Takehiko Amano

Optical monitoring of excitatory synaptic activity between cultured hippocampal neurons by a multi-site Ca2+ fluorometry

The fluorescence intensities from cultured hippocampal neurons loaded with a Ca2+-indicator (fura-2), showed spontaneous, periodical oscillations, which were synchronized among the cells. Tetrodotoxin or 2-aminophosphonovalerate, a glutamate-receptor antagonist, blocked the oscillation. The rising phase of the fluorescence was accompanied by a burst of inward currents, as monitored by a patch electrode. It is thus suggested that the fluorescence elevation represents an increase in intracellular Ca2+ concentration accompanied by excitation of neurons which formed synaptic connections between each other. This method for detection of neuronal activity including synaptic excitation escapes from the limitations of the conventional technique employing microelectrodes.

Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

A 50‐base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Production of nerve growth factor in rat skeletal muscle.

Production of the nerve growth factor (NGF) was confirmed by Northern blot hybridization using a specific probe of synthetic cDNA. In normal rat skeletal muscle, this probe clearly showed a band equivalent to 1.3 kilobases (kb) of messenger RNA (mRNA) of NGF of male mouse submaxillary gland. By denervation, the density of the bands derived from muscles increased by a factor of more than 3 at 4 and 6 days later compared to the control. The synthesis of mRNA of NGF in muscle was also confirmed following tetrodotoxin (TTX) blockade of sciatic nerve without denervation.

Sexual dimorphism in α-bungarotoxin binding capacity in the mouse amygdala

A sex difference in alpha-bungarotoxin binding capacity in the mouse amygdala has been demonstrated by quantitative light microscopic autoradiography. The difference persisted even under widely different steroid-hormonal environment. In addition, it was observed that the binding capacities in both sexes were reversibly activated by administration of either testosterone or estradiol. Neonatal castration, on the other hand, permanently altered the toxin binding capacity in the adult male mouse. These data suggest the possibility that neonatal sex steroids irreversibly modify the cholinergic nicotinic mechanism in the developing mouse amygdala, while the hormones reversibly modulate the mechanism when applied in adulthood.

A mouse neuroblastoma × rat glioma hybrid cell produces immunoreactive substance P-like material

A new hybrid cell line, designated NG115-401, produced a significant amount of immunoreactive (IR) substance P-like material. The production was measured by radioimmunoassay using specific antisera raised to substance P. The production rate was dependent upon cell growth, and maximum production occurred at the same stage (late logarithmic stage) as the time of appearance of choline acetyltransferase activity, one of the characteristic neuronal properties of the cells. On the other hand, no IR-substance P was found in hybrid cells other than NG115-401, i.e. NG115-301 and NG115-303 cells, which were derived from the same parent mouse neuroblastoma N115TG-2 cells. Furthermore, in an attempt to survey IR-substance P production in several clonal cells, consisting of some mouse neuroblastoma cells and a rat glioma cell, and several of their hybrid cells, we could find no cell line producing a significant amount of IR-substance P except for NG115-401 hybrid cells. The cellular production of IR-substance P in NG115-401 hybrid cells was also confirmed by detecting the material in all 14 subpopulations of this hybrid cells. Partial characterization of IR-substance P-like material produced by NG115-401 hybrid cells was carried out by its application to gel-filtration column chromatography. Only high molecular weight material (approximately 26,000) appeared in the column eluates.

Serotonin-receptor coupled with membrane electrogenesis in a rat glioma clone

The electrophysiological consequences of serotonin application to glial cells in culture are described. Membrane hyperpolarization due to K+ permeability-increment was induced by serotonin in rat clonal glioma (C6-BU-1) cells. Similar response was recognized in some of rat cerebral cortical glial cells. Pharmacological examinations in the C6-BU-1 glioma cells suggested a participation of serotonin-receptors sharing properties known in neuronal receptors.

Intracellular Ca2+ elevation induced by a neurotransmitter in a glial cell clone

By fluorometry using a Ca2+-indicator Quin 2, we found an elevation of intracellular Ca2+ concentration ([Ca2+]i) in response to an application of serotonin in a rat clonal glial cell (C6BU-1). The [Ca2+]i rise depended on the dose of applied serotonin and the level of environmental Ca2+. The possibility was suggested that neuron-glia interactions might be controlled by a receptor-coupled [Ca2+]i-regulation system.

A glycoprotein resembling a peripheral nicotinic acetylcholine receptor that binds [125I]-α-bungarotoxin in mouse brain

A protein that binds alpha-bungarotoxin (alpha-BGT) was found in mouse brain and purified by means of Sepharose 6B column chromatography. The binding was inhibited by d-tubocurarine but not by atropine. This component was a glycoprotein with a molecular weight of 7 x 10(5) determined by gel chromatography, an apparent S value of approximately 11 determined by sucrose density gradient centrifugation and an isoelectric point of 5.6. The results showed that the component resembled a nicotinic acetylcholine receptor (AChR) in skeletal muscle and in electric organs.

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation.

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.

Subunit Structure of α‐Bungarotoxin Binding Component in Mouse Brain

Abstract: The α‐bungarotoxin binding component in mouse brain was purified by affinity chromatography with toxin‐Sepharose, gel‐chromatography on Sepharose 6B, and ion‐exchange chromatography with DE52 resin. The iodinated product of the last step produced one major and one minor band on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The molecular weight of the minor peak was twice as large as that of the major one. The iodinated product could bind α‐bungarotoxin, and this binding was inhibited by a nicotinic antagonist, d‐tubocurarine, which demonstrated that the iodinated product was a true α‐bungarotoxin binding component. The molecular structure of the product was analysed by cross‐linking followed by SDS‐PAGE. The results fitted the model for an α‐bungarotoxin binding component in the mouse brain composed of six identical or very similar subunits of 51,000‐52,000. One subunit carrying the binding site for toxin bound one molecule of toxin. This subunit structure of an α‐bungarotoxin binding component in the brain is discussed in comparison with that of a nicotinic acetylcholine receptor in the electric organ.