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T J Millar

Publications and source records attributed to T J Millar.

46 records · Page 3Linked to original sources
PubMed1985

Embryonic rat adrenal glands in organ culture: effects of dexamethasone, nerve growth factor and its antibodies on pheochromoblast differentiation.

In the present study we sought to determine the developmental potentialities and restrictions of adrenal medullary cells (pheochromoblasts) by investigating their morphological and biochemical response to nerve growth factor (NGF), anti-NGF antibodies and dexamethasone (DEX) after explantation into culture at different embryonic stages. With the exception of explants taken at embryonic day 15 (E 15) cultures of embryonic adrenal glands showed neurite outgrowth, which was not influenced by the addition of NGF, anti-NGF antibodies or DEX to the culture medium during the 4-day-culture period. Pheochromoblasts in E 17 + 4 explants showed spontaneous ultramorphological and biochemical maturation in terms of an increase in the number of catecholamine storage vesicles (CSVs) per micron 2 of cytoplasmic area, diameters of the cores of CSVs, percentages of electron-lucent cores of CSVs indicative of increased storage of adrenaline, overall catecholamine (CA) content and relative amount of adrenaline. NGF did not significantly affect this maturational process. Anti-NGF antibodies slightly decreased the proportion of adrenaline. The most pronounced maturation was seen in response to DEX and DEX plus NGF, although a maturational state equivalent to the E 21 stage was not achieved. E 21 + 4 explants showed neither spontaneous nor drug-induced biochemical maturation. Medullary cells in NGF-treated E 21 explants frequently retained the morphological features of pheochromoblasts. Treatment with anti-NGF antibodies significantly reduced the portion of adrenaline as compared to any other treatment. We conclude that under the culture conditions employed (1) a few pheochromoblasts spontaneously express a neuronal phenotype, (2) differentiation of pheochromoblasts towards chromaffin cells is enhanced by glucocorticoids but not by NGF, and (3) anti-NGF antibodies do not impair spontaneous neuritic growth and morphological maturation of pheochromoblasts, but cause a small reduction in the relative amount of adrenaline.

Adrenal Medulla↗
PubMed1984

Substance P in the chick retina: effects of light and dark.

Monoclonal antibodies against substance P were used to study the distribution of the peptide in the retinae of chicks that had been kept in either total darkness or brightly lit conditions. In retinae from light-adapted chicks, substance P was found to be in normal sized amacrine cells and giant cells in the inner nuclear layer, in fibres in sublaminae 1,3-5 in the inner plexiform layer, in cells in the ganglion cell layer which may have been mostly displaced amacrine cells and in a few fibres in the optic nerve fibre layer. There was no apparent concentration of immunoreactive cell bodies in any part of the retina. After dark-adaption of the birds, the immunoreactivity in the retinae was much reduced; there were fewer visible cells in all parts of the retina and there was no reaction in the inner plexiform layer. When birds were taken from the dark and kept in the light for different periods, there was a gradual increase in the number of cells and in the overall immunoreaction. Substance P is thus like several other putative retinal neurotransmitters in that its concentration in the retina is regulated by light/dark. These experiments do not indicate how the changes occur, but they underline the need for a strict control of lighting conditions in experiments with these relatively slowly metabolized neurotransmitters.

Animals↗
PubMed1984

Treatment of sections of chick retina with acetylcholinesterase increases the enkephalin and substance P immunoreactivity.

Frozen sections 10 microns thick were cut from the retina of chicks which had been kept either in total darkness or in a well lit room. The sections were incubated with acetylcholinesterase before antibodies to [Leu] enkephalin, substance P or somatostatin were applied. Sections of bovine adrenals were treated similarly but they were developed only with antibodies to [Leu]enkephalin. There were low numbers of immunoreactive amacrine cells and processes when any of the three antibodies were used on sections of dark-adapted retinae. When the sections were treated with acetylcholinesterase, however, the enkephalin-like and substance P-like immunoreactivity was enhanced while there was no effect on somatostatin. Counts of immunofluorescent cells indicated that the numbers had increased to levels like those found in light-adapted retinae. The adrenal also showed an enhanced enkephalin-like immunoreaction after treatment with the enzyme. Incubation with buffer alone or with enzyme together with 10 mM acetylcholine abolished the reaction. Acetylcholinesterase treatment of sections from light-adapted retinae had no discernible effect on the already high immunoreaction found using any of the three antisera. It is concluded that the peptidase activity of acetylcholinesterase has the capacity to hydrolyze proteins of which some may be the precursor molecules for the enkephalins and substance P. Since the amacrine cells that contain the enkephalin-like and the substance P-like immunoreactivity were found to contain acetylcholinesterase, it is possible that the action found here in vitro represents a physiological function of the enzyme. The immunoreactivity on which there was no effect, somatostatin, does not co-exist with acetylcholinesterase. A second conclusion that may be drawn from these data is that the dark-adapted retinae lose immunoreactive peptide because of the rate of processing; the results suggest that there is adequate precursor molecule available to maintain "control" levels.

