Ethanolic Phosphotungstic Acid Research Articles

Synapses in developing cingulate and hippocampal cortices in undernourished rats

Differential patterns of developmental changes were observed between 10 and 150 days of age in numerical densities of ethanolic-phosphotungstic acid stained synapses in the molecular layer of cingulate cortex (area 29 c,d) and hippocampus (CA1)-dentate region in rat. Chronic caloric undernutrition commencing prenatally caused considerable reduction of synaptic densities up to 45 days of age in both the cortices, but showed opposite trends subsequently; the density attained was higher than control in the cingulate area, but lower than control in hippocampus-dentate area. Post-weaning rehabilitatory nutrition resulted in a reduced deviation from normal in the cingulate area but not in the hippocampus-dentate area.

Spermiogenesis in Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae): Ultrastructural and cytochemical studies of proteins using E-PTA.

Spermiogenesis in the South American leptodactylid frog Odontophrynus cultripes was analyzed ultrastructurally. The spermatids undergo morphological modification while still enclosed in microtubule-rich processes of Sertoli cells. Electron-dense plates resembling junctional structures appear in regions at which the spermatids lie in close contact with the surface of Sertoli cell processes. Spermatid differentiation can be divided into five distinct stages based mainly on chromatin condensation. In the late stages, the densely compacted chromatin loses reactivity to ethanolic phosphotungstic acid (E-PTA). Helical arrangements of microtubules appear in the cytoplasm that surrounds the spermatid nucleus after the second stage. The acrosomal vesicle differentiates into a cone-shaped acrosome that caps the anterior region of the nucleus. The connecting piece, located in the flagellum implantation zone, has transverse striations, and is continuous with the axial rod. The tail is formed by a 9 + 2 axoneme, an undulating membrane, and an axial rod that is rich in basic proteins as demonstrated by E-PTA staining.

Neurobiology of the aging brain: morphological alterations at synaptic regions

Computer-assisted morphometric studies have been carried out on nerve cell terminal regions in rats of different ages. The numerical density (Nv), the average area (S) and the surface density (Sv) of ethanol phosphotungstic acid stained (E-PTA) synaptic junctions were evaluated in cerebellar glomeruli and dentate gyrus supragranular layers. The volume density (Vv), the average volume (V) and the numerical density (Nvm) of synaptic mitochondria were measured in the cerebellar glomeruli of young (3 months), adult (11 months) and old (28 months) animals. We found that during aging Nv and Sv undergo a significant decrease, whereas S is significantly increased. The mitochondrial Vv is unchanged in all the age groups analysed, whereas in old rats V is increased and Nvm decreased, respectively. We interpret our results as supporting that the old CNS retains part of its remodelling activity and is capable of adaptive morphological response at synaptic terminal regions.

Effects of Low-Power Gallium Aluminium Arsenide Diode Laser Irradiation on the Development of Synapses in the Neonatal Rat Hippocampus

The effect of low-power gallium aluminium arsenide diode laser irradiation on the development of synapses was studied in the radiatum layer and the lacunosum-molecular layer of field CA3 of the neonatal rat hippocampus. Neonatal rats were irradiated with a low-power diode laser (830 nm, 60 mW) at two points located above the hippocampi, for 15 s, respectively, twice per day from birth (day 1) to day 5. The mean body weights of the laser-irradiated animals were found to be lower than those of the control animals, the deficit at day 20 being 22.6%. Moreover, the density of synaptic junctions stained by ethanolic phosphotungstic acid per unit area of the radiatum layer and the lacunosum-molecular layer of the neonatal rat hippocampus was significantly reduced at day 20. It was suggested that the low-power diode laser irradiation affected the development of synapses in the neonatal rat brain.

