L-type calcium channels in the photoreceptor ribbon synapse: localization and role in plasticity.

Calcium (Ca(2+)) influx through voltage-gated Ca(2+)channels stimulates a variety of neural activities, including process outgrowth, neurotransmission, and synaptic plasticity. In general, L-type channels control Ca(2+) influx into the soma and dendrites, whereas other Ca(2+) channel types control presynaptic activities. Neurons that make ribbon synapses, however, are among a select group of nerve cells whose presynaptic Ca(2+)-dependent secretion is linked to L-type channels. Recently, photoreceptor ribbon synapses have been shown to be capable of dramatic structural remodeling and neuritic outgrowth. Here, we have examined 1) the distribution of dihydropyridine (DHP)-sensitive (L-type) Ca(2+) channels in photoreceptor presynaptic structures and 2) the role of these channels in axonal plasticity and process outgrowth in culture. Using anti-alpha(1C) and the fluorescent dihydropyridine, (-)-DM-BODIPY DHP, L-type channels were localized in the outer plexiform layer of retinal sections and in presynaptic terminals of freshly isolated photoreceptors. In the rod terminal, dense patches of label were present; their distribution and number matched that of synaptic ribbons. After 1-7 days in vitro, punctate alpha(1C) staining occurred along newly formed neurites and presynaptic varicosities. Functional channels were present throughout the culture period, as determined by fura-2 imaging. Channel blockage by nicardipine, a DHP antagonist, inhibited axonal remodeling. Specifically, it prevented axon retraction and lamellipodium formation, reduced neurite growth, and produced long, thin processes on some, primarily cone, photoreceptors. L-type Ca(2+) channel activity, therefore, not only stimulates neurotransmission but contributes to presynaptic structural plasticity at the ribbon synapse.

Micromanipulation of retinal neurons by optical tweezers.

Micromanipulation by optical tweezers has been tested in cultures of mature isolated retinal cells to determine its potential for use in creating synaptic circuits in vitro. Rod and cone photoreceptors as well as other retinal nerve cell types could be optically trapped with a 980 nm diode laser mounted on an inverted light microscope using a 40x oil immersion objective numerical aperture of 1.3. Manipulation was done under sterile conditions using transparent culture dishes. To form cell groups, one half of a culture dish was made less adhesive by application of a thin layer of silicone elastomer. Unattached cells were trapped and relocated next to cells lying on an adhesive culture substrate. Optical trapping did not affect the ability of neurons to subsequently attach to the culture substrate. Up to 60% of trapped cells survived for 2 or more days. The pattern and rate of process outgrowth for manipulated cells was comparable to unmanipulated cells and by 2 days, cell-cell contacts were observed. Cultures were fixed at 2 and 5 days for electron microscopy. Organelle, nuclear and cytoplasmic structure of manipulated cells was completely normal and in photoreceptors, synaptic vesicles and ribbons were intact. Optical tweezers, therefore, provide a benign technique with which to micromanipulate whole neurons. The procedures also bestow increased precision to the study of cell-cell interactions by allowing the selection of potentially interacting cell types at a single cell level.

Age-related changes in the tiger salamander retina.

Tiger salamanders have been used in visual science because of the large size of their cells and the ease of preparation and maintenance of in vitro retinal preparations. We have found that salamanders over 27 cm in length show a variety of visual abnormalities. Compared to smaller animals (15-23 cm), large animals exhibited a decrease in visual responses determined by tests of the optomotor reflex. Small animals responded correctly an average of 84.5% of the time in visual testing at three light levels compared to an average of 68.4% for the large animals with the poorest visual performance at the lowest level of illumination. In addition, large animals contained (i) histological degeneration of the outer retina, in particular, loss and disruption of outer segments and abnormalities of the retinal pigmented epithelium, (ii) loss of cells, including photoreceptors, by apoptosis as evaluated with the TUNEL technique, and (iii) an increase in the number of macrophages and lymphocytes within the retina as determined by morphological examination. These histological changes were present in all large animals and all quadrants of their retinas. In contrast, small animals showed virtually no retinal degeneration, no TUNEL-positive cells, and few immune-like cells in the retina. Since large animals are also older animals. the visual changes are age-related. Loss of visual function and histological degeneration in the outer retina also typify aged human eyes. Thus, we propose that large salamanders serve as an animal model for age-related retinal degeneration. In addition to providing a source of aging retina that is readily accessible to experimental manipulation, the salamander provides a pigmented retina with a mixed (2:1, rod:cone) population of photoreceptors, similar to the degeneration-prone parafoveal region of the human eye.

