Effect of glutamate on extracellular potassium activity in isolated retina of cyprinid fish

The effect of ( -)glutamate on extracellular K⁺ activity of the isolated retina of the cyprinid fish, roach, was investigated using double-barrelled K⁺-sensitive micro-electrodes. Application of μM-mM glutamate to the retina as a “puff” from an atomizer induced a transient rise in extracellular K⁺ activity, which was maximal 50–100 μm below the photoreceptor surface. The effect was concentration-dependent, but not related to the state of light adaptation of the retina. In the presence of dinitrophenol or ouabain, the glutamate-induced increase in extracellular K⁺ activity was maintained.

The following conclusions are made. (1) The most likely cellular origin(s) of the glutamate-induced rise in extracellular K⁺ activity are the photoreceptors and/or the Muller (glial) cells. (2) The mechanism responsible for returning the extracellular K⁺ activity to normal depends strongly on metabolic, Na⁺, K⁺ pump activity. (3) The effect parallels the action of endogenous glutamate, and may be important for modulation of neurotransmission in the intact retina.     

Glutamate-induced cytoplasmic Ca2+ transients in neurones isolated from the rat dorsal cochlear nucleus

Extracellular application of glutamate elicited cytoplasmic Ca2+ transients in freshly dissociated rat neurones of the dorsal cochlear nucleus (DCN) (identified as pyramidal cells) with half-maximal concentration of 513 micromol/l while saturating doses (5 mmol/l) of this neurotransmitter caused transients of 46.1 +/- 3.0 nmol/l on an average. The genesis of these glutamate-evoked Ca2+ transients required extracellular Ca2+. When [Mg2+]o was 1 mmol/l, the NMDA receptor antagonist AP5 (100 micromol/l) had no effects while 100 micromol/l CNQX and 10 micromol/l NBQX, inhibitors of the AMPA receptors, greatly decreased the glutamate-induced Ca2+ transients (a decrease of 92 and 57%, respectively). When facilitating the activation of the NMDA receptors (50 micromol/l glycine, 20 micromol/l [Mg2+]o) in the presence of 100 micromol/l CNQX, Ca2+ transients of 55.4 +/- 13.1 nmol/l could be produced. Block of the voltage-gated Ca2+ channels (200 micromol/l Cd2+) decreased the Ca2+ transients to approx. 50%. The data indicate that under our control experimental circumstances the glutamate-induced Ca2+ transients of the isolated DCN neurones are produced mainly by Ca2+ entry through voltage-gated Ca2+ channels and AMPA receptors. However, when the activation of the NMDA receptors may take place, these receptors also contribute significantly to the genesis of the glutamate-evoked cytoplasmic [Ca2+] elevations.     

Visual pigment and photoreceptor sensitivity in the isolated skate retina

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.     

Two components of extracellularly-recorded photoreceptor potentials in the cephalopod retina: Differential effects of Na+, K+ and Ca2+

