon morphologies in different groups (Tipulidae, Chironomidae, Culicidae) indicates that this pattern must represent an ancestral condition of the dipteran visual system and is thus a precursor of neural superposition found in flies (Brachycera) in which only one set of photoreceptors R1–R6 converges onto each cartridge instead of the four sets found in Chaoborus. It can be concluded that specific axonal input channels from the large array of innervating photoreceptors found in midges have been retained during the evolution of neural superposition.

Transcellular openings through frog microvascular endothelium

Reconstructions from serial ultrathin sections of microvascular endothelium suggest that gaps, which are induced by a range of stimuli, may pass through endothelial cells as well as between them. To address the possibility that the transcellular gaps are not artefacts of aldehyde fixation, we have reconstructed fourteen gaps, induced by the ionophore A23187, in frog mesenteric microvessels where the primary fixative was osmium tetroxide. All fourteen gaps were transcellular. The different actions of osmium tetroxide and glutaraldehyde lead us to consider that it is highly unlikely that transcellular gaps are fixation artefacts.

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%)of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.

Ultrastructural and confocal laser scanning microscopic examination of TUNEL-positive cells.

TdT-mediated dUTP-biotin nick end labelling (TUNEL) has been widely used for detecting cells with DNA fragmentation or apoptotic cells. However, since the concept of apoptosis is based on cellular ultrastructure, it is important to identify the morphological features of TUNEL-positive cells. In this study, we performed TUNEL and electron microscopic observation on serial semithin and ultrathin sections of pancreas from bilaterally adrenalectomized rats with caerulein-induced pancreatitis. TUNEL-positive cells were identified with two different ultrastructural patterns. One was characteristic of apoptosis, with condensed nuclei, intact mitochondria, and zymogen granules. The other pattern was one of marked cellular degeneration, possibly representing the end stage of cell death. Cells which did not demonstrate these ultrastructural patterns were not labelled by the TUNEL method. The three-dimensional structure of TUNEL-positive cells was also investigated by confocal laser scanning microscopy (CLSM), which showed the apoptotic nuclei exhibited various three-dimensional structures. These results confirm the utility of the TUNEL method in detecting apoptosis; application of the technique reported in this study will contribute to the further characterization of individual TUNEL-positive cells.

Ultrastructural and confocal laser scanning microscopic examination of TUNEL‐positive cells

TdT-mediated dUTP-biotin nick end labelling (TUNEL) has been widely used for detecting cells with DNA fragmentation or apoptotic cells. However, since the concept of apoptosis is based on cellular ultrastructure, it is important to identify the morphological features of TUNEL-positive cells. In this study, we performed TUNEL and electron microscopic observation on serial semithin and ultrathin sections of pancreas from bilaterally adrenalectomized rats with caerulein-induced pancreatitis. TUNEL-positive cells were identified with two different ultrastructural patterns. One was characteristic of apoptosis, with condensed nuclei, intact mitochondria, and zymogen granules. The other pattern was one of marked cellular degeneration, possibly representing the end stage of cell death. Cells which did not demonstrate these ultrastructural patterns were not labelled by the TUNEL method. The three-dimensional structure of TUNEL-positive cells was also investigated by confocal laser scanning microscopy (CLSM), which showed the apoptotic nuclei exhibited various three-dimensional structures. These results confirm the utility of the TUNEL method in detecting apoptosis; application of the technique reported in this study will contribute to the further characterization of individual TUNEL-positive cells. © 1997 John Wiley & Sons, Ltd.