Atrial specific granules: The intracellular calcium store

It is well known that certain organells in cytosol contain high calcium concentration and they are named intracellular Ca stores. These stores regulate the intracellular Ca concentration. The present project aims at exploring whether the release of atrial natriuretic peptide (ANP) from the atrial specific granules(ASG) is dependent on the release of calcium from the ASG. For this purpose, we have to know first whether the ASG contain high Ca concentration. In this work, positive results were obtained by quantitative electron probe x ray microanalysis (EPMA) and Ca-ATPase cytochemical localization.Adult female Sprague Dawley rats were used. The auricle samples were prepared in five different procedures: 1 )preparation of routine ultrathin sections; 2)ethanolic phosphotungstic acid(EPTA) staining ultrathin sections for ASG showing; [1] 3) cytochemical localization of Ca-ATPase ;[2] 4) preparation of ultrathin cryosections of fresh rapid frozen auricles; [3] 5) quantitative EPMA with Link AN10000 and Link Quantem/FLS quantitative analysis software.

Quantifying synaptic number and structure: effects of stain and post-mortem delay

Current research indicates the importance of synaptic number and structure in plastic processes such as development, learning and memory, and aging. As such, the examination of these neural features has become an important factor in research on human conditions such as mental retardation, aging and Alzheimers disease. Synaptic research in human tissue typically involves delayed post-mortem fixation, therefore the current research was designed to examine the effect of post-mortem delay on synaptic number and structure in tissue stained with either routine osmium lead citrate/uranyl acetate (osmium) or ethanol phosphotungstic acid (EPTA). Results indicate that synaptic density shows either a gradual decline (EPTA) or an initial marked drop followed by a plateau (osmium) up to 10–15 h post-mortem depending on the stain used. The number of synaptic vesicles per synapse also undergoes a gradual decline. Measures of synaptic structure were more stable, with the primary change being an initial increase in the cross-sectional length of the synapse. Maximal height of the pre- and postsynaptic dense elements were not affected by post-mortem delay. The EPTA stain gave the best estimates of synaptic parameters with short post-mortem delays. These results indicate that different synaptic measures (and stains) show different responses to post-mortem fixation delay, and that experimental or statistical methods must be used to control for postmortem effects.

Morphological adaptive response of the synaptic junctional zones in the human dentate gyrus during aging and Alzheimer's disease

A computer-assisted morphometric study has been carried out on ethanol phosphotungstic acid (E-PTA) stained synaptic junctions in the human dentate gyrus supragranular layer from adult, old and Alzheimer's disease (AD)-affected patients. The number of synapses per unit volume of tissue (Nv = numerical density), the average area of the single junction (S) and the total area of the synaptic contact zones in a unit volume of tissue (Sv = surface density) were the 3 parameters taken into account. The synapse to neurone ration was also calculated for each patient. During physiological aging, Nv and Sv significantly decreased and S increased, respectively. In the AD hippocampi, Nv and Sv underwent a further decrease which was in the range of more than 40% with reference to the adult values. S was the same as the old control group. In comparison with the adult values, the number of synapse/neurone decreased by 15.6 and 48% in old and AD patients, respectively. Nv, S and Sv, while reporting on discrete ultrastructural features of the synaptic junctional zones, are closely related to each other and, taken together per group of patients, may represent a reliable index of the morphological adaptive changes taking place at the synapses. Thus, the significant increase of S both in old and AD hippocampi may be regarded as a CNS plastic response to aging and disease, although the marked decrease of Nv and Sv supports that in AD synaptic ultrastructural alterations proceed beyond a critical threshold for functional recovery.

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosomephase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late capphase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Effects of low and high relative molecular protein mass on four methods for total protein determination in urine

Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.

Cytochemistry of trichohyalin granules: a possible role for cornification of inner root sheath cell in the hair follicle.

