Ethanolic Phosphotungstic Acid Research Articles

Successive episodes of synapses production in the developing rat nucleus tractus solitarii.

In the rat nucleus tractus solitarii (NTS), synaptogenesis is thought to occur both pre- and postnatally. The present study was performed to precisely define the timetable of synapse formation in the NTS after birth. Changes in synapse morphology and densities were analyzed between postnatal day 3 (P3) and P28 using electron microscopy and ethanol phosphotungstic acid (E-PTA) staining. The proportion of morphologically immature synapses was high at P3 (38%) and P14 (30%) and low (8-14%) at the other ages investigated (P7, P21, and P28). Synaptic density significantly increased between P7 and P14 (60%) and between P21 and P28 (54%), but did not significantly change between P3 and P7 and between P14 and P21. Mean synaptic diameter also increased over the first postnatal month. Significant increases in synaptic size occurred between P3 and P7 (28%) and between P14 and P21 (15%). The present data indicate that, in the NTS, synaptogenesis occurs over a protracted period of time and involves distinct successive episodes of synapse production.

Morphometry of E-PTA stained synapses at the periphery of pathological lesions

We carried out a novel application of the disector sampling and counting method, in a biopsy material from the pathologic human brain, to estimate the synaptic structural dynamics, quantitatively. Parietal cortex biopsies of adult (mean age: 40.0 years) and old (mean age: 66.2 years) patients having undergone surgical intervention were investigated. The tissue samples were excised at the periphery of meningioma masses. Synaptic contact zones were stained en bloc by the ethanol phosphotungstic acid (E-PTA) preferential technique which selectively enhances both the pre- and post-synaptic paramembranous material separated by a sharp cleft against a very faint background, thus facilitating and objectifying synaptic morphometry. The disector method, associated with currently used morphometric formulas, enabled us to measure the number of synapses/μm3 of tissue (numeric density: Nv); the total area of the synaptic contact zones/μm3 of tissue (surface density: Sv) and the average synaptic size (S). In old vs. adult patients, Nv decreased by 7.5% (Mean (SEM): Adult 2.0040(0.0452); Old 1.6780(0.0623)), while S increased by 17.5% (Adult 0.0203(0.0026); Old 0.0246(0.0035)). Sv did not show any age-related difference. The same negative correlation between Nv and S has also been reported in physiological aging, and this suggests the active presence of age-related synaptic restructuring mechanisms in the nervous tissue surrounding a tumoral mass.

Total regional and global number of synapses in the human brain neocortex.

An estimator of the total number of synapses in neocortex of human autopsy brains based on unbiased stereological principles is described. Each randomly chosen cerebral hemisphere was stratified into the four major neocortical regions. Uniform sampling with a varying sampling fraction in each region of neocortex was performed. The total volume of each neocortical region was estimated using point counting according to Cavalieri's principle. The ethanolic phosphotungstic acid staining technique was modified for synapses in human autopsy brains. The numerical density of synapses in each neocortical region studied was estimated using the disector at the electron microscopical level. The total number of neocortical synapses in each region was estimated as the product of the total volume of neocortex and the numerical density of synapses. The influence of the postmortem fixation delay on the number of synapses was investigated in five large mammals (one dog, one cow, and three pigs), the brains of which were kept under conditions similar to those under which human corpses are normally kept. The apparent decrease of 3.9% in the numerical density of synapses in the large mammals following a 2-day fixation delay was not significant. The average total number of synapses in the neocortex of five young male brains was 164 x 10(12) (CV = 0.17). An analysis of the precision of the estimate of the total number of synapses in neocortex indicates that blocks represent both the major source of variation and the largest workload. Using eight blocks per brain the imprecision of the estimate is, however, only 66% of the total variance.

Protein Aggregation after Transient Cerebral Ischemia

Protein aggregates containing ubiquitinated proteins are commonly present in neurodegenerative disorders and have been considered to cause neuronal degeneration. Here, we report that transient cerebral ischemia caused severe protein aggregation in hippocampal CA1 neurons. By using ethanolic phosphotungstic acid electron microscopy (EM) and ubiquitin immunogold EM, we found that protein aggregates were accumulated in CA1 neurons destined to die 72 hr after 15 min of cerebral ischemia. Protein aggregates appeared as clumps of electron-dense materials that stained heavily for ubiquitin and were associated with various intracellular membranous structures. The protein aggregates appeared at 4 hr and progressively accumulated at 24 and 48 hr of reperfusion in CA1 dying neurons. However, they were rarely observed in dentate gyrus neurons that were resistant to ischemia. At 4 hr of reperfusion, protein aggregates were mainly associated with intracellular vesicles in the soma and dendrites, and the nuclear membrane. By 24 hr of reperfusion, the aggregates were also associated with mitochondria, the Golgi apparatus, and the dendritic plasmalemma. High-resolution confocal microscopy further demonstrated that protein aggregates containing ubiquitin were persistently and progressively accumulated in all CA1 dying neurons but not in neuronal populations that survive in this model. We conclude that proteins are severely aggregated in hippocampal neurons vulnerable to transient brain ischemia. We hypothesize that the accumulation of protein aggregates cause ischemic neuronal death.