The structure of the Xyp sex chromosome complex in male meiosis of two beetles: Tenebrio molitor (Tenebrionidae) and Chrysolina graminis (Chrysomelidae)

Sex chromosome association was studied in male meiosis of Tenebrio molitor, a darkling beetle, using chromosome spreads prepared from testes. Staining with a DNA-specific fluorescent dye showed that the sex chromosomes formed a cone-shaped element at pachytene. The two chromosomes were closely associated. Concomitantly with chromatin condensation at late prophase I, a slender unstained gap developed between the large X and the tiny y chromosome, indicating that the sex chromosome pair is achiasmatic. The gap persisted in metaphase I spermatocytes. The electron microscopic analysis of ultrathin serial sections through metaphase I spermatocytes also revealed one asymmetric bivalent per metaphase plate. This bivalent contained a block of electron-dense material located between a large and a small mass of chromatin. It is plausible to assume that the asymmetric body represented the XY pair. The electron-dense mass corresponded in location to the chromosomal portion not stained by the DNA-specific dye and is believed to consist of non-chromatin material. In order to determine whether electron-dense material is routinely found in the achiasmatric sex chromosome pairs in male meiosis of beetles, primary spermatocytes of a leaf beetle, Chrosolina graminis, were studied as well. As in T. molitor, a homogeneously textured mass was detected in one bivalent, the sex chromosome pair, but in contrast to this species transparent vacuoles were scattered throughout the material in C. graminis.

Three-dimensional Dynamics of the Golgi Apparatus in Mitotic Parotid Acinar Cells: Computer-aided Reconstruction from Cytochemically-marked Ultrathin Serial Sections.

For clarification of the structural and cytochemical dynamics of the Golgi apparatus of the parotid acinar cell during mitotic division in vivo induced by repeated injections of isoproterenol, computer-generated three-dimensional reconstruction was conducted of serial section electron micrographs in conjunction with Golgispecific enzyme cytochemistry. Outlines of the cells, nuclei, thiamine pyrophosphatase (TPPase) -positive Golgi elements (trans Golgi) and TPPase-negative ones (middle/cis Golgi) could be clearly seen through the use of a commercially available computer software program.In interphase acinar cells, the Golgi apparatus could be seen to spread out in the supranuclear region of the cytoplasm as a single continuous reticular structure with many branches and anastomosis. TPPase-positive trans elements were associated with the entire surface of the interphase Golgi stack. With the start of the mitotic phase, gradual disorganization of the stack into a loose structure followed by dispersion throughout the cytoplasm and marked disappearance of TPPase activity became evident. Up to the anaphase, the Golgi apparatus was completely disorganized as small TPPase-negative clusters comprised of tubulo-vesicular membranes were distributed throughout the cytoplasm. Recovery of the stack, single reticular continuity, supranuclear location and TPPase-reactivity of Golgi apparatus was apparent in daughter cells during the telophase. Distributional change of the Golgi apparatus of parotid cell during mitotic division was clearly confirmed by the present study. Dispersion and reassembly may be significant factors in the equal partitioning of the Golgi apparatus into daughter cells. TPPase activity would appear quite essential to the expression of the normal cellular secretory functions.

Topography and immunocytochemical characterization of nerve fibers in the leptomeningeal compartments of the rat. A light- and electron-microscopical study.

The localization of peptidergic, catecholaminergic, and nitroxidergic nerve fibers in the ventral leptomeningeal connective tissue compartment was studied in whole-mount preparations and serial semithin and ultrathin sections. For immunocytochemistry, whole-mount preparations of the leptomeninges and ventral brain slices with the meninges were incubated as free-floating specimens with primary antibodies against protein gene product 9.5 (PGP 9.5), substance P (SP), calcitonin gene-related peptide (CGRP), dopamine beta-hydroxylase (DbetaH), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and nitric oxide synthase (NOS) using the avidin-biotin-peroxidase method. Based on the regional differences of the connective tissue organization, the leptomeninx is subdivided into the pial, trabecular, and adventitial leptomeninx. The antibody PGP 9.5 stains all unmyelinated nerve fibers in the leptomeninx. Although the highest density of nerve fibers occurs in the adventitial leptomeninx, nerve fibers, and terminals are additionally present in the trabecular and pial leptomeninx. DbetaH-, NPY-, VIP- and NOS-immunoreactive (IR) nerve fibers occur exclusively in the adventitial leptomeninx forming neuromuscular junctions. CGRP- and SP-IR nerve fibers are localized in all three leptomeningeal compartments where they terminate close to the subarachnoid space (type 1) or within the connective tissue (type 2). Due to their morphological and immunocytochemical characterization a possible chemo-, mechano- or nociceptive function is discussed in the context of pathophysiological aspects.

