Electron-dense Product Research Articles

A skin test for latent tuberculosis

To the Editor: Penn-Nicholson et al.[1] have suggested using a blood test for diagnosing early tuberculosis. It may be possible, however, to use a skin test to diagnose latent tuberculosis.[2,3] An autoimmune model of tuberculoid leprosy has been developed, using peripheral nerve as antigen. Rabbits were injected with a homogenate of human sensory nerve plus adjuvant. Some of the rabbits developed a state of granulomatous hypersensitivity; i.e. skin testing with a dilute solution of nerve in saline produced an epithelioid cell granuloma, in which the cytoplasm contained dilated rough endoplasmic reticulum filled with an electron-dense product.[4] The antigen is a non-myelin protein, active in doses of 1 μg. The ultrastructural appearances are similar to those in human tuberculoid leprosy. [5]

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

Recruitment of resting vesicles into recycling pools supports NMDA receptor‐dependent synaptic potentiation in cultured hippocampal neurons

Most presynaptic terminals in the central nervous system are characterized by two functionally distinct vesicle populations: a recycling pool, which supports action potential-driven neurotransmitter release via vesicle exocytosis, and a resting pool. The relative proportions of these two pools are highly variable between individual synapses, prompting speculation on their specific relationship, and on the possible functions of the resting pool. Using fluorescence imaging of FM-styryl dyes and synaptophysinI-pHluorin (sypHy) as well as correlative electron microscopy approaches, we show here that Hebbian plasticity-dependent changes in synaptic strength in rat hippocampal neurons can increase the recycling pool fraction at the expense of the resting pool in individual synaptic terminals. This recruitment process depends on NMDA-receptor activation, nitric oxide signalling and calcineurin and is accompanied by an increase in the probability of neurotransmitter release at individual terminals. Blockade of actin-mediated intersynaptic vesicle exchange does not prevent recycling pool expansion demonstrating that vesicle recruitment is intrasynaptic. We propose that the conversion of resting pool vesicles to the functionally recycling pool provides a rapid mechanism to implement long-lasting changes in presynaptic efficacy.

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H(2)O(2) accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl(3) staining at ultrastructure level. When the 3-week-old rice plants were affected by 100mM NaCl for 14days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H(2)O(2) accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl(3) were mainly observed especially around the swelling of thylakoids. H(2)O(2) accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H(2)O(2) accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H(2)O(2) under salinity is responsible for the changes in chloroplast ultrastructure. H(2)O(2) accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H(2)O(2) and the production is light dependent under salinity.

Synapsin regulates vesicle organization and activity-dependent recycling at Drosophila motor boutons

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. We investigated synapsin function in mediating synaptic activity during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin(−) boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin (+) boutons, while in synapsin (−) boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin(−) but not in synapsin(+) boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K+ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation.

The ultrastructure of malpighian tubules and the chemical composition of the cocoon of Aeolothrips intermedius Bagnall (Thysanoptera)

The secretory activity of the two branched malpighian tubules (MTs) of the second-instar larva in Aeolothrips intermedius is described. MTs of adult thrips have the typical ultrastructure of excretory epithelium with apical microvilli containing long mitochondria and a rich system of basal membrane infoldings. In the second-instar larva just before pupation, the ultrastructure of MT epithelial cells is dramatically different, and there are numerous huge Golgi systems in the cytoplasm. These cells are involved in an intense secretory activity to produce an electron-dense product which is released into the MTs lumen. This secretion is extruded from the hindgut and used by the second-instar larva to build an elaborate protective cocoon for pupation. Electron-spray-ionization mass spectrometry analysis of the cocoon revealed the presence of a beta-N-acetyl-glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera.

