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Biomedical subjects

M M Bird

Publications and source records attributed to M M Bird.

At least 19 recordsLinked to original sources

Patterns of expression of brain-derived neurotrophic factor and tyrosine kinase B mRNAs and distribution and ultrastructural localization of their proteins in the visual pathway of the adult rat.

We have examined the cellular and subcellular distribution and the patterns of expression of brain-derived neurotrophic factor (BDNF), and of its high affinity receptor, tyrosine kinase B (TrkB), in retinorecipient regions of the brain, including the superior colliculus, the lateral geniculate nucleus and the olivary pretectal nucleus. In the retinorecipient layers of the superior colliculus, BDNF protein and mRNA were present in the cell bodies of a subpopulation of neurons, and BDNF protein was present in the neuropil as punctate or fiber-like structures. In the lateral geniculate nucleus, however, BDNF mRNA was not detected, and BDNF protein was restricted to punctate and fiber-like structures in the neuropil, especially in the most superficial part of the dorsal lateral geniculate nucleus, just below the optic tract. At the ultrastructural level, BDNF protein was localized predominantly to axon terminals containing round synaptic vesicles and pale mitochondria with irregular cristae, which made asymmetric (Gray type I) synaptic specializations (R-boutons). Enucleation of one eye was followed by loss of BDNF immunoreactivity and disappearance of BDNF-positive R-boutons in the contralateral visual centers, confirming the retinal origin of at least most of these terminals. TrkB was present in postsynaptic densities apposed to immunoreactive R-boutons in the superior colliculus and lateral geniculate nucleus, and was also associated with axonal and dendritic microtubules. These findings suggest that BDNF is synthesized by a subpopulation of retinal ganglion cells and axonally transported to visual centers where this neurotrophin is assumed to play important roles in visual system maintenance and/or in modulating the excitatory retinal input to neurons in these centers.

Animals↗

Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils.

Neutrophils play a key role in host-defence mechanisms against invading pathogens, using their capacity to migrate, engulf micro-organisms and produce toxic radicals. Protein kinase C (PKC) isotypes are important intracellular regulators of these processes in neutrophils. PKC isotypes themselves are controlled by interactions with lipids, Ca(2+) and proteins. The C2-like domain of PKC-delta (deltaC2) has been identified as a protein-interaction domain in this PKC isotype. In the present paper we have investigated the contribution of protein interactions at this domain to the regulation/function of PKC-delta in neutrophils. Using affinity chromatography we identified actin as a deltaC2 binding partner in these cells. Fluorescein-labelled deltaC2, microinjected into immobilized neutrophils, interacts with filamentous actin (F-actin) inside the cell. PKC-delta co-localizes with F-actin in neutrophils, in lamellipodia at the leading edge of the cell. Stimulation with phorbol ester or IgG-opsonized Staphylococcus aureus results in co-ordinated redistribution of PKC-delta and F-actin, and a PKC-delta inhibitor inhibits these changes. Microinjection of deltaC2 also inhibits F-actin redistribution. Thus PKC-delta binds to F-actin through its C2 domain, and these interactions are important in regulating actin redistribution in neutrophils.

Actins↗

The effect of calcium ionophore A23187 on neurites from embryonic mouse spinal cord explants in culture.

The calcium channel ionophore A23187 has been shown to cause a striking reduction in both the rate of neurite outgrowth and in growth cone motility; it has also been shown that these changes may be reversed when the ionophore is removed from the culture medium. The evidence supports the view that a specific concentration range of calcium is essential for outgrowth and motility and that calcium influences neuronal growth by an effect on actin structures within the growth cone. Using time-lapse video microscopy techniques, the effects of calcium ionophore A23187 on the rate of neurite outgrowth and growth cone motility in cultured embryonic mouse spinal cord neurons were examined. Concentrations in the range of 1-100 microM resulted in an inhibition of neuronal outgrowth, a loss of filopodia and a significant reduction in survival after seven hours. The results show that although the treatment with A23187 inhibited growth at all concentrations used, motility was inhibited only at the highest concentration indicating that the optimum calcium concentration for motility is higher than that for growth and that calcium mediates its effects on growth and motility at a very local level.

Animals↗

The effects of diethylcarbamazine on the morphology and ultrastructure of foetal mouse cerebellar neurons in culture.

This paper examines morphological and ultrastructural changes in foetal mouse cerebellar neurons treated with a culture medium containing diethylcarbamazine (DEC), a widely used anti-parasitic drug known to interfere with vesicle transfer to and from the Golgi apparatus. The effects of DEC were drastic, with the complete disorganization of the Golgi apparatus and the appearance of large vacuoles within the cytoplasm. Quantitative data comparing normal and DEC-treated neurons in culture demonstrated significant differences between the length of Golgi cisternae and numbers of clear vesicles and secretory vesicles. The evidence therefore suggests that the various synthetic and secretory activities involving these organelles are severely impaired.

Animals↗

Cellular uptake of the prion protein fragment PrP106-126 in vitro.