The ultrastructure of mitosis in the free-living kinetoplastid Bodo curvifilus

Summary The nuclear division of a free-living bodonid flagellate was investigated at the ultrastructural level for the first time. Transmission electron microscopy of serial ultrathin sections shows that nuclear division of Bodo curvifilus occurs by closed mitosis with an intranuclear spindle, as in trypanosomatids and cryptobiids. The nucleolar material of B. curvifilus persists during the nuclear division and seems to be shared between the daughter nuclei. The spindle consists of one compact bundle of approximately 30 microtubules running through the nuclear centre: most of the microtubules continue from one pole to another. Microtubule-organising centres of the mitotic spindle lie at the opposite poles, and they are not distinguished morphologically. The spindle of B. curvifilus is associated with at least 10 pairs of kinetochore-like dense plaques. In sections only the plaques can be seen at different stages of migration towards the poles as chromosomes are not condensed during mitosis of kinetoplastids.

Ultrastructure of a sensillum associated with the pharynx of the cockroachPeriplaneta americana

A sensillum associated with the pharynx of the cockroach Periplaneta americana was examined in serial ultrathin sections using electron microscopy. This sensillum consisted of a group of 10–20 similar sensillar subunits. Each sensillar subunit possessed one 60- to 70-μm long dendritic sheath that made direct contact with the cuticle. The dendritic sheath enclosed 3–5 sensory cilia arising from 3–5 sensory cells located in a cluster approximately 30 μm proximal to the base of the sheath. Between the sensory cell body and the base of the sheath the dendrites were wrapped by the sheath-forming cell. Before entering the dendritic sheath itself, the dendrites crossed through an extracellular space, the ciliary sinus. No cuticular specializations, such as a well-defined sensory hair or pore, were observed. The structure of this sensillum suggests that it responds poorly to mechanical distortion of its surroundings. This characteristic supports the hypothesis that this sensillum measures the osmotic concentration of the ingested food.

Three-dimensional analysis of cerebellar terminals and their postsynaptic components in the ventral lateral nucleus of the cat thalamus.

Relationships among cerebellar terminals (CTs), dendrites of thalamocortical projection neurons (TCNs), and dendrites of local circuit neurons in the ventral lateral nucleus of the cat thalamus were analyzed quantitatively by observing several series of serial ultrathin sections and by using a computer-assisted program for the three-dimensional reconstruction from serial ultrathin sections. In pentobarbital-anesthetized cats, CTs were labeled either by injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the cerebellar nuclei or by intra-axonal injection of HRP after electrophysiological identification. By using two series of 133 and 73 serial sections, mutual relationships between 43 WGA-HRP-labeled CTs and their postsynaptic structures were analyzed based on their synaptic specializations and shapes of synaptic vesicles. Thirty-nine of these CTs formed a synapse with one TCN dendrite, whereas only four CTs formed synapses with two TCN dendrites. These CTs also synapsed on dendrites containing pleomorphic synaptic vesicles (presynaptic dendrites). Single CTs synapsed on 0-6 presynaptic dendrites (2.2 +/- 1.5, N = 43) through their whole extents, and about 40% of these presynaptic dendrites that were contacted by CTs established synaptic contacts with the same TCN dendrites on which the CTs synapsed. Thus, a CT, a presynaptic dendrite, and a TCN dendrite formed a triadic arrangement. Triadic arrangements were identified in approximately 60% of these 43 CTs. However, they rarely had a glomerulus-like appearance, as described previously in the ventral lateral nucleus and other main thalamic relay nuclei. In another series of 83 and 43 serial sections along dendrites of TCNs, observations were focused on the triadic arrangement. Triadic arrangements were located evenly on the primary and secondary dendrites of TCNs. Computer-assisted three-dimensional reconstructions were made on one WGA-HRP-labeled CT and two intra-axonally labeled CTs (a bouton en passant and a bouton terminal) with their surrounding neuronal elements, and complex spatial arrangement of neuronal processes became obvious. These results provide the quantitative assessment of synaptic arrangements among CTs, presynaptic dendrites, and TCN dendrites and reveal their spatial interrelations in the cat ventral lateral nucleus.

