Granule Cell Membrane Research Articles

Evidence for the colocalization of the axonal mitogen for Schwann cells and oligodendrocytes

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodendrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemma-enriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma-enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is colocalized with the neuronal mitogen for Schwann cells.

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells.

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.

Distribution of Thy-1 in the avian nervous system: Immunohistochemical and absorption analyses with a monoclonal antibody

A monoclonal antibody against chicken Thy-1 has been used to study the histochemical localisation of Thy-1 in chicken nervous and lymphoid tissues and to quantitate the relative amounts of Thy-1 in different brain subregions, subcellular fractions and non-neural tissues. An indirect ELISA using chicken brain membranes as a target established that the highest levels of Thy-1 were present in chicken forebrain, followed by midbrain, brainstem, spinal cord, cerebellum, retina and sciatic nerve. Analyses of subcellular fractions of chicken forebrain revealed a generalised localisation of Thy-1 on membranes comprising both the junctional and extrajunctional components of the synaptosome. Consistent with this finding are the immunohistochemical studies on cryostat sections where Thy-1 was localised to certain axonal and synaptic regions of chicken nervous tissue. Strong monoclonal antibody binding was found in the molecular layer and white matter of chicken cerebellar sections with fibrous staining on the axons running through the granule cell layer. No continuous staining could be seen on the perikaryal membranes of Purkinje cells or granule cells and no staining was present within the cell bodies. The Bergmann glia of the cerebellum were Thy-1-negative. The monoclonal antibody showed preferential binding to the inner plexiform and optic fibre layers of the chicken retina, suggesting a retinal ganglion cell localisation for chicken Thy-1, as has been suggested for the rat and mouse homologues. Surprisingly the lymphocytes of both the bursa and thymus gland were Thy-1-negative, however some extracellular staining was observed of interlobular connective tissue of the bursa.

Pharmacologically distinct glutamate receptors on cerebellar granule cells

Cultured cerebellar granule cells were found to exhibit calcium-dependent release of 3H-D-aspartate when stimulated with excitatory amino acids. L-glutamate and L-aspartate were found to be potent stimulators of 3H-D-aspartate release, D-aspartate was weaker and only minor effects were seen with D-glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) and L-α-aminoadipate (L-αAA). It was also found that only L-glutamate and L-aspartate showed high affinity for the 3H-L-glutamate binding sites on granule cell membranes. Stimulation by L-glutamate of 3H-D-aspartate release could be blocked by various excitatory amino acid antagonists. From the relative potencies of agonists and antagonists on D-aspartate release it is suggested that cerebellar granule cells express functionally active glutamate receptors with pharmacological characteristics different from all known excitatory amino acid receptors.

Rectification of single GABA-gated chloride channels in adult hippocampal neurons

The properties of single chloride channels activated by gamma-aminobutyric acid (GABA) were investigated with hippocampal slices from adult guinea pigs. After the slices were treated with proteolytic enzymes, gigaseal recordings were made from excised patches of pyramidal or granule cell membranes. This newly developed preparation permits the application of patch-clamp techniques to the adult mammalian central nervous system. Guinea pig hippocampal slices were prepared in a conventional manner. Once prepared, the slices were treated with two different enzymes for brief periods and gently agitated. Slices generally split apart along the boundaries of the cell body regions, exposing neuronal somata. Standard patch-clamp techniques were used for the gigaseal recordings from excised patches. Solutions for both sides of the patches consisted of symmetrical concentrations of chloride, with all cation channels blocked. GABA at concentrations of 0.5-1.0 microM was added to the solution for the extracellular side of the patches. At transmembrane potentials negative to the chloride reversal potential (0 mV), the conductance through the GABA-gated chloride channels was approximately 20 pS. When the transmembrane potential was changed to positive values, the chloride conductance increased dramatically. For example, at +40 mV the conductance through the GABA-gated channels was almost 40 pS. Ramp-clamp commands were used to measure the current-voltage (I-V) relationship through single open channels. The open-channel I-V curves displayed outward rectification. The relationship between open-channel conductance and voltage could be fitted reasonably well by a single energy-barrier model for the channel, with the higher energy barrier placed near the cytoplasmic side of the membrane (at a fractional distance through the membrane of 0.73).(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in baclofen-sensitivity between CA3 neurons and granule cells of the guinea pig hippocampus in vitro