Stimulation-Induced Formation of the Reserve Pool of Vesicles in Drosophila Motor Boutons

We combined electron microscopy (EM), synaptic vesicle staining by fluorescent marker FM1-43, photoconversion of the dye into an electron dense product, and electrical recordings of synaptic responses to study the distribution of reserve and recycling vesicles and its dependence on stimulation in Drosophila motor boutons. We showed that, at rest, vesicles are distributed over the periphery of the bouton, with the recycling and reserve pools being intermixed and the central core of the bouton being devoid of vesicles. Continuous high-frequency stimulation followed by a resting period mobilized the reserve vesicles into the recycling pool and, most notably, produced an increase in vesicle abundance. Recordings of synaptic activity from the temperature-sensitive endocytosis mutant shibire during continuous stimulation until complete depression provided an independent estimate of the increase in vesicle abundance on intense stimulation. EM analysis demonstrated that continuous stimulation produced an increase in the vesicle density, whereas during a subsequent resting period, vesicles filled empty areas of the bouton, spreading toward its central core. Although the observed structural potentiation did not alter basal transmitter release, it produced an increased synaptic enhancement during high-frequency stimulation. The latter effect was not observed when the boutons were potentiated using high-frequency stimulation without a subsequent resting period. We concluded therefore that the newly formed vesicles replenish the reserve pool during a resting period following intense stimulation.

Neuronal circuit reconstruction using serial block-face scanning electron microscopy

Event Abstract Back to Event Neuronal circuit reconstruction using serial block-face scanning electron microscopy A fundamental limitation to understanding the function of neuronal circuits is the lack of complete wiring (connection) diagrams. In many nervous systems, local circuits are comprised of neurons extending several hundreds of microns. The tissue volumes that are required to reconstruct such circuits are on the order of 10^8 um3 representing several terabytes of pixel information at the pixel size (20-30nm) needed to trace all neuronal processes. It is therefore necessary to automate both the acquisition and analysis of the data. Data acquisition of 3D data with the required resolution is possible in a fully automated way by using serial block-face scanning electron microscopy (SBFSEM). Images are acquired from the face of a tissue block with slices as thin as 25 nm removed by ultra-thin sectioning with an oscillating diamond knife. This technique replaces the difficult and labor-intensive process of manually cutting, mounting, and imaging sections in a transmission electron microscope. We acquire data with nearly isotropic resolution at lateral resolutions of ~20 nm and 25 nm section thickness. We envision the ability to automatically collect data from volumes large enough to encompass complete circuits at a resolution capable of capturing all the fine, convoluted neuronal processes. The segmentation of neurons from images is critically dependent on a high signal contrast between structures of interest. We have developed a staining strategy that labels cell surfaces with an electron dense product. This method leaves the cytosol and organelles largely unstained and thus results in a high contrast between intracellular space and cell surfaces, allowing the profiles of neuronal processes to be easily identified. The manual tracing of each and every neuron through a large volume is prohibitively slow, hence the tracing of neurons must be automated. We first manually segment a representative sub-volume into intracellular and cell surface regions. This segmented sub-volume becomes a training dataset for machine learning algorithms. Local cubes of data (5-7 voxels^3) are used to generalize voxel connectivity from the training to test data sets. A crucial step, however, is the validation of the obtained segmentations so as to obtain an estimate of the reconstruction reliability. We have developed tools that allow browsing through large 3D data sets and the efficient creation of 3-dimensional skeletons by human tracers. The comparison between skeletons and automated segmentations allows the estimation of break and merger probabilities, which are needed for the optimization of the classification and segmentation algorithms. Also, this data provides a measure for the reliability of the reconstructed circuitry. Finally, we are attempting to correlate functional recordings with circuit structure. Proof of principle experiments have shown that bulk loading of calcium indicators in the mammalian retina allow functional signals to be imaged without significantly damaging tissue ultrastructure. Conference: Computational and systems neuroscience 2009, Salt Lake City, UT, United States, 26 Feb - 3 Mar, 2009. Presentation Type: Poster Presentation Topic: Poster Presentations Citation: (2009). Neuronal circuit reconstruction using serial block-face scanning electron microscopy. Front. Syst. Neurosci. Conference Abstract: Computational and systems neuroscience 2009. doi: 10.3389/conf.neuro.06.2009.03.103 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Feb 2009; Published Online: 02 Feb 2009. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Google Google Scholar PubMed Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Localization of intracellular lipid hydroperoxides using the tetramethylbenzidine reaction for transmission electron microscopy.

