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Biomedical subjects

G R Dutton

Publications and source records attributed to G R Dutton.

At least 37 records · Page 2Linked to original sources
PubMed1983

Tetanus toxin inhibition of K+-stimulated [3H]GABA release from developing cell cultures of the rat cerebellum.

The effect of tetanus toxin pretreatment on K+-stimulated [3H]gamma-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K+-evoked [3H]gamma-aminobutyric acid release from 7- and 14-day-old cultures (approximately 80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.

Animals↗
PubMed1983

Induction of a stress protein in developing cell cultures of the rat cerebellum.

We examined the ability of developing cerebellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd2+ treatment. The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used. Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd2+ was effective only in cultures at 7 days of age and older. These findings are discussed with reference to the development of various cell types in these cultures.

Animals↗
PubMed1982

Autoreceptors modify the evoked release of [3H]GABA from cerebellar neurons in dissociated cell culture.

We investigated the effect of GABA, muscimol and THIP on the K+ -stimulated and spontaneous release of [3H]GABA from neuron-enriched cell cultures of the rat cerebellum. Each agonist produced significant reductions in evoked [3H]GABA without causing marked changes in spontaneous release. The agonist-induced inhibition of K+ -stimulated [3H]GABA release was reversed by the GABA antagonists bicuculline and picrotoxin. It is suggested that GABAergic neurons in cerebellar cell cultures possess GABA receptors which are involved in the regulation of evoked transmitter release.

Animals↗
PubMed1982

Development of Thy-1 antigen on cerebellar neurons in culture.

The Thy-1.1 cell surface antigen was demonstrated by indirect immunofluorescence on two types of neurons in dissociated cell cultures of developing rat cerebellum. In cultures from postnatal rats, small cells predominated. They bound tetanus toxin, a neuronal marker, and did not have the capacity to take up gamma-aminobutyric acid (GABA) as measured by autoradiography. From these properties, we conclude that they are granule cells. These neurons began to express the surface antigen Thy-1.1 by 2 weeks in culture, and by 4 weeks, the antigen was detected on about 40% of all of the surviving neurons. The second type of neuron, which may be a heterogeneous mixture of cerebellar neurons, was numerous only in cultures prepared from embryonic rats. They were larger than granule cells and expressed Thy-1.1 antigen after a few days in culture. Such cells bound tetanus toxin and were negative for the glial markers galactocerebroside and glial fibrillary acidic protein. Thy-1-positive cells and cells with high GABA uptake were most frequent in embryonic day 19 cultures, where 40 to 50% of all of the neurons were positive for either property. They survived about 1 week in culture. The size and frequency of Thy-1 cells and the frequency of similar cells with high GABA uptake suggest that the embryonic rat cultures included many Purkinje cells, which express Thy-1.

Animals↗
PubMed1981

Potassium-stimulated, calcium-dependent release of [3H]GABA from neuron- and glia-enriched cultures of cells dissociated from rat cerebellum.

The release of [3H]GABA from cultures of cells dissociated from the rat cerebellum was investigated. The culture system contained a population of neurons (stellate and basket cells) which were capable of accumulating [3H]GABA and releasing it in a calcium-dependent manner in response to 50 mM potassium. In addition, the release of [3H]GABA for cultured astrocytes was found to be insensitive to potassium depolarization.

Animals↗
PubMed1981

An improved method for the bulk isolation of viable perikarya from postnatal cerebellum.

A modified method for the isolation of large numbers of viable perikarya from postnatally developing rat cerebellum is described in comparison with an earlier published version. Advantages of the modified preparation include greater cell yields which emphasize the preservation of the larger sized perikarya (inhibitory interneurons and Purkinje cells). In addition, the use of cytotoxic EDTA is eliminated, and by combining several steps and using a simpler buffer system, the preparation time is reduced by half to less than 1 h.

Animals↗
PubMed1981

Microvessels isolated from rat brain: localization of astrocyte processes by immunohistochemical techniques.

Microvascular networks were isolated from rat telencephalon by density gradient centrifugation. The vessels prepared were characterized morphologically at the light and electron microscope level and immunohistochemically by the localization of glutamine synthetase and glial fibrillary acidic protein, two proteins found almost exclusively in astrocytes. The vast majority of the vessels prepared contained more than just endothelial cells surrounded by a basement membrane. Many arterioles were found still retaining their smooth muscle cells. Pericytes were found in association with most of the venules and many of the capillaries. Astrocyte processes remained attached to most of the microvessels. These results show that vessels prepared from rat brain still maintain most of their complex intercellular contacts and must be viewed as a heterogeneous network of cells.