Morphologic and neurochemical target selectivity of regenerating adult photoreceptors in vitro.

Regenerating adult central nervous system (CNS) neurons must re-establish synaptic circuits in an environment very different from that present during development. However, the complexity of CNS circuitry has made it extremely difficult to assess the selectivity and mechanisms of synaptic regeneration at the cellular level in vivo. The synaptic preferences of adult photoreceptors were examined by using a defined cell culture system known to support regenerative process growth, presynaptic varicosity formation, and establishment of functional synapses. Immunolabeling for synaptic vesicle protein 2 and ultrastructural analysis demonstrated that cell-cell contacts made by photoreceptors were synaptic in nature. Target selectivity was determined by quantitative analysis of contacts onto normal and novel target cell types in cultures in which opportunities to contact all retinal cell types were present. Target cells were identified by morphology and immunolabeling for the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine. Regenerating photoreceptors showed a strong preference for novel multipolar cell targets (amacrine and ganglion cells) over normal photoreceptor, horizontal, and bipolar cell targets. Additionally, photoreceptors were selective for targets containing the transmitter GABA. These results indicate first, that the normal synaptic partners for photoreceptors are not intrinsically the optimal targets for regenerative synapse formation, and second, that GABA may modulate synaptic targeting by adult photoreceptors.

Properties of acidified compartments in hippocampal neurons.

We have studied cultured rat hippocampal neurons using electron microscopic procedures based on the accumulation of DAMP (dinitroanilino-aminomethyl-dipropylamine) to identify acidified locales, fluorescence procedures to provide information about the pHs within certain endocytic compartments, and the uptake of horseradish peroxidase to evaluate effects of altering pH on membrane cycling in the axonal varicosities. We find that the endocytic compartments related to the lysosome-endosome system in the cell bodies and dendrites of these neurons maintain pHs in the range of about 5 to 6.5. This is the range that would be expected for structures participating in lysosomal digestion and for such functions as the endosomal dissociation of ligands from receptors. We also find that, as judged by uptake of horseradish peroxidase, exposure of the preparations to weak bases that neutralize intracellular compartments does not abolish the endocytic labeling of synaptic vesicles in the axonal varicosities. This suggests that passage through a markedly acidified compartment or stage is not obligatory for the endocytic phase of the cycling of synaptic vesicles.

Features of the polarity of the Golgi apparatus of frog photoreceptors: studies with lectins and with Brefeldin A.

The cis-trans polarity of the Golgi apparatus is important in Golgi functioning in glycosylation, sorting and other processes. The present work extends our prior studies on the polarity of the Golgi apparatus of frog (Rana) rod photoreceptors. We demonstrate that Golgi structures with the morphology and distribution of cis elements show the heaviest deposition of osmium. Elements with morphology and cytochemical reactivities resembling trans Golgi structures remain discernible as discrete arrays after exposure of the cells to Brefeldin A. These properties strengthen our identification of cis and trans elements since they are shared with corresponding Golgi structures in other cell types. We have also investigated the binding of lectins to sections prepared by cryoultramicrotomy. We find that Concanavalin A, with probable chief affinity for core mannoses in oligosaccharides, localizes to cis, medial and trans elements of the photoreceptor's Golgi apparatus. Wheat germ agglutinin, with likely affinity at least partly for terminal N-acetylglucosamines, localizes to trans and medial elements. A trans localization is seen with Ricinus communis agglutinin (RCA 120), but this lectin binds extensively only after neuraminidase treatments suggesting that its chief affinity is for galactose residues that are penultimate to sialic acids (neuraminic acids) in the native oligosaccharides. Overall, the pattern of lectin binding to Golgi structures that we observe resembles that seen in a variety of other cell types. The distribution of glycosylated molecules we detect in the photoreceptor's Golgi apparatus may bear upon such matters as the unusual features of the glycosylation of mature opsin.

Frog cones as well as Müller cells have peroxisomes.

We have localized D-amino acid oxidase in peroxisomes of frog retina using cerium procedures on tissue fixed in mixtures containing lower concentrations of glutaraldehyde than we had previously used in our cytochemical studies of this enzyme. We find the Müller cells of these preparations contain a more striking population of peroxisomes than had previously been thought: the D-amino acid oxidase-containing bodies are especially concentrated near the outer limiting membrane, but appreciable numbers are also found in the outer plexiform layer and near the inner limiting membrane. In addition, we find peroxisomes to be present in frog cone photoreceptors, particularly in zones near the ellipsoid. To our knowledge peroxisomes have not been described hitherto in vertebrate photoreceptors. Possible roles for the peroxisomes of the neural retina include participation in the metabolism of lipids (e.g. those of the cones' oil droplets, or of the outer segment) and involvement in oxidation of transmitter-related amino acids and of other small molecules.