Fine structural organization of spinothalamic and trigeminothalamic lamina I terminations in the nucleus submedius of the cat.

We examined lamina I trigemino- and spinothalamic tract (TSTT) terminals labeled with Phaseolus vulgaris leucoagglutinin in the nucleus submedius (Sm), a nociceptive relay in the cat's thalamus. Volume-rendered (three-dimensional) reconstructions of ten lamina I TSTT terminals identified with light and electron microscopy were built from serial ultrathin sections by computer, which enabled the overall structures of the terminal complexes to be characterized in detail. Two fundamentally different terminations were observed: compact clusters of numerous boutons, which predominate in the dense focus of a lamina I terminal field in the Sm, and boutons-of-passage, which are present throughout the terminal field and predominate in its periphery. Reconstructions of cluster terminations reveal that all boutons of each cluster make synaptic contact with protrusions and branch points on a single dendrite and involve presynaptic dendrites (PSDs) in triadic arrangements, providing a basis for the secure relay of sensory information. In contrast, reconstructions show that boutons-of-passage are generally characterized by simple contacts with PSDs, indicating an ascending inhibitory lamina I influence. These different synaptic arrangements are consistent with physiological evidence indicating that the morphologically distinct nociceptive-specific and thermoreceptive-(cold)-specific lamina I TSTT neurons terminate differently within the Sm. Thus, a suitable structural substrate exists in the cat's Sm for the inhibitory effect of cold on nociception, a behavioral and physiological phenomenon of fundamental significance. We conclude that the Sm is more than a simple relay for nociception, and that it may be an integrative comparator of ascending modality-selective information that arrives from neurons in lamina I.

Three-dimensional analysis of the whole mesangium in the rat

The three-dimensional structure of the mesangium was analyzed by means of reconstruction from serial semithin and ultrathin sections of the rat glomerulus. The mesangial domains traced on light micrographs of semithin sections were transferred to styrene models, which were stacked up to reconstruct the whole mesangium. The reconstructed mesangium was tree-like in shape and was divided into three lobes that were connected to the vascular pole by a slender neck. The glomerulus contained no islets of mesangium which were not connected to the vascular pole. The mesangium contained 64 mesangial loops that were penetrated by capillaries. Reliability of the findings on the mesangial loops was ascertained by various methods including reconstruction of part of the mesangium from ultrathin sections. Electron microscopic observations revealed that the arms of the mesangial loops were frequently very slender and consisted of mesangial cell processes containing prominent bundles of actin filaments. The mesangial loops were distributed evenly within the mesangium. Considering previous reports showing about 400 capillary branches in the rat glomerulus as well as the present findings, we concluded that the mesangial loops may change the distribution of intraglomerular blood flow by dynamic contraction of the mesangial cells, or serve as an additional safety device to prevent the expansion of glomerular capillaries.

Frequency and dendritic distribution of autapses established by layer 5 pyramidal neurons in the developing rat neocortex: Comparison with synaptic innervation of adjacent neurons of the same class

Synaptic contacts formed by the axon of a neuron on its own dendrites are known as autapses. Autaptic contacts occur frequently in cultured neurons and have been considered to be aberrant structures. We examined the regular occurrence, dendritic distribution and fine structure of autapses established on layer S pyramidal neurons in the developing rat neocortex. Whole-cell recordings were made from single neurons and synaptically coupled pairs of pyramidal cells, which were filled with biocytin, morphologically reconstructed, and quantitatively analyzed. Autapses were found in most neurons (in 80% of all cells analyzed; n = 41. On average 2.3 ± 0.9 autapses per neuron were found which were located primarily on basal dendrites (64%; 50–70 μm from the soma), to a lesser extent on apical oblique dendrites (31%; 130–200 μm from the soma), and rarely on the main apical dendrite (5%; 480–540 μm from the soma). About three times more synaptic than autaptic contacts (ratio 2.4:1) were formed by a single adjacent synaptically coupled neuron of the same type. The dendritic locations of these synapses were remarkably similar to those of autapses. Electron microscopic examination of serial ultrathin sections confirmed the formation of autapses and synapses respectively, and showed that both types of contacts were either located on dendritic spines or shafts. The similarities between autapses and synapses suggest that autaptic and synaptic circuits are governed by some common principles of synapse formation. We would therefore assume that autapses may contribute to local self-excitation of a neuron.

