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R Cumming

Publications and source records attributed to R Cumming.

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

Characterisation of microtubule-associated proteins at the synapse: absence of MAP 2.

The high molecular weight microtubule-associated proteins MAP 1 and MAP 2 are major components of brain cytosol and can be readily identified using polyacrylamide gel electrophoresis on the basis of heat-stability and co-sedimentation with microtubules. An examination of synaptosomal cytosol, synaptic plasma membrane and postsynaptic density fractions showed that MAP 2 is absent from these fractions and thus from both pre- and postsynaptic sites. All of the fractions contained polypeptides that comigrated with MAP 1 and a MAP 1 like polypeptide was identified in a microtubule preparation from synaptosomal cytosol. The absence of MAP 2 from synaptosomal cytosol was confirmed by immunoblotting using an antibody directed against MAP 2. Immunocytochemistry using this antibody showed that MAP 2 was present in cell bodies and dendrites but absent from axons.

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PubMed1983

Axonal sub-populations in the central nervous system demonstrated using monoclonal antibodies against alpha-tubulin.

Using two monoclonal antibodies that specifically recognise alpha-tubulin we describe differences in their light and electron microscopic immunoperoxidase staining of axons in cerebellum, hippocampus, and cerebral cortex. In the molecular layer of the cerebellar cortex at the light microscopic level, one of the antibodies (YOL/34) labelled parallel fibre axons (in an identical manner to a beta-tubulin monoclonal antibody) while the other antibody (YL1/2) failed to label them. Extending these studies to the electron microscopic level in the cerebellum we have determined the sub-cellular localisation of alpha-tubulin in microtubules and the postsynaptic density, and also demonstrated a sub-population of parallel fibres and myelinated axons labelled with antibody YL1/2. The monoclonal antibodies were further characterised using immunoblotting against alpha-tubulin separated by isoelectric focusing gels. The results suggest that the contrasting staining patterns between the alpha-tubulin antibodies may reflect axonal sub-populations containing different isotypes of alpha-tubulin.

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PubMed1982

The enigma of microtubule coils in brain synaptosomes.

When synaptosomes are prepared from rat brain and incubated in Krebs solution, the presynaptic bulb develops a coil of microtubules (mts). Various considerations indicate that the coil does not have a cytoskeletal supportive function. Synaptosome coil mts show certain peculiarities, e.g. they thrive during incubation in Krebs solution (dendritic mts are depolymerized in Krebs solution) and they show no protofilament molecular substructure with tannic acid. Dendritic mts show clearly a 13 protofilament substructure when processed in the same way. Synaptosomal coil mts are sensitive to micromolar calcium and are depolymerized by treatment of the synaptosomes with veratridine or A23187. Our evidence indicates that coil mts of synaptosome and synaptic vesicle clothed mts of 'intact' albumin-treated synapses are different morphological and functional entities. As mentioned above, the function of coil mts remains enigmatic, while the mts seen in albumin-treated synapses could well have a role in synaptic vesicle translocation.

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PubMed1982

Evidence for the presence of high-Mr microtubule-associated proteins and their Ca2+-dependent proteolysis in synaptosomal cytosol.

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of Mr 340 000 and 300 000. These latter polypeptides comigrated with high-Mr microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca2+-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the Mr 340 000 and 300 000 polypeptides was seen in synaptosomal cytosol at less than 10 microM free Ca2+.

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PubMed1982

A technique combining intracellular dye-marking, immunocytochemical identification and ultrastructural analysis of physiologically identified single neurons.

A new method which produces an insoluble osmophilic polymer within Lucifer Yellow-injected neurons has allowed us to develop a technique for the ultrastructural examination of electrophysiologically characterized, immunocytochemically identified single neurons. In this initial report, we examine the light- and electron-microscopic features of neurophysin-containing, pituitary-projecting neurons in the goldfish nucleus.

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PubMed1982

Post-natal ontogenesis of calmodulin and cyclic AMP-dependent protein kinase subunits in the Purkinje cell using immunofluorescence.

Specific immunofluorescent techniques were utilized to demonstrate the regulatory (RI and RII) and catalytic (C) subunits of cyclic AMP-dependent protein kinase, and calmodulin, in the rat cerebellar Purkinje cell during post-natal ontogenesis. Whereas these second messenger receptor proteins were not detectable at 5 days, an increase in staining intensity occurred from this time until adult levels and distribution were attained at 25 days. Differences in immunofluorescent staining were noted between these proteins during ontogenesis. The relationship of these immunocytochemical changes to synaptogenesis and cellular maturation are discussed, including possible interactions between cyclic AMP and calcium messenger systems.

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PubMed1982

Ultrastructural immunocytochemical characterization of isotocin, vasotocin and neurophysin neurons in the magnocellular preoptic nucleus of the goldfish.