In the slice preparation of the guinea pig hippocampus, the effects of (±) baclofen added to the Krebs-Ringer solution on dentate granule cells and CA3 pyramidal cells were investigated by means of intracellular recording techniques. In a 10–25 μM concentration, baclofen reduces the inhibitory postsynaptic potentials of the granule cells evoked by electrical stimulation of the perforant path and hyperpolarizes the granule cell membrane slightly. The reduction of both, the excitatory and inhibitory postsynaptic potentials of CA3 pyramidal cells evoked by mossy fiber stimulation, however, is accompanied by a strong hyperpolarization and conductance increase. Further, repetitive discharges of granule cells elicited in the presence of the convulsant bicuculline (25 μM) are hardly affected by baclofen (50 μM), whereas those of CA3 neurons are blocked.

Neuronal transmission through hippocampal pathways dependent on behavior

1. In chronically prepared, freely moving rats, electrical stimulation was applied to the angular bundle, and responses were recorded extracellularly at a variety of sites in the ipsilateral hippocampal formation. At each recording site the stimulus-response relationship was tested during four different behavioral states. These were slow-wave sleep (SWS), REM sleep ( REM), and also two waking behaviors consisting of the still, alert condition (labeled SAL), and voluntary movement (AW theta). 2. Two varieties of evoked responses were recorded: those due to the synchronous firing of neuronal action potentials (EAPs) and those produced by excitatory synaptic activity (ESPs). The overall pattern of monosynaptic, di-, and trisynaptic responses found was similar in the rat to that found by Andersen et al. (3-5) in cat and rabbit. 3. When the trisynaptic EAP was recorded in CA1, the threshold was similar during all four behavioral states. However, suprathreshold stimuli evoked a greater response during SWS than during the other three states. The trisynaptic ESP was also greater during SWS. 4. Disynaptically, EAPs were recorded in CA3. These were greater in magnitude during SWS than during SAL, but were intermediate in mean amplitude during AWtheta and REM. Response variability was much greater during AWtheta and REM. 5. The monosynaptic EAP recorded in the upper blade of the dentate gyrus (DG) exhibited the same behaviorally correlated properties found disynaptically in CA3. 6. The monosynaptic ESP recorded in the DG, in contrast to the EAP, was greater in magnitude during SAL than during SWS. 7. The primary afferent volley was also recorded at high gain in the DG. The amplitude of this was found to be dependent solely on stimulus intensity and not on behavioral state. 8. The results are interpreted as suggesting that the granule cell membranes in the DG are relatively hyperpolarized during SAL compared with SWS as the result of either tonic excitatory bombardment occurring during SWS or tonic inhibitory bombardment during SAL.

The electron microscopic localization of cations to pancreatic islets of langerhans and their possible role in insulin secretion

Earlier antimonate techniques for electron microscopic localization of sodium were applied to isolated islets of Langerhans obtained by partial collagenase digestion of mouse and rabbit pancreas. Incubation of islets in low or high glucose media followed by fixation in either (a) glutaraldehyde-osmium-antimonate mixtures or (b) osmium-antimonate mixtures resulted in deposition of precipitation particles in association with organelles directly involved in insulin secretion: beta granules (saccules) and cell membranes. Precipitation particles increased at these two cell sites following glucose incubation. X-ray microprobe analysis of precipitation particles using the AEI-EMMA-4 analytical microscope revealed the presence of other ions in addition to sodium, confirming the nonspecific nature of the antimonate technique. The predominant cation associated with the beta granule saccule and cell membrane was calcium, and the increased precipitation following glucose stimulation suggests an active role of this cation in the insulin secretion process as proposed earlier by biochemical methods.