K. Negishi

Dual actions of some amino acids on spike discharges in the carp retina

The effects of some amino acids (Glu, Gly and GABA), applied in 3 different manners (electrophoretically, in the superfusate and by pressure-microinjection), were investigated on spontaneous and light-induced spike discharges in the isolated carp retina. When applied electrophoretically or by pressure-microinjection in the inner plexiform layer (IPL), the agents acted directly on spike-generating units. Electrophoretic application of Glu at IPL consistently increased while Gly and GABA always decreased spike discharges regardless of the light-induced response patterns, when the tangential distance between the recording and injection electrodes was 25--100 micron. Increasing the distance up to 400 micron diminished the effects, but did not invert them. When added to the superfusate, the amino acids produced a dual action (two different sequential effects); Glu (5 mM) initially decreased and increased spike discharges, while Gly and GABA (5 mM) produced opposite effects. Gly and GABA tended to suppress selectively off-discharges (of ON--OFF units and certain OFF-center units), leaving on-discharges (of ON--OFF units and certain ON-center units) unaffected. The amino acids produced different effects on some units, when applied by pressure-microinjection into OPL or IPL. When injected in OPL Glu suppressed, while in IPL it activated spike discharges, whereas Gly and GABA caused opposite changes to those observed with Glu. Therefore, the action of the agents when pressure-microinjected in OPL is equivalent to the initial action of the agents applied in the superfusate. The dual actions of the agents are assumed to be mediated by bipolar cells, resulting in disfacilitation (Glu) or in disinhibition (Gly or GABA) of spike-generating units.

Lateral spread of light-induced potentials along different cell layers in the teleost retina

Abstract By displacing a spot of white light (0.25 mm in dia.), in steps of 0.25 mm along a straight line over the receptor surface, the extent of the lateral spread of light-induced potential recorded intracellularly and the related spike discharges were investigated in the isolated retina of fishes ( Gerridae, Centropomidae and Mugilidae ). Hyperpolarizing S-potentials were found to spread laterally along each layer of horizontal cells and of a class of amacrine cells. The S-potential amplitude was maximal at the center of the light spot, and decayed to one half of its maximum when the light spot was displaced 0.5 to 1.5 mm away from the recording point; differences in the half decay distance along different horizontal and amacrine cell layers were statistically significant. In the inner plexiform layer, various types of light-induced (sustained or on-off transient) potentials were recorded intracellularly. Although some of these sustained potentials were C-type S-potentials , the others were assumed to arise from spike-producing cells. The lateral distribution of these potentials varied widely from cell to cell, and some of the sustained potentials showed polarity reversal with distance, from the central hyperpolarizing to the peripheral depolarizing response, or vice versa. The spatial changes observed in spike discharge pattern were closely related to the changes in the sustained potentials exhibiting the polarity reversal or in the depolarizing on-off transients.

Effects of anoxia, CO2 and NH3 on S-potential producing cells and on neurons

SummaryComparative studies were carried out concerning effects of anxoia, CO2 and NH3 on the plasma membrane potential recorded from “controller cells” (S-potential producing cells, isolated fish retina) and from “conductor cells” (spike producing neurons, isolated frog dorsal root ganglion). The controller cells were observed to be surprisingly sensitive to oxygen deprivation, the cell function stopping in seconds without O2, whereas conductor cell function was not directly dependent on oxidative metabolism, the spike potential being evoked by sciatic nerve stimulation during a period of anoxia lasting for 30–60 min. It is assumed that a component of the controller cell membrane potential is coupled to the respiration of the plasma membrane.CO2 hyperpolarized the controller cell membrane but depolarized the conductor cell, whereas NH3 caused the contrary effects on the two types of cell membranes. CO2 and NH3 had more pronounced effects on the controller cell than on the conductor cell; however, the membrane potential changes in the two classes of cells displayed approximately the same time-courses. It is suggested that in the dorsal root ganglion CO2 and NH3 primarily affect the plasma membrane respiration of the satellite cells, which then influence the conductor cell membrane behavior by means of non-synaptic cellular interactions, analogous to those suggested to occur in the fish retina.

