Tubular localization of silent calcium channels in crustacean skeletal muscle fibers.

Ca2+-induced Ca2+ release (CICR) in the superficial abdominal flexor muscle of the crustacean Atya lanipes appears to be mediated by a local control mechanism similar to that of vertebrate cardiac muscle, but with an unusually high gain. Thus, Ca2+ influx increases sufficiently the local concentration of Ca2+ in the immediate vicinity of the sarcoplasmic reticulum Ca2+ release channels to trigger the highly amplified release of Ca2+ required for contraction, but is too low to generate a macroscopic inward current (i.e., the Ca2+ channels are silent). To determine the localization of the silent Ca2+ Channels, the mechanical, electrophysiological and ultrastructural properties of the muscle were examined before and after formamide treatment, a procedure that produces the disruption of transverse tubules of striated muscle. We found that tubular disruption decreased tension generation by about 90%; reduced inward current (measured as Vmax, the maximum rate of rise of Sr2+ action potentials) by about 80%; and decreased membrane capacitance by about 77%. The results suggest that ca. 80% of the silent Ca2+ channels are located in the tubular system. Thus, these studies provide further evidence to support the local control mechanism of CICR in crustacean skeletal muscle.

Astrocytes and regenerating axons at the proximal stump of the severed frog optic nerve.

We have studied the growing tip of the severed frog optic nerve, a central nervous system pathway that successfully regenerates. Since reconnection with the distal stump was prevented, guidance of the growing axons along anterogradely degenerating axons and their debris was precluded. One week after nerve section, there was vigorous mononuclear macrophage activity at the cut end, which quickly subsided. Phagocytosis of the remaining debris of retrograde degeneration in the proximal stump was carried out by astrocytes. Regenerating axons appeared at the tip of the stump 3 weeks after the cut. They were preferentially located near the periphery of the stump, in close proximity to astrocytes of the glia limitans. Eight weeks after the cut, regenerating axons formed a region of outgrowth protruding from the tip of the proximal stump. They were always accompanied by astrocytes, and no myelin-producing oligodendrocytes were seen in the outgrowth.

Patch-clamp recording from Müller (glial) cell endfeet in the intact isolated retina and acutely isolated Müller cells of mouse and guinea-pig.

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K+ homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K+ conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K+ currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K+ currents by Ba2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K+ via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

Changes in ultrastructure and voltage-dependent currents at the glia limitans of the frog optic nerve following retinal ablation.

The surface of the frog optic nerve consists of astrocytic processes separated by narrow extracellular clefts underlying a pial sheath of loose connective tissue. Macroscopic voltage dependent currents can be recorded from this surface using the loose patch-clamp technique. In this study the changes in ultrastructure and voltage dependent Na currents have been studied for up to 1 year following removal of the retina. During the first 1-4 weeks, many of the myelinated and unmyelinated axons of the retinal ganglion cells degenerate, and the debris is phagocytosed by macrophages and glial cells. However, some morphologically intact axons remain even 12 weeks after surgery. Finally, after 16 weeks all the axons have disappeared, leaving a nerve consisting only of glial cells, some of which contain phagosomes. At 40-52 weeks after enucleation, the nerve persists, at 20-40% of the normal diameter, consisting mostly of normal looking astrocytes. The amplitude of the voltage dependent Na currents recorded from nerves during the first 1-4 weeks after enucleation, with the pial sheath intact, decreases by about 50%. After 8 weeks, the Na current recorded from the surface is about 30% of control. At 16-52 weeks after removal of the retina, when there are no intact axons, the Na current is reduced by 90%. If, however, the pial sheath is stripped away, the Na currents recorded from the glial surface are 40-50% of control during this same 16- to 52-week period, suggesting that in the all-glia nerve, the currents are shunted by the relatively thicker pial sheath.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and mechanical properties of electrically inexcitable skeletal muscle fibers of the crustacean Atya lanipes.

