Salamander Rod Photoreceptors Research Articles

Sema3A Reduces Sprouting of Adult Rod Photoreceptors In Vitro

PurposeRod photoreceptor terminals respond to retinal injury with retraction and sprouting. Since the guidance cue Semaphorin3A (Sema3A) is observed in the retina after injury, we asked whether Sema3A contributes to structural plasticity in rod photoreceptors.MethodsWe used Western blots and alkaline phosphatase (AP)-tagged neuropilin-1 (NPN-1) to detect the expression of Sema3A in an organotypic model of porcine retinal detachment. We then examined Sema3A binding to cultured salamander rod photoreceptors using AP-tagged Sema3A. For functional analysis, we used a microspritzer to apply a gradient of Sema3A-Fc to isolated salamander rod photoreceptors over 24 hours.ResultsSema3A protein was biochemically detected in porcine retinal explants in the retina 7, 24, and 72 hours after detachment. In sections, NPN-1 receptor was bound to the inner and outer retina. For isolated rod photoreceptors, Sema3A localized to synaptic terminals and to neuritic processes after 1 week in vitro. In microspritzed rod photoreceptors, process initiation occurred away from high concentrations of Sema3A. Sema3A significantly decreased the number of processes formed by rod photoreceptors although the average length of processes was not affected. The cellular orientation of rod photoreceptors relative to the microspritzer also significantly changed over time; this effect was reduced with the Sema3A inhibitor, xanthofulvin.ConclusionSema3A is expressed in the retina after detachment, binds to rod photoreceptors, affects cell orientation, and reduces photoreceptor process initiation in vitro. Our results suggest that Sema3A contributes to axonal retraction in retinal injury, whereas rod neuritic sprouting and regenerative synaptogenesis may require a reduction in semaphorin signaling.

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.

Large-Conductance Calcium-Activated Potassium Channels Facilitate Transmitter Release in Salamander Rod Synapse

Large-conductance calcium-activated potassium (BK) channels are colocalized with calcium channels at sites of exocytosis at the presynaptic terminals throughout the nervous system. It is expected that their activation would provide negative feedback to transmitter release, but the opposite is sometimes observed. Attempts to resolve this apparent paradox based on alterations in action potential waveform have been ambiguous. In an alternative approach, we investigated the influence of this channel on neurotransmitter release in a nonspiking neuron, the salamander rod photoreceptors. Surprisingly, the BK channel facilitates calcium-mediated transmitter release from rods. The two presynaptic channels form a positive coupled loop. Calcium influx activates the BK channel current, leading to potassium efflux that increases the calcium current. The normal physiological voltage range of the rod is well matched to the dynamics of this positive loop. When the rod is further depolarized, then the hyperpolarizing BK channel current exceeds its facilitatory effect, causing truncation of transmitter release. Thus, the calcium channel-BK channel linkage performs two functions at the synapse: nonlinear potentiator and safety brake.

Imaging of Ca2+Dynamics Within the Presynaptic Terminals of Salamander Rod Photoreceptors

Although the overall importance of Ca(2+) as a mediator of cell signaling and neurotransmitter release has long been appreciated, the details of Ca(2+) dynamics within the inner segments of vertebrate rod photoreceptors are just beginning to be elucidated. Even less is known regarding Ca(2+) dynamics within the rod presynaptic terminal compartment. Using fura-2 to report changes in intracellular Ca(2+), we imaged the responses of enzymatically dissociated salamander rod photoreceptors retaining intact axons and presynaptic terminals stimulated with a brief depolarizing puff of KCl (30 mM pipette concentration). In the vast majority of cells, the response was a large increase in Ca(2+) levels in the terminal compartment, but not in the soma. In contrast, rods exhibited a substantial elevation in somatic Ca(2+) levels when depolarized with a brief puff of 100 mM KCl (pipette concentration). These data are consistent with previously reported differences in Ca(2+) buffering mechanisms within the somatic and terminal compartments. Additionally, they may reflect the presence of Ca(2+) channels having distinct properties within the membranes of the two compartments. Consistent with this hypothesis, fluorescent immunocytochemistry using an antibody against the L-type Ca(2+) channel Ca(v)1.2 (alpha1C) subunit and semiquantitative confocal microscopy revealed a high concentration of immunoreactivity in the membranes of terminals of intact rods compared with the somata. Further investigations using enzymatically dissociated preparations of intact rod photoreceptors retaining their presynaptic terminals will allow further testing of these and other hypotheses regarding the compartmentalized regulation of Ca(2+) dynamics within rod photoreceptors.

Modulation of rod photoreceptor potassium Kx current by divalent cations.