Electron Microscopic Contrast of the Cytoskeleton and Junctional Complexes of Intestinal Epithelial Cells by Ethanolic Phosphotungstic Acid

Electron Microscopic Contrast of the Cytoskeleton and Junctional Complexes of Intestinal Epithelial Cells by Ethanolic Phosphotungstic Acid

Modification of Postsynaptic Densities after Transient Cerebral Ischemia: A Quantitative and Three-Dimensional Ultrastructural Study

Abnormal synaptic transmission has been hypothesized to be a cause of neuronal death resulting from transient ischemia, although the mechanisms are not fully understood. Here, we present evidence that synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the hippocampus of rats subjected to 15 min of ischemia followed by various periods of reperfusion. Postsynaptic densities (PSDs) from both area CA1 and the dentate gyrus were thicker and fluffier in postischemic hippocampus than in controls. Three-dimensional reconstructions of selectively stained PSDs created using electron tomography indicated that postsynaptic densities became more irregular and loosely configured in postischemic brains compared with those in controls. A quantitative study based on thin sections of the time course of PSD modification indicated that the increase in thickness was both greater and more long-lived in area CA1 than in dentate gyrus. Whereas the magnitude of morphological change in dentate gyrus peaked at 4 hr of reperfusion (140% of control values) and declined thereafter, changes in area CA1 persisted and increased at 24 hr of reperfusion (191% of control values). We hypothesize that the degenerative ultrastructural alteration of PSDs may produce a toxic signal such as a greater calcium influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it causes or contributes to neuronal damage during the postischemic phase.

Asynchronous changes in size, number, and shape of synapses in the developing superior colliculus

The ultra-structural development of synapses in retino-receptive layers of the opossum superior colliculus was studied by the ethanolic phosphotungstic acid (E-PTA) method. There was a tendency for a slight reduction in the diameter of synaptic disks, a rise and fall of numerical densities and, except for an ephemeral period, a general increase in the proportion of ‘‘frown’’ among curve synapses. The lack of strict synchrony and the occurrence of different patterns of changes suggest that multiple factors contribute to synaptic maturation.

Ultrastructural localization of basic proteins ofBlastocystis hominis

Basic proteins ofBlastocystis hominis were detected by the ammoniacal silver and ethanolic phosphotungstic acid techniques using electron microscopy. The central vacuole showed many silver grains when treated with ammoniacal silver and an increased electron density when treated with phosphotungstic acid. The intensity of positive reactions correlated with the electron density of the central vacuole, because cells having an electron-lucent central vacuole showed no silver grain deposits. Since it is known that the concentration of electron-dense materials in the central vacuole increases during log phase of growth, and then decreases in stationary phase, this organelle must accumulate basic proteins during cell growth.

The Intracellular Calcium Store Function of Platelet Granules.

The atrial specific granule has been described as an intracellular Ca store (ICaS). It is reasonably proposed that other polypeptidesecretory granules could also play the function of ICaS. In the present work, high Ca concentration in the dense granule (dG) and α granule (αG) of platelets was confirmed by using x-ray microanalysis in both ultrathin cryosections and ethanolic phosphotungstic acid staining sections, and Ca2+-ATPase activities were found on the membrane of both the granules. Furthermore, when platelets were activated by A 23187 1 mM, thrombin 2 U/ml or ADP 200μM, both the Ca content and number of the granules decreased, which means that the Ca2+ was released and secretion of the granules increased. It is suggested that the release Ca2+ raise the cytosolic Ca2+ level and, thus, the secretion of the granules is promoted. In addition, it was described that the ultrastructure of the platelet granules in ultrathin cryosections was somewhat different from that in routine sections.