Transient hyperglycosylation of rhodopsin with galactose.

Rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and N-acetylglucosamine. In the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. Treatment of bovine rod outer segments (ROS) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. Rats injected intravitreally with [3H]galactose and [14C]leucine and maintained in darkness were killed 1 hr, 6 hr, 1, 3 or 5 days following the injection. Retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by ConA sepharose chromatography and SDS-PAGE. During the first 6 hr, galactose selectively labeled rhodopsin in the Golgi-enriched fraction resulting in increased [3H]/[14C] ratios in both Golgi and ROS. The data suggested that trimming was occurring at the transition from Golgi to ROS. Furthermore, a decrease in isotope ratio in the ROS between 6 hr and 1 day suggested further trimming of rhodopsin after membrane assembly in the ROS. Additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. Rats injected intravitreally with [35S]methionine were dark-adapted for 2 hr. Following subcellular fractionation of retinas, ConA purified rhodopsin from ROS was applied to one of two additional lectin columns: Ricinus communis agglutinin (RCA) or Griffonia simplicifolia I (GSA). Eight to nine percent of the labeled rhodopsin was bound to and eluted from RCA, whereas none bound to GSA, indicating the presence of a beta-galactoside. The RCA agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. Galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. Binding of rhodopsin's oligosaccharide to RCA was abolished by pre-treatment with beta-galactosidase. Decreased binding of rhodopsin to RCA was observed following intravitreal injection of castanospermine but not swainsonine. Of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. The association of galactose with rat rhodopsin appeared to be a transient one. At 2 hr, 8-9% of rhodopsin contained galactose, at 6 hr only 2.2% had galactose and by 24 hr less than 1% did. The galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. Separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that 75% of the galactose-containing rhodopsin was in the plasma membrane and only 25% was in the discs. These findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed.

Peroxisomal oxidation of thiazolidine carboxylates in firefly fat body, frog retina, and rat liver and kidney.

D-amino acid oxidase is a widely distributed peroxisomal enzyme whose principal natural substrates are still unknown. Thiazolidine carboxylates, their derivatives and relatives, and the intermediates in their metabolism are among the more plausible substrate candidates. Using a cytochemical procedure, we have explored the distribution of peroxide-generating enzymatic activity against two thiazolidine carboxylates. We find that these compounds are effective substrates for peroxisomal oxidation in a variety of tissues that contain peroxisomal D-amino acid oxidase. Reaction was seen in the "classical" peroxisomes of rat liver and kidney, the peroxisomes of the fat body of firefly and of Drosophila and the peroxisomes of frog retina. Interestingly, both with the thiazolidine compounds and with more traditional D-amino acid oxidase substrates, the fireflies' photocyte granules, which are peroxisomes, lack activity.

Peroxisomes in the head of Drosophila melanogaster.

Peroxisomes were localized in the head of wild-type and mutant strains of Drosophila melanogaster by use of a cytochemical method for the demonstration of D-amino acid oxidase activity. With similar techniques we had found previously that vertebrate photoreceptors have few, if any, bodies with cytochemically demonstrable oxidase activity, but that the pigment epithelial cells adjacent to the photoreceptors have a substantial population of such bodies. Peroxisomes in Drosophila were very abundant in the fat body. Probable peroxisomes were also present in the peripheral retina of the eye, including in retinular (retinula) and pigment cells, but there were very few of them. Thus, our results suggest that the fat body, which lies adjacent to the eye, is the principal site of peroxisomal function in the head. Peroxisome functions in the Drosophila head may include participation in the genesis of eye pigments.

The localization and timing of post-translational modifications of rat rhodopsin.