Similarities between photoreceptor Kx channels and EAG and KCNQ channels suggest that the Kx channel may be related to one or both of these families of potassium channels. To provide a basis for further comparison of these channels, as well as to reveal the role of zinc (endogenously present in photoreceptors) in Kx channel modulation, we studied the effects of divalent cations on activation and kinetic properties of the noninactivating potassium current, I(Kx), in salamander rod photoreceptors. We found that divalent cations slowed the activation of I(Kx) and shifted its activation curve to positive potentials, effects attributable to surface charge screening. The effect of zinc on I(Kx) kinetics was considerably stronger than that expected from the activation curve shift alone. None of the divalent ions studied changed the monoexponential nature of I(Kx) activation. A simple model of a rod photoreceptor was created using NEURON software. In the model, zinc accelerated and sharpened the light response, broadening the frequency response of rod photoreceptors. Our results show that although the overall effects of divalent cations are in part due to surface charge screening effects, zinc also induced a specific change in I(Kx) activation kinetics, and this action profoundly alters rod response properties.

Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors.

The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.

Linear transduction of natural stimuli by dark-adapted and light-adapted rods of the salamander, Ambystoma tigrinum.

1. We examined signal, noise and response properties of salamander rod photoreceptors by measuring: (a) the circulating current of rods which were adapted to darkness and to a wide range of backgrounds; (b) contrasts of natural environments; (c) the effect of adaptation on the linear response range of rods; and (d) the behaviour of rods responding to dynamically modulated stimuli having a range of contrasts found in nature. 2. In the dark, the circulating current contained two noise components analogous to those described in toad. A discrete noise component consisted of events occurring at a rate of 1 event per 32 s (21 degrees C) and had a variance of 0.036 pA2. A continuous noise component contributed 0.022 pA2 to the dark current, roughly equal to the discrete noise variance. 3. Exposure to a wide range of steady backgrounds (suppressing up to 80% of the circulating current), elicited a sustained fluctuating photocurrent having a power spectrum which resembled those of single photon responses and was consistent with the linear summation of single photon events; this indicates that the primary source of noise in the current is caused by the light. 4. Eighty-nine per cent of the contrasts (C) measured in natural environments had magnitude of C < 50%, where C = magnitude of I - Imean/magnitude of Imean. The linear response range elicited by brief flashes expanded with brighter backgrounds, well-encompassing flash contrasts of 100%. 5. Dynamically modulated stimuli and incremental flashes having contrasts similar to those in natural scenes elicited small currents which deviated by a few picoamps about the mean and the transfer functions computed from each type of stimulus-response pair closely corresponded to one another. These results indicate that in natural environments, rods behave as linear small-signal transducers of light.

The action of cytoplasmic calcium on the cGMP-activated channel in salamander rod photoreceptors.

1. Truncated salamander rod photoreceptors were internally perfused to investigate the action of cytoplasmic Ca2+ on cGMP-activated channels in the outer segment. 2. Switching from 1 microM Ca2+ to 0 Ca2+ increased the cGMP-activated current by a factor of 7.1 +/- 0.5 when measured in the first 60 s after the outer segment was opened to the bath, but only 2-fold after 5 min or more. This was attributed to the loss from the outer segment of a soluble factor required for Ca2+ to inhibit the cGMP-activated channel. 3. Short exposures to 0 Ca2+ caused an irreversible increase in the cGMP-activated current measured in 1 microM Ca2+, indicating that lowering [Ca2+] accelerated the loss of the channel inhibitor from the outer segment. 4. Channel activation occurred with a half-time of 6.7 s on switching to 0 Ca2+. Replacing 1 microM Ca2+ inhibited the current again with a half-time of 11.0 s. 5. The inhibition of the cGMP-activated current by Ca2+ could be described by a Hill curve with half-maximal suppression at 55 +/- 13 nM Ca2+ and a Hill coefficient of 1.4 +/- 0.4. 6. Addition of calmodulin (1 microM), or the calmodulin inhibitors mastoparan and calmidazolium (5 microM), did not alter the action of Ca2+ on the cGMP-activated current. 7. The increased affinity of the cGMP-activated channels in response to a fall in [Ca2+] has the magnitude, speed and Ca2+ dependence to suggest that it will promote recovery of the cGMP-activated current in response to the light-induced fall in [Ca2+] that normally occurs inside the outer segment.

Kinetics of oxygen consumption and light-induced changes of nucleotides in solitary rod photoreceptors.

We made simultaneous measurements of light-induced changes in the rate of oxygen consumption (QO2) and transmembrane current of single salamander rod photoreceptors. Since the change of PO2 was suppressed by 2 mM Amytal, an inhibitor of mitochondrial respiration, we conclude that it is mitochondrial in origin. To identify the cause of the change of QO2, we measured, in batches of rods, the concentrations of ATP and phosphocreatine (PCr). After 3 min of illumination, when the QO2 had decreased approximately 25%, ATP levels did not change significantly; in contrast, the amount of PCr had decreased approximately 40%. We conclude that either the light-induced decrease of QO2 is not caused by an increase in [ATP] or [PCr], or that the light-induced change of [PCr] is highly heterogeneous in the rod cell.

Effects of lowered cytoplasmic calcium concentration and light on the responses of salamander rod photoreceptors.