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

Dynamic morphology of the synaptic junctional areas during aging: the effect of chronic acetyl- l-carnitine administration

The ultrastructural features of hippocampal synaptic contact zones have been investigated by means of computer-assisted morphometry in rats of 6, 12 and 22 months of age and in age-matched animals chronically treated with ALCAR at a daily dose of 50 mg/100 g body weight from the age of 1 month up to the day of sacrifice. The number of synapses/μm 3 (Nv), the average size of the junctional areas (S) and the total area of the synaptic contact zones/μ m 3 (Sv) were measured in tissue samples stained by means of the ethanol phosphotungstic acid (E-PTA) preferential technique for synaptic membranes. In control animals Nv was constant between 6 and 12 months of age, but significantly decreased in 22-month-old rats; S did not show significant differences due to age; Sv was unchanged between 6 and 12 months, but it decreased significantly in the old animals. In ALCAR treated rats Nv increased and S decreased significantly vs. age-matched controls. Sv showed a lifespan constancy among the groups of age analysed. In ALCAR treated rats the number of contact areas smaller than 0.08 μm 2 increased by 18, 9 and 10% at 6, 12 and 22 months of age, respectively. ALCAR administration resulted in a lifespan modulation of synaptic structural dynamics. A proper metabolism at nerve terminals is accounted to play a crucial role in synaptic remodelling potential: on the basis of current research data, it is suggested that ALCAR may improve neuronal bioenergetic mechanisms.

Sudden Infant Death Syndrome

The medulla was sampled from nine cases of sudden infant death syndrome (SIDS) and from six age-matched control cases without neurological disease. Morphometric analyses were performed on serial Nissl sections through the left half of each medulla to determine the total volume of the hemimedulla and the numerical density of neurons (Nv, neurons per mm3) in the central reticular nucleus. Blocks of tissue from the right half were prepared for electron microscopy using the ethanolic phosphotungstic acid (EPTA) method to stain synaptic contacts. Stereological analyses were performed to determine the Nv of synapses in the central reticular nucleus. Total volume of the hemimedulla did not differ significantly between SIDS cases and controls. However, in the central reticular nucleus of SIDS cases, the Nv of neurons was significantly less than in controls (30%), while the mean profile area of reticular neurons was significantly greater (39%). Synaptogenesis in control cases was characterized by a gradual decrease in the Nv of synapses from approximately 150 million at 40 postconceptional weeks to 110 million at 84 weeks. In SIDS cases the Nv of synapses was significantly greater (38%). These results demonstrate a disorder of synaptogenesis in the central reticular nucleus of SIDS cases. In view of previous reports of a persistence of dendritic spines on reticular neurons in SIDS, the results are consistent with a failure to eliminate normally extraneous synapses during early development.

Long-term effects of intermittent versus continuous ethanol exposure on hippocampal synapses of the rat.

The hippocampus is known to be very sensitive to a large spectrum of different neurotoxins including ethanol. Ethanol administered continuously or intermittently may affect the hippocampus in different ways. Intermittent administration of ethanol has many features in common with the low level electrical stimulation protocols which lead to the functional changes associated with the phenomenon of kindling. In this study, the differential effects of intermittent intraperitoneal ethanol injections (3 g/kg twice daily) and continuously administered ethanol in drinking water (20%) on hippocampal synapses in the rat were studied using ethanolic phosphotungstic acid staining and electron microscopy. After 1 month of intermittent exposure a significant reduction (18%) of synapses was seen in the stratum lucidum of the CA3 region. Continuously treated animals showed no significant change over this time despite a higher total ethanol intake. In the dentate gyrus, a compensatory increase in supragranular synaptic number was seen only in continuously treated animals. These findings demonstrate the sensitivity of synapses of the hippocampus to the presence of ethanol and the larger effects of peaking ethanol concentrations compared to more constant levels. These results emphasize the need to consider the differential effects of various types of ethanol consumption also on the human brain.

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

Nuclear changes during spermiogenesis in two chrysomelid beetles

Ultrastructural and cytochemical studies were carried out on sperm nucleus of the beetles, Coelomera lanio and Diabrotica speciosa. Nuclear development involves changes in the shape and in the degree of chromatin condensation, with specific aggregation patterns of DNA-histone complex occurring during this process. Lamellar and paracrystalline arrangements of the nuclear material were observed in the Diabrotica speciosa spermatid and in the Coelomera lanio spermatozoon, respectively. Ethanolic-phosphotungstic acid technique suggest the presence of basic proteins in the nuclear material of spermatids. This reaction disappears during chromatin condensation. Chromatin condensation patterns may reflect specific intranuclear mechanisms and offers protection to the genome during spermatozoon transport to the oocyte.

Cytochemical localization and calcium quantitation of intracellular calcium store in rat atrial cardiocytes.

