Cytochemical localization of Ca(2+)-ATPases and demonstration of ATP-dependent calcium sequestration in giant smooth muscle fibres of Beroe.

A cytochemical analysis of the mechanisms underlying cytosolic calcium regulation was undertaken in the giant smooth muscle fibres of the marine invertebrate Beroe. The ability of the sarcoplasmic reticulum to accumulate Ca2+ was demonstrated on living skinned single cells. In the presence of oxalate, and physiological concentrations of Ca2+, calcium oxalate crystals were formed in the lumen of tubules and cisternae of the sarcoplasmic reticulum. The subcellular distribution of Ca(2+)-ATPase was studied with a cytochemical technique; a dense precipitate resulting from Ca(2+)-ATPase activity was found on the plasma membrane, on the membranes of tubules and cisternae of the sarcoplasmic reticulum, and in mitochondria.

Two distinct distribution patterns of sarcoplasmic reticulum in two functionally different giant smooth muscle cells of Beroe ovata.

The sarcoplasmic reticulum has been studied in radial and longitudinal giant smooth muscle fibres of the marine planktonic invertebrate Beroe. Impregnation with heavy metals has revealed that the smooth component is organised in a longitudinally oriented three-dimensional network of tubules running along the myofilaments. An ultrastructural morphometric analysis has shown that the relative volume of the sarcoplasmic reticulum is the same (1% of the myofilament volume) in both fibres but that the size, number and distribution of the sarcoplasmic reticulum tubules differ significantly. The longitudinal fibres are characterised physiologically by an action potential with a short calcium-dependent plateau that can trigger a short contraction; radial fibres produce action potentials without a plateau and their contraction requires a train of spikes. The sarcoplasmic reticulum tubules in longitudinal fibres are thinner (132 nm in diameter) and more numerous than those in radial fibres (160 nm in diameter). Moreover, the tubules are homogeneously distributed among the myofilaments in radial fibres, whereas they are more numerous in the centre of longitudinal muscles.

The abundance of Aplysia gliagrana depends on Ca2+ and/or Na+ concentrations in sea water.

The glial cells surrounding the identified giant nerve cell bodies R2 or LP1 of Aplysia punctata were studied by quantitative electron microscopy. They contain specific, electron-dense but non-osmiophilic membrane-bound granules, approximately 0.3 microns in diameter, called gliagrana. Similar glial granules are more often found in marine than in freshwater molluscs, possibly because they represent a calcium store used to compensate excess Na+ in the extracellular milieu of marine species and to regulate perineuronal calcium concentration. In agreement with this hypothesis, the abundance of gliagrana (= number of glial granules per microns 2) is found to be higher in animals adapted to low Ca2+ artificial sea water than in animals kept in high Ca2+ (or low Na+) conditions. This finding is not observed after 1 week but after 2 weeks of adaptation.

Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.

The Desheathed Periphery of Aplysia Giant Neuron. Fine Structure and Measurement of [Ca2+]o Fluctuations with Calcium-selective Microelectrodes.

The visceral ganglion of Aplysia was mechanically desheathed after protease softening of the connective tissue to permit the positioning of ion-selective electrodes in the vicinity of the neuronal membrane. The effects of this treatment on satellite glia and neuronal cytology were observed by electron microscopy. The intracellular alterations were not suggestive of serious membrane damage but the cohesion between glial and neuronal membranes was affected-the glial processes appeared to retract from the trophospongium and in some cases the neuronal membrane was completely naked. The external calcium activity [Ca2+]o at the surface of identified giant neuron, R2, was measured using double-barrelled calcium-selective microelectrodes. A decrease of approximately 1 mM in [Ca2+]o could be recorded only during trains of action potentials induced by intracellular depolarizing current injection, and when the electrode was pushed firmly against the neuron surface. A recovery from this decrease in [Ca2+]o could sometimes, but not always, be observed during the phase of induced neuronal activity.