We describe the ultrastructural localization of isotocin, vasotocin and neurophysin in the magnocellular preoptic nucleus of the goldfish. With the aid of immunocytochemical techniques, we see staining both in classical neurosecretory granules and in diffuse agranular form throughout somata and processes. Signs of cellular and synaptic interactions between chemically identified neurons include axon terminals which contain vasotocin immunoreactivity and membrane specializations (puncta adhaerentia) between adjacent somata. Our investigations provide an anatomical basis for neuroendocrine and neurotransmitter-like functions of peptidergic neurons in the teleost preoptic nucleus.

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PubMed1982

Light- and electron-microscopic characterization of electrophysiologically-identified, horseradish peroxidase-injected magnocellular neuroendocrine cells in goldfish preoptic nucleus.

We recorded intracellularly from neurons in the goldfish preoptic nucleus which were antidromically identified by electrical stimulation of the pituitary gland and marked by intracellular injection of horseradish peroxidase for subsequent localization. At the light-microscopic level, labeled neurons resembled profiles of Golgi-impregnated neurons and lay in the magnocellular portion of the preoptic nucleus. Densely labeled axons and dendrites projected to the lateral forebrain bundle, the medial forebrain bundle, fiber tracts in the preoptico-hypophysial tract, small blood vessels and capillaries, the ependymal lining of the third ventricle and toward the preoptic neurons. Occasionally, a lightly-labeled, large perikaryon lay adjacent to a large, heavily-labeled magnocellular neuron. Ultrastructural examination of these identified cells revealed dense reaction product in neuronal perikarya and processes. Heavily labeled perikarya had elaborate networks of endoplasmic reticulum, extensive Golgi apparatus, occasional somatic spines and infrequent axo-somatic contacts from unlabeled neurons. These labeled perikarya which were frequently in close somatic apposition with unlabeled profiles were sometimes adjacent to a large, lightly-labeled perikaryon. A thin glial sheath separated most labeled neurons and processes from brain capillary endothelium. Labeled dendrites had heavily labeled spines and axo-dendritic contacts from unlabeled neurons. Labeled axons abutted unlabeled-axons and -dendrites. Synaptic boutons innervating labeled structures always contained small clear synaptic vesicles and some boutons also contained large dense-core vesicles. These results demonstrate the complex connections of goldfish preoptic magnocellular neuroendocrine cells with other neurons, fiber systems, brain capillaries, ventricular ependyma and the pituitary and provide further support for non-endocrine as well as endocrine functions of magnocellular neurons.

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PubMed1981

Ultrastructural immunocytochemical localization of enkephalin in the goldfish preoptic nucleus.

This study describes the ultrastructural localization of the opioid peptide enkephalin (ENK) in the preoptic nucleus of the goldfish. Using immunocytochemical techniques, ENK could be seen in neurosecretory granules and throughout the cytoplasm of magnocellular neurons with an agranular distribution. ENK was also associated with small clear vesicles and large dense-core vesicles within certain axon terminals in the preoptic nucleus. In this report, we discuss the cellular and synaptic relationships of ENK neurons in the preoptic nucleus of the teleost.

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PubMed1981

Immunofluorescent localization of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase in the choroid plexus.

An immunofluorescent technique has demonstrated that tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase are localized within the cytoplasm of the epithelial cells of the choroid plexus. In all other cell types and regions of the central nervous system previously examined, however, these molecules have shown contrasting immunofluorescent localization. These results suggest that the interaction in the choroid plexus may be related to the specialized physiological functions of the neuroglial epithelial cell.

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PubMed1980

Selection of antibodies for the immunofluorescent localisation of cyclic GMP in the central nervous system.

Radioimmunoassay techniques have been used to determine why only a small number of antibodies against cyclic GMP are able to show immunofluorescent staining in the central nervous system. However, neither the titre, avidity nor specificity as determined in liquid-phase radioimmunoassay employing [3H]cyclic GMP, have been able to predict whether a given antibody will show specific staining, or account for the different sites of immunofluorescent localisation observed with these antibodies. These observations are discussed in terms of the populations of antibody molecules employed in the radioimmunoassay and immunofluorescent procedures.

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PubMed1979

Characterization of immunofluorescent cyclic GMP-positive fibres in the central nervous system.

The cyclic GMP immunofluorescent fibres in the rat central nervous system have been characterized as processes of fibrous astrocytes, on the basis of distribution and similarity to the localization of glial fibrillary acidic protein. The neuroglial localization of the nucleotide is discussed together with the surprising observation that these cyclic GMP positive fibres are absent from the central nervous system of the adult mouse.

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PubMed1977

Immunohistochemical localization of cyclic GMP in rat cerebellum.

The technique of cyclic nucleotide fluorescence immunohistochemistry has been applied for the specific localization of cyclic GMP in rat cerebellum. We report immunofluorescence associated with fibres and membranes, contrasting with previously reported cytoplasmic localization of cyclic AMP in different cell populations, using a similar technique. We have been unable to detect changes in cyclic GMP staining in response to post-mortem changes, harmaline and pentobarbitone administration. A role of cyclic GMP is suggested in membrane ion transport.

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