We studied ultracytochemically how trichohyalin granules (TGs) were involved in the cornification of the inner root sheath (IRS) cells in the hair follicle. After storing unfixed mouse skin samples in glycerol and then low ionic strength salt solution (G-LISS), the TGs reduced electron-dense amorphous material (EDM) to various degrees, with the appearance of filamentous material in routine staining. As the IRS cells were differentiating, these changes in the TGs became successively prominent; Some TGs consisted of the filamentous material and residual granular EDM, showing a "filamentogranular structure". In the cells in transition to cornified cells, or transitional cells, the EDM of the TGs disappeared and consisted of only filamentous material which was intertwined with keratin filaments (KFs). The internal substructure of the TGs induced by the G-LISS treatment stained well with ethanolic phosphotungstic acid, which reacts with basic proteins, and the KFs associated with the TGs also stained well. In skin samples not treated with G-LISS, the TGs of the transitional cells often exhibited fibrogranular structure after PTA staining to detect tissue basic proteins, suggesting a release of PTA stainable proteins(s) by the TGs in living transitional cells. The KFs of the cornified cells were more intensively stained and thicker (about 20 nm wide) than those of the differentiating IRS cells. These findings suggest that the TGs may contain at least two kinds of basic proteins; One is G-LISS-soluble, and the other is a very insoluble filamentous protein. These protein materials may be added to or incorporated into the KFs in the cornified cells with the disappearance of the TGs, since the KFs are markedly thick with PTA staining.

'Blebbing' of the nuclear envelope of mouse zygotes, early embryos and hybrid cells.

In the mouse zygote and in two-cell stage embryos the inner leaflet of the nuclear envelope of pronuclei and that of blastomere and polar body II nuclei evaginate, forming multiple blebs within the perinuclear space, which contains a granular material. Blebbing exists only in oocytes activated by sperm in vivo or in vitro, or parthenogenetically by treatment with ethanol or puromycin. The germinal vesicle and an interphase nucleus formed after treatment of the oocyte at metaphase I by puromycin do not form blebs. Formation of blebs is specifically located in the cell cycle. The burst of the blebbing activity occurs during the first half of the cell cycle in one-cell embryos and in the earliest interphase period in the second cell cycle. Blebbing ceases from the beginning of the third cell cycle. The occurrence in the cytoplasm of 'double-layered' vesicles containing granular material resembling bleb contents and the disappearance of blebs from the nuclear envelope by the end of the cell cycle provide evidence that blebs represent a step in the transport of some material from the nucleus to the cytoplasm. Ethanolic phosphotungstic acid does not stain blebs, suggesting the absence of basic protein in their contents. Blebbing can be induced in somatic (thymocyte) and embryonic (blastomere of 8-cell stage embryo) nuclei following cell hybridization with activated oocytes. Their response to the oocyte cytoplasm by initiating blebbing depends on: (1) the position of the host cell in its cell cycle at the moment of hybridization, and (2) the time spent by the foreign nuclei in the host cytoplasm following cell fusion. If donor nuclei are introduced close to the time of activation, they start to produce blebs at the time corresponding to the initiation of blebbing by the female pronucleus in the first cell cycle. If foreign nuclei are introduced a few hours after activation they must be incubated in the host cytoplasm for some time before initiation of bleb formation, provided that the host pronucleus has initiated blebbing by that time. The existence of blebbing in nuclei formed only after oocyte activation, and the timing and the general occurrence of this event during the earliest cleavage stages of almost every mammalian embryo, suggest that this special nucleocytoplasmic transport plays a specific role at the beginning of development.

Structural aspects, potassium stimulation and calcium dependence of nonsynaptic neuropeptide release by the egg laying controlling caudodorsal cells of Lymnaea stagnalis

