Motohiko Murakami

Spectral response curves of single cones in the carp

Abstract Single cone spectral response curves were obtained with the aid of specially designed equipment. Statistical analysis of 142 records selected from hundreds, based solely on the greatest signal-to-noise ratio, revealed three groups of cones; the red cones (74 per cent) with the peaking wavelength at 611±23mμ, green cones (10 per cent) at 529±14mμ , and blue cones (16 per cent at 462±15mμ). These peaking wavelengths are in close accord with those of single cone absorption spectra measured by Marks and MacNichol in the goldfish by a microspectrophotometer. Apparently, Youngs trichromatic theory applies at the photoreceptor level in Cyprinidae.

Effects of chemicals on receptors and horizontal cells in the retina

1. By applying atomized chemical solutions on to gecko and carp retinae, neuropharmacological reactions of the photoreceptors and horizontal cells were observed.

Effects of aspartate and glutamate on the bipolar cells in the carp retina.

In the fish retina. two types of bipolar cell have been identified: on-center and off-center bipolar cells. The former responds to a small light spot with depolarization and to an annular light with hyperpolarization, and vice versa for the latter (Kaneko. 1970; Toyoda. 1973). One approach to study the synaptic mechanisms underlying these responses is to observe alterations in behavior of the bipolai cells caused by externally applied neurotransmitters. Although identification of the transmitter at the outer synaptic layer has yet to be made. aspartate or glutamate has been known to have a potent effect on the horizontal cells and to mimic a transmitter action (Cervetto and MacNichol, 1972; Murakami. Ohtsu and Ohtsuka. 1972; Sugawara and Negishi. 1973). As the synapses of both the horizontal and bipolar cells are located in very close proximity at the receptor terminals (Stell. 1967; Witkovsky and Dowling. 1969). these amino acids are expected to exhibit effects on the bipolar cells as well. The experiments were performed on retinas isolated from the carp (Cyprirus carpio), which had been darkadapted for more than 30 min. The retina was mounted receptor-side up in a moist chamber, through which a stream of moist oxygen continuously flowed. The retina was illuminated by a two-channel photostimclator (Tomitn. Kaneko. Murakami and Pautler. 1967). which first gave a small spot of white light (dia. 200 pm on the retina) from one channel, and after a short interval. an annular white light (concentric to the spot. i.d. I mm and o.d. 4 mm) from the other channel. This sequence was repeated during the experiments. Intensity of light from each channel was adjusted by neutral density filters to produce maximal responses in the bipolar cells under observation. Micropipettes were filled with 3 M KC1 or 4 M potassium acetate. A microelectrode. located at the center of the light spot. was advanced from the receptor-side into the retina. Identification of cell types in the carp retina was carried out according to the physiological criteria established by Toyoda (1973), who based his identification on intracellular dye stainings. The bipolar cells in the carp retina are so small that holding of the electrode tip inside the cell is very difficult. Thus. aspartate or glutamate was applied not by a perfusion method. but by means of a nebulizing system. After an intracellular recording was obtained (Receiued 15 July 1974)

Arginine blocks gap junctions between retinal horizontal cells

Functions of nitric oxide are of common interest among a variety of tissues, since it activates soluble guanylate cyclase to produce cGMP. Here we report that intracellular application of L-arginine, the precursor of nitric oxide, blocked gap junctions between horizontal cells of the turtle retina. The input resistances of the cells were greatly increased and the cells were thereby easily polarized by current injections through microelectrodes. This procedure enables us to plot precise I-V curves and the reversal potential of light responses was estimated at around 0 mV. These results were quite similar to those obtained by intracellular application of cGMP, suggesting that the L-arginine:nitric oxide:cGMP pathway is present in retinal horizontal cells.

Differentiation of PIII subcomponents in cold-blooded vertebrate retinas

Abstract The P III fraction was analysed on isolated inverted retinas obtained from frogs and turtles by means of the coaxial microelectrode. It was found that the conventional P III consisted of at least two subcomponents; the distal P III arising in the receptor cell layer and the proximal P III originating in a more proximal layer. Latency measurements revealed that the onset of the distal P III occured sooner than that of the proximal P III. Furthermore, these two subcomponents differed in resistivity to ammonia vapor. Utilizing the excised opened frog eye, electrode placement between the origins of the distal and proximal P IIIs resulted in a biphasic deflexion prior to the onset of the b-wave. This deflexion was a consequence of differences in latency and polarity between the P III subcomponents.