1. In order to study the interactions between cytoplasmic calcium concentration ([Ca2+]i) and light in modulating the responses of rod photoreceptors, [Ca2+]i was held at different levels by manipulating Ca2+ fluxes across the outer segment membrane. 2. If [Ca2+]i was reduced by the removal of external Ca2+ in the continued presence of Na+, and then held near this reduced level by exposure to 0 Ca(2+)-0 Na+ solution, the onset of the recovery phase of the response to a bright flash delivered just before the return to Ringer solution was accelerated, much as is the case during light adaptation, provided that precautions were taken to minimize Na+ influx. 3. If the rod was first allowed to adapt to steady light, [Ca2+]i held near the appropriate light-adapted level by superfusion with 0 Ca(2+)-0 Mg(2+)-0 Na+ solution and the light extinguished, the onset of the recovery phase of the bright flash response varied with the original background intensity in the same way as in the continued presence of steady light. These results indicate that reduction of [Ca2+]i is sufficient to induce this manifestation of light adaptation in darkness. 4. When [Ca2+]i was held at a reduced level in darkness, not only was the sensitivity to dim flashes reduced, but the response rising phase was also delayed and its amplitude increased supralinearly with flash intensity, neither of which changes is seen during light adaptation. However, similar changes in response kinetics resulted when [Ca2+]i was held near its normal dark level and the phosphodiesterase was partially inhibited by 3-isobuty-1-methylxanthine (IBMX), suggesting that they arose indirectly from an elevated cyclic GMP concentration rather than from a direct effect of Ca2+. 5. If [Ca2+]i was held near the normal dark level and bright steady light presented, the circulating current was completely suppressed. Partial inhibition of the phosphodiesterase by superfusion with 0 Ca(2+)-0 Na+ solution including IBMX resulted in restoration of the circulating current. Dim flash responses recorded under these conditions exhibited kinetics similar to those recorded in 0 Ca(2+)-0 Na+ solution in darkness, in contrast to the response acceleration seen when [Ca2+]i was held near the appropriate light-adapted level. These results indicate that the kinetics of the flash response depend on [Ca2+]i rather than on the steady light intensity.

Proton modulation of M-like potassium current (I Kx) in rod photoreceptors

The effect of extracellular pH (pH e 6.9–8.1) and intracellular pH (pH i 6.4–8.1) on the non-inactivating voltage-sensitive M-like potassium current (I Kx) was studied in patch-clamped salamander rod photoreceptors. The midpoint of the I Kx activation curve shifted by 6.6 mV per pH e unit, with acidification producing positive shifts and alkalinization producing negative shifts. The time constant of I Kx activation shifted with pH e in a manner consistent with the shifts in the activation curve. Maximum conductance and gating charge were unaffected by changes in pH e. I Kx did not depend on pH i. Given the importance of I Kx in rod function, these results suggest that pH e could affect the signal transmitted from rods by changing I Kx activation parameters.

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.

Effect of potassium ions and membrane potential on the Na-Ca-K exchanger in isolated intact bovine rod outer segments.

Two recent studies reported that Na-Ca exchange in the outer segments of tiger salamander rod photoreceptors (Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W., and McNaughton, P. A. (1989) Nature 337, 740-743) and of bovine rod photoreceptors (Schnetkamp, P. P. M., Basu, D. K., and Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-157) requires and transports K+ in a 4Na/(1Ca+1K) stoichiometry. In this study, we have examined the effects of K+ ions and membrane potential on the kinetics of Na-Ca and Ca-Ca exchange in rod outer segments isolated from bovine retinas. The objective was to establish the ion selectivity and voltage dependence of the different cation binding sites on the Na-Ca-K exchange protein. Potassium ions activated Na-Ca exchange when present on the Ca2+ side, although the extent of activation decreased with decreasing Na+ concentration. Potassium ions inhibited Na-Ca exchange when present on the Na+ side; inhibition arose from competition between Na+ and K+ for a common single cation-binding site. Activation of Na-Ca exchange by K+ displayed a different ion selectivity than that observed for inhibition of Na-Ca exchange by K+. The results are interpreted in terms of a three-site model for the rod Na-Ca-K exchanger. The rate of forward Na-Ca exchange decreased by 1.75-fold for a 60 mV depolarization of the plasma membrane but only at lower Na+ concentrations. The rate of Ca-Ca exchange was not affected by changes in membrane potential.

Characterization of a voltage-gated K + channel that accelerates the rod response to dim light

In this study a K + current, I Kx, in isolated salamander rod photoreceptors was characterized and its role in shaping small photovoltages was examined. I Kx is a standing outward current of about 40 pA at −30 mV that deactivates slowly when the cell is hyperpolarized ( τ max = 0.25 s). The voltage and time dependence of I Kx are similar to that of M-current, but I Kx can be distinguished from M-current because it is not suppressed by acetylcholine and is “blocked” by external Ba 2+ in a surprising manner: the activation range of I Kx is shifted strongly in the positive direction. Using current-clamp recordings and a computer simulation of the photo-response, we show that I Kx figures prominently in setting the dark resting potential and accelerates the voltage response to small photocurrents.