Molecular origin of continuous dark noise in rod photoreceptors.

Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.     

Transduction in photoreceptors with bistable pigments: Intermediate processes

The prolonged depolarizing after potential (PDA) in the R1–6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA.By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA ^H52and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Adaptive potentiation in rod photoreceptors after light exposure

Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10–35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP^−/−), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide–gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)₃) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca²⁺/Mg²⁺-sensitive GCAPs.     

Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.     

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.     

Two types of arrestins expressed in medaka rod photoreceptors

Globular protein thermostability is characterized the cold denaturation, maximal stability (Tms) and heat denaturation temperatures. For mesophilic globular proteins, Tms typically ranges from -25 degrees C to +35 degrees C. We show that the indirect estimate of Tms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (delta Cp) alone is shown to accurately predict Tms. Delta Cp and hence Tms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of Tms is 1.0 kcal mol(-1), which is small compared to typical values of the total free energy of unfolding.     

Time course of opsin expression in developing rod photoreceptors

We have investigated the time course of rod photoreceptor determination in the goldfish retina. Rod precursor cells located in the outer nuclear layer of the mature retina continuously generate rod photoreceptors. In this study, we asked when rod precursor cells begin to express opsin, which would signal their commitment to the rod pathway of differentiation. There are three possibilities: a rod precursor could express opsin while still mitotic, at or shortly after the terminal mitosis but before differentiation, or during differentiation. We used immunocytochemistry with antibodies against bromodeoxyuridine, BrdU (a thymidine analogue) and against opsin to determine when during the mitotic history of a cell the expression of opsin first occurred, taking a double labelled cell to be evidence of commitment to the rod cell fate. We found that the first double labelled cells appeared at 4 days after BrdU injection. The number of double labelled cells increased to peak at 10 days, and then fell. These results support the hypothesis that dividing rod precursor cells are probably multipotent stem cells not committed to the rod cell fate.     

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.     

Vigabatrin-induced retinal toxicity is partially mediated by signaling in rod and cone photoreceptors

Vigabatrin (VGB) is a commonly prescribed antiepileptic drug designed to inhibit GABA-transaminase, effectively halting seizures. Unfortunately, VGB treatment is also associated with the highest frequencies of peripheral visual field constriction of any of the antiepileptic drugs and the mechanisms that lead to these visual field defects are uncertain. Recent studies have demonstrated light exposure exacerbates vigabatrin-induced retinal toxicity. We further assessed this relationship by examining the effects of vigabatrin treatment on the retinal structures of mice with genetically altered photoreception. In keeping with previous studies, we detected increased toxicity in mice exposed to continuous light. To study whether cone or rod photoreceptor function was involved in the pathway to toxicity, we tested mice with mutations in the cone-specific Gnat2 or rod-specific Pde6g genes, and found the mutations significantly reduced VGB toxicity. Our results confirm light is a significant enhancer of vigabatrin toxicity and that a portion of this is mediated, directly or indirectly, by phototransduction signaling in rod and cone photoreceptors.     

Demonstration by lectin cytochemistry of rod and cone photoreceptors in the lamprey retina

Summary Lectin cytochemical analysis was undertaken to examine the distribution of glycoconjugates associated with the short and long photoreceptor cells in the lamprey retina. Concanavalin A bound preferentially to the outer segment region of the short cells. Wheat germ agglutinin bound weakly to both long and short cells. The outer segment regions of the long cells were stained intensely with peanut agglutinin. Pretreatment with neuraminidase to remove sialic acid resulted in decreased binding of wheat germ agglutinin throughout the retina and increased binding of peanut agglutinin to the outer segment region of the short cells and the region of myoid process of the long cells. These results suggest that there is a difference in the distribution of glycoconjugate residues between the long and short cells. A rod-like character of the short cell and a cone-like character of the long are tentatively discussed. Lectin-binding patterns in other retinal regions is also examined.     

A clockwork hypothesis: synaptic release by rod photoreceptors must be regular

We can see at light intensities much lower than an average of one photon per rod photoreceptor, demonstrating that rods must be able to transmit a signal after absorption of a single photon. However, activation of one rhodopsin molecule (Rh*) hyperpolarizes a mammalian rod by just 1 mV. Based on the properties of the voltage-dependent Ca2+ channel and data on [Ca2+] in the rod synaptic terminal, the 1 mV hyperpolarization should reduce the rate of release of quanta of neurotransmitter by only 20%. If quantal release were Poisson, the distributions of quantal count in the dark and in response to one Rh* would overlap greatly. Depending on the threshold quantal count, the overlap would generate too frequent false positives in the dark, too few true positives in response to one Rh*, or both. Therefore, quantal release must be regular, giving narrower distributions of quantal count that overlap less. We model regular release as an Erlang process, essentially a mechanism that counts many Poisson events before release of a quantum of neurotransmitter. The combination of appropriately narrow distributions of quantal count and a suitable threshold can give few false positives and appropriate (e.g., 35%) efficiency for one Rh*.     

Block of the cGMP-gated cation channel of catfish rod and cone photoreceptors by organic cations.

Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.     

Germinal cells in the goldfish retina that produce rod photoreceptors

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina). Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P.R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Müller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.