Histochemical reactions for lipid hydroperoxides (LHP) using indophenol, benzidine, or phenylendiamine as the electron donor have been described previously for auto-oxidized adipose () and neuronal () tissue. More recently, tetramethylbenzidine (TMB) has been proposed as another chromagen (). The usefulness of the TMB reaction for ultrastructural studies of lipid peroxidation was demonstrated in retina where LPH was generated by incubation with exogenous lipoxygenase. The glutaraldehyde fixed tissue, which was reacted with TMB and then postfixed in osmium tetroxide, showed an electron-dense product (). This technique allows intra- and extracellular localization, as well as a comparison of relative intensity amoung various cell types and subcellular organelles (). In light-induced lipid peroxidation, discs of the outer segments, which are rich in oxidizable long-chain polyunsaturated fatty acids, stain strongly and appear as bubble-like structures (). These are however, quite similar to fingerprint profiles seen acutely in outer segments and chronically in neurons, which are visualized without TMB following exogenous exposure to LHP (,). A possible caveat to the reported method is that peroxidized protein and carbohydrates many also react and so the TMB method has not been proven to be specific for LHP only.

Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion.

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3′-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.

Simultaneous ultrastructural identification of growth hormone and its messenger ribonucleic acid using combined immunohistochemistry and non-radioisotopic in situ hybridization: a technical note.

The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopical in situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.

Ultrastructure of Merkel-like cells labeled with carbocyanine dye in the non-taste lingual epithelium of the axolotl

Fluorescent carbocyanine dye (diI), applied to the glossopharyngeal (IX) nerve of the axolotl, transneuronally labeled solitary cells in the non-taste lingual epithelium. With diaminobenzidine (DAB), the diI was photoconverted to a dark, electron-dense product. The labeled cell had a large nucleus with invaginations, dense-cored vesicles in the cytoplasm, and finger-like processes. These are reminiscent of morphological features of cutaneous Merkel cells, suggesting that solitary cells innervated by the IX nerve are associated with mechanosensory function of the IX nerve system.

The unipolar brush cells of the rat cerebellar cortex and cochlear nucleus are calretinin-positive: a study by light and electron microscopic immunocytochemistry.

Cell class-specific markers are powerful tools for the study of individual neuronal populations. The peculiar unipolar brush cells of the mammalian cerebellar cortex have only recently been definitively identified by means of the Golgi method, and we have explored markers of cerebellar neurons with the purpose of facilitating the analysis of this new cell population and, especially, its distribution and ultrastructural features. By light microscopic immunocytochemistry, we demonstrate that, in the rat, the unipolar brush cells are the cortical neurons that are most densely immunostained with antiserum to calretinin, a recently discovered calcium-binding protein. The unipolar brush cells are highly concentrated in the flocculo-nodular lobe, the ventral uvula and the ventral paraflocculus, occur at relatively high density in the lingula, at moderate-to-low density in other folia of the vermis and in the narrow intermediate cortex, and at low to very low density, with the exception of a few hot spots, in the lateral regions of the cerebellar hemispheres and in the dorsal paraflocculus. Unipolar brush cells are also found in the cochlear nucleus. In addition to the unipolar brush cells, calretinin antibody distinctly stains certain mossy fibers, and weakly to moderately stains other cerebellar elements, such as granule neurons and climbing fibers. In the lobules containing high densities of unipolar brush cells, the granule cell bodies and the parallel fibers are much less immunoreactive, and there are many more densely immunostained mossy fibers than in the lobules, where these cells are rare, which suggests some relationships between these elements. In the cerebellar nuclei, small neurons are densely immunostained, while large neurons are immunonegative. The unipolar brush cells reside nearly exclusively in the granular layer. They are small neurons, intermediate in size between granule cells and Golgi cells, and their features are remarkably similar across all lobules. They usually have a single, relatively thick dendrite of varying length that terminates in a brush-like tip consisting of several short branchlets. Utilizing a pre-embedding protocol, we have identified unipolar brush cells with the electron microscope. The cytoplasm of these cells is partially obscured by the electron dense product of calretinin immunoreaction in all regions of the soma and processes. The cells are often covered with non-synaptic appendages and contain a peculiar cytoplasmic inclusion consisting of ringlet subunits. Other characteristic components are numerous neurofilaments, mitochondria and large, dense-core vesicles. Individual brushes enter one or two glomeruli, where the dendritic branchlets establish an unusually extensive synapse with mossy fiber rosettes.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAergic regulation of noradrenergic spinal projection neurons of the A5 cell group in the rat: An electron microscopic analysis

