Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.     

Calcium-sensitive downregulation of the transduction chain in rod photoreceptors of the rat retina

In vertebrate rod outer segments phototransduction is suggested to be modulated by intracellular Ca. We aimed at verifying this hypothesis by recording saturated photosignals in the rat retina after single and double flashes of light and determining the time t(c) to the beginning of the signal recovery. The time course of Ca(i) after a flash was calculated from a change of the spatial Ca(2+) concentration profile recorded in the space between the rods. After single flashes t(c) increased linearly with the logarithm of flash intensity, confirming the assumption that t(c) is determined by deactivation of a single species X* in the phototransduction cascade. The photoresponse was shortened up to 45% if the test flash was preceded by a conditioning preflash. The shortening depended on the reduction of Ca(i) induced by the preflash. The data suggest that the phototransduction gain determining the amount of activated X* is regulated by a Ca(i)-dependent mechanism in a short time period (<800 ms) after the test flash. Lowering of Ca(i) by a preflash reduced the gain up to 20% compared to its value in a dark-adapted rod. The relation between phototransduction gain and Ca(i) revealed a K(1/2) value close to the dark level of Ca(i).     

Regeneration of goldfish retina: rod precursors are a likely source of regenerated cells

This study describes regeneration of the neural retina in juvenile goldfish. The retina was destroyed with an intraocular injection of ouabain, a technique introduced by Wolburg and colleagues (Maier and Wolburg, 1979; Kurz-Isler and Wolburg, 1982). We confirmed their observation that the level of damage produced by the toxin was graded, in that neurons in the inner retinal layers were preferentially destroyed, and only in the more severely affected retinas were cells in the outer nuclear layer (i.e., photoreceptor cells) damaged. Evidence of retinal regeneration could be seen beginning about 2 weeks after the injection of ouabain. In contrast to previous studies (Maier and Wolburg, 1979), we found that regeneration took place only in those retinas in which photoreceptors had been destroyed. In cases in which the outer nuclear layer was spared, no regeneration of inner layers occurred, even after 6 months. Thymidine autoradiography was used to document the regeneration of new retinal neurons and to show that rod precursors, like other dividing cells, were not destroyed by the ouabain, but in contrast showed an increased mitotic activity. Regeneration did not proceed uniformly, but was initiated at neurogenic foci scattered across the retina. These foci consisted of clusters of dividing neuroepithelial-like cells. The evidence is consistent with the proposal that these cells were derived from rod precursors. These results imply that rod precursors are capable of a wider range of developmental fates than they normally express.     

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.     

Demonstration of rod and cone photoreceptors in the lamprey retina by freeze-replication and immunofluorescence

Summary In common with other cyclostomata, the Japanese river lamprey (Lampetra japonica) has a retina consisting of distinct types of photoreceptor cells called long and short photoreceptor cells. After freeze-fracture, disc membranes of these photoreceptor cells were characterized in common by a homogeneous distribution of intramembrane particles on the protoplasmic fracture faces, in contrast to those of the myeloid bodies bearing scattering particles.Immunofluorescent examination was applied to the retina with monoclonal antibodies raised against bovine and chicken rhodopsins. Positive immunoreactivity was found to be limited to outer segments of the short cell, leaving the entire body of the long cell and all other components of the retina negative. The results suggest that the short cell is more closely related to a rod-type photoreceptor cell characterized by rhodopsin as its visual pigment.     

Measurement of potassium turnover in rod photoreceptors in toad isolated retina using ion-selective microelectrodes

Ion-selective microelectrodes (ISMs) were used to measure the turnover of intracellular K+ (Ki+) in rods in the isolated retina of the toad, Bufo marinus. The light-evoked hyperpolarization of rods decreases their passive K+ efflux, which in combination with active K+ uptake, decreases extracellular K+ concentration, Ko+.Rb+ substitutes for K+ in these processes. The turnover of Ki+ was measured as Rb+ and K+ were exchanged, using ISMs that were approximately five times more sensitive to Rb+ than to K+. When Ko+ was replaced by Rbo+, the light-evoked decrease in K+ efflux produced only a small change in ISM voltage, delta VISM, owing to the background of Rbo+. As Rbi+ replaced Ki+, the efflux shifted from K+ to Rb+ and delta VISM grew in amplitude. After loading the rods with Rbi+, Rbo+ was replaced by Ko+. The light-evoked decrease in Rb+ efflux lead transiently to a large delta VISM, since the change in Rbo+ was superimposed upon a background of Ko+. As Ki+ replaced Rbi+, the amplitude of delta VISM declined. When measured using this technique, the turnover of Ki+ was 95% complete in approximately 15 min. In low Ca2+ solutions, transmembrane fluxes of K+ (Rb+) increased and turnover of Ki+ occurred more rapidly. During background illumination, transmembrane fluxes of K+ (Rb+) decreased and turnover of Ki+ was slowed. These experiments have provided independent corroboration of earlier observations concerning rod K+ fluxes. This ISM-based technique also may be useful in measuring K+ turnover in other cell types.     

