Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

Is a mosaic embryo also a mosaic of communication compartments?

We have studied the pathways of cell communication in embryos of the mollusc Lymnaea stagnalis in which the developmental fate of a cell or a group of cells is known from cell lineage studies. We iontophoretically injected Lucifer Yellow CH and followed the spread of fluorescence between cells interconnected via gap junctions. In early stages all blastomeres appear to be dye-coupled, but later on communication is restricted within compartments. The pattern of cell communication corresponds with the development of compartments with specific cell fates. Dye-spread is limited by communication boundaries which completely or mostly prevent the passage of dye to adjacent compartments with different developmental fates. These boundaries appear progressively during development. Our results suggest that, during the development of Lymnaea, the progressive changes in the pattern of dye spread correspond with the progressive restrictions of the developmental fates of individual cells or groups of cells. We conclude that changes in the pattern of cell communication and in the appearance of communication compartments are not exclusive features of regulative embryos.     

Microanatomy of the subumbrellar motor innervation inAglantha digitale (Hydromedusae: Trachylina)

Summary The hydrozoan medusaAglantha digitale (Müller 1776) has eight syncytical giant motor axons, up to 40 m in diameter, running from the margin, up the inside of the bell towards the apex. Giant motor axons injected with Lucifer Yellow CH are connected with lateral neurons running circumferentially across the subumbrellar muscle. These processes fill with the dye. Bundles of 20 to 50 small dye-coupled neurons extend circumferentially along the margin for up to 0.85mm. Giant motor axons injected with horseradish peroxidase divide into a few short branches on entering the inner nerve ring. Here the giant motor axon forms both chemical synapses and gap junctions with neurons that also send their axons into the inner nerve ring. In this region the inner and outer nerve ringe are connected by axons passing through openings in the intervening mesoglea.     

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.     

Quantitative determination of gap junction intercellular communication by scrape loading and image analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.     

Iontophoretic injection of lucifer yellow CH into zygotes and blastomeres of the hydroidHydractinia echinata

The dye Lucifer yellow CH was iontophoresed into recently fertilized eggs and early blastomeres ofHydractinia echinata. Iontophoresis was carried out on the stage of an inverted microscope in order to follow filling of the injected cells by short pulses of epifluorescent illumination. Lucifer yellow proved to be nontoxic and development in embryos with injected blastomeres proceeded normally. When zygotes were injected all the cells of the forming embryo contained dye. When one of the first two blastomeres was injected all the progeny of the injected cell also contained dye. Dye-coupling between injected and uninjected blastomeres did not occur in two-cell embryos nor between descendants of either line. Development of Lucifer-yellow-filled blastomeres or zygotes could be stopped by blue light irradiation. In a number of injected cells, the dye tended to accumulate forming brightly shining spots. The dye did not penetrate the nuclear envelope of injected cells.     

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Synaptogenesis in the giant-fibre system of Drosophila: interaction of the giant fibre and its major motorneuronal target

The tergotrochanteral (jump) motorneuron is a major synaptic target of the Giant Fibre in Drosophila. These two neurons are major components of the fly's Giant-Fibre escape system. Our previous work has described the development of the Giant Fibre in early metamorphosis and the involvement of the shaking-B locus in the formation of its electrical synapses. In the present study, we have investigated the development of the tergotrochanteral motorneuron and its electrical synapses by transforming Drosophila with a Gal4 fusion construct containing sequences largely upstream of, but including, the shaking-B(lethal) promoter. This construct drives reporter gene expression in the tergotrochanteral motorneuron and some other neurons. Expression of green fluorescent protein in the motorneuron allows visualization of its cell body and its subsequent intracellular staining with Lucifer Yellow. These preparations provide high-resolution data on motorneuron morphogenesis during the first half of pupal development. Dye-coupling reveals onset of gap-junction formation between the tergotrochanteral motorneuron and other neurons of the Giant-Fibre System. The medial dendrite of the tergotrochanteral motorneuron becomes dye-coupled to the peripheral synapsing interneurons between 28 and 32 hours after puparium formation. Dye-coupling between tergotrochanteral motorneuron and Giant Fibre is first seen at 42 hours after puparium formation. All dye coupling is abolished in a shaking-B(neural) mutant. To investigate any interactions between the Giant Fibre and the tergotroachanteral motorneuron, we arrested the growth of the motorneuron's medial neurite by targeted expression of a constitutively active form of Dcdc42. This results in the Giant Fibre remaining stranded at the midline, unable to make its characteristic bend. We conclude that Giant Fibre morphogenesis normally relies on fasciculation with its major motorneuronal target.     

