Ethanolic Phosphotungstic Acid Research Articles

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.

Chronic dietary choline modulates synaptic plasticity in the cerebellar glomeruli of aging mice

A morphometric investigation was carried out on ethanolic phosphotungstic acid (E-PTA) stained synaptic junctions in the cerebellar glomeruli of adult, old, old choline-deficient and old choline-supplemented mice. Numerical (Nv) and surface (Sv) density as well as average length ( L ) of the synapses were calculated on 100 pictures per group. A significant reduction of NV and SV, as well as an increase of L was found during aging. Choline deficient animals did not show any change as compared to old animals of the same age. In choline supplemented mice Nv and Sv were significantly increased and L significantly decreased, respectively, as compared to old control littermates. No differences was found between adult and choline supplemented mice. In the cerebellar glomeruli only a small fraction of fibers are cholinergic, therefore the present findings support the idea that dietary choline can influence systems other than cholinergic. The possible role of choline supplementation in the modulation of synaptic plasticity via the synthesis and/or turnover of neuronal membrane choline phospholipids, is discussed.

Age-related changes in the subiculum of Macaca mulatta: Synaptic density

Dendritic spines and synapses were quantitated in the subiculum of 12 Macaca mulatta from 7 to 29 years of age. Dendritic spines were analyzed in six dendritic foci of pyramidal neurons impregnated by the Golgi-Cox method. The dendritic foci examined were the apical shaft, apical oblique, apical tuft, apical terminal, basal terminal, and basal dendrites. Electron microscopic observations were made on synapses stained with ethanolic phosphotungstic acid. There was no difference in spine density between a group of young (7 to 12 years of age) and middle-aged (18 to 21 years) animals. The mean spine density was 1.09/μm for the young and 1.15/μm for the middle-aged animals. In contrast, the spine density of the old animals was 0.87/μm, which was significantly lower than the spine density found in the young and middle-aged animals. A loss of dendritic spines in the old animals occurred equally in all six dendritic foci. A decline in synaptic density in the aged animals were further evidenced by an ultrastructural analysis. The synaptic density of the old animals was 8.3 × 10 8/mm 3, significantly lower than 9.5 × 10 8/mm 3 and 10.2 × 10 8/mm 3 found in the young and middle-aged animals, respectively. This study on synaptic density demonstrated the decline in synaptic number as a function of age in M. mulatta.

The size and curvature of synapses in the cerebellar cortex of the cat.

At the supra Purkinje layer and the subpial level in the molecular layer of lobules V and VI of the cerebellar cortex of the cat, synaptic profiles were measured in ultrathin serial sections treated with either osmium tetroxide (OsO4) or ethanolic phosphotungstic acid (E-PTA). From trace and chord lengths of the intercepts of synaptic profiles the curvature and the mean caliper diameter of the synapse was calculated. In OsO4-material the curvature of synapses yielded an average angle of about 47 degrees and about 36 degrees in E-PTA material. Although these values are contrary to the assumption of a flat disc, which is commonly required in stereological procedures to estimate caliper diameter, the effect of the curvature on the estimation of the mean caliper diameter is limited. This is shown by serial reconstruction analysis of the largest diameter of synapses from maximal arc and chord length measurements at the subpial and supra Purkinje level. The results provide quantitative data concerning synaptic size, curvature and the frequency of intercepts per synapse at the subpial and supra Purkinje level in the cerebellar cortex in OsO4 and E-PTA material. In addition the advantages and disadvantages of E-PTA and osmiumtetroxide staining in quantitative analysis are discussed.

Ultrastructural investigation into the influence of ethanol on synaptic maturation in rat neocortex. II. Quantitative analysis.

Male rats were exposed to ethanol vapour daily from 3 days of age, and their brains were examined at 7, 14, 21, 56 and 76 days postnatally. Control animals were examined at each age, with ethanol-rehabilitated animals being examined at 56 and 76 postnatal days. Tissue, stained with osmium tetroxide and with ethanolic-phosphotungstic acid, from the parietal cortex of each animal was analyzed by quantitative ultrastructural techniques. Most of the statistically significant findings occurred at days 56 and 76. When compared with controls, ethanol-treated material at 56 days was characterized by a decrease in the number of synapses which were larger and more spherical, with a greater number of synaptic vesicles and a decrease in dense projections. At 76 days there was an increase in the percentage of axospinous-symmetrical terminals in the ethanol-treated population. In ethanol-rehabilitated tissue at 56 days the synaptic terminals were relatively immature, and were smaller, with a reduced number of synaptic vesicles coupled with an increase in large cisternae and with more positively curved junctions. By 76 days this material was practically indistinguishable from control tissue. It is proposed that at 56 days of age synaptic remodelling is under way in the ethanol-treated material, whereas following rehabilitation the terminals are relatively immature and appear to be actively functioning. Low to moderate levels of ethanol administered postnatally have a limited effect on synaptic maturation.