The cerebral peptidergic caudodorsal cells of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying behaviour by releasing peptides into (1) the haemolymph, from neurohaemal axon terminals in the periphery of the cerebral commissure and (2) the intercellular space of the central nervous system, from collaterals in the inner compartment of this commissure. Recently, it was shown that collateral release occurs from nonsynaptic release sites, which lack the morphological specializations that are characteristic of classical synapses. Probably, these sites enable the caudodorsal cells to communicate with central neurons in a nonsynaptic (“paracrine”, “diffuse”, “hormone-like”) fashion. The structural and ionic bases of nonsynaptic release were studied using the tannic acid-Ringer incubation-method for the detection of exocytotic release of secretory granule contents in vitro. Elevation of the extracellular potassium concentration strongly stimulates exocytotic activity in the collaterals. No stimulation was found in the absence of extracellular calcium ions. Similar results have been obtained for the neurohaemal axon terminals. Electron-dense material occurs apposed at the cytoplasmic side of the axolemma of collaterals (ethanolic phosphotungstic acid method). This material appears homologous with the presynaptic dense projections forming the “vesicular grid” in classical synapses. Such projections are also present in the neurohaemal axon terminals. It is concluded that secretion from nonsynaptic release sites in caudodorsal cell collaterals shares fundamental characteristics with secretion from conventional neuronal release sites (neurohaemal axon terminals and classical synapses); release occurs by exocytosis of secretory granules, is associated with a vesicular grid, is stimulated by membrane depolarization, and depends on the presence of extracellular calcium ions. The results underline the importance of nonsynaptic release sites for interneuronal communication within the central nervous system.

Synaptic connections of an interneuron with axonal arcades in the cat visual cortex

A single, isolated interneuron with axonal arcades in the cat visual cortex was analysed in detail by both light and electron microscopy. The neuron was impregnated by the Golgi-Kopsch method, gold-toned, and processed for electron microscopy using the ethanolic phosphotungstic acid (PTA) staining method of Bloom & Aghajanian (1968). These methods, in combination, resulted in the successful identification of a large number of synaptic boutons arising from the axon of the cell under study. We examined serially at the electron microscope level 210 boutons of the axonal arborization of the cell. Of these, 152 formed identifiable symmetrical synaptic contacts with a variety of postsynaptic elements. The vast majority of the postsynaptic targets were dendritic profiles, which represented 95.7% of all the synaptic contacts identified. Only one example was observed of two labelled boutons making contacts with the same postsynaptic element; the rest were apparently on different elements. This distribution of synapses, characterized by the lack of convergence, is very similar to that reported by other authors for a certain kind of double bouquet cell which, in turn, shares some morphological features with the neurons with axonal arcades. It is suggested that fine details of the geometry of the axonal arborization of a given cell are an important reflection of the distribution of its synapses.

Feline ‘C’-type terminals possess synaptic sites associated with a hypolemmal cistern and Nissl body

Previous findings suggest that axosomatic ‘C’-type terminals on rat spinal motoneurones possess ‘active’ synaptic sites associated with the characteristic subsynaptic cistern and postsynaptic Nissl body, but ‘C’-terminals in cat do not. A re-examination of feline ‘C’-terminals undertook to compare the synaptic ultrastructure in aldehyde-fixed material stained by either osmium or ethanolic phosphotungstic acid (E-PTA). In agreement with previous findings osmicated cat ‘C’-terminals failed to reveal synaptic complexes in regions possessing the subsynaptic cistern (‘cisternal regions’). In contrast, cisternal regions of E-PTA stained ‘C’-terminals exhibited a linear array of presynaptic dense projection all opposed by a single extended postsynaptic density which abutted directly onto both postsynaptic membrane and the cistern. This close topographical relationship is suggested to be functionally significant in the trans-synaptic trophism previously demonstrated for feline ‘C’-terminals.

Synaptic structural changes during development and aging

Although a great deal is known about the development of synaptic number, comparatively little is known about the effects of development, and particularly aging, on the structure of the synapse. The present study examined synaptic structure in the molecular layer of the motor-sensory neocortex during early development (postnatal days (P) 1, 3, 5, 7, 10, 15, 20, 30), adulthood (P60, 90), and old age (28 months). Tissue was stained with osmium tetroxide (osmium) or ethanol phosphotungstic acid and the following synaptic characteristics were quantified: (1) presynaptic element length, area, thickness, maximal projection height and smoothness, and number and size of vesicles adjacent to the presynaptic element; (2) postsynaptic element length, area, and thickness; and (3) cleft width. There is an early developmental increase in synaptic element length, followed by an increase in thickness into adulthood. During development the height and width of the presynaptic dense projections increase, after which they remain stable. While the number of adjacent synaptic vesicles increases throughout the lifespan, there is a parallel decrease in their size. During the period of rapid synaptogenesis in this brain region there are no decreases in any of the synaptic structural parameters examined, indicating that newly generated synapses are either formed the same size as the existing mature synapses, or are extremely plastic and grow very rapidly. Unlike age-associated changes in synaptic number, no changes were found in synaptic structure during aging.