The lacunar glial zone at the periphery of Aplysia giant neuron: volume of extracellular space and total calcium content of gliagrana.

The relative volume of perineuronal extracellular space, the number of gliagrana and their total calcium content have been measured in Aplysia punctata and A. californica, at the periphery of giant neurons R2 and LP1. After chemical fixation, the extracellular space amounts to 26% of the periganglionic glial zone, but this increases to 36% after quick freezing and freeze-substitution. The glial cytoplasm contains gliagrana, membrane-bound granules approximately 0.3 micron in diameter. The number of gliagrana per micron 2 of section, defined as "abundance", was counted in electron micrographs of chemically fixed tissues. The abundance of gliagrana appears to be directly proportional to the volume of the extracellular space when the values are averaged per individual Aplysia. The total calcium concentration of the gliagrana is measured by X-ray microanalysis on sections of ganglia processed by rapid freezing and freeze-substitution in the presence of oxalic acid: it was found to be very high. An individual granule may contain 100 mM Ca in A. californica and 50 mM in A. punctata but in both species the calcium concentration varies along a wide range as if there were different functional states of the granules with respect to this concentration. The total calcium stored in the specific granules of the glial zone was estimated. It was calculated that should the glial calcium store be entirely diluted in the extracellular space of the glial zone, it would raise the calcium concentration of this space by approximately 1 mM (0.1-2.7 mM). These findings are discussed with regard to the hypothesis of glial cells regulating the perineuronal calcium concentration.

X-ray microanalysis of calcium containing organelles in resin embedded tissue.

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites: calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images: it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types.

Membrane currents that govern smooth muscle contraction in a ctenophore.

Ctenophores are transparent marine organisms that swim by means of beating cilia; they are the simplest animals with individual muscle fibres. Predatory species, such as Beroe ovata, have particularly well-developed muscles and are capable of an elaborate feeding response. When Beroe contacts its prey, the mouth opens, the body shortens, the pharynx expands, the prey is engulfed and the lips then close tightly. How this sequence, which lasts 1 s, is accomplished is unclear. The muscles concerned are structurally uniform and are innervated at each end by a neuronal nerve net with no centre for coordination. Isolated muscle cells studied under voltage-clamp provide a solution to this puzzle. We find that different groups of muscle cells have different time-dependent membrane currents. Because muscle contraction depends upon calcium entry during each action potential, these different currents produce different patterns of contraction. We conclude that in a simple animal such as a ctenophore, a sophisticated set of membrane conductances can compensate for the absence of an elaborate system of effectors.

Electron microscopy and x-ray microanalysis of calcium-binding sites of the plasma membrane of Beroe giant smooth muscle fibre.

When they are fixed with glutaraldehyde in the presence of calcium ions, the plasma membranes of Beroe giant smooth muscle fibres display micropapillae filled with an electron-dense deposit. After freeze-fracturing of fixed tissue, the micropapillae are still present, therefore their shape and size are determined during or before glutaraldehyde fixation, and are not due to rearrangement during subsequent steps of tissue processing; intramembranous particles are seen at the periphery rather than at the top of micropapillae. In conventional stained sections, the surface of most micropapillae is surrounded by fine fibrils; when the fuzzy coat is separated from the muscle cell by a clear space, this fibrillar material becomes conspicuous and links the micropapillae to the coat. After calcium-free (EGTA) fixation, the plasma membrane is completely free of electron-dense sites but "empty' micropapillae can be seen. X-ray microanalysis of single electron-dense deposits by wavelength-dispersive spectrometry reveals a high calcium content. A weak osmiophily is suspected, but does not seem to interfere with this analysis of calcium. The highest peak-to-background ratios for calcium were obtained using the smallest aperture of the Wehnelt of the analytical microscope. In the Discussion, the micropapillae are compared to similar structures described by other authors in a variety of cell types.

Isolation of functional giant smooth muscle cells from an invertebrate: structural features of relaxed and contracted fibers.

