Biomedical subjects

R W Burry

Publications and source records attributed to R W Burry.

At least 37 records · Page 2Linked to original sources

PubMed1990

A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.

The availability of 1-nm gold particles permits the use of a particulate label with standard pre-embedding electron microscopic immunocytochemical techniques. We have employed these particles to localize a synaptic vesicle protein, p65, and a growth-associated protein, GAP-43, in neuron cell cultures. To be detected by standard transmission electron microscopy, these ultra-small gold particles must be enlarged. We have applied a commercially available silver development kit (IntenseM), the method of Danscher, and a neutral pH development procedure which we developed to effect this enlargement. Although IntenseM permits development with good preservation of morphology, it is limited by lack of reproducibility and by variability of final particle size. The method of Danscher provides well-controlled and reproducible enlargement, but is limited with respect to preservation of ultrastructural details. The neutral pH development procedure reproducibly enlarges gold particles with superior preservation of morphology. The use of this development procedure in conjunction with 1-nm gold probes should permit precise ultrastructural localization of a variety of intracellular antigens.

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PubMed1989

Highly basic 30- and 32-kilodalton proteins associated with synapse formation on polylysine-coated beads in enriched neuronal cell cultures.

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.

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PubMed1988

Serum and fibroblast growth factor stimulate quiescent astrocytes to re-enter the cell cycle.

An in vitro model was used to study the cytokinetics of astroglial cells derived from neonatal rat cerebellum. Confluent monolayers of astrocytes (85% astroglial as assessed by GFAP immunoreactivity) were subcultured at low cell density and after 2-3 days growth were rendered quiescent by shifting them to low serum medium (0.25%) for several days. Cells could be stimulated to re-enter the proliferative compartment by challenging them with high concentrations of fetal bovine serum (5-10% FBS) or fibroblast growth factor (FGF). FGF added alone at a concentration of 25 ng/ml caused quiescent astrocytes to re-enter the cell cycle nearly as effectively as 5-10% serum. Moreover, when FGF (25 ng/ml) was combined with 0.5% serum there was a potentiation of the mitogenic effect seen with FGF alone. This synchronization scheme is an important tool for continuing studies of the growth factor and hormonal requirements for astroglial cell proliferation and differentiation.

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PubMed1986

Development and elimination of presynaptic elements on polylysine-coated beads implanted in neonatal rat cerebellum.

Polylysine-coated sepharose beads were implanted in the cerebellum of neonatal rats and examined at 3 hr, 3 days, 7 days, 14 days, and 21 days after surgery. Previous studies at 5 or 8 days after implantation showed that axons formed neuronal swellings that appeared to be presynaptic elements, with the bead surface in the position of a postsynaptic element. Results reported here show that no beads at 3 hr had presynaptic elements, whereas the number of beads with presynaptic elements increased to high levels at 3 and 7 days but dropped to low values at 14 and 21 days after implantation. Presynaptic elements were seen on beads regardless of their distance from cerebellar tissue except at 3 hr, when no axons were seen in the implant, indicating that axons first grew into the implant and then formed presynaptic elements. The morphological measurements of presynaptic elements on beads at 3 to 7 days after implantation showed increases in area and number of synaptic vesicles, which then decreased at 14 and 21 days after implantation. These results show that axons can grow into implants of polylysine-coated beads and form presynaptic elements that do not survive with increased time after implantation. The survival of presynaptic elements on beads can be used as a model for investigations into regeneration of axons and presynaptic elements in the injured brain.

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PubMed1986

Presynaptic elements formed on polylysine-coated beads contain synaptic vesicle antigens.

Cell cultures of the rat cerebellum were immunostained with antibodies to synaptic vesicle antigens, Synapsin I and SV48. Light microscopic immunocytochemistry showed that the initial appearance of demonstrable SV48 and Synapsin I immunoreactivity occurred at different times. Synapsin I immunostaining, unlike SV48 immunostaining, was first seen at 3 days in vitro as occasional punctate immunofluorescence in neurites, while SV48 immunostaining was first seen at 5 days in vitro. Both SV48 and Synapsin I punctate immunostaining became frequent at 7 days in vitro. Double labelling experiments showed coexistence of the above proteins in punctate swellings and growth cones. Using the electron microscope, either SV48 or Synapsin I immunostaining was demonstrated within presynaptic elements in the neuropil. When cultures were incubated with polylysine-coated beads, both types of immunostaining were found in the vesicle containing presynaptic elements formed on the bead surface. It is concluded that Synapsin I and SV48 are co-localized in the same populations of presynaptic elements, co-localized in some growth cones and found in presynaptic elements on beads.