Examination of the ultrastructure and mechanical activation of the ventro-abdominal flexor muscle of the freshwater crustacean Atya lanipes shows that the fibers are of the long sarcomere, tonic type. The fibers possess an ample and well-organized internal membrane system, with extensive regions of T/SR dyad contacts near the ends of the A bands. An orbit of 10-12 thin filaments surrounds each thick filament. The lanthanum tracer method reveals a highly regular organization of the T-system, Z-tubules, and multiple internal clefts. Tension generation responds to extracellular potassium in a concentration dependent manner and is very slow. Mechanical activation is strictly dependent on extracellular Ca2+, even though these muscle fibers do not generate Ca2+ currents when depolarized. Tension development responds to caffeine and is also dependent on extracellular Na+, suggesting that Ca2+ release from the SR and Ca2+ influx via the Na/Ca exchanger intervene in mechanical activation.

Voltage-gated currents recorded from the surface of the frog optic nerve.

Macroscopic voltage-dependent currents were recorded from the surface of the intact optic nerve of Rana pipiens using the loose patch clamp technique. Depolarizing steps of more than 40 mV produced sodium-dependent TTX sensitive inward currents and a 4-AP and sodium sensitive fast outward current in addition to a slower outward current. Since the surface of the nerve is a glia limitans, it appears that the membranes of these astrocytes contain both voltage-sensitive sodium and potassium channels.

Optical recording of electrical activity from axons and glia of frog optic nerve: potentiometric dye responses and morphometrics.

Voltage-sensitive dyes were used to study the changes in membrane potential in axons and glial cells of the frog optic nerve following electrical stimulation. The lack of a signal in the unstained nerve and the multiphasic action spectra after staining indicated that the optical responses were from the extrinsic dyes. Changes in dye absorption and fluorescence had rapid and slow phases. The rapid phases resulted from action potentials in myelinated and unmyelinated axons. The kinetics of the slow phase of the optical response were similar to the depolarization recorded from the glial cells with intracellular electrodes. The ratio of the amplitudes of the fast and slow phases was characteristic for each type of dye. Pharmacological analysis of the action potential of the unmyelinated axons revealed tetrodotoxin-sensitive sodium channels and 4-aminopyridine-sensitive potassium channels. Repeated exposure of the stained preparation to light led to photodynamic damage as shown by a block of recovery of the glial depolarization. An electron microscopic morphometric study of the nerve was carried out in an effort to understand the contribution of the various anatomical elements to the compound optical response. The ratio of unmyelinated axon membrane to glial membrane was much greater than was the ratio of the fast and slow components of the signal, suggesting that the dyes either had a higher affinity for glial membrane or did not penetrate the nerve uniformly.

Luminal and basolateral surface membranes of secretory acinar cells: electrophysiological comparison of cationic sensitivities.

Cation sensitivities (K+, Na+, and Ca2+) of luminal and basolateral membrane surfaces of secretory acinar cells were compared using a luminally perfused and externally superfused salivary gland from the aquatic snail, Helisoma trivolvis. Tight junctions delimiting the two membrane surfaces were observed near the acinar lumen suggesting that the total membrane area exposed to the superfusion solution exceeded that in contact with the luminal perfusion solution. The resting membrane potential of acinar cells was found to be dependent upon the K+ concentration in both the external superfusion and the luminal perfusion solutions. Unilateral K+ elevation at either membrane surface produced a rapid and sustained depolarization of the acinar cell. For a given K+ concentration, the level of depolarization produced by K+ elevation at the basolateral surface was significantly higher than at the luminal surface. The highest level of membrane depolarization was observed following simultaneous K+ elevation at both membrane surfaces. The ability of acinar cells to generate overshooting action potentials in response to electrical field stimulation was dependent upon both Na+ and Ca2+. Complete blockade invariably occurred following bilateral removal of either cation. The effects of unilateral removal of either Na+ or Ca2+ proved to be somewhat variable. In general, unilateral removal of Na+ was more effective in reducing the regenerative response than Ca2+ while removal of either cation from the basolateral surface was more effective in reducing the regenerative response than its removal from the luminal surface. Electrically evoked action potentials in acinar cells could also be blocked with unilateral application of the Ca2+ antagonist, cadmium (Cd2+), at either membrane surface. However, higher Cd2+ concentrations were required to achieve complete blockade when applied to the luminal than to the basolateral gland surface. This result fails to support a hypothesis of voltage-sensitive Ca2+ channels being spatially restricted to the luminal cell surface in this preparation.