Ultrastructural Investigation into the Influence of Ethanol on Synaptic Maturation in Rat Neocortex

Male rats were exposed daily to ethanol vapour from 3 days of age, and their brains were examined at 7, 14, 21, 56 and 76 days postnatally. Control animals were examined at each age, and ethanol-rehabilitated animals were examined at 56 and 76 days postnatally. Tissue from the parietal cortex of each animal stained with osmium tetroxide and with ethanolic-phosphotungstic acid (E-PTA) was analyzed by qualitative ultrastructural techniques. The ethanol flow rate in the incubation chamber was adjusted to maintain the blood ethanol level as close as possible to 0.1 g/100 ml. Ethanol-treated rats weighed less than ethanol-rehabilitated animals at days 56 and 76. At day 7 synapses were formed between axons and dendritic growth cones, dendritic shafts and filopodia in control and ethanol-treated tissue. At days 14 and 21 well-developed axodendritic and axospinous synapses were evident in both groups. The neuropil of 56- and 76-day-old material was similar in the control and treatment groups, except that there was an enlargement of dendritic profiles in the 56-day-old ethanol-treated material. Perforated synapses were most common in 56-day-old ethanol-treated material, with degenerating synapses most common in 56-day-old (and to a lesser extent 76-day-old) ethanol-rehabilitated material. No obvious differences were detected between any of the groups of the E-PTA-stained material. The presence of degenerating and perforated synapses suggests that synaptic remodelling is occurring, and this may be a means of adapting synaptic mechanisms to the functional demands imposed by ethanol.

Synaptic density in chronic animals with experimental neurofibrillary changes

Synaptic density was quantitated in the cerebral cortex and subiculum of rabbits with experimental neurofibrillary changes. Animals were subjected to subcutaneous injection of aluminum tartrate for 90 days, and synapses stained with ethanolic phosphotungstic acid were analyzed in animals killed 100, 200, or 300 days postinjection with aluminum tartrate. A significant difference was found in synaptic density between animals injected with aluminum tartrate and their age-matched controls. This difference was a result of a low synaptic density present in animals killed 200 or 300 days postinjection of aluminum tartrate. In contrast, animals killed 100 days postinjection of aluminum revealed the same synaptic density as their control. The data suggest that the synaptic depopulation associated with experimental neurofibrillary changes is a gradual process, and such changes are demonstrable only long after the initial appearance of neurofibrillary changes.

Variations in presynaptic grid size in the granular and molecular layer of the cerebellar cortex of the cat: I. A quantitative ultrastructural study on semithin E-PTA sections

The size distribution of synapses in the cerebellar cortex of the cat was defined on 0.5-μm semithin sections stained with ethanolic phosphotungstic acid (E-PTA). The surface area (SA) of synaptic grids was measured and the number of dense projections per grid (NDP) was counted. The results show large differences in mean values between molecular and granular layer. Within the molecular layer the differences in mean values at different levels below the pial surface were small; however, the frequency distributions differed significantly. In the granular layer a confined unimodal frequency distribution of SA and NDP was observed (mean NDP, 7.02 ± 0.10), in the molecular layer a considerable variation in the size of the synaptic discs was observed (mean NDP, 2.40 ±_0.43). Only a small percentage of the synaptic discs have less than 5 or more than 47 DPs. The sharply defined differences in synaptic size between the granular and molecular layer and the smaller differences within the granular and molecular layer are discussed in the context of the congruity hypothesis of Chan-Palay 10.

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy.

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.

Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.

In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin.

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons.

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.

Fine structure and cytochemistry of the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi.

Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6-8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/microns2 of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.

The localization of Ca2+‐ATPase and Ca2+ binding proteins in the flagellum of guinea pig sperm

AbstractFerritin‐labeled antibody to calmodulin was localized in flagella of guinea pig sperm. Ferritin granules were present on the surface of the medial portion of the coarse fibers, and in the matrix immediately surrounding this segment of the fibers. The results demonstrate that calmodulin is confined to regions close to the tubulin‐dynein systems os the axoneme. A calcium‐dependent ATPase was localized in the same region by trapping the reaction product with cadmium. The presence together of calmodulin and the Ca2+‐ATPase close to the axonemal complex suggests that they function to control the concentration of Ca2+, which in turn may regulate the action of the dynein ATPase, and sperm motility. The remainder of the coarse fiber surface also contains specialized material as evidenced by intense staining with ethanolic phosphotungstic acid. These data suggest that the role of the coarse fibers is complex and may not be solely structural.

A quantitative study of visual cortex synapses during the postnatal development of dark-reared rats.