The ERG of the isolated, superfused half-eye of the cephalopod Sepiola atlantica, evoked by a brief (10 s) light flash, has been studied by recording intraretinal potentials with glass microelectrodes. The intensity-response characteristics of the potentials recorded at an electrode fixed at the surface (V _s ) can be fitted by a simple equation derived from an equivalent circuit model based on a sodium conductance increase mechanism. Raising the external potassium level reduces the maximal response (V _m ), but does not alter the half-saturation intensity value (I ₀). Reducing external sodium does not affect (V _m ), but increases I ₀. Reducing external calcium also does not affect (V _m ), but decreases I ₀. These effects are adequately described by the model if it is also assumed (a) that changing the external sodium does not significantly alter the transmembrane sodium gradient, and (b) that sodium and calcium ions compete for the sensitivity control mechanism.Differential-depth recording between the fixed electrode at the surface and another electrode that could be moved into the retina revealed that the two component appearance of the transretinal ERG arose from the superposition of two vitreal-negative waveforms. An initial fast component was mainly recorded in the photoreceptive distal segments while a slow component was prominent in the more proximal regions of the retina. Perfusion with high K⁺ salines resulted in a decrease in the amplitudes of both fast and slow components of the response whereas reducing external Na⁺ reduced the amplitude of the fast component at all light intensities but reduced the amplitude of the slow component only at low intensities. The amplitudes of both the fast and slow components increased on reducing external calcium, but the rate of rise and fall of the fast component was independent of external calcium. The rate of rise of the slow component was also independent of the external Ca²⁺ level but a minimum in the recovery time (t _F ) was shifted to a lower intensity value at lower calcium concentrations. The shift of the minimum was to a higher intensity value with lowered sodium perfusing solutions. On the basis of the differential sensitivity of the two components to ion changes, as well as stimulus intensity and intraretinal distribution of the components, it is suggested that they reflect two distinct processes in the light-evoked potential of the photoreceptor cells.List of the more important symbols V _s Potential of fixed electrode at the surface of retina relative to grounded electrode - V _p Potential of moveable electrode relative to ground - V _D V _s - V _p - V _m Maximum value of V _s produced by saturating light intensities - I _o Stimulus required to evoke a response of half-maximal amplitude - g _K Membrane potassium conductance - g _Na Membrane sodium conductance - E _K Nernst potential for potassium - E _Na Nernst potential for sodium - G Coefficient connecting light intensity and light-induced change of conductance - t _R Time, measured from the stimulus onset, for the response to reach 50% of peak amplitude - t _F Time, measured from the time to peak, for the response to fall to 50% of its maximal value     

Functional anatomy of the photoreceptor and second-order cell mosaics in the retina of Xenopus laevis

Mosaics of photoreceptors, and horizontal and bipolar cells of the Xenopus laevis retina were studied in whole-mount preparations applying lectin-cytochemical, immunocytochemical and intracellular labeling techniques. The combined density of all photoreceptor types was about 13700/mm2, of which rods represented 53%. Of the cones, the large long-wavelength-sensitive (86% of all cones) and the miniature ultraviolet-wavelength-sensitive (4%) ones could be labeled with peanut agglutinin, whereas the large short-wavelength-sensitive (10%) cones remained unlabeled. There were no significant regional differences in photoreceptor distribution. Bipolar cells were selectively labeled with antibodies against calretinin. Their density was between 4000 and 6000 cells/cm2, with slightly elevated numbers in the superior nasal quadrant. Two types of horizontal cell were injected intracellularly. The luminosity-type cells were more frequent (approximately 1000 cells/mm2) than the chromaticity cells (approximately 450 cells/mm2). The dendritic field size of the latter cell type was threefold bigger than that of the luminosity cells. The coverage factors were estimated to be 3.3 for the luminosity cells and 5.2 for the chromaticity cells. The luminosity cells contacted all photoreceptor types, whereas chromatic horizontal cells received their inputs from the short-wavelength-sensitive cones and from some, but not all, rods. Luminosity cells encounter about 50-60 potential synaptic partners within their dendritic fields, whereas chromatic horizontal cells only about 20. Chromatic horizontal cells form multiple synaptic contacts with the short-wavelength-sensitive cones. The results indicate that the overall photoreceptor to bipolar and bipolar to ganglion cell convergence in Xenopus retina is similar to that in the central retinal specialized regions of mammals, predicting comparable spatial resolutions.     

Amacrine cells in the retina of a cyprinid fish: functional characterization and intracellular labelling with horseradish peroxidase

Summary Forty amacrine cells in retinae of a cyprinid fish, the roach, were intracellularly labelled with horseradish peroxidase following electrophysiological identification as sustained depolarizing, sustained hyperpolarizing or transient units. Labelled cells were analysed by light microscopy and compared with a catalogue of amacrine cells established in a previous Golgi study on the same species. About 30% of the cell types characterized by the Golgi method were encountered in the present study. When intracellularly labelled cells were differentiated on the basis of their dendritic organization in the plane of the retina, a given electrophysiological response pattern was found to be generated by different morphological types, and vice versa. However, examination of the ramification patterns of the dendrites within the inner plexiform layer (i.e. in the radial dimension of the retina), showed that this morphological parameter of a given amacrine cell could be correlated with its light-evoked response. Several amacrine cell types were found to possess special distal dendrites which arose from the main dendritic branches and extended well over a mm in the retina. Distal dendrites were oriented tangentially with respect to the optic nerve papilla, but did not appear to be involved in any synaptic connectivity. It is concluded that the Golgi-based classification is a valuable tool for identifying intracellularly labelled amacrine cells. However, although the correlation between layering of dendrites in the inner plexiform layer and electrophysiology was generally good, additional physiological parameters would be required to determine whether more extensive parallels exist between structural and functional characteristics of amacrine cells. Alternatively, the considerable morphological diversity of amacrine cells may be of limited physiological significance.A preliminary account of the present findings was presented to the Physiological Society (Djamgoz et al. 1984)     

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.     