Recent studies have demonstrated an important contribution of the A5 noradrenergic cell group of the rostral medulla in the regulation of nociceptive messages at the level of the spinal cord. These noradrenergic controls parallel those arising from the serotonin-containing neurons of the nucleus raphe magnus. In the present study, we used postembedding immunogold staining to identify GABA-immunoreactive terminals that synapse upon identified spinally projecting noradrenergic neurons of the A5 cell group in the rat. A5 projection neurons were identified by Fluoro-Gold transport from the spinal cord; sections containing retrogradely labelled cells were then immunoreacted for tyrosine hydroxylase (TH) to identify the catecholamine-containing, presumed noradrenergic, neurons. Double-labelled A5 cells were intracellularly filled with Lucifer Yellow (LY) and then the LY was photo-oxidized to an electron-dense product. Seven intracellularly filled TH-immunoreactive projection neurons were studied with postembedding immunocytochemistry. Each A5 neuron received a significant GABA-immunoreactive terminal input. Out of a pooled total of 151 terminal profiles found in apposition to intracellularly labelled somatic and dendritic profiles, 31 (20.5%) were GABA-immunoreactive. The proportion of GABA-immunoreactive terminals that contacted somatic profiles (12/72; 17%) was similar to the proportion that contacted TH-labelled dendritic profiles (19/79; 24%). There was a discernible synaptic specialization in about 50% of the labelled terminals that contacted the TH projection neuron. Both symmetric and asymmetric synaptic specializations were found. Labelled terminals contained round or pleiomorphic vesicles, but not flat vesicles; many also contained dense-core vesicles. Our results indicate that noradrenergic neurons of the A5 cell group, which contribute to both antinociceptive and cardiovascular controls through their projection to the spinal cord, are regulated by local GABAergic, presumably inhibitory, mechanisms. Whether the initiation of A5 neuron activity results from a lifting of tonic GABAergic inhibitory control, as has been proposed for the neurons of the nucleus raphe magnus, remains to be determined.

Photoconverted carbocyanine DiI allows direct visualization of transplanted glial cells at the ultrastructural level

We have used the carbocyanine fluorochrome, DiI, to trace living glial cells (astrocytes, oligodendrocytes and immortalized Schwann cells) after their transplantation into the newborn shiverer and normal mouse brain. DiI fluorescence first detected on vibratome sections, was photoconverted into a stable, non-diffusible and electron-dense diaminobenzidine product. Both fluorescence and precipitate were found in the same cells and were detectable until 60 days after transplantation. At the ultrastructural level, DiI precipitate was contained within cytoplasmic vesicles scattered in the transplanted cell bodies and processes. Photoconversion did not interfere with the cell fine structure or predicted post-transplantation behavior. DiI is thus a suitable marker to trace, at the ultrastructural level, living cells after their transplantation.

Synaptic Contacts and the Transient Dendritic Spines of Developing Retinal Ganglion Cells.