Neocortical synaptogenesis, aging, and behavior: Lifespan development in the motor-sensory system of the rat

Little evidence presently exists on the development and aging of synaptic contacts and their relationship to behavior, particularly in nonvisual brain areas. To investigate this interrelationship, rats at a series of developmental ages [postnatal day 1 (P1) to P90] were initially examined on a battery of motor tasks. The battery, ranging from simple reflexive tests to tests of complex locomotor capacities, consisted of tactile-induced forelimb placing, chin-induced placing, body righting, climbing an inclined plane, traversing a narrow beam, and keeping up with a revolving wheel. Following completion of the behavioral testing, the animals, together with an additional group of aged (28- to 29-month-old) rats, were killed and their motor-sensory cortex was removed, stained with osmium tetroxide or ethanol phosphotungstic acid (EPTA), and examined under electron microscopy for density of synaptic contacts. Simple motor abilities such as tactile-induced placing was present by the end of the first postnatal week, with locomotor performance reaching a mature level by the end of the third postnatal week, and intermediate task abilities maturing within this range. Paralleling the development of complex locomotor skills was a sharp increase in synaptic density in the molecular layer of the motor-sensory cortex, commencing in the second postnatal week and peaking at P30. After P30 there was a sharp decline in synaptic density as well as a decline in performance on some motor tasks, although these two functions seemed to be occurring independently. There was a continued, but less dramatic synaptic loss evident in the aged rats.

Selective electronmicroscopical contrast of the ring canals joining germ cells by ethanolic phosphotungstic acid and uranyl-EDTA-lead

The edge of ring canals joining germ cells in mouse testis show a high and selective electronmicroscopical contrast after application of ethanolic phosphotungstic acid and uranyl-EDTA-lead. The contrasting reaction of this fibrous structure, which originates from the contractile ring during previous cytokinesis, is briefly discussed.

Basic proteins in the nuage of growing Chinese hamster oocytes.

Two types of nuage aggregates, the mitochondria-associating nuage and the freely-existing nuage, appeared in the cytoplasm of the growing Chinese hamster oocytes in a growing stage-specific manner. Enzyme digestion studies on the ultrathin sections obtained from the glycol methacrylateembedded materials showed that both types of nuage contain RNA and proteins. The ethanol-phosphotungstic acid staining and also the cobalt thiocyanate staining used to examine the localization of basic proteins at the ultrastructural level revealed that the mitochondria-associating nuage contains a considerable amount of basic proteins, whereas only a few may be present in the freely-existing nuage.It was considered that the mitochondria-associating nuage may lose its basic proteins and transform into the freely-existing nuage, along with the oocyte's growth.

Ethanol-Phosphotungstic Acid Staining of Basic Proteins in the Nucleolar Condensation Occurring in Growing Mouse Oocytes

Ethanol-Phosphotungstic Acid Staining of Basic Proteins in the Nucleolar Condensation Occurring in Growing Mouse Oocytes

An improved EPTA staining method for synapses in rat cerebral cortex cultures

Ethanolic phosphotungstic acid (EPTA) staining has proved to be a useful tool in electron microscopic studies on synapse formation and maturation in the developing brain. However, the available staining procedures did not give satisfactory contrast between synaptic structures and background in reaggregated cerebral cortex tissue grown in vitro. Such contrast is a prerequisite for reliable quantitative studies. We therefore varied several steps of the staining procedure and have succeeded in obtaining a modification which gives the required contrast. A short rinsing time after EPTA staining turned out especially to be very crucial.