GABA transaminase in cyprinid fish retina: Localization and effects of inhibitors on temporal characteristics of S-potentials

Cellular localization and physiology of GABA-transaminase have been studied in the cyprinid fish retina. Immunohistochemical localization of the enzyme in the goldfish retina revealed a broad distribution pattern, including both outer and inner plexiform layers. Well known inhibitors of GABA-transaminase activity, such as gabaculine and γ-vinyl-GABA, caused an improvement of the temporal frequency transfer functions of luminosity type horizontal cells in the roach retina. In contrast, bicuculline had the opposite effect.Results suggest that GABA-transaminase has a physiological role in the retina consistent with the hypothesis that GABAergic (negative) feed-back from horizontal cells to cones regulates the temporal characteristics of light-evoked electrical activity in the outer plexiform layer.     

Sildenafil,N-desmethyl-sildenafil and Zaprinast enhance photoreceptor response in the isolated rat retina

Here we report that the active component of Viagra, Sildenafil and the first metabolite, N-desmethyl-sildenafil (UK-103, 320) increased the amplitude of flash-evoked electroretinogram (ERG) of dark-adapted albino rat retina. Effects of Sildenafil and N-desmethyl-sildenafil were comparable to those of the known phosphodiesterase inhibitor, Zaprinast. The photoreceptor cell response was isolated by blocking the glial K(+) ion-buffering and the on-bipolar components of the ERG with the use of BaCl(2) (500 microM) and the specific type VI metabotropic glutamate receptor agonist, DL-2-amino-4-phosphonobutyric acid (25 microM), respectively. Zaprinast, Sildenafil and N-desmethyl-sildenafil (1 microM each) increased the amplitude of photoreceptor cell response either. Besides, Sildenafil was significantly more effective than N-desmethyl-sildenafil. These findings suggest an increased sensitivity of photoreceptor cells in the presence of Sildenafil and it is metabolite.     

Correlation of spinule dynamics and plasticity of the horizontal cell spectral response in cyprinid fish retina: quantitative analysis

Summary A negative feedback interaction between luminosity type horizonatal cells (HCs) and green-sensitive cones generates the long-wavelength-sensitive depolarizing response in biphasic chromaticity type HCs. This interaction is suppressed in the dark and is potentiated by light adaptation of the retina. HCs are morphologically plastic; during light adaptation, their dendritic terminals within cone pedicles extend, giving rise to spinules. This paper examines whether there is a quantitative correlation between the time course of light-dependent formation of the spinules and enhancement of the feedback interaction. The strength of the feedback interaction in isolated retinac of the roach was determined as the neutral wavelength at which reversal of spectral response polarity occurred in biphasic HCs. A good correlation was found between the neutral wavelength and the spinule/ribbon ratios of retinae. Biphasic HCs were intracellularly stained with horseradish peroxidase and the correlative ultrastructure of the contacted pedicles was examined. Neutral wavelength was found to be correlated with the spinule number, weighted according to the number of synaptic contacts mediating feed-forward transmission. The latter was estimated from the total number of labelled C_b/H2 HC processes (central and lateral) at synaptic triads. A model in which spinules mediate the negative feedback interaction of HCs in the retina of cyprinid fish is presented.     

A horizontal cell selectively contacts blue-sensitive cones in cyprinid fish retina: intracellular staining with horseradish peroxidase

A horizontal cell selectively contacting blue-sensitive cones has been intracellularly stained with horseradish peroxidase in the retina of a cyprinid fish, the roach. The light microscopical morphology of the cell belonged to the H3 category of horizontal cells found in cyprinid fish retinae. In response to spectral stimuli, the cell generated chromaticity-type S-potentials that were hyperpolarizing to blue and depolarizing to yellow-orange. A red-sensitive hyperpolarizing component was absent possibly because of suppression of the negative feedback pathway between luminosity-type (H1) horizontal cells and green-sensitive cones.     