Opposite effects of ammonia and carbon dioxide on dye coupling between horizontal cells in the carp retina

Effects of ammonia (NH3) and carbon dioxide (CO2) on the membrane potential of horizontal cells and on dye coupling between the cells in isolated retinas of the carp (Cyprinus carpio) were investigated. Ammonia (less than 300 ppm NH3 in air) initially depolarized and subsequently hyperpolarized, while CO2 (10% in air) hyperpolarized the membrane potential of horizontal cells, accompanied by a diminution of both center and surround responses to spot and annular light stimuli. During the course of amplitude diminution, the center response consistently became smaller with NH3 and larger with CO2 than the surround response. In the presence of intravitreally applied DA (50 microM) or amphetamine (100 microM), a fluorescent dye Lucifer Yellow CH (LY) was found to be restricted to single injected horizontal cells. The presence of intravitreal haloperidol (100 microM) for 20-25 min or an exposure of the retina to NH3 for 5-10 min diffused the restricted LY from single injected cells to numerous neighboring cells. On the other hand, CO2 was found to restrict the injected dye to single cells, an effect similar to that of DA and opposite to that of NH3 and haloperidol. The results suggest that NH3 appears to act as a coupler while CO2 acts as an uncoupler on gap junctions between horizontal cells in the carp retina, presumably by changing the intracellular pH. In addition, a brief exposure of cells, marked with LY in the presence of DA, to the exciting light 426 nm was found to prevent the NH3-induced dye diffusion from single cells to their neighbors; the reason is unknown.

Effects of anoxia and metabolic inhibitors on theS-potential of isolated fish retinas

Abstract Large and rapid effects are produced by anoxia (N 2 ), azide and cyanide (N 3 H, HCN gases), on the membrane potentials and light-induced responses of the cells originating the S-potentials in the isolated light-adapted retina of Centropomidae fish. Anoxia hyperpolarizcs the membrane potential and abolishes the light-induced responses. Depolarization sometimes precedes the hyperpolarization, which may increase the normal low membrane potential by more than 60 mV. The pre-anoxia membrane potential has high temperature sensitivity Q = 2–3 ), while the anoxic-hyperpolarized membrane potential has low temperature dependence. The results suggest that for a cell originating s-potentials there is a labile component of the membrane potential strongly dependent on aerobic metabolism. If aerobic metabolism is blocked, or its control of membrane potential is disrupted, the resultant hyperpolarized level possibly represents a diffusion (Donnan) potential. Results are also included that suggest that there are structures in or close to the receptor layer analogous to the cells originating the s-potentials .

Effects of alcohols and volatile anesthetics on S-potential producing cells and on neurons

SummaryComparative studies were carried out regarding the effects of alcohols (ethyl- and methyl-alcohols) and of volatile anesthetics (ethyl-ether, Fluothane and chloroform) on the plasma membrane potential recorded from “controller cells” (S-potential producing cells, isolated fish retina) and from “conductor cells” (spike producing neurons, isolated frog dorsal root ganglion). The results clearly demonstrate that alcohols and volatile anesthetics in low concentrations suppress controller cell function, whereas the photoreceptor, conductor cells and synaptic transmission have a great resistance to these agents. The depolarizing component of the C-type S-potentials was selectively depressed by ethanol. A fundamental difference was found between the effects of ethanol and of ether on the controller cell membrane behavior.

Effects of temperature on S-potential producing cells and on neurons

SummaryComparative studies were carried out regarding the effect of temperature on the plasma membrane potential recorded from “controller cells” (S-potential producing cells, isolated fish retina) and from “conductor cells” (spike conducting neurons, isolated frog dorsal root ganglion). The effect of temperature changes was pronounced and immediate on the controller cells but was small on the conductor cells, the results well agreeing with those presented in the preceding paper as to the effects of anoxia, CO2 and NH3. The resting potential of the horizontal cells was always hyperpolarized by heating from 20° (room temperature) to 30° C, and depolarized by cooling from 20° to 10° C, the light-induced responses being diminished at low and high temperatures. The temperature range between 18° and 23° C was optimal for the controller cell function, within this range the Q10 being 2–3 (4–5 mV/C°) for the horizontal cell resting potential. The hyperpolarized resting potential of the controller cell during anoxia was much less sensitive to temperature changes. The resting potential of the conductor cells in fresh preparations was slightly depolarized by heating and hyperpolarized by cooling, this membrane behavior being opposite to that of the controller cells. A strong temperature dependence, however, was observed in the spike duration of the conductor cells. The results suggest that there exist non-synaptic cellular interactions between adjacent amacrine cells in the retina, and between the conductor cell and its satellite cells in the dorsal root ganglion.