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PubMed1986

Presynaptic elements on artificial surfaces. A model for the study of development and regeneration of synapses.

Recently a model has been developed to study the synapse formation in which the components of a synapse can be isolated and examined independently. The observation of neurites forming presynaptic elements on polylysine-coated surfaces is a model for which the formation of presynaptic elements can be studied independently of a cellular postsynaptic element. Studies with neurons from both cell cultures and the intact cerebellum have shown that beads coated with poly-basic proteins can serve as a "postsynaptic element." With use of this system, observation have shown that the presynaptic element can form quickly, within 3 h, and contain many of the characteristics of a mature presynaptic element, such as synaptic vesicle antigens. Additional studies have shown that astrocytes appear to be involved in the loss or removal of the presynaptic elements on beads. Thus, synaptogenesis may involve the development of inappropriate synaptic contacts, which are eliminated by astrocytes. The lack of regeneration in the central nervous system (CNS) also may involve the astrocyte's ability to remove immature and/or inappropriate presynaptic elements and growth cones as they attempt to cross the lesion site.

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PubMed1985

Enhanced survival of apparent presynaptic elements on polylysine-coated beads by inhibition of non-neuronal cell proliferation.

Increased survival of presynaptic-like neuronal profiles was found in cell cultures of rat cerebellum when the non-neuronal cell numbers were reduced with an antimitotic drug. In both treated and untreated cell cultures, neurites grew onto the polylysine-coated surface of sepharose beads and formed a swelling. The neuronal swelling contained an accumulation of synaptic vesicles and a membrane density at the site of contact with the bead and was called an apparent presynaptic element. The apparent presynaptic elements in untreated cultures increased in number from the time the beads were added to the culture to 7 days incubation and then showed a decrease to one half the 7-day value at 14 days incubation. A 75% reduction in cell division of non-neuronal cells was seen in cultures exposed to a 5 X 10(-6)M cytosine arabinoside (Ara-C) for 2 days. Adding polylysine-coated beads to cultures treated with Ara-C showed at 14 days incubation a 7-fold increase in the number of apparent presynaptic elements as compared to untreated cultures. Additional experiments examined the numbers of neurites on the beads and found only small differences between treated and untreated cultures. A decrease, however, was shown in the number of glial fibrillary acidic protein staining astrocytes on the surface of the beads in treated cultures. The reduction of astrocytes by Ara-C appeared to enhance the survival of apparent presynaptic elements but did not enhance the growth of neurites. These results suggest that proliferating non-neuronal cells at a site of injury in the central nervous system may inhibit the formation of synaptic contacts and the growth of neurites through the site of injury.

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PubMed1985

Protein synthesis requirement for the formation of synaptic elements.

The formation of synapses in cell cultures of rat cerebellum was examined in the presence of the protein synthesis inhibitor cycloheximide. First, cell survival in the presence of 25 micrograms/ml cycloheximide was determined by phase contrast microscopy, trypan blue exclusion, total protein and uptake of [3H]gamma-aminobutyric acid (GABA). Neurons with 24 h incubation in cycloheximide appeared normal with little cell death, but by 48 h incubation the first signs of cell death were found. Some viable neurons were still found in cultures incubated continuously in cycloheximide for 72 h. Normally, the number of synapses seen in cerebellar cultures with the electron microscope shows an increase during the first several weeks in culture. However, the number of synapses in cultures treated with cycloheximide decreased, indicating that inhibition of protein synthesis at least partially inhibited synaptogenesis. Cycloheximide also inhibited the maintenance of synapses already formed as seen by the decrease in the number of synapses from the time the cycloheximide was added. To determine the sensitivity of the forming presynaptic element to cycloheximide, the development of apparent presynaptic elements was investigated. In cultures treated with polylysine-coated sepharose beads, neurites grew and formed apparent presynaptic elements with the bead taking the position of the postsynaptic element. Cultures pretreated with cycloheximide for 1 h followed by 24 h incubation with both cycloheximide and coated beads showed a normal number of apparent presynaptic elements. The first decrease in numbers was seen after 12 h preincubation and 12 h incubation with both cycloheximide and coated beads. Even after 72 h continuous incubation some apparent presynaptic elements could be formed although at reduced levels. Results presented here suggest that continuous protein synthesis is not necessary for the formation of the presynaptic element, but that active protein synthesis is required for neurons to form and maintain postsynaptic elements.

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PubMed1985

Glucocorticoid hormones inhibit DNA synthesis in glial cells cultured in chemically defined medium.