Acetylcholinesterase↗
PubMed1984

The concentration of enkephalin-like material in the chick retina is light dependent.

The enkephalin-like immunoreactivity in the retina of chicks has been studied using immunohistochemical and radioimmunoassay techniques. The histochemical experiments showed that the immunoreactivity was confined to a subpopulation of amacrine cells in the inner nuclear layer which projected processes into sublaminae 1 and 3-5 of the inner plexiform layer. The distribution of the immunoreactivity was markedly influenced by the ambient lighting conditions: it was reduced in the dark and restored by a period in the light. The reactivity was lost from both cell soma in the inner nuclear layer and from the processes. Radioimmunoassays showed that the quantity of enkephalin-like material was reduced by more than 60% after 12 h in the dark. Attempts to entrain a rhythm by keeping chicks on 12/12 h light/dark cycles for up to 4 days were largely unsuccessful. A rhythm may have been partially entrainable, but the major factor involved was light. These results highlight the lability of the neuropeptide in the retina and the need for controlled lighting conditions in studies of this kind. They also indicate that this system may be a fruitful model to explore two important issues: (i) it could allow studies of neuropeptide metabolism in a physiologically intact system; (ii) the role of particular amacrine cells in visual processing could be determined by depleting them of their neurotransmitter/neuromodulator.

Animals↗
PubMed1983

A simple and cheap method for removing glass coverslips from neuronal cultures and relocating identified cells.

A simple and cheap method is described for removing glass coverslips from neuronal cultures grown on collagen and embedded into Epoxy resins for electron microscopy. The method involves scoring the coverslip with a scalpel and rubbing an ice block over the scored coverslip. A thin film of water then runs between the glass and the resin, which expands during cooling and causes the coverslip to spring away. This method is superior to the application of liquid nitrogen, which often causes damage of the embedded tissue.

Adrenal Medulla↗
PubMed1982

Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing's sarcoma with special reference to differential diagnosis from neuroblastoma.

The history of a 6-year-old girl with a tumor originating from thoracic spine and finally becoming resistant to surgery, radio-, and chemotherapy is reported. Tumor-biopsy material was studied by light and electron microscopy, in cell culture, by acetylcholinesterase ultracytochemistry, and by quantitative catecholamine analysis and this led to the rejection of the initial diagnosis of a neuroblastoma. Light microscopy revealed a uniform population of undifferentiated cells incompletely lobulated by broad fibrovascular septa. Using the electron microscope, cells were characterized by large intracellular pools of glycogen, little cytoplasm with an abundance of free ribosomes and a paucity of organelles. A few cells displayed desmosome-like attachment sites. Staining for specific and unspecific acetylcholinesterase was negative with light and electron microscopy, as were the results of catecholamine histofluorescence using the glyoxylic acid method. The latter result was confirmed by the negative outcome of quantitative analyses of dopamine, noradrenaline, and adrenaline with high pressure liquid chromatography nd electrochemical detection in tissue samples. Tumor cells could easily be maintained in culture for up to 4 weeks. None of a variety of treatments that are known to favor expression of neuronal characteristics in neuroblastoma cells (serum withdrawal, nerve growth factor, dbcAMP, dexamethasone) induced morphological differentiation in cultured tumor cells. On the basis of the clinical history, morphology, and of our experiments with tumor cells, the diagnosis of a so-called extraskeletal Ewing's sarcoma is most likely. Our results strengthen the view that a cell biology approach may be valuable in neuroblastoma differential diagnosis.

Acetylcholinesterase↗
PubMed1982

Nerve growth factor requirement of postnatal rat adrenal medullary cells in vitro for survival, aggregate formation and maintenance of extended neurites.

Rat adrenal medullary cells, taken at different postnatal stages (1, 4, 10 and 14 days), were cultured in serum-containing medium with and without nerve growth factor (NGF 2.5 S, 50 ng/ml) to investigate their dependence on NGF for survival and maintenance of extended neurites. It was found that medullary cells depend on NGF for survival at day 1 and, to a lesser extent, at day 4. Neurite-like processes extended in response to NGF treatment breakdown when NGF is withdrawn. NGF-treated adrenal medullary cells displayed a pronounced tendency to form aggregates. It was also shown that plating adrenal medullary cells at higher cell densities, which largely increased the numbers of fibroblast-like cells present, may substitute for NGF with respect to survival.

Adrenal Medulla↗
PubMed1981

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat. Acetylcholinesterase as a means for early discrimination of cell types.

The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80-120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eight postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed. By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining with the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative. It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.

Acetylcholinesterase↗
PubMed1981

Ultrastructure of growth cones formed by isolated rat adrenal medullary chromaffin cells in vitro after treatment with nerve growth factor.

Growth cones formed by adrenal chromaffin cells from young postnatal rats cultured in the presence of nerve growth factor (NGF) were studied at an electron microscopic level. These growth cones are similar in many respects to those formed by sympathetic neurons in vitro supporting the view that NGF-treated chromaffin cells may undergo neuronal transdifferentiation.

Adrenal Medulla↗