Characteristics of bipolar-bipolar coupling in the carp retina

ON and OFF bipolar cells were identified in the light-adapted carp retina by means of intracellular recording and Lucifer yellow dye injection. The receptive field centers, determined by measuring the response amplitudes obtained by centered spots of different diameters, were 0.3-1.0 mm for ON bipolar cells and 0.3-0.4 mm for OFF bipolar cells. These central receptive field values were much larger than the dendritic field diameters measured by histological methods. Simultaneous intracellular recordings were made from pairs of neighboring bipolar cells. Current of either polarity injected into one member of a bipolar cell pair elicited a sign-conserving, sustained potential change in the other bipolar cell. The coupling efficiency was nearly identical for both depolarizing and hyperpolarizing currents. The maximum separation of coupled bipolar cells was approximately 130 microns. This electrical coupling was reciprocal and summative, and it was observed in cell types of similar function and morphology. Dye coupling was observed in 4 out of 34 stained cells. These results strongly suggest that there is a spatial summation of signals at the level of bipolar cells, which makes their central receptive fields much larger than their dendritic fields.     

Comparison of dark- and light-adapted carp retinas with NADPH diaphorase staining

The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,     

Strongly pH-buffered ringer's solution expands the receptive field size of horizontal cells in the carp retina

By intracellular recordings, we studied the effects of pH buffering on the size of the receptive field and the extent of dye coupling of horizontal cells (HCs) in the light-adapted carp retina. These parameters were compared between data obtained in fortified Ringer's solution and those obtained in control bicarbonate Ringer's of the same pH (7.60). In Ringer's fortified with 10 mM HEPES or 15 mM Tris, the dye-coupling ratio of HCs increased by 71% and 70%, respectively. These fortified Ringer's solutions also depolarized the dark membrane potential and increased the light-evoked response. The HC receptive field profile could be described by the exponential decline in peak response amplitude to a slit of light moved tangentially from the recording electrode. Thus, the receptive field size was determined as a space constant proportional to (gj/gm)(1/2), where gj and gm denote gap and non-gap-junctional conductances. The HEPES- or Tris-fortified Ringer's significantly increased the space constant by 43% and 41%, respectively. Since dye coupling was increased in the fortified Ringer's, it is likely that gj increased more than gm as a result of alkalinization of the cytosol. Since HEPES has an aminosulfonate moiety, it has been assumed to close the hemi-channels of connexin 26, but the pH-buffering effects were essentially the same as those of Tris that has no aminosulfonate moiety. Therefore, it is unlikely that the closure of connexin 26 hemichannels is responsible for the change in the receptive field size of HCs.     

The role of retinal bipolar cell in early vision: an implication with analogue networks and regularization theory

A linear analogue network model is proposed to describe the neuronal circuit of the outer retina consisting of cones, horizontal cells, and bipolar cells. The model reflects previous physiological findings on the spatial response properties of these neurons to dim illumination and is expressed by physiological mechanisms, i.e., membrane conductances, gap-junctional conductances, and strengths of chemical synaptic interactions. Using the model, we characterized the spatial filtering properties of the bipolar cell receptive field with the standard regularization theory, in which the early vision problems are attributed to minimization of a cost function. The cost function accompanying the present characterization is derived from the linear analogue network model, and one can gain intuitive insights on how physiological mechanisms contribute to the spatial filtering properties of the bipolar cell receptive field. We also elucidated a quantitative relation between the Laplacian of Gaussian operator and the bipolar cell receptive field. From the computational point of view, the dopaminergic modulation of the gap-junctional conductance between horizontal cells is inferred to be a suitable neural adaptation mechanism for transition between photopic and mesopic vision. Received: 20 December 1996 / Accepted in revised form: 16 May 1997     

Dye-coupling visualizes networks of large-field motion-sensitive neurons in the fly

In the fly, visually guided course control is accomplished by a set of 60 large-field motion-sensitive neurons in each brain hemisphere. These neurons have been shown to receive retinotopic motion information from local motion detectors on their dendrites. In addition, recent experiments revealed extensive coupling between the large-field neurons through electrical synapses. These two processes together give rise to their broad and elaborate receptive fields significantly surpassing the extent of their dendritic fields. Here, we demonstrate that the electrical connections between different large-field neurons can be visualized using Neurobiotin dye injection into a single one of them. When combined with a fluorescent dye which does not cross electrical synapses, the injected cell can be identified unambiguously. The Neurobiotin staining corroborates the electrical coupling postulated amongst the cells of the vertical system (VS-cells) and between cells of the horizontal system (HS-cells and CH-cells). In addition, connections between some cells are revealed that have so far not been considered as electrically coupled.     