Pharmacological manipulation of spatial properties of S-potentials

The morphology and spatial properties of horizontal cells (HC’s) make them the best suited elements to convey, at the level of the outer plexiform layer of the retina, information about illumination conditions in distant areas. The S-potentials which they generate in response to illumination have a characteristic large summation area (Naka and Rushton. 1967), particularly in the fish retina. Different tangential layers of HC’s show different summation areas (Negishi and Sutija, 1969) and spectral sensitivities (Laufer and Millan, 1970) and their properties depend upon the illumination conditions of the surround (Laufer and Negishi. 1978). Thus, they can contribute to the integration of information which is to flow through bipolar cells to the proximal retina (Naka and Witkovsky, 1972), at the same time that they can modulate, through feedback (Baylor et al., 1971) the sensitivity of photoreceptors. The extent of the receptive field of HC’s has been shown to be modified by several drugs. Marshall and Werblin (1978) showed that, in the retina of the tiger salamander, 5 mM acetylcholine (ACh) produces a contraction of the receptive field of HC’s. Testing responses evoked by local spot and surround (annulus) illumination, Negishi and Drujan (1978, 1979a) reported a corresponding effect, characterized by an increase of the locally evoked response and a reduction of the distantly evoked response, in a fish retina perfused with lOtS2OOpM dopamine (DA) or other catecholamines. A similar effect was reported (Negishi and Drujan, 1979b) to occur upon perfusion with a high dose of ACh. In another fish retina, Hedden and Dowling (1978) reported depolarization and response reduction when a DA solution was applied by means of an atomizer. The effects of DA were reported by both groups to be blocked by the g-adrenergic blocking agent phentolamine. Negishi and Drujan (1979b) further reported that phentolamine also blocked the effects of ACh and that hexamethonium blocked the effects of the latter but not those of DA. These findings led to the idea (Drujan et al., 1980) that the effects of ACh are exerted through the liberation of DA from retinal interplexiform cells, present in the fish retina. with processes at the level of the HC’s, and known to be dopaminergic (Dowling and Hedden, 1978). The studies to be reported briefly here were aimed at further exploring this possibility. If true, it should be possible to deplete dopaminergic cells of their DA content by repeated application of a liberating agent which would, thus, progressively lose its effect. The effect should return upon loading dopaminergic cells with DA or a precursor. Blocking agents that interfere with the action of DA should block the effect of any other agent which would exert its action through liberation of DA.

[3H]-Dopamine uptake in the dark-adapted fish retina

Dopamine (DA) is a putative neurotransmitter in some atnacrine cells of the retina. Exposure to light increases the release of DA from the perfused cat eye (Kramer, 1971) and enhances the rate of formation of DA in the rat retina (Iuvone et al.. 1978). Furthermore, it has been demonstrated by both quantitative and histochemical methods that retinal DA content and fluorescence in rats was decreased by dark adap tation (Nichols er al., 1967; Kato er al.. 1980). Up to date. however, there is only histochemical work dealing with DA uptake in the dark-adapted rat retina (Nguyen-Legros and Berger, 1977). No quantitative study of light and dark influence on the retinal DAuptake system has been reported. In the present experiment, we have compared the effects of light and dark adaptation on the activity and kinetic properties of C3H]-DA accumulation in the fish retina. It is well known that dopaminergic neurons (DAcells) of the vertebrate retina belong to two distinct classses of cells located in the innermost level of the inner nuclear layer (Hlggendal and Malmfors, 1965; Ehinger, 1976). DA-cells of the fish retina (as well as the Cebus monkey retina) extend their processes towards both the outer (OPL) and inner plexiform layer (IPL), innervate bipolar and external horizontal cells in the inner nuclear layer (INL), and reciprocally contact amacrine ceils in the IPL. Thus, the DA-cells form a feedback loop from the IPL to OPL, and for this reason are refered to as “interplexiform cells” (Dowling et al., 1976). This morphological feature differs from that of DA-amacrine cells in other vertebrates; the latter class of amacrine cells extend processes exclusively in the IPL (Ehinger, 1976; Kato et al.. 1980). Recent histochemical studies on the fish retina (Negishi et al., 1979, 1980a. b; Hayashi, 1980) have revealed that exogenously applied DA is accumulated specifically by interplexiform ceils in either in uiuo or in vitro preparations. Therefore, the present series of experiments concerns the DA-uptake system which is related to interplexiform cells of the carp retina. METHODS

Enhancement of hyperpolarizing S-potentials by surround illumination in a teleost retina.

Abstract In the isolated retina of the teleostEugerres plumieri, the presence of a light annulus enhances the hyperpolarizing response of horizontal cells to center light spots. Surround sensitization thus occurs already at the level of horizontal cells. Cells with maximal response at 470 nm show large enhancement of their responses to red center lights in the presence of blue or red annuli. but their responses to blue center lights are not enhanced by either surround, while cells with maximal response at 605 nm show enhanced responses to both red and blue center lights with either color in the surround. The magnitude of the enhancement depends upon the intensities of both center and surround stimuli at light levels below saturation of the responses. During the first second after onset of the annulus. the enhancement remains within 80% of its maximum value observed with a given wavelength and intensity combination, and decays slowly thereafter.