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP(c)). This pathological isoform (PrP(Sc)) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP(Sc) in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP(Sc)-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP(c) expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP(Sc) in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.

Animals↗

The effect of castanospermine on embryonic mouse cerebellar neurons in culture.

This study examines ultrastructural changes in explants of fetal mouse cerebellum following exposure to castanospermine, an inhibitor of N-linked glycosylation and therefore likely to interfere with cell-cell interactions in the developing nervous system. Explants were grown in castanospermine enriched medium for a period of 10 days and compared with identical explants maintained in control culture medium. Both control and treated explants contained many neurons and numerous synaptic profiles. A detailed analysis of 75 thin sections from each group of explants did however reveal highly significant differences between the two. The castanospermine-treated cells contained smaller numbers of cisternae in each Golgi stack; much larger numbers of secretory vesicles which were also substantially larger in size and four times as many coated vesicles per section as control cells. In addition, at 10 days in culture the first changes in the rough endoplasmic reticulum were observed, with individual profiles of reticulum occasionally associated with large vacuoles. Thus, although cerebellar neurons in culture are able to survive prolonged exposure to castanospermine, there are marked ultrastructural changes in subcellular organelles associated with glycoprotein synthesis, packaging and secretion.

Animals↗

Role of glutamate in the regulation of the outgrowth and motility of neurites from mouse spinal cord neurons in culture.

The excitatory amino acid glutamate has been shown to be toxic to a number of neuronal cell types both in vitro and in vivo. It has also been shown to be capable of controlling the development of neurons grown in vitro. Using time-lapse video microscopy techniques the effects of glutamate on the rate of neurite outgrowth and growth cone motility were examined on cultured mouse spinal cord neurons. Concentrations in the range of 1 to 100 microM caused a significant inhibition of neurite outgrowth and concentrations of 10 and 100 microM significantly inhibited growth cone activity. In addition it was shown that the kainate/AMPA receptor antagonist (+/-)3-(2-carbvoxypiperazin-4-yl)-propyl-l-phosphonic acid, but not the NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione, was capable of blocking the inhibitory actions of glutamate on both outgrowth and motility. These results show that, at least in the culture system employed, glutamate might have a role in regulating neuronal development and function.

Animals↗

The effects of exogenous nerve growth factor on foetal rat adrenal cells in culture.

Foetal rat adrenal cells, identified as chromaffin cells, maintained in culture in the presence of exogenous NGF, express a neuronal phenotype. Once this neuronal phenotype has been established bundles of outgrowing neurites commonly form links with adjacent reaggregates, and also develop synaptic contacts with the cell bodies of chromaffin cells in the adjacent reaggregates. Subsequent withdrawal of exogenous NGF from the culture medium did not result in the chromaffin cells losing their neuronal phenotype if this had been established before NGF was withdrawn. Tyrosine hydroxylase immunocytochemistry revealed that all neurites developed in culture were positive for the enzyme including those in cultures only transiently exposed to exogenous NGF. These results suggest that exogenous NGF may be essential only until the chromaffin cells develop neurites which make contact with appropriate target cells and that once these links, however immature, have been established, neurites are retained for many weeks in the absence of exogenous NGF.

Adrenal Glands↗

Changes in the amount and distribution of neuronal alkaline and acid phosphatase after chronic exposure of cultures of cingulate cortex to antidepressant drugs.

Enzyme histochemistry was used to examine alkaline and acid phosphatases in cultures of embryonic rat cingulate cortex after 14 days exposure in vitro to two tricyclic antidepressants (amitriptyline and desipramine) and two non-tricyclic antidepressants (mianserin and citalopram). An increased amount of acid phosphatase reaction product was observed in lysosomes of neurons in cultures treated chronically with the non-tricyclic antidepressants, mianserin or citalopram. More strikingly, reaction product was also present in the inner lamellae of the Golgi apparatus after this treatment, but never in controls. These observations suggest that non-tricyclic antidepressants significantly increase the rate of degradative processes in cingulate neurons. In cultures, treated chronically with desipramine or amitriptyline, pre- and postsynaptic membranes contained heavy deposits of alkaline phosphatase reaction product, whereas in control cultures not exposed to these drugs the corresponding membranes were entirely devoid of reaction product. An increase in the amount of alkaline phosphatase reaction product was also observed on the plasma membranes of neuronal cell bodies. These observations suggest that chronic exposure to antidepressants may influence transmembrane transport in cingulate neurons.

Acid Phosphatase↗

Changes in filipin-sterol binding in the rat cingulate cortex after the administration of antidepressant drugs. A freeze-fracture study.

The amount of cholesterol in cell membranes of cingulate cortex nerve cells from rats treated with Imipramine (tricyclic antidepressant drug) or mianserin (non-tricyclic antidepressant) was investigated using filipin-sterol binding and freeze-fracture. The number of filipin-sterol complexes decreased within membranes of neurons from cingulate cortex chronically treated with imipramine or mianserin. These results suggest that the decreased content of cholesterol causes the cell membrane to become more fluid. This increased fluidity of the cell membrane may play an important role in regulating the interaction of transmitters and drugs with their membrane receptors.