Histotypic pattern formation in cerebellar reaggregate cultures in the presence of antibodies to L1 cell surface antigen.

Reaggregate cultures of cerebella from 5-day-old C57BL/6J mice were cultured in the presence of Fab fragments of polyclonal antibodies against the cell surface adhesion molecule L1. Light microscopic examination showed that histotypic differentiation, as observed by the appearance of radially oriented glial processes and the sorting out of mature and immature neurons, was not affected by the antibody. Electron microscopic observation showed no effect on the synapse formation and the packing density of fasciculated neurites. These observations show that under the culture conditions used, L1 antibodies do not alter the particular cell interactions investigated in this study.

Coupling and uncoupling of amphibian neuroglia.

Glial cells in the optic nerve of Necturus are coupled to each other by low resistance pathways which also permit the diffusion of the fluorescent dye Lucifer Yellow CH among the cells. The spread of dye is readily observed as nuclei of cells distant from the site of intracellular injection are stained. By contrast, horseradish peroxidase does not traverse the intercellular pathways. This protein remains in the injected cell. The addition of weak acids (carbonic or propionic) to the bathing medium reversibly uncouples the glia; it blocks the spread of ionic current and Lucifer Yellow among the cells. A block of ionic coupling will block the spatial buffering of potassium by the glial syncytium.

Specificity of histiotypic organization and synaptogenesis in reaggregating cell cultures of mouse cerebellum.

The fine structure of reaggregating cultures of cells from 6- to 7-day-old mouse cerebellum was studied at intervals between 3 and 21 days in vitro (DIV). The resulting aggregates consisted mainly of small neurons (granule, stellate and basket cells), neuroglial cells and their processes. Large neurons were rarely present. By 7 DIV the previously loosely packed components had tightened into a more compact mass. A peripheral plexiform layer had formed which had many fine axons arranged into fascicles of parallel fibers. Deep to this zone was a cellular region containing clusters of neurons interspersed with small areas of neuropil. Axosomatic synapses appeared on neurons which resembled stellate or basket cells but not on granule cells. Axo-dendritic synapses formed in the neuropil of the cellular zone and, less frequently, in the outer plexiform layer. After 3 weeks glial cell processes had increased in volume at the expense of neurons. When cerebellar cells were cultured with cells from pons and medulla, which are normal sources of mossy fiber input, aggregates formed in which synaptic glomeruli were found. They were not seen in aggregates containing cells from retina and olfactory bulb cultured with cerebellum. Our observations suggest: that natural histogenetic mechanisms persist after dissociation and reaggregation of cerebellar cells resulting in a separation of an outer, 'molecular'-like layer from an inner granule cell layer and that neurons retain specificity of their synaptogenic capabilities both with regard to appropriate cell types and the morphological form that the synapses take.

Membrane properties of neuroglia in the optic nerve of Necturus.

The optic nerve of Necturus has proved a useful preparation for the study of glial cell membranes in vivo and in vitro with anatomical relations to axons intact and isolated following axon degeneration. The glial membrane potential behaves as a selective potassium diffusion potential; there is no evidence of a significant permeability to other naturally occurring ions. The specific membrane resistance of the glial cells is high compared to that of neurones; there are low-resistance intercellular connexions among the cells which permit the passage of both ions and the dye Lucifer Yellow. The cells are readily and reversibly uncoupled by procedures which decrease the intracellular pH. There is no evidence for voltage-sensitive sodium channels in the membrane. Following sodium gain and potassium loss the membrane displays a potassium-dependent strophanthidin-sensitive electrogenic sodium pump. The glial membrane is depolarized by potassium released from active axons as well as by glutamate. The glial depolarization contributes to potentials recorded with surface electrodes. Depolarization by K+ plays a role in the redistribution of K+ which locally accumulates around active neurones and also affects glial metabolism and glucose uptake.

Surface specializations of the olfactory epithelium of rainbow trout, Salmo gairdneri.