Primary cultures of glial cells from 2-day-old rat cerebellum were used to examine the growth control properties of steroid hormones. Immunocytochemical staining with antiglial fibrillary acidic protein (anti-GFAP) demonstrated that the cultures were highly enriched for astrocytes (90%). In an effort to avoid the potential influence of serum-borne steroids, cultures were switched from serum-supplemented to serum-free, chemically defined medium prior to experimentation. Assays for DNA synthesis used [3H]thymidine incorporation with either liquid scintillation counting of TCA-insoluble material or light microscopic autoradiography. Glial cells grown in serum-free, chemically defined medium (F12 basal medium supplemented with putrescine, selenium, insulin, transferrin, and BSA) replicated their DNA to a limited extent even in the absence of serum mitogens. When the glial cells were shifted to defined medium supplemented with various steroid hormones (corticosterone, dexamethasone, hydrocortisone, 17 beta-estradiol, progesterone, or testosterone) at a concentration of 10(-7) M, it was found that the glucocorticoids, corticosterone and dexamethasone inhibited synthesis of DNA by 49.6 and 56.9%, respectively. Hydrocortisone, another glucocorticoid, caused only a small reduction in DNA synthesis. The growth-controlling activity of the glucocorticoids was dose-dependent with concentrations of 10(-7) -10(-6) M showing maximal effect on DNA synthesis. These results suggest that physiological concentrations of glucocorticoid hormones may exert negative control over DNA synthesis of glial cells in the developing or injured central nervous system.

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PubMed1984

The distribution and synaptic organization of serotoninergic elements in the inferior olivary complex of the opossum.

Immunohistochemistry and high-resolution autoradiography were used to analyze the distribution and synaptic organization of serotonin (5HT) - containing elements in the inferior olivary complex of the opossum. Immunoreactive beaded varicosities are present throughout the olivary complex. The densest 5HT immunostaining is present in subnucleus b of the caudal medial accessory nucleus. The rostral principal olive is sparsely populated with immunoreactive elements. Fine beaded fibers arborize throughout the neuropil of all the olivary nuclei except in subnucleus b of the caudal medial accessory nucleus where they also circumscribe neuronal cell bodies. In addition, a distinct population of large beaded fibers are occasionally encountered in the neuropil of the medial accessory nucleus. Ultrastructurally, labeled profiles that correspond in size to the smaller beads (less than 1 micron) contain tubulovesicular elements, large dense-cored vesicles, and clear vesicles. In contrast, larger profiles (greater than 2 microns) are characterized by numerous clear synaptic vesicles. Synaptic junctions were encountered in only 2% of the labeled elements. The majority of the labeled profiles were in juxtaposition to small-diameter dendrites (less than 2 microns) except in the caudal medial accessory nucleus, where they also were found in apposition to olivary cell bodies. Our results, when compared with other accounts, indicate that rather than major differences in the nuclear distribution of serotonin between species, there are differences in the density of serotoninergic elements in specific nuclei of the mammalian inferior olive. Based on the size of the labeled profiles and the distinct vesicle populations, our data suggest there are at least two populations of 5HT varicosities that are in juxtaposition to olivary neurons. Further, boutons containing 5HT primarily interact with the distal dendrites of olivary neurons except in the caudal medial accessory nucleus where cell bodies are in apposition to 5HT varicosities.

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PubMed1983

Postnatal rat neurons form apparent presynaptic elements on polylysine-coated beads in vivo.

Polylysine-coated sepharose beads implanted in the cerebellum of 3-4-day-old rats resulted in the formation of neuronal swellings on the surface of the beads. These neuronal swellings resembled presynaptic elements and contained numerous 40 nm vesicles that accumulated at the site of contact with the bead. Uncoated beads did not show apparent presynaptic elements on the surface of the beads. The in vivo finding of apparent presynaptic elements on polylysine-coated beads suggests that polybasic surfaces may be involved in recognition leading to the formation of presynaptic elements. This system offers an excellent model to test developing or regenerating axons to determine their ability to form presynaptic elements.

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PubMed1983

Antimitotic drugs that enhance neuronal survival in olfactory bulb cell cultures.