Animals↗

Glycoprotein expression in mouse cerebellum: effects of inhibitors of N-linked glycosylation.

1. Explants of cerebellum from foetal mouse were cultured in vitro for 10 days in the presence of one of 4 inhibitors of N-linked glycosylation (castanospermine, deoxymannojirimycin, swainsonine, and tunicamycin). 2. The effects of the inhibitors were compared with respect to: (a) the activity of enzymes involved in glycoprotein biosynthesis and degradation; (b) the expression of N-linked glycoproteins; (c) the morphology and ultrastructure of the treated cerebellar explants.

1-Deoxynojirimycin↗

Glycoprotein expression in mouse cerebellum: effects of inhibitors of vesicle function.

1. Explants of cerebellum from foetal mouse were cultured in vitro for 10 days in the presence of one of 5 inhibitors of vesicle function (ammonia, brefeldin A, carbonyl cyanide m-chlorophenylhydrazone, diethylcarbamazine, and monensin). 2. The effects of the inhibitors were compared with respect to: (a) the activity of enzymes involved in glycoprotein biosynthesis and degradation; (b) the expression of N-linked glycoproteins; (c) the morphology and ultrastructure of the cerebellar explants.

Ammonia↗

The long-term effects of taxol on explants of developing chick optic tectum in culture.

The long term effects on cellular ultrastructure and survival of exposure to the antimitotic drug taxol were investigated using explant cultures of embryonic chick optic tectum. After 7-8 days of continuous exposure the major changes were similar to those previously reported for shorter periods of exposure (Bird, 1984), i.e. the presence of increased numbers of microtubules in large bundles, associated with networks of filamentous cytoskeletal elements (inter-microtubule substance). With longer periods of exposure microtubule numbers increased further, the inter-microtubule substance became displaced towards the periphery of the microtubule bundles and filamentous cross-bridges between microtubules, other microtubules and membranous organelles became more numerous. Further microtubule accumulation, darkening of the cytoplasmic matrix, compression of endoplasmic cisterns and neuronal cell death were increasingly evident after 15 days and longer, and the numbers of synaptic profiles fell drastically. Few neurons survived beyond 28 days and return of cultures to normal medium after 7-14 days continuous exposure to taxol had limited effects on neuronal survival and none on synaptic profile numbers.

Alkaloids↗

Glycoprotein expression in foetal and adult mouse cerebellum.

1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.

Animals↗

The effects of various antidepressant drugs on the fine-structure of neurons of the cingulate cortex in culture.

We have examined the effects of antidepressant drugs on the morphology and fine-structure of cingulate cortex neurons and synapses in vitro. Dissociated cell cultures from 16-day-old rat fetuses were maintained for up to three weeks in the presence of amitriptyline and desipramine (tricyclic antidepressants) mianserin and citalopram (non-tricyclic antidepressants) or without drugs as controls. Synapses in cultures exposed to amitriptyline and desipramine displayed increased numbers of presynaptic vesicles and more extensive membrane specializations than in control cultures, and increased numbers of coated vesicles both pre- and postsynaptically. In mianserin- and citalopram-treated cultures, synaptic specializations were increased in length, and aggregates of mitochondria at the base of neurites were observed. Our results indicate that long-term exposure to antidepressant drugs results in significant changes in intracellular structure, including changes in synaptic ultrastructure.

Amitriptyline↗

Formation of synapses in cerebellar explants by axons from co-cultured medulla.

Explants of cerebellum from E12-E14 chick embryos were co-cultured with pieces of medulla from the same embryos for periods of up to 4 weeks and examined by EM with or without prior transection of the fibre bundles that formed rapidly between the co-cultured explants. The cerebellar explants developed a rich and complex neuropil within which a variety of presynaptic endings could be recognised, including some resembling climbing fibre endings and some mossy fibre endings. Four to six hours after transection of linking fibre bundles about 5% of preterminal and terminal profiles were undergoing intense degeneration indicating their origin from cell bodies in the adjacent explant of medulla and showing that most of the synaptic contacts are of intrinsic origin. The degenerating terminals contained spherical synaptic vesicles and made Gray Type I synaptic contacts. They appeared to be predominantly small-medium sized en passant terminals from long, thin, branched preterminal axons, and most closely resembled climbing fibre terminals.

Animals↗

Retinal axons innervate the embryonic chick diencephalon in an in-vivo-like pattern in explant co-cultures.

Explants of chick embryo diencephalon co-cultured with explants of retina display areas of complex neuropil containing large retinal-like endings which establish synaptic contact with conventional and presynaptic dendrites. Transection of fibre bundles linking retinal to diencephalic explants results in the degeneration of endings of this type, suggesting that axons of extrinsic (retinal) origin innervate the diencephalic explants in an in-vivo-like manner.

Animals↗