Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Duke University

Cone photoreceptors respond to light with less sensitivity, faster kinetics and adapt over a much wider range of intensities than do rods. These differences can be explained, in part, by the quantitative differences in the molecular processes that regulate the cytoplasmic free Ca2+ concentration in the outer segment of both receptor types. Ca2+ concentration is regulated through the kinetic balance between the ions' influx and efflux and the action of intracellular buffers. Influx is passive and mediated by the cyclic-GMP gated ion channels. In cones, Ca2+ ions carry about 35% of the ionic current flowing through the channels in darkness. In rods, in contrast, this fraction is about 20%. We present a kinetic rate model of the ion channels that helps explain the differences in their Ca2+ fractional flux. In cones, but not in rods, the cGMP-sensitivity of the cyclic GMP-gated ion channels changes with Ca2+ at the concentrations expected in dark-adapted photoreceptors. Ca2+ efflux is active and mediated by a Na+ and K+-dependent exchanger. The rate of Ca2+ clearance mediated by the exchanger in cones, regardless of the absolute size of their outer segment is of the order of tens of milliseconds. In rod outer segments, and again independently of their size, Ca2+ clearance rate is of the order of hundreds of milliseconds to seconds. We investigate the functional consequences of these differences in Ca2+ homeostasis using computational models of the phototransduction signal in rods and cones. Consistent with experimental observation, differences in Ca2+ homeostasis can make the cone's flash response faster and less sensitive to light than that of rods. In the simulations, however, changing Ca2+ homeostasis is not sufficient to recreate authentic cone responses. Accelerating the rate of inactivation (but NOT activation) of the enzymes of the transduction cascade, in addition, to changes in Ca2+ homeostasis are needed to explain the differences between rod and cone photosignals. The large gain and precise kinetic control of the electrical photoresponse of rod and cone retinal receptors suggested a long time back that phototransduction is mediated by cytoplasmic second messengers that, in turn, control membrane ionic conductance. (1) The unquestionable identification of cyclic GMP as the phototransduction messenger, however, did not come until the mid 1980's with the discovery that the light-regulated membrane conductance in both rods and cones is gated by this nucleotide (2-4) and is, in fact, an ion channel. (7) The cyclic nucleotide gated (CNG) channels, now we know, are not just the compliant targets of light-dependent change in cytoplasmic cGMP, but actively participate in the regulation transduction through Ca2+ feedback signals. The precise magnitude and time course of the concentration changes of cGMP and Ca2+ in either rods or cones remains controversial. It is clear, however, that whereas cGMP directly controls the opening and closing of the plasma membrane channels, Ca2+ controls the light-sensitivity and kinetics of the transduction signal. (8,9) The modulatory role of Ca2+ is particularly apparent in the process of light adaptation: in light-adapted rods or cones, the transduction signal generated by a given flash is lower in sensitivity and faster in time course than in dark-adapted cells. Light adaptation is compromised if Ca2+ concentration changes are attenuated by cytopiasmic Ca2+ buffers (8,10,11) and does not occur if Ca2+ concentration changes are prevented by manipulation of the solution bathing the cells. (2,4) Several Ca2+-dependent biochemical reactions have been identified in photoreceptors, among them: 1. ATP-dependent deactivation. (15,16) 2 Phodopsin phospshorylation, through the action of recoverin (S-modulin). (17-19) 3. Catalytic activity of guanylyl cyclase, (20-22) through the action of GCAP proteins. (23,24,25) 4. cGMP-sensitivity of the CNG channels. (26-29,30) A challenge in contemporary phototransduction research is to understand the details of these reactions and their role in the control of the phototransduction signal. Transduction signals in cone photoreceptors are faster, lower in light sensitivity, and more robust in their adaptation features than those in rods (for review see refs. 31;32). A detailed molecular explanation for these differences is not at hand. However, biochemical and electrophysiological (33) studies indicate that the elements in the light-activated pathway that hydrolyzes cGMP are quantitatively similar in their function in rods and cones and unlikely to account for the functional differences. Also, within the limited exploration completed todate, the Ca2+-dependence of guanylyl cyclase (34) and visual pigment phosphorylation (19) do not differ in rods and cones. On the other hand, data accumulated over the past few years indicate that cytoplasmic Ca2+ homeostasis, while controlled through essentially identical mechanisms it is quantitatively very different in its features in the two photoreceptor types. Both Ca2+ influx through CNG channels and the rate of Ca2+ clearance from the outer segment differ between the two receptor cells. Also, the Ca2+-dependent modulation of cGMP sensitivity is larger in extent in cones than in rods. Most significantly, the concentration range of this Ca2+ dependence overlaps the physiological range of light-dependent changes in cytoplasmic Ca2+ level in cones, but not in rods. We briefly review some of the evidence that supports these assertions and we then provide a quantitative analysis of the possible significance of these known differences. We conclude that while differences in Ca2+ homeostasis contribute importantly to explaining the differences between the two receptor types, they are alone not sufficient to explain the differences in the photoreceptor's response. It is likely that Ca2+-independent inactivation of the transduction cascade enzymes is more rapid in cones than in rods.

Tuning outer segment Ca2+ homeostasis to phototransduction in rods and cones.

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP-gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is approximately 2.3 microM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of approximately 480 ms time constant at 20-21 degrees C and 138 ms at 32 degrees C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.

In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods.

Vertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel alpha subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both alpha (CNGA3) and beta (CNGB3) channel subunits. For alpha, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For beta, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca(2+)-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species.

Cloning and molecular characterization of cGMP-gated ion channels from rod and cone photoreceptors of striped bass ( M. saxatilis ) retina.

The transduction current in several different types of sensory neurons arises from the activity of cyclic nucleotide-gated (CNG) ion channels. The channels in these sensory neurons vary in structure and function, yet each one demonstrates calcium-dependent modulation of ligand sensitivity mediated by the interaction of the channel with a soluble modulator protein. In cone photoreceptors, the molecular identity of the modulator protein was previously unknown. We report the discovery and characterization of CNG-modulin, a novel 301 aa protein that interacts with the N terminus of the β subunit of the cGMP-gated channel and modulates the cGMP sensitivity of the channels in cone photoreceptors of striped bass (Morone saxatilis). Immunohistochemistry and single-cell PCR demonstrate that CNG-modulin is expressed in cone but not rod photoreceptors. Adding purified recombinant CNG-modulin to cone membrane patches containing the native CNG channels shifts the midpoint of cGMP dependence from ∼91 μM in the absence of Ca(2+) to ∼332 μM in the presence of 20 μM Ca(2+). At a fixed cGMP concentration, the midpoint of the Ca(2+) dependence is ∼857 nM Ca(2+). These restored physiological features are statistically indistinguishable from the effects of the endogenous modulator. CNG-modulin binds Ca(2+) with a concentration dependence that matches the calcium dependence of channel modulation. We conclude that CNG-modulin is the authentic Ca(2+)-dependent modulator of cone CNG channel ligand sensitivity. CNG-modulin is expressed in other tissues, such as brain, olfactory epithelium, and the inner ear, and may modulate the function of ion channels in those tissues as well.

CNG-modulin: a novel Ca-dependent modulator of ligand sensitivity in cone photoreceptor cGMP-gated ion channels.

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca(2+)-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.

Tatiana I. Rebrik

Department of Ophthalmology

Duke University

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

Tatiana I. Rebrik

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology

Duke University

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

Department of Ophthalmology