Duality of conduction mechanism in bipolar cells of the frog retina

Abstract After impulse activity in the frog retina was suppressed by tetrodotoxin, the graded postsynaptic potentials were still observed inside the ganglion cells. Impulse activity, therefore, may be dispensable for information transmission in the bipolar cells. In short-axon neurons such as the bipolar cells, the postsynaptic potentials arising at the receptor-bipolar synapses appears to spread electrotonically over the whole distance of the cells and, in this manner, transmit information to the ganglion cells. Based on the above observation, electrophysiological properties of the bipolar cells are discussed and compared with those of the multipolar neurons in the central nervous system.

Calcium action potential and its use for measurement of reversal potentials of horizontal cell responses in carp retina.

1. In the carp retina perfused with a solution containing high‐Ca2+, Ba2+ and some K+‐channel blockers, the horizontal cell produced a regenerative Ca2+ action potential when the cell was depolarized by bath application of L‐glutamate (Glu) or L‐aspartate (Asp). The action potential was triggered also by a transretinal electrical stimulation which evoked an e.p.s.p. in the horizontal cell. In this solution, some cells produced the action potential spontaneously. 2. The action potential had an overshoot of about 20 mV which lasted for several seconds or even minutes. It had a threshold and showed refractoriness. In addition, it was insensitive to tetrodotoxin, but was blocked by Co2+. These observations revealed, in horizontal cells in situ, the presence of a voltage‐dependent Ca2+ channel similar to that found in dissociated cells. It is supposed that, in a physiological environment, the Ca2+ channel is prevented from becoming regenerative probably because it is counteracted by K+ channel activities. 3. Simultaneous recordings from two separate horizontal cells showed full synchronization of the Ca2+ action potentials whose amplitudes were identical. The potential uniformity thus formed in the S‐space (Naka & Rushton, 1967) enabled us to measure reversal potentials of horizontal cell responses irrespective of the electrical coupling between the cells. 4. During an overshoot of the Ca2+ action potential, an electrically evoked e.p.s.p. as well as a light response appeared with polarities reversed to those elicited at the resting state. Their reversal potentials could be estimated within a very narrow range between ‐5 and ‐10 mV. At this range, both Glu‐ and Asp‐induced potentials reversed the polarity, too. 5. These observations suggest that the ionic mechanisms are identical in the three kinds of horizontal cell response: light response, e.p.s.p. and amino acid‐induced potentials.

Synaptic inputs to turtle horizontal cells analyzed after blocking of gap junctions by intracellular injection of cyclic nucleotides

Intracellular injection of cAMP or cGMP into turtle horizontal cells significantly increased the input resistances, and the cells could thus be easily polarized by current injection, suggesting that the cyclic nucleotides blocked gap junctions between cells. Then, synaptic inputs onto triphasic chromaticity-type cells were analyzed. Hyperpolarizing and depolarizing light responses were all reduced with depolarizing current, and their polarities were reversed by further depolarization. Their reversal potentials coincided at around 0 mV. This level was the same as observed in luminosity-type and biphasic chromaticity-type cells, suggesting that the ionic mechanisms of synaptic transmission are common among horizontal cell types.

Characterization of the light-induced increase in the Michaelis constant of the cGMP phosphodiesterase in frog rod outer segments

Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (Km) of the phosphodiesterase was measured with pH assay method. On illumination, the Km increased from the value of the dark (130 microM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced Km increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the Km in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the Km increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light Km of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced Km increase and that the factor is a membrane-bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the Km increase after light stimulation was less than 2 s.

Calcium action potential in ON-OFF transient amacrine cell of the carp retina.

For accurate measurement of a reversal potential of a postsynaptic potential, it is essential to polarize a postsynaptic neuron uniformly at equipotential levels. So far as the conventional intracellular current injection is employed, uniform polarization cannot be achieved in such neurons as retinal amacrine cells which have extensive dendritic arborizations, and a reversal potential value is inevitably overestimated. In the present experiment, we employed a new technique; carp amacrine cells were polarized by a Ca2+-action potential produced in the cells themselves. To evoke the action potential, the retina was superfused with a Ringer solution containing tetraethylammonium chloride, and amacrine cells were depolarized either by intracellular or by extracellular electrical stimulation. The action potential appeared in a regenerative manner, and showed a refractoriness. In addition, Co2+ application suppressed the action potential, indicating its Ca2+-dependent nature. The Ca2+ action potential was more readily evoked or occurred even spontaneously in a solution containing high Ca2+, Ba2+ and some K+-channel blockers. It showed an overshoot and its duration was several seconds. During the overshoot, the transient light responses appeared in reversed, hyperpolarizing polarity, and their reversal potentials were measured at -10 mV. Based on the above results, physiological roles of the Ca2+-channel are discussed. Our technique is promising for wide application to neurons in other nervous systems if the superfusion technique is available for preparations.