Animals↗
PubMed1980

[3H]GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb.

Uptake of [3H]GABA into cell cultures of rat cerebellum and olfactory bulb was studied by autoradiography, using beta-alanine and aminocyclohexane carboxylic acid to distinguish neuronal-specific and glial-specific uptake. Neurons and astrocytes were also labeled by tetanus toxin and anti-GFAP respectively. This combination of markers allowed identification and quantification of several cell types. Cerebellar cultures were found to contain 77% granule neurons, 7.5% inhibitory neurons (probably stellate and basket cells) and 15% astrocytes. Olfactory bulb cultures were over 50% in small neurons which accumulated GABA, the olfactory bulb granule neuron being GABAergic in vivo.

Animals↗
PubMed1979

Monolayer cultures of perikarya isolated from postnatal rat cerebellum.

Viable cerebellar perikarya of mixed cell type obtained from 7--9-day-old rats were maintained in monolayer cutlure for up to 12 days. During this time extensive neurite formation and outgrowth occurred. The large majority of the cells developing in culture were tentatively identified as granule neurons. This identification was based on the large number isolated from the starting tissue, and the cells' general morphological features in culture such as perikaryal and nuclear size, the bipolar nature of neurite extension, and their migratory behaviour.

Animals↗
PubMed1978

Separation of cell types from the developing cerebellum.

The heterogenous population of perikarya (cells) obtained by dissociating cerebellar tissue of developing postnatal rats was separated by sedimentation at unit gravity. Peak fractions, defined by monitoring the distribution of different size classes with a particle analyzer, were enriched in ultrastructurally well-preserved and metabolically competent specific cell types. These fractions included the peak of rapidly sedimenting cells comprising large neurones, such as Purkinje cells, which accounted for about 50% of the cells (vs. 2% in the initial cell suspension) and for a much greater proportion of the total cell mass (over 80%). More slowly sedimenting peak fractions contained 2-5-fold enrichments in replicating cells (assessed in terms of [3H]thymidine incorporation into DNA or by the frequency of mitotic cells), astroglia-like cells and external and differentiating granule cells, respectively. Separated, replicating cells continued synthesizing DNA in vitro; the [3H]thymidine incorporation rate was about 5-fold greater in the fraction enriched in proliferating cells than in the total cell suspension. Besides their structural integrity, the viability of the cells was also indicated by the finding that the proportion of trypan blue-excluding cells in all fractions exceeded 80%. Moreover, protein synthesis, in terms of incorporation of labelled amino acids, continued in the separated cells at a linear rate for a relatively long time. The rate per cell was highest in the large neuronal fraction, and lowest in the astroglia-like fraction.

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PubMed1976

Preparation of cell bodies from the developing cerebellum: structural and metabolic integrity of the isolated cells.

A method is described for the isolation from the developing rat cerebellum of cell bodies which display a high level of ultrastructural organization. The procedure, which utilizes isotonic conditions throughout, begins with a brief trypsinisation at low enzyme concentration (0.025%). Proteolysis is terminated by trypsin inhibitor and followed by short exposure to EDTA. The technique is effective with cerebella from rats up to 2 weeks after birth. Recoveries of cell bodies vary from 130-410 million/g wet weight of tissue, depending on age: this represents, in terms of recovered DNA, a mean value for yield of 33%. Suspensions contain little debris, free nuclei are rare and about 80% of the perikarya excludes trypan blue. Survey electron micrographs show that most cell bodies possess uninterrupted plasma membrane profiles and retain highly organised cytoplasmic and nuclear ultrastructure. Structural preservation is highlighted in the case of Purkinje cell bodies in which may characteristic features survive including, most notably, perisomatic spines. Metabolic integrity appears to parallel morphological preservation as judged by several functional criteria, including the ability to metabolise glucose, accumulate K+ ions and synthesize proteins. SDS-polyacrylamide gel electrophoresis shows that the tissue dossociation technique does not lead to major deletions of cell proteins and that the pattern of perikaryal protein synthesis in vitro closely resembles that in vivo. These perikaryal preparations therefore hold out great promise as a simplified system for metabolic studies and as a starting material for the derivation of purified sub-populations of cell bodies from developing cerebellum.

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