成年大鼠视皮层(17区)神经元间的染色耦合--离体脑片研究

在离体脑片上,用生物胞素（biocytin）对成年大鼠视皮层神经元进行胞内注射。在标记成功的49例中,21例（42．9％）有染色耦合现象,耦合细胞的个数从2个到5个不等,平均2．8±1．1个。其中,18例为锥体-锥体神经元之间的耦合,2例为锥体-非锥体神经元之间的耦合,1例为非锥体神经元之间的耦合。在耦合细胞中,只有5例的胞体紧靠在一起,其它细胞的胞体之间都有一定的距离,其中有1例达到了635μm。大多数情况下（19／21例）,耦合细胞的胞体位于同一层次内,只有2例胞体位于不同层次。在Ⅱ／Ⅲ、Ⅳ和Ⅴ层内都观察到染色耦合神经元,但以Ⅱ／Ⅲ层内发生染色耦合的比率为最高。比较了染色耦合与非耦合神经元的某些电生理特性,没有观察到在它们之间有明显的差别。     

Roles of subcellular Na+ channel distributions in the mechanism of cardiac conduction

The gap junction and voltage-gated Na⁺ channel play an important role in the action potential propagation. The purpose of this study was to elucidate the roles of subcellular Na⁺ channel distribution in action potential propagation. To achieve this, we constructed the myocardial strand model, which can calculate the current via intercellular cleft (electric-field mechanism) together with gap-junctional current (gap-junctional mechanism). We conducted simulations of action potential propagation in a myofiber model where cardiomyocytes were electrically coupled with gap junctions alone or with both the gap junctions and the electric field mechanism. Then we found that the action potential propagation was greatly affected by the subcellular distribution of Na⁺ channels in the presence of the electric field mechanism. The presence of Na⁺ channels in the lateral membrane was important to ensure the stability of propagation under conditions of reduced gap-junctional coupling. In the poorly coupled tissue with sufficient Na⁺ channels in the lateral membrane, the slowing of action potential propagation resulted from the periodic and intermittent dysfunction of the electric field mechanism. The changes in the subcellular Na⁺ channel distribution might be in part responsible for the homeostatic excitation propagation in the diseased heart.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Changing patterns of ganglion cell coupling and connexin expression during chick retinal development

We have used dye injection and immunolabeling to investigate the relationship between connexin (Cx) expression and dye coupling between ganglion cells (GCs) and other cells of the embryonic chick retina between embryonic days 5 and 14 (E5-14). At E5, GCs were usually coupled, via soma-somatic or dendro-somatic contacts, to only one or two other cells. Coupling increased with time until E11 when GCs were often coupled to more than a dozen other cells with somata in the ganglion cell layer (GCL) or inner nuclear layer (INL). These coupled clusters occupied large areas of the retina and coupling was via dendro-dendritic contacts. By E14, after the onset of synaptogenesis and at a time of marked cell death, dye coupling was markedly decreased with GCs coupled to three or four partners. At this time, coupling was usually to cells of the same morphology, whereas earlier coupling was heterogeneous. Between E5 and E11, GCs were sometimes coupled to cells of neuroepithelial morphology that spanned the thickness of the retina. The expression of Cx 26, 32, and 43 differed and their distribution changed during the period studied, showing correlation with events such as proliferation, migration, and synaptogenesis. These results suggest specific roles for gap junctions and Cx's during retinal development.     

Spectral sensitivity of the weakly discharging electric fish Gnathonemus petersi using its electric organ discharges as the response measure

The spectral sensitivity of the weakly electric mormyrid fish Gnathonemus petersi was investigated under dark- and light-adapted conditions using a transient change (startle) in its electric organ discharge (EOD) rate as response measure. The startle was resistant to habituation and graded with light intensity. Under both lighting conditions, the fish responded optimally to a monochromatic light of 525 nm. A porphyropsin pigment (520–540₂) appears to mediate spectral sensitivity over most of the visible spectrum. However, G. petersi responded more strongly to 625- and 675-nm lights (dark- and light-adapted fish) and a 725-nm light (light-adapted fish only) than predicted by the presence of a single rod pigment. These data suggest that at least one additional visual pigment (most likely of cone cells) maximally absorbing long wavelength light (600 nm or longer) is present. The spectral sensitivity data are consistent with the sensitivity hypothesis in that heightened sensitivity to long wavelength light is predicted for fish living in blackwater habitats which are characterized typically by low light levels and transmission of predominantly long wavelengths. Histology of the retina showed photoreceptors grouped into bundles and ensheathed by pigment epithelial cells. Our results demonstrated a functional visual sense in a species of fish much better known and studied for its electrosensory and electromotor abilities.     

Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.     

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate in dark- and light-adapted rat retinas

Light-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) to 1,2-diacylglycerol and D-myo-inositol 1,4,5-triphosphate (IP3) has been reported in the visual photoreceptor cells. We have investigated the localization of the TPI antigenic sites in dark- and light-adapted rat retinas using rabbit anti TPI antibodies (Ab). Sprague-Dawley rats were dark-, or light-adapted, or were exposed to a light flash. The eyes were fixed immediately and the tissue sections stained with the rabbit anti TPI Ab. The peroxidase-antiperoxidase method was used to find the localization of the TPI antigenic site. Image analysis of the sections was performed to obtain optical density profiles of the stain. Dark-adapted retinas showed intense staining of the rod outer segment (OS) layer but much less staining of the rod inner segment layer. Compared with the OS of dark-adapted retinas, those of the flash-bleached retinas were stained much less. The OS of fully bleached retinas showed little or no staining. The anti TPI Ab-protein A-gold conjugate intensely stained disks from dark-adapted retina but those from bleached retina much less. Our results suggest that rapid hydrolysis of TPI in rat rod outer segments occurs in vivo in response to light.     

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.     

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.     

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.     

The morphology and topographic distribution of AII amacrine cells in the cat retina

When cat retina is incubated in vitro with the fluorescent dye, 4',6-diamidino-2-phenyl-indole (DAPI), a uniform population of neurons is brightly labelled at the inner border of the inner nuclear layer. The dendritic morphology of the DAPI-labelled cells was defined by iontophoretic injection of Lucifer yellow under direct microscopic control: all the filled cells had the narrow-field bistratified morphology that is distinctive of the AII amacrine cells previously described from Golgi-stained retinae. Although the AII amacrines are principal interneurons in the rod-signal pathway, their density distribution does not follow the topography of the rod receptors, but peaks in the central area like the cone receptors and the ganglion cells. There are some 512 000 AII amacrines in the cat retina and their density ranges from 500 cells per square millimetre at the superior margin to 5300 cells per square millimetre in the centre (retinal area is 450 mm2). The isodensity contours are kite-shaped, particularly at intermediate densities, with a horizontal elongation towards nasal retina. The cell body size and the dendritic dimensions of AII amacrines increase with decreasing cell density. The lobular dendrites in sublamina a of the inner plexiform layer span a restricted field of 16-45 microns diameter, while the arboreal dendrites in sublamina b form a varicose tree of 18-95 microns diameter. The dendritic field coverage of the lobular appendages is close to 1.0 (+/- 0.2) at all eccentricities whereas the coverage of the arboreal dendrites doubles within the first 1.5 mm and then remains constant at 3.8 (+/- 0.7) throughout the periphery.     

Membrane current responses of skate photoreceptors

Light-evoked membrane currents were recorded with suction electrodes from the outer segments of individual photoreceptors enzymatically dissociated from the skate retina. The intensity-response relation of dark-adapted cells closely followed a Michaelis function for which a half-saturating response was elicited by a flash intensity that produced about 36 photoisomerizations. Dim-light responses, as well as the early rising phase of the responses to a wide range of flash intensities, could be described by a reaction scheme that involved a series of four first-order delay stages. The number of delay stages required to model the rising phase of the photocurrents did not change in light adaptation. However, background illumination that reduced sensitivity by 1.5 log units, or a bleaching exposure that resulted in a nearly equivalent desensitization, shortened significantly the time scale of the responses. In both instances there were two- to threefold increases in the rate constants of the transitional delays, and almost complete suppression of the tail current that characterized the response of the dark-adapted cell. These findings suggest that although light adaptation alters the gain and kinetics of the transduction mechanism, the nature of the intervening processes is the same in dark- and light-adapted photoreceptors. Moreover, the results show clearly that there is no need to postulate the existence of a second class of cone-like rods to account for the remarkable ability of skate photoreceptors to respond to incremental stimuli presented on "saturating" background fields or after exposure to an intense bleaching light.     

Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse horizontal cells do not express connexin26 or connexin36

Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a beta-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.