Fluctuations of the Ca2+-activated K+ current in Aplysia neurones

Fluctuations of the Ca2+-activated K+ current were measured in identified Aplysia neurones under voltage clamp conditions. The amplitude of IK,Ca was manipulated by ionophoretic injections of Ca2+. At small amplitudes of Ca2+-activated outward currents the variance of the Ca2+-activated current fluctuations increases linearly with the mean outward current. The single-channel conductance estimated from the variance of the fluctuations and the mean outward current is 11 +/- 3 pS at -30 mV. Power spectra of the Ca2+-activated K+ current can be fitted by the sum of two Lorentzian components with corner frequencies of about 10 Hz and 120 Hz.     

Synaptic organization of the indoleamine-accumulating neurons in the cyprinid retina

The distribution and synaptic connections of the indoleamine-accumulating neurons in the retinae of the goldfish and carp were studied by means of fluorescence and electron microscopy. The indoleamine-accumulating neurons were visualized after intravitreal injection and uptake of the indoleamine 5,6-dihydroxytryptamine. This labeling procedure produced a characteristic yellow fluorescence of the indoleamine-accumulating neurons and also characteristic ultrastructural changes in these cells. To avoid interference from the dopaminergic neurons of the retina, their processes were either removed by prior treatment with 5-hydroxydopamine or prevented from taking up 5,6-dihydroxytryptamine by the simultaneous injection of the catecholamine alpha-methyl-noradrenaline. Fluorescence-microscopic studies confirmed earlier reports that the indoleamine-accumulating perikarya and processes are distributed similar to those of amacrine cells. The indoleamine-accumulating processes ramify in three bands in the inner plexiform layer, the outermost one being the densest. Electron-microscopic investigations showed the indoleamine-accumulating neurons to have synapses of the conventional type, similar to amacrine cells. Their main synaptic contacts are with other amacrine cells, but synapses with bipolar cell terminals are also present. Both the distribution of the indoleamine-accumulating processes and their synaptic arrangement in the cyprinid retina differ from those found in mammalian retinae investigated previously.     

Mitochondrial K + transport: effect of N-ethyl maleimide on 42 K flux

The energy-linked flux of K⁺ into rat liver mitochondria is found to be stimulated by the sulfhydryl reagent, N-ethyl maleimide. The stimulation of K⁺ influx by N-ethyl maleimide is observed only at alkaline external pH. N-ethyl maleimide also stimulates efflux of K⁺ from the mitochondria. The stimulation by N-ethyl maleimide of K⁺ influx, but not K⁺ efflux, is dependent on the availability of metabolic energy. It is suggested that the effect of N-ethyl maleimide on K⁺ influx may be secondarily the result of an inhibition of phosphate-hydroxyl exchange. The dependence of energy-linked K⁺ influx on the external pH may be interpreted as evidence for a role of OH^? as a counterion accompanying K⁺ through the mitochondrial pump mechanism.     

Graded differential photoreceptor orientation: ramifications for ultramicrotomy of retina.

When sectioning small blocks of tissue from the retina of the eye, it is sometimes difficult to obtain sections which simultaneously cut squarely across the inner retinal layers and are on the long axis of the photoreceptors. This difficulty is, at least partially, due to the fact that the receptors tilt progressively relative to the tangent to the retinal curve at progressively more peripheral loci. Consideration of the graded differential orientation of the receptors indicates that, in order for the section to be simultaneously coaxial with the receptors and the inner retinal layers, the plane of the section must be parallel to and include the anterior-posterior axis of the eye, as is the case when the whole eye is sectioned through its center. It is illustrated that this criterion can be met for small blocks of retina if the block is excised along a parallel of the eye and the plane of section is perpendicular to the tangent to the retinal curve and the parallel. An approach which accomplishes this is described. Theoretical analysis suggests that distortion of apparent size of structures in the retina can become significant within a few degrees of the posterior pole if this condition is not met.