The giant smooth muscle fibers of a ctenophore were isolated by enzymatic digestion. These fibers are multinucleated cells, up to 50 micrometers in diameter and 2 cm in length. Their ultrastructure and membrane electrical properties are similar to those of in situ fibers. Relaxed, coiled (partially contracted), and fully shortened states were distinguished in isolated cells and studied by scanning and transmission electron microscopy. Calcium-containing mitochondrial granules were found in the coiled cells but not in either the relaxed or the fully shortened cells. The relaxed cell is characterized in cross section by the density of myosin filaments (457 +/- 15 per micrometer2) and the thin-to-thick filament ratio (5.2 +/- 0.2). In the coiled cell, the muscle lattice does not expand uniformly, as shown by the variability of myosin spacing, and the thin-to-thick filament ratio decreases. Both clockwise and counterclockwise coiling occur along the same fiber. The implications of these findings with respect to the structure of the contractile apparatus are discussed.

Giant smooth muscle cells of Beroë. Ultrastructure, innervation, and electrical properties.

Beroë muscle fibers are single cells which may be 20-40 micrometer in diameter in mature specimens. Longitudinal muscles may be 6 cm or more long. There is no striation pattern and the muscles were observed to contract in a tonic fashion when stretched. They are innervated by a nerve net, and external recording revealed what are probably nerve net impulses. Intracellular stimulation of the muscles themselves was found to initiate large propagating action potentials which were recorded intracellularly. The action potentials were insensitive to tetrodotoxin (10(-5) g/ml), tetraethylammonium ions (50 mM), MnCl2 (25 mM), and low concentrations of verapamil (2 X 10(-6) g/ml). Full-size action potentials were recorded in sodium- or calcium-deficient salines, but were small and graded in salines deficient in both sodium and calcium. Cable analysis yielded mean values for lambda (1.95 mm), Ri (154 omega cm), Rm (9,253 omega cm2), and tau m (13.9 ms). The conduction velocity depended primarily on fiber diameter and maximum rate of rise of the action potential and could be predicted from the theoretical analysis of Hunter et al. (1975 Prog. Biophys. Mol. Biol. 30: 99-144). The calculated membrane capacity (less than microF/cm2) indicates little infolding of the surface membrane, a conclusion which is in agreement with anatomical studies.

The cat locus coeruleus. Light and electron microscopic study of the neuronal somata.

The neuronal cell bodies of the locus coeruleus (LC) and subcoeruleus (SC) of the cat were investigated using Nissl and Golgi preparations, and electron microscopy. On the basis of morphological criteria--size and shape of cell body, branching pattern of dentrites, distribution of cytoplasmic organelles and number of axosomatic synapses--four types of neuronal perikarya were recognized in each region: medium-sized, small-sized and two groups of intermediate-sized neurons. The medium-sized neurons (30--50 micron) had an elongated cell body, thick dendrites with a moderate number of branchings, abundant organelles arranged in concentric rings around the nucleus and a moderate number of axosomatic synapses. They were found throughout the LC and SC and most probably correspond to the larger class of catecholaminergic neurons demonstrated by fluorescence histochemistry. The small neurons (10--25 micron) were also seen in both LC and SC and are believed to represent non-monoaminergic local interneurons. They displayed sparsely branching dendrites and a thin rim of cytoplasm containing few organelles. In the SC, some of these cells occurred in closely associated pairs. Ultrastructural analysis of such pairs revealed a close apposition (80--100 A degrees) of the cell membranes for long distances (up to 10 micron) and a narrowing of the intercellular space (30--40 A degrees) at some discrete points, perhaps indicative of an electrical interaction. The intermediate-sized neurons exhibited some regional morphological differences, but two distinct subgroups could be distinguished. One was characterized by a low number of axosomatic synapses, while the other exhibited a high number of such contacts. It may be assumed that the two subgroups of intermediate-sized neurons comprise catecholaminergic and indolaminergic neurons.