Frequency and Dendritic Distribution of Autapses Established by Layer 5 Pyramidal Neurons in the Developing Rat Neocortex: Comparison with Synaptic Innervation of Adjacent Neurons of the Same Class

Synaptic contacts formed by the axon of a neuron on its own dendrites are known as autapses. Autaptic contacts occur frequently in cultured neurons and have been considered to be aberrant structures. We examined the regular occurrence, dendritic distribution, and fine structure of autapses established on layer 5 pyramidal neurons in the developing rat neocortex. Whole-cell recordings were made from single neurons and synaptically coupled pairs of pyramidal cells, which were filled with biocytin, morphologically reconstructed, and quantitatively analyzed. Autapses were found in most neurons (in 80% of all cells analyzed; n = 41). On average, 2.3 +/- 0.9 autapses per neuron were found, located primarily on basal dendrites (64%; 50-70 microns from the soma), to a lesser extent on apical oblique dendrites (31%; 130-200 microns from the soma), and rarely on the main apical dendrite (5% 480-540 microns from the soma). About three times more synaptic than autaptic contacts (ratio 2.4:1) were formed by a single adjacent synaptically coupled neuron of the same type. The dendritic locations of these synapses were remarkably similar to those of autapses. Electron microscopic examination of serial ultrathin sections confirmed the formation of autapses and synapses, respectively, and showed that both types of contacts were located either on dendritic spines or shafts. The similarities between autapses and synapses suggest that autaptic and synaptic circuits are governed by some common principles of synapse formation.

Reevaluation of synaptic relationships of cerebellar terminals in the ventral lateral nucleus of the rhesus monkey thalamus based on serial section analysis and three-dimensional reconstruction.

Terminals of cerebellar afferents (CB) to different regions of the ventral lateral nucleus (VL) of the rhesus monkey thalamus were labeled with wheat germ agglutinin-horseradish peroxidase following injections into the dentate nucleus. Synaptic relationships of 17 CB with projection neuron dendrites (PNd) and local circuit neuron dendrites (LCNd) were analyzed in serial ultrathin sections from dorsal and ventral VL regions, which are known to differ cytoarchitecturally and functionally. Three terminals were reconstructed using three-dimensional (3D) computer image analysis techniques to obtain volumetric and planar measurements. CB in the ventral VL were often flat and elongated with synaptic vesicles arranged in clusters. Each CB was engaged with one PNd and one to four LCNd. A single bouton formed 8-50 synaptic contacts, with those on PNd outnumbering the ones on LCNd 4.1:1. Only some CB in the ventral VL were engaged in complex synaptic arrangements such as triads and serial synapses. Most CB in the dorsal VL displayed a roundish shape and numerous uniformly distributed synaptic vesicles. They formed 5-25 synaptic contacts with a 3:1 ratio of contacts on PNd compared with those on LCNd. CB in the dorsal VL participated in a variety of complex synaptic arrangements. Two types of triads were found: classic with CB, PNd and LCNd, and unconventional with CB and two LCNd. CB were also involved in serial synapses with two LCNd or LCNd and another PNd, and serial sequential synapses with two LCNd and a PNd. Three glomerulus-like structures were encountered in the dorsal VL. 3D reconstruction and volumetric measurements revealed that synaptic contacts formed by CB on PNd had varying shapes and sizes (0.022-0.274 microns2). Synapses formed on LCNd were larger (0.09-0.407 microns2). The total area of all active zones of a single CB on LCNd was either equal to or about 40% smaller than that of synapses on PNd. The entire active zone area comprised 1-1.6% of the total CB surface area and did not seem to correlate with the volume. Synaptic contacts formed by associated LCNd on PNd in complex arrangements were usually small (0.021-0.044 micron2). The results suggest that: synapses formed by CB on PNd and LCNd, and synapses formed by LCNd on PNd may differ in strength; a variety of different circuits participate in the processing of cerebellar afferent information in the primate VL; and these circuits differ in functionally different VL subdivisions.

Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin.

In contrast to normal microvessels, those that supply tumors are strikingly hyperpermeable to circulating macromolecules such as plasma proteins. This leakiness is largely attributable to a tumor-secreted cytokine, vascular permeability factor (VPF). Tracer studies have shown that macromolecules cross tumor vascular endothelium by way of a recently described cytoplasmic organelle, the vesiculo-vacuolar organelle or VVO (VVOs are grapelike clusters of interconnected, uncoated vesicles and vacuoles). However, equivalent VVOs are also present in the cytoplasm of normal venules that do not leak substantial amounts of plasma protein. To explain these findings, we hypothesized that VPF increased the permeability of tumor blood vessels by increasing VVO function and that the VVOs of normal venules were relatively impermeable in the absence of VPF stimulation. To test this hypothesis, VPF was injected intradermally in normal animals after intravenous injection of a soluble macromolecular tracer, ferritin, whose extravasation could be followed by electron microscopy. VPF caused normal venules to leak ferritin, and, as predicted by our hypothesis, ferritin extravasated by way of VVOs, just as in hyperpermeable tumor microvessels. Ultrathin (14-nm) serial electron microscopic sections and computer-aided three-dimensional reconstructions better defined VVO structure. VVOs occupied 16-18% of endothelial cytoplasm in normal venules. Individual VVOs were clusters of numerous (median, 124) interconnected vesicles and vacuoles that formed complex pathways across venular endothelium with multiple openings to both luminal and abluminal surfaces. Like VPF, histamine and serotonin also stimulated ferritin extravasation across venules by way of VVOs. Together, these data establish VVOs as the major pathway by which soluble plasma proteins exit venules in response to several mediators that increase venular hyperpermeability. These same mediators also increased the extravasation of colloidal carbon, but this large particulate nonphysiological tracer exited venules primarily through endothelial gaps.

Primary Spermatocytes inAgelastica alni(Chrysomelidae, Coleoptera, Insecta): A Fine Structure Study with Emphasis on the Behavior of the Mitochondria

The present fine structure study deals with the behavior of the mitochondria during meiosis I in males of a leaf beetle,Agelastica alni.For the sake of completeness, the restructuring of cytoplasmic elements, spindles, and nuclei is also described. Serial ultrathin sections through prophase I spermatocytes in diverse developmental stages and metaphase and telophase I spermatocytes were studied. While mitochondria were scattered throughout the cytoplasm in pachytene, they aggregated in the vicinity of the nucleus in diplotene and formed prominent stacks. It was clear that individual mitochondrial stacks were interconnected by elongated mitochondria, but the three-dimensional relationship of mitochondria within the stacks was difficult to assess on account of their dense packing. Filamentous and granular material was found between the individual mitochondria and may link them. In diakinesis, the stacks gave rise to elongated mitochondria located in invaginations of the nuclear envelope. When the nuclear envelope opened in prometaphase I, some mitochondria entered the spindle area but the majority of them remained lateral to the forming spindle apparatus. Spindles in metaphase I spermatocytes were characterized by a spindle envelope consisting of two closely neighboring cisternae of smooth endoplasmic reticulum and some elongated mitochondria inside this sheath. The bulk of the mitochondria was arranged at the cytoplasmic face of the spindle envelope. Telophase cells possessed elongated mitochondria oriented parallel to the spindle apparatus. A volumetric approach showed that the entire mitochondrial mass of prophase I spermatocytes associated with the spindle apparatus in metaphase I. The close association of mitochondria with the spindle apparatus may guarantee roughly bisection of the chondriome during meiotic division. The association between mitochondria and prophase I nuclei may be a preparatory step for the imminent alignment with the spindle but my also serve the high energy demands of the nuclear compartment in this system.