Juan I. Korenbrot

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

In teleost fish, unlike other vertebrates, the retina continues to grow throughout the animals life both by stretching of the mature tissue and by the addition of new cells. Following larval development, new retinal cell birth is known to occur in a rim at the periphery of the mature retina and in the outer nuclear layer (ONL). We have now found that cell birth and proliferation also occurs in the inner nuclear layer (INL) of the mature fish retina. In rainbow trout (Onchoryncus mykiss), proliferative cells exist in the INL of fish of all ages, at least up to 2 years posthatching. The proliferative cells form clusters in the INL that align in radial columns, reaching from the inner to the outer plexiform layers. The density of proliferative cell clusters changes along the equatorial plane of the retina and is highest near both the nasal and temporal poles. Our data suggest that, after birth, the proliferative cells migrate away from the INL and into the ONL, with a half‐time of about 3 days, and their cell bodies can be seen in the outer plexiform layer. Once they are in the ONL, the proliferative cells continue to divide and likely give rise to the precursor cells that differentiate into new rod photoreceptors. J. Comp. Neurol. 394:271–282, 1998.

Calcium and calcium-dependent chloride currents generate action potentials in solitary cone photoreceptors

Vertebrate rod and cone photoreceptors hyperpolarize when illuminated. However, synaptic input from horizontal cells can depolarize cones and even elicit action potentials. Using the whole-cell tight-seal recording technique, we determined that, in solitary cones isolated from a lizard retina, action potentials can be generated by depolarizing current steps under conditions where only two ionic currents are activated. A dihydropyridine-sensitive, inward Ca2+ current that activates at potentials positive to -40 mV can regeneratively depolarize the cell. Subsequently, a SITS-sensitive, Ca2(+)-dependent outward Cl- current repolarizes the cell. We suggest that these ionic currents may help explain lateral inhibition in the retina.

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

SummaryA suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2∶1 to 10∶1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented, with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.

Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors.

We studied the ionic permeability of cGMP-dependent currents in membrane patches detached from the outer segment of retinal cone and rod photoreceptors. Reversal potentials measured in membranes exposed to symmetric Na+ but with varying cytoplasmic Ca2+ concentrations reveal that the permeability ratio, PCa/PNa, is higher in the cGMP-gated channels of cones (7.6 +/- 0.8) than in those of rods (3.1 +/- 1.0). Ca2+ blocks both channels in a voltage-dependent manner. At any Ca2+ concentration, the channel block is maximal near the ionic reversal potential. The maximal block is essentially identical in channels of cones and rods with respect to its extent and voltage and Ca2+ dependence. The Ca2+ block is relieved by voltage, but the features of this relief differ markedly between rods and cones. Whereas the Boltzmann distribution function describes the relief of block by hyperpolarizing voltages, any given voltage is more effective in relieving the Ca2+ block in cones than in rods. Similarly, depolarizing voltages more effectively relieve Ca2+ block in cones than in rods. Our results suggest that channels contain two binding sites for Ca2+, one of which is similar in the two receptor types. The second site either interacts more strongly with Ca2+ than the first one or it is located differently in the membrane, so as to be less sensitive to membrane voltage. The channels in rods and cones differ in the features of this second site. The difference in Ca2+ permeability between the channels is likely to result in light-dependent changes in cytoplasmic Ca2+ concentration that are larger and faster in cones than in rods. The functional differences between channels, therefore, may be critically important in explaining the differences in the phototransduction signal of the two photoreceptor types.

Retinal network adaptation to bright light requires tyrosinase

The visual system adjusts its sensitivity to a wide range of light intensities. We report here that mutation of the zebrafish sdy gene, which encodes tyrosinase, slows down the onset of adaptation to bright light. When fish larvae were challenged with periods of darkness during the day, the sdy mutants required nearly an hour to recover optokinetic behavior after return to bright light, whereas wild types recovered within minutes. This behavioral deficit was phenocopied in fully pigmented fish by inhibiting tyrosinase and thus does not depend on the absence of melanin pigment in sdy. Electroretinograms showed that the dark-adapted retinal network recovers sensitivity to a pulse of light more slowly in sdy mutants than in wild types. This failure is localized in the retinal neural network, postsynaptic to photoreceptors. We propose that retinal pigment epithelium (which normally expresses tyrosinase) secretes a modulatory factor, possibly L-DOPA, which regulates light adaptation in the retinal circuitry.

Cytoplasmic free calcium concentration in dark-adapted retinal rod outer segments

We measured the cytoplasmic free Ca concentration in the outer segment of intact, dark-adapted rods of the toad retina. The Ca indicator dye, Quin2, was loaded at concentrations of 0.273 +/- 0.06 mM into the rod cytoplasm by incubation of isolated retinas in the hydrophobic ester, Quin2AM. Quin2 did not alter the cytoplasmic Ca concentration in the dark, but it buffered light-dependent concentration transients and, hence, modified the rod photoresponse. In the presence of 1 mM external Ca, the cytoplasmic Ca concentration in the dark was 273 +/- 129 nM.

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates.

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.

Proton transport by bacteriorhodopsin through an interface film

SummaryInterface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.

Speed, sensitivity, and stability of the light response in rod and cone photoreceptors: Facts and models

The light responses of rod and cone photoreceptors in the vertebrate retina are quantitatively different, yet extremely stable and reproducible because of the extraordinary regulation of the cascade of enzymatic reactions that link photon absorption and visual pigment excitation to the gating of cGMP-gated ion channels in the outer segment plasma membrane. While the molecular scheme of the phototransduction pathway is essentially the same in rods and cones, the enzymes and protein regulators that constitute the pathway are distinct. These enzymes and regulators can differ in the quantitative features of their functions or in concentration if their functions are similar or both can be true. The molecular identity and distinct function of the molecules of the transduction cascade in rods and cones are summarized. The functional significance of these molecular differences is examined with a mathematical model of the signal-transducing enzymatic cascade. Constrained by available electrophysiological, biochemical and biophysical data, the model simulates photocurrents that match well the electrical photoresponses measured in both rods and cones. Using simulation computed with the mathematical model, the time course of light-dependent changes in enzymatic activities and second messenger concentrations in non-mammalian rods and cones are compared side by side.

Differences in transduction between rod and cone photoreceptors: an exploration of the role of calcium homeostasis

Rod and cone photoreceptors respond to light with distinct sensitivity and kinetics. Recent biochemical and electrophysiological studies demonstrate that the enzymes of the phototransduction cascade are similar, but not identical, in these two photoreceptor types. In contrast, light or voltage stimulation generates changes in the cytoplasmic concentration of Ca2+ in the outer segment that are far larger and faster in cones than in rods. This distinction reflects rod-cone differences in each of the elements that control Ca2+ homeostasis: cell volume, the rate of Ca2+ clearance from the outer segment, the cytoplasmic Ca2+ buffering, and the Ca2+ influx through cGMP-gated ion channels.