The method of Aghajanian and Bloom (1967) was applied to the visual cortex of normal and neonatally visually deprived rats. The rats were kept with their mothers in total darkness since birth, in a ventilated and temperature-controlled animal quarter. Controls were rats from the same stock, maintained in a regular 12-h-light/12-h-dark rhythm. The animals were killed at 15, 23, 40, and 65 days of age, and the visual cortices fixed in 4% glutaraldehyde and processed for electron micrography with ethanol-phosphotungstic acid. A total of 6249 synaptic profiles were counted and their numerical density (DS) determined in both conditions. Features of the presynaptic grid were used for classifying the synaptic profiles in: type A [with one presynaptic dense projection (PsDP)]; type B (with two or three PsDPS); and type C (with four or more PsDPS). In the visually deprived rats the DS increases with a rate similar to controls, but the values for each age are slightly lower (P less than 0.01). Type B synapses predominate in the visually deprived group while types A and C are scarcer. The differences found between types were maximal at 65 days of age and the results were highly significant (P less than 0.001). It is concluded that major effects of dark-rearing are manifested when the individual features of the presynaptic grid are considered. It seems that a selected population of synapses is affected by the alteration of the normal epigenetic influence of early visual experience.

The effect of acute and chronic centrophenoxine treatment on the synaptic plasticity of old rats

The cerebellar glomerulus was studied by electron microscopic morphometry in female Wistar rats. Age-dependent alterations have been revealed from 3 to 28 mth of age, and the effect of centrophenoxine (CPH) was analyzed in two different patterns of administration. First, 27-mth-old rats were treated daily for 6 wk (acute treatment), and second, 18-mth-old rats were treated 3 times per week for 5 months (chronic treatment). The dose was 100 mg CPH/kg body weight, injected intraperitoneally. The surface density ( S v), the numerical density ( N v) and the average length ( L ) of the synaptic junctions were calculated from data obtained on ethanol-phosphotungstic acid stained ultrathin sections. An age-dependent reduction of S v and N v of the synaptic contact zones was found, and the L increased in the oldest animals. CPH-treatment resulted in a marked increase of S v in both types of application, whereas the other two parameters behaved differently in the two groups. The chronic treatment resulted in a significant slowing down of the decrease of N v, whereas L remained invariate. On the contrary, the acute treatment increased L but did not alter significantly N v. The results and the differences between the treatment types are discussed in terms of synaptic plasticity and are interpreted as different manifestations of the same reactive synaptogenetic process.

The kinetochores of Caenorhabditis elegans.

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 micrometers in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18-0.2 micrometer in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 micrometer, and an electron lucent middle layer of 0.03 micrometer. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.

Tritrichomonas foetus: Ultrastructural localization of basic proteins and carbohydrates

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver proteinate technique was used to localize carbohydrates. With the AS technique reaction product was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa, in the hydrogenosomes, and at the region of the recurrent flagellum's adhesion to the cell body. Previous acetylation of the cells under conditions that block most free amino groups abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex, and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA techniques to detect basic proteins is based on observations made with T. foetus as well as with other cell types.

The effect of prenatal and postnatal lead exposure on neonatal synaptogenesis in rat cerebral cortex.

Pregnant rats were exposed to drinking water with lead (Pb) concentrations of 0, 30, or 200 mg/l. The resultant pups were sacrificed at 11, 15, and 21 d of postnatal age for the determination of synapses/mm3 in parietal cortex. Synaptic counts from electron micrographs of ethanol phosphotungstic acid stained cortical slices were counted by four observers who were blinded as to treatment (control or 200 mg Pb/l drinking water). A greater than fourfold increase in synaptic counts was observed in layers I, II, and III of rat pups parietal cortex between 11 and 21 d of age. Pb treatment depressed synaptic counts maximally at 15 d of age. However, Pb-exposed pups displayed essentially the same synaptic counts as controls by 21 d of age. In a cross-fostering design, it was shown that prenatal exposure to Pb completely accounted for the delays in synaptogenesis. No significant depression of synaptic counts was observed in pups exposed only during the postnatal period. Blood lead concentrations (Pb X B) were determined during gestation and suckling in both mother and offspring. A dramatic peripartum (partum plus and minus 4 d) peak in Pb X B was seen in mother and pup. Pup Pb X B peaked at 80 micrograms/dl at exposures of 200 mg Pb/l drinking water. In addition to being dose-dependent, blood Pb levels resulting from the same concentration of Pb in drinking water displayed a significant dependence on litter at time-points between birth and 1 yr of age. These data indicate that the substantially elevated blood Pb concentrations that are evident at partum in pups prenatally exposed to Pb might be responsible for the postnatally observed delay in synaptogenesis.

Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues.

A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25 degrees C) which is known to methylate and acetylate--respectively--free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30 degrees C) eliminated the UA staining without affecting that due to PTA. It is concluded that--COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.

Dense material associated with apparent presynaptic elements in cell cultures of the CNS

In cell cultures of the rat cerebellum, electron-dense material has been found occasionally between adjacent cells. More often than not, presynaptic elements on one side of the dense material faced either neuronal or nonneuronal cells on the other side. The 20 nm thick material was stained either with the osmium-uranyl-lead (OsUL) procedure or with the ethanolic phosphotungstic acid (E-PTA) procedure. To determine the source of the dense material, various compounds were added to cultures at 7 days in vitro. Only a crude nuclear fraction was able to duplicate the appearance of the dense material associated with the apparent presynaptic elements. It was concluded that apparent presynaptic elements were associated with the polybasic dense material and that this type of association may duplicate an interaction in the normal development of synaptic contacts.