Ethanolic phosphotungstic acid (EPTA) method and G-6-Pase method were used to localize cytochemically the atrial specific granules (ASG) and sarcoplasmic reticulum (SR) of rat atrial cardiocytes under the electron microscope, and at the same time, the calcium (Ca) in ASG and SR was analyzed quantitatively by analytical electron microscopy (AEM). In these methods, the procedure of osmium tetroxide fixation was shortened or even abolished to keep more combined Ca. The Ca concentration in ASG and in the terminal cisterns of SR (TSR) was 64±16 and 99±21mM/kg dry wt respectively. These values are approximate to those reported in earlier works that were done on cryosections. It is suggested that these cytochemical methods could reserve a large part of combined Ca in the intracellular Ca store, and they are much simpler than the low temperature techniques of biological specimen preparation. Therefore, these methods are useful for AEM microanalysis of elements in organelles.

Cytochemical and immunocytochemical ultrastructural study of nucleoproteins during nucleologenesis in 8-cell bovine embryos.

The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, basic lysine-rich and histone proteins in 8-cell bovine embryos. Two silver staining techniques, ethanolic phosphotungstic acid (PTA) and immunocytochemical methods using specific antibodies, were applied at the ultrastructural level. The silver-stained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus precursor bodies (NPBs). The amounts of these proteins increased during the transformation of the NPBs into the fibrillogranular nucleolus. At this stage the well-developed dense fibrillar components encircling fibrillar centres showed intense staining. PTA-positive (basic lysine-rich) proteins were present within most nucleolar structures during nucleologenesis as well as in the chromatin. Histones H2B, H3 and H4 were concentrated throughout the chromatin including the nucleolus-associated chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multivacuolated NPBs, both in the fibrous mass as well as inside the vacuoles. Nucleolar histones appeared with the massive penetration of DNA into the NPBs. We suggest that nucleologenesis may serve as a criterion of normal early embryonic development and that the proteins involved in the process of nucleologenesis and transcription could be used as chemical markers of nucleolar function.

Quantitative aspects of synapses on Golgi-impregnated neurons.

With the classical Golgi techniques, numerous types of neurons can be distinguished in the cerebral cortex, each with a specific dendritic geometry and pattern of axonal ramifications. In the present review we describe two techniques which allow quantification of synapses on identified neurons: (1) Golgi-rapid impregnation-gold toning-electron microscopy, and (2) Golgi-Kopsch impregnation-gold toning-electron microscopy in combination with staining of the tissue with ethanolic phosphotungstic acid (E-PTA). Both techniques were applied on neurons in the visual cortex of young and adult rabbits. By means of rotating and tilting specimens in the electron microscope, the nondistinctive ultrastructure of obliquely sectioned synapses can be circumvented, leading to precise estimates of asymmetrical vs. symmetrical synapses without complete reconstruction of the neuron.

Mitochondrial derivatives of Culex quinquefasciatus (culicidae) spermatozoon: Some new aspects evidenced by cytochemistry and image processing

Spermatozoa of the mosquito Culex quinquefasciatus have two mitochondrial derivatives of similar size and shape, containing a periodic matrix embedded by an amorphous region and surrounded by a membrane complex. The presence of cytochrome C oxidase activity was shown in the cortical zone. The use of the ethanolic-phosphotungstic acid technique suggests the presence of basic proteins in the paracrystalline structure. The analysis of the paracrystalline structure by digital image processing suggests that the cylindric-like subunits are empty structures and appear connected to each other by thin filaments. This approach showed that longitudinal filaments present an undulation disposition alongside the spermatozoon flagellum.

Effects of Total Asphyxia on the Development of Synaptic Junctions in the Brains of Mice

Using ethanolic phosphotungstic acid staining, we studied the development of synaptic junctions in the brains of mice subjected to total asphyxia. One-day-old mice, Std:ddY strain, were put into a chamber continuously flushed with 100% CO2-gas for 35 min. The surviving mice (28.1%) were used as experimental models, while unasphyxiated littermates served as controls. The synaptic junctions in the frontal motor cortex and hippocampal cortex were studied at 20 and 60 days of age using E-PTA staining, and the numbers of synaptic junctions in both areas were counted. In comparison with the control animals, the frontal motor cortex of the subject mice showed a smaller increase in the number of synaptic junctions, both at 20 and at 60 days of age. In the hippocampal cortex, the number of synaptic junctions seen in the experimental mice brains was similar to that in the controls at 20 days of age; however, the number of synaptic junctions in the treated mice brains showed a slower rate of increase than in the controls at 60 days of age.