Receptors for olfactory stimulus molecules appear to be located at the surface of olfactory receptor cells. The ultrastructure of the distal region of rainbow trout (Salmo gairdneri) olfactory epithelium was examined by transmission electron microscopy. On the sensory olfactory epithelium, which occurs in the depressions of secondary folds of the lamellae of the rosettes, five cell types were present. Type I cells have a knob-like apical projection which is unique in this species because it frequently contains cilia axonemes within its cytoplasm in addition to being surrounded by cilia. Type II cells bear many cilia oriented unidirectionally on a wide, flat surface. Type III cells have microvilli on a constricted apical surface and centrioles in the subapical cytoplasm. Type IV cells contain a rod-like apical projection filled with a bundle of filaments, and type V cells are supporting cells. Cilia on the sensory epithelium contain the 9 + 2 microtubule fiber pattern. Dynein arms are clearly present on the outer doublet fibers, which suggests that the cilia in the olfactory region are motile. Their presence in olfactory cilia of vertebrates has been controversial. The cilia membrane in this species is unusual in often showing outfoldings, within which are included small, irregular vesicles or channels. In addition, cilia on type II cells frequently contain dense-staining bodies closely apposed to the membranes, along with a densely stained crown at the cilia tip. Previous biochemical evidence indicates that odorant receptors are associated with the cilia.

Electrical properties and structure of the frog arachnoid membrane.

We have used an in vitro preparation of the frog arachnoid membrane to study the role of this membrane in the maintenance of the "blood-cerebrospinal fluid (csf) barrier". Electron microscopy showed that the membrane was made up of 10-15 layers of flat epithelial cells joined together by numerous cell junctions. The electrical resistance of the preparation was about 2000 ohms cm2. The steady-state transmural potential difference (pd) ranged up to 45 mV, csf positive, and this eliminated by either the addition of ouabain to the csf, or by replacing the NaC1 with TEA C1. The pd across the membrane increased when bicarbonate was added to the external bathing solutions. We conclude that this pd is due to the active transport of sodium from the subural fluid to the csf. In some preparations the transmural pd was reversed, i.e., csf negative, and this was also abolished by the addition of ouabain to the csf, or by replacing chloride with isethionate. We conclude that this pd is related to active chloride transport. These, and other experiments, lead us to the conclusion that the arachnois membrane is involved in the production of the cerebrospinal fluid and the maintenance of the blood-cerebrospinal fluid barrier.

Neuromuscular junctions in the buccal mass of Aplysia: fine structure and electrophysiology of excitatory transmission.

The lower extrinsic protractor muscle in the buccal mass of Aplysia consists of bundles of muscle fibers 4--12 mu in diameter, containing thick and thin filaments that are not arranged in a transversely striated pattern. Individual fibers come close to one another and form specialized junctional regions. Electrophysiological evidence indicates that the muscle fibers form an electrical cyncytium. Muscle bundles are innervated by more than one excitatory axon at a number of points along their length. The presynaptic terminals contain spherical electron-lucent vesicles and a few larger electron-dense vesicles. There are no obvious structural postsynaptic specializations. Graded contraction can result from summation of excitatory junctional potentials in separate axons or from summation and facilitation of junctional potentials from a single axon. The buildup of facilitation during a train of stimuli results from the linear summation of facilitation remaining from preceding impulses.

A further study of the fine structure and membrane properties of neuroglia in the optic nerve of Necturus.

The optic nerve of Necturus maculosus consists of a homogeneous population of astroglia and bundles of unmyelinated axons. The glial cell processes ramify within the nerve roughly delineating fascicles of axons and come together at the periphery to form a complete external limiting membrane interrupted only by narrow clefts between adjacent processes. They are frequently "attached" to one another, forming specialized junctions. Blood vessels are entirely outside the nerve which is surrounded by a basal lamina. The temperature dependence of the glial membrane potential is accurately predicted by the Nernst relation. The membrane potential is unaffected by changes in Cl, Na, Li, and guanidinium which are apparently impermeant. The permeability of the glial membrane to other cations is in the sequence Tl greater than K greater than Rb greater than Cs greater than NH4. This suggests that the chemical nature of the site of potassium permeability in glial cells is similar to that in the neuron.