Cell cultures of olfactory bulb from 2-3-day-old rats were used to evaluate 6 antimitotic drugs for their effects on neuronal survival. In cultures grown in medium without drugs for 7 days, neurons died as a layer of non-neuronal cells proliferated. A series of experiments were performed to determine dilutions and exposure times that allow maximum neuronal survival for each of the antimitotic drugs. Use of bromodeoxyuridine (BUdR) at 10(-5) M with a 5 day exposure gave cultures with large aggregates of neurons and an extensive network of interconnecting neurites. Cytosine arabinoside (Ara-C) at 8 X 10(-6) M for 5 days of exposure was also effective but the aggregates contained cellular debris. Fluorodeoxyuridine (FUdR) at 10(-4) for 5 days was less effective in flowing neurons to survive. Methotrexate (MTX), high thymidine and hydroxyurea were all not effective in allowing neuronal survival. In all cases there was an inverse relationship between the survival of neurons and proliferation of non-neuronal cells. Additional experiments were performed to determine how proliferating non-neuronal cells can lead to the death of neurons. In a series of transfer experiments, cultures were exposed to conditioned medium. Cultured olfactory bulb neurons grown on small cover slips were exposed to BUdR under optimal conditions and after 7 days were transferred to cultures treated or not treated with BUdR. In cultures not treated with BUdR, most of the transferred BUdR treated neurons died, while in cultures treated with BUdR the BUdR treated neurons survived. These results suggest that antimitotics enhance olfactory bulb neuronal survival by reducing the number of non-neuronal cells. In addition, it appears that proliferating non-neuronal cells are responsible for neuronal cell death by a medium factor and not by contact with the dividing non-neuronal cells.

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PubMed1982

Development of apparent presynaptic elements formed in response to polylysine coated surfaces.

Formation of apparent presynaptic elements on polylysine-coated surfaces was examined with both scanning and transmission electron microscopy. Neurons from 2- or 8-day-old rat cerebellums were used in dissociated cell cultures as a source of growing neurites. An apparent presynaptic element is a defined swelling on a neurite with 40 nm diameter vesicles accumulated at the membrane which has a slight thickening. Apparent presynaptic elements do not have a normal postsynaptic element. In place of the postsynaptic element was the polylysine-coated surface of a large diameter Sepharose bead. The first apparent presynaptic elements were seen at 2 h of incubation, suggesting that morphologically identifiable synapses may form in this short a time. The number of apparent presynaptic elements on beads increased from 2 h to 24 h incubation and decreased from 5 to 9 days incubation. At all times non-neuronal cells grew up on to the beads and often covered both neurites and apparent presynaptic elements. In the longer incubations degenerating apparent presynaptic elements were seen engulfed by non-neuronal cells, suggesting that non-neuronal cells may have the ability to remove presynaptic elements that are not functioning. The number of synaptic vesicles per apparent presynaptic elements increased continuously between 2 h and 9 day incubation, eventually surpassing the number of synaptic vesicles seen in other presynaptic elements in cultures. This result suggests that an interaction between presynaptic and postsynaptic elements may be necessary to limit the number of synaptic vesicles found in presynaptic elements. Cultures grown for only 5 days in vitro (DIV) and incubated for 1 day with coated beads had the most apparent presynaptic elements, while those at 28 DIV and incubated for 1 day had the least. In the cultures at 5 DIV, neurons formed many apparent presynaptic elements, but these neurons could form only a very few normal synapses between themselves. Thus the beginning of the formation of synaptic contacts may depend on the availability of future postsynaptic elements and not on the lack of future presynaptic elements.

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PubMed1982

A unit density method of grain analysis used to identify GABAergic neurons for electron microscopic autoradiographs.

The distribution of electron microscopic autoradiographic grains over neurons in cerebellar cultures incubated with [3H]gamma-aminobutyric acid ([3H]GABA) was examined. With the unit density method of grain analysis, the number of grains over each structure was tested against the total grain density for the entire section. If an individual structure has a grain density higher than the expected grain density, it is considered one of the group of heavily labeled structures. The expected grain density for each structure is calculated based on the area for that structure, the total grain density and the Poisson distribution. A different expected grain density can be calculated for any P value required. The method provides an adequate population of structures for morphological analysis but excludes weakly labeled structures and thus may underestimate the number of labeled structures. The unit density method of grain analysis showed, as expected, a group of cell bodies and synapses that was labeled heavily. Cultures incubated with other [3H]amino acids did not have any heavily labeled synaptic elements. In addition, serial section analysis of sections showed that synapses heavily labeled with [3H]GABA are seen in adjacent section. The advantage of the unit density method of grain analysis is that it can be used to separate two groups of metabolically different neurons even when no morphological differences are present.

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PubMed1980

Formation of apparent presynaptic elements in response to poly-basic compounds.

Neuronal cell processes in dispersed cell culture grow out onto Sepharose beads coated with poly-basic proteins. Apparent presynaptic elements are formed by these neuronal processes with the bead serving as a postsynaptic element. It is suggested that electrostatic binding between poly-acidic groups on the presynaptic element and poly-basic troups on the postsynaptic element is one of the initial steps in the formation of a synaptic contact in culture.

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PubMed1980

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.

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PubMed1979

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.

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PubMed1978

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography.

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.

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