Dye-coupling among frog (Rana catesbeiana) taste disk cells

• 1.1. Dye-coupling among taste disk cells in the bullfrog fungiform papillae was examined histologically by injecting a fluorescent dye (Lucifer yellow) into the cell, and the effects of the dye-coupling on depolarizing responses induced by taste stimuli were studied electrophysiologically.
• 2.2. With dye injection into a taste cell, dye-coupling was found between taste cells (23%) or between taste cell and supporting cell (28%). With dye injection into a supporting cell, dye-coupling was found between supporting cells (34%) or between supporting cell and taste cell (27%).
• 3.3. Depolarizing responses recorded from either a taste cell or a supporting cell to stimulation with 0.5 M NaCl or 10 mM quinine-HCl were the same in amplitude whether the dye-coupling to another cell was present or not. On the other hand, depolarizing responses recorded from a taste cell for 0.5 mM acetic acid became significantly larger when dye-coupled to a supporting cell.
• 4.4. It is concluded that gustatory transduction for acid stimuli is influenced by supporting cells coupled to taste cells.

     

Symplastic Transport in Ipomea tricolor Source Leaves : Demonstration of Functional Symplastic Connections from Mesophyll to Minor Veins by a Novel Dye-Tracer Method

A novel method for the delivery of the fluorescent dye Lucifer Yellow CH to the cytosol of a source leaf mesophyll cell was devised which utilized a preencapsulation of the dye in phospholipid vesicles (liposomes). The liposomes were easily injected into the vacuoles of leaf cells of Beta vulgaris or Ipomea tricolor, where fusion with the tonoplast resulted in the release of the dye into the cytosol. Subsequent cell-to-cell movement of the dye was readily followed by fluorescence microscopy. Using this liposome technique symplastic continuity from the the mesophyll to the minor veins of the source leaf of Ipomea tricolor was demonstrated. This agreed with ultrastructural studies which demonstrated the presence of plasmodesmata between all cells from the mesophyll to the minor veins. The symplastic movement of dye from the injected mesophyll cell to the minor veins was unaffected by pretreatment of the leaf tissues with 2 millimolar p-chloromercuribenzenesulfonic acid. Pretreatment of the leaf tissues at alkaline pH (3-[N-morpholino] propanesulfonic acid-KOH, pH 8.0) had no apparent effect on dye movement between adjacent mesophyll cells but inhibited the movement of dye into and along the minor veins. Thus, although there were no apparent barriers to symplastic solute movement in this leaf, symplastic barriers could be imposed by the experimental conditions used.     

Central generation of swimming activity in the hydrozoan jellyfish Aequorea aequorea

Swimming in Aequorea is controlled by a network of electrically coupled neurons (swim motorneurons) located in the inner nerve ring. The network is made up of the largest neurons in the ring, up to 22 microns in diameter. Intracellular recordings from swim motorneurons reveal slow membrane potential oscillations and a superimposed barrage of synaptic "noise." The synaptic noise, but not the slow oscillations, is eliminated in seawater containing an elevated Mg++ concentration. The swim motorneurons produce a rapid burst of two to eight action potentials preceding each contraction of the subumbrella. Spontaneous bursting persists in high-Mg++ seawater. Injected ramp currents indicated a "bursty" character of the swim motorneurons as suprathreshold depolarizations produced repetitive bursting with an increasing burst frequency with increased depolarization. Hyperpolarizing currents locally blocked spiking in swim motorneurons. Intercellular coupling was demonstrated with Lucifer Yellow injection and dual electrode recordings. In dye fills, only the large neurons of the inner nerve ring were dye-coupled. Two pieces of evidence suggest that swim motorneurons activate the overlying epithelial cells via chemical synapses. First, direct synaptic connections have been noted in ultrastructural examination of the inner nerve ring region. Second, dual recordings from a swim motorneuron and an epithelial cell reveal a 1:1 correspondence between neuron spikes and epithelial synaptic potentials. The synaptic potentials occur with a latency as short as 3 ms which is constant in any one recording session. The results suggest that the swim motorneuron network of Aequorea not only performs a motorneuron function, but also serves as the pattern generator for swimming activity.     