Enkephalin in the goldfish retina

Enkephalin-like immunoreactive amacrine cells were visualized using the highly sensitive avidin-biotin method. The somas of these cells were situated in the inner nuclear and ganglion cell layers. Enkephalin-stained processes were observed in layers 1, 3, and 5 of the inner plexiform layer. The biosynthesis of sulfur-containing compounds in the goldfish retina was studied by means of a pulse-chase incubation with 35S-methionine. A 35S-labeled compound, which comigrated with authentic Met5-enkephalin on high-performance liquid chromatography (HPLC), was synthesized and was bound competitively by antibodies to enkephalin and by opiate receptors. This compound was tentatively identified as "Met5-enkephalin." The newly synthesized 35S-Met5-enkephalin was released upon depolarization of the retina with a high K+ concentration. This K+-stimulated release was greatly suppressed by 5 mM Co2+, suggesting that the release was Ca2+ dependent. Using a double-label technique, enkephalin immunoreactivity and gamma-aminobutyric acid (GABA) uptake were colocalized to some amacrine cells, whereas others labeled only for enkephalin or GABA. The possible significance of enkephalin-GABA interactions is also discussed.     

Light-stimulated protein movement in rod photoreceptor cells of the rat retina

We examined the intracellular distribution of three proteins involved in the cyclic GMP cascade of visual transduction; cGMP phosphodiesterase, the alpha-subunit of G-protein and arrestin. In adult rats, light-induced changes in the amounts of G and arrestin in the photoreceptor cell outer segments were observed both by polyacrylamide gel analysis of purified ROS and by immunocytochemical localization on retinal sections. In dark conditions, G was concentrated in the outer segments of photoreceptor cells while in the light G alpha was seen in the inner segments and the outer nuclear layer. Arrestin had the opposite distribution, appearing in the inner segments and outer nuclear layer under dark conditions and in the ROS under light conditions. In contrast, PDE, the enzyme which is activated by G and inhibited by arrestin showed no light-stimulated movement. In both light- and dark-adapted retinas, PDE was localized primarily in the outer segments of the photoreceptor cells.     

Model feedback mechanism between horizontal cells and photoreceptors of the vertebrate retina

A model with nonlinearity of the photoreceptor presynaptic membrane as its important distinguishing feature was created on the basis of the hypothesis that feedback between the horizontal cells and photoreceptors is effected by a current generated by the subsynaptic membrane of the horizontal cells and leaking partly into the photoreceptors. Measurements with the model also reproduced experimental observations such as depolarization of the cone during hyperpolarization of the horizontal cell in response to the showing of a ring of light or passage of an electric current, and also certain special features of the current-voltage curves of the cones.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 86–94, January–February, 1977.     

Local regeneration in the retina of the goldfish

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transsclearal surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2′-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithiallike cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.     

Double-labeling techniques demonstrate that rod bipolar cells are under GABAergic control in the inner plexiform layer of the rat retina

The synaptic connectivity between rod bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina was studied using two immunocytochemical markers. Rod bipolar cells were stained with an antibody specific for protein kinase C (PKC, α isoenzyme), and GABAergic neurons were stained with an antiserum specific for glutamic-acid decarboxylase (GAD). Some amacrine cells were also labeled with the anti-PKC antiserum. All PKC-labeled amacrine cells examined showed GABA immunoreactivity, indicating that PKC-labeled amacrine cells constitute a subpopulation of GABAergic amacrine cells in the rat retina. A total of 150 ribbon synapses established by rod bipolar cells were observed in the IPL. One member of the postsynaptic dyads was always an unlabeled AII amacrine cell process, and the other belonged to an amacrine-cell process showing GAD immunoreactivity. The majority (n=92) (61.3%) of these processes made reciprocal synapses back to the axon terminals of rod bipolar cells. In addition, 78 conventional synapses onto rod bipolar axons were observed, and among them 52 (66.7%) were GAD-immunoreactive. Thus GABA provides the major inhibitory input to rod bipolar cells.     

Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.     

Local regeneration in the retina of the goldfish.

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transscleral surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2'-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithial-like cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

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.