The dendrites of ganglion cells in the mammalian retina become extensively remodelled during synapse formation in the inner plexiform layer. In particular, after birth in the cat, many short spiny protrusions are lost from the dendrites of ganglion cells during the time when ribbon, presumably bipolar, synapses appear in the inner plexiform layer and when conventional, presumed amacrine, synapses increase significantly in number. It has therefore been postulated that these transient spines may be the initial or preferred substrates for competitive interactions between amacrine or bipolar cell terminals that subsequently result in the formation of appropriate synapses onto the ganglion cells. If so, the majority of synapses made onto developing ganglion cells should be found on these dendritic spines. To test this hypothesis, we determined the synaptic connectivity of identified ganglion cells in the postnatal cat retina during the period of peak spine loss and synapse formation. The dendritic trees of ganglion cells were intracellularly filled with Lucifer yellow that was subsequently photo-oxidized into an electron-dense product suitable for electron microscopy. In serial reconstructions of the dendrites of a postnatal day 11 (P11) alpha ganglion cell and a P14 beta ganglion cell, conventional and ribbon synapses were found predominantly on dendritic shafts. Only three out of a total of 341 dendritic spines from the two cells received direct presynaptic input, all of which were conventional synapses. Thus, our observations suggest that the transient dendritic spines are not the preferred postsynaptic sites as previously suspected. However, it is possible that these structures play a different role in synaptogenesis, such as mediating interactions between retinal neurons that may lead to cell - cell recognition, a necessary step prior to synapse formation at the appropriate target sites (Cooper and Smith, Soc. Neurosci. Abstr., 14, 893, 1988).

Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo.

We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide, which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

Ultrastructure of somatic tissues in the ovaries of a chaetognath (Ferosagitta hispida).

Transmission electron microscopy reveals that the ovaries of Ferosagitta hispida contain four somatic tissues. A myoepithelial ovary wall, continuous with a thin layer of peritoneocytes lining the coelomic cavity, encloses a fluid-filled ovarian space in which oocytes develop. Lamellar extensions of a "follicular reticulum" branch throughout the ovarian space and ensheath developing oocytes. This tissue has been overlooked in most previous studies of chaetognath ovaries. A bipartite oviductal complex extends the length of each ovary just within the lateral ovary wall. It consists of a flattened, blindly ending cellular tube, herein referred to as the cellular sheath, and an enclosed syncytium. Sheath cells secrete an electron-dense product into the ovarian space. Those sheath cells directly bordering the syncytium are contractile and are joined to the to the syncytium by gap junctions and microvillar interdigitations. The syncytium contains a complex of membrane-bounded lumina. The latter sometimes enclose sperm received during mating or ovulated eggs. Thus the syncytium serves both as a seminal receptacle and as a duct for passage of eggs to the outside. Contrary to several classical reports, the cellular sheath and syncytium of the oviductal complex do not separate at ovulation to form a temporary oviductal lumen.

Biogenic amine localization in cardiac ganglion intrinsic neurons: Electron microscopic histochemistry of SIF cells

The parasympathetic cardiac ganglion in the mudpuppy, N. maculosus, contains postganglionic nerve cells and intrinsic neurons, many of which are small intensely fluorescent (SIF) cells. Several bioactive substances have been localized in the intrinsic nerve cells which may have integrative effects at synapses within the ganglion. Ganglionic intrinsic neurons can be identified electron microscopically by the presence of numerous cytoplasmic granular vesicles 80–120 nm in diameter. Throughout the ganglion there are bundles of unmyelinated fibers some of which are filled with granular and agranular vesicles and axosomatic terminals with similar vesicles synapsing on principal parasympathetic nerve cells. To understand the aminergic contribution to ganglionic synaptic circuitry the chromaffin reaction was used. The intrinsic neurons (i.e., SIF cells) were readily identified by their characteristic intracellular granule population. All intrinsic nerve cells identified showed granules which were positively labelled by the chromaffin reaction. Granular vesicles in synaptic profiles on principal cells (P cells) were also labelled indicating a direct aminergic synaptic innervation to these cells. The cell bodies of intrinsic neurons, ensheathed with supportive glial-like cellular processes, rarely received synapses. Elemental microanalysis was used to verify the chromium content of the electron dense product within the granular vesicles. These studies demonstrated direct aminergic synaptic input to at least a subpopulation of principal parasympathetic cells in the cardiac ganglion of mudpuppy.