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current “two-cell model” of the stria vascularis.     

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Different Patterns of Intercellular Transport of Lucifer Yellow in Young and Mature Antheridia of Chara vulgaris L.

Intra- and intercellular movement of the fluorochrome Lucifer Yellow (LY), microinjected into the shield cells of male sex organs, was examined at different stages of spermatogenesis in Chara vulgaris L. Two distinct stage-associated types of probe movement were noted: (I) a fast and uniform spreading of fluorescence within the shield cell injected, followed by centripetal transport of LY into the adjoining manubrium and the capitular cells, and subsequent redistribution into the other manubria and shield cells in young antheridia (up to the stage of 4-cellular filaments), and (II) limited spreading of fluorescence, restricted to the triangle-shaped area of the impaled shield cell in more mature antheridia with multicellular filaments. These results confirm earlier electron microscopic observations on plasmodesmatal connections indicating an intensive solute movement between all cell types in young antheridia of Chara. Developmental changes appearing at later stages, during differentiation of sperm cells, strongly reduce the capacity for intercellular symplasmic transport.     

TNF-alpha plus IFN-gamma induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses

In this work, the effects of bacterial LPS, TNF-alpha, and IFN-gamma on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence, immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-alpha, or IFN-gamma alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 alpha-glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF-alpha significantly reduced the LPS plus IFN-gamma-induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18-alpha-glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF-alpha plus IFN-gamma. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates.     

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata

Summary Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK₂ cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were either one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK₂ cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.     

Cell communication compartments in molluscan embryos

Early embryos of Patella vulgata have been injected with Lucifer Yellow. No restriction of dye spread was found. We show that later in the development, the larval trochophore stage present evidence of compartments of cell communication. These dye compartments coincide with different presumptive regions.     

Morphology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Electrophysiologically identified cells of the cockroach pars intercerebralis (Periplaneta americana) were injected with the dye Lucifer Yellow for morphological examination and with horseradish peroxidase for ultrastructural marking. In addition to this, uninjected cells were also studied to elaborate the findings from the injected material. The two electrophysiologically distinct classes of cells (type I and type II) correspond to two distinct morphological and ultrastructural classes. Type I cells are the medial neurosecretory cells of the pars intercerebralis, which project their axons to the retrocerebral neuro-hemal complex. Their cell bodies have a mean diameter of 17 microns, and they contain neurosecretory granules 200 nm in diameter. Arborizations emanate from the axon in the anterior part of the protocerebral neuropil. The type II cell bodies are larger (38 microns in diameter). Their axons project into the contralateral circumesophageal connective. These cells were usually multipolar, having somatic arborizations in the anterior portocerebral neuropil. The cell bodies contain vesicles 40 nm in diameter, numerous trophospongia, and a multi-layered glial envelope.     

Gap junctions between odontoblasts revealed by transjunctional flux of fluorescent tracers

Summary Cell communication between odontoblasts was investigated with the use of fluorescent-dye tracers; Lucifer Yellow CH (molecular weight = 457.3), and dextran-Lucifer Yellow CH (average molecular weight = 10000). Dyes were injected into cell bodies of individual odontoblasts via an intracellular microelectrode or into a group of cells through their processes, and passage to adjacent cells was examined with a fluorescence microscope. Lucifer Yellow CH appeared to diffuse very easily among odontoblasts, while dextran-Lucifer Yellow remained within the injected cell or cells. This efficient migration of Lucifer Yellow CH can be considered a functional manifestation of gap junctions between odontoblasts.