Rat retinas were labeled by incubation with, or intravitreal injection of, [14C]leucine along with tritiated palmitic acid, glucosamine or galactose. At selected intervals, subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted and purified by affinity chromatography and gel electrophoresis. Time courses revealed that leucine rapidly and transiently labeled the rhodopsin in the rough endoplasmic reticulum (RER), with a maximum at 1.5 hr post-injection. Subsequently, the rod outer segments (ROS) contained the labeled rhodopsin, with the ROS labeling maximally at 6-12 hr. Palmitate labeling followed the same pattern but was subject to a delay, presumably because of a large intracellular pool of the fatty acid. With palmitate the RER rhodopsin was not maximally labeled until 12 hr. The acylation of rhodopsin takes place in the RER sometime after the polypeptide has been translated but before transport to the Golgi. Glucosamine labeling was also delayed because of intracellular pools of the sugar or its metabolic derivatives. But because of secondary glycosylation in the Golgi, the rhodopsin in the ROS also labeled maximally with glucosamine at about 6 hr. Administration of [3H]galactose resulted in the labeling of rhodopsin both in vivo and in vitro, in part possibly because of its conversion to mannose and subsequent insertion into the core oligosaccharide on the RER. However, in the ROS the ratio of tritium, derived from [3H]galactose, to [14C]leucine decreased by a factor of 2 between 6 and 24 hr post-injection. Moreover, between 6 and 12 hr post-injection, labeled rhodopsin molecules in the ROS underwent a shift in mobility on gels indicative of trimming to a lower molecular weight. Thus some sugar residues may be added to the rhodopsin in the inner segment and removed in the ROS.

Addition of the chromophore to rat rhodopsin is an early post-translational event.

Rat retinas were labeled either by intravitreal injection of [14C]leucine or by incubation with [3H]-leucine or [35S]-methionine. Subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted with detergent and purified by chromatography on ConA-Sepharose. A fraction enriched in rough endoplasmic reticulum (RER) and substantially free of rod outer segments (ROS) was found to contain a light-sensitive protein exhibiting the properties of rhodopsin on ConA-Sepharose or Agarose chromatography and on SDS-polyacrylamide gel electrophoresis, as well as immunologically. Intravitreal injection of [3H]-retinol also labeled the rhodopsin in the RER under conditions in which the rhodopsin in the ROS was not heavily labeled. Thus the chromophore appears to be attached to opsin shortly after the apoprotein is translated in the RER.

Cytochemical localization of a D-amino acid oxidizing enzyme in peroxisomes of Drosophila melanogaster.

A peroxide generating oxidase is demonstrated cytochemically in the peroxisomes of adult and larval Drosophila melanogaster, Oregon R and Rosy-506 strains. This enzyme activity is demonstrable using D-pipecolate or D-proline, but not L-proline, as substrate and is inhibited by kojic acid. Thus this enzyme shares cytochemical characteristics with vertebrate D-amino acid oxidase.

Acylation of disc membrane rhodopsin may be nonenzymatic.

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.

The acylation of rat rhodopsin in vitro and in vivo.

Rat retinas were incubated with [3H] palmitate and [14C] leucine and subsequently detergent-extracted. Glycoproteins were isolated on Con A-Sepharose columns and separated by gel electrophoresis. Leucine labeled the newly synthesized opsin but palmitate was esterified to both mature rhodopsin and newly synthesized opsin which migrated more slowly because of its untrimmed oligosaccharide chains. Crude rod outer segments were found to contain most of the palmitate-labeled mature rhodopsin, while the retinal debris contained most of the doubly labeled newly synthesized opsin. Homogenization of double-labeled retinas followed by centrifugation in a linear sucrose gradient gave rise to several bands of particles including purified rod outer segments, a Golgi-enriched fraction and a pellet enriched in endoplasmic reticulum. Newly synthesized opsin was first found in the pellet at the earliest incubation times and subsequently appeared in the Golgi fraction and finally in the rod outer segments. Palmitate-labeled mature rhodopsin was found only in the rod outer segments. It appeared at the earliest time points and increased with time. Thus the acylation of opsin occurs in the endoplasmic reticulum, shortly after polypeptide synthesis, and in the rod outer segments, the latter possibly as an exchange reaction. Most of the newly synthesized opsin remained in the pellet and did not pass through the Golgi to the rod outer segments. Intravitreal injection of [3H] palmitate and [14C] leucine gave rise to doubly labeled opsin that appeared to remain untrimmed for at least 6 hr in vivo. After 17 hr, both labels were found only in mature rhodopsin, thus accumulation of new molecules in the endoplasmic reticulum may occur in vivo. In addition, leucine maximally labeled the opsin-rhodopsin pool early in the first day whereas palmitate did not maximally label rhodopsin until 2- or 3 days post injection. Moreover, while leucine label was lost at day 9 because of rod outer-segment renewal and shedding, the palmitate label in rhodopsin remained unchanged. Thus, palmitate labeling in vivo reflects the pattern seen in vitro with a prolonged equilibration of rod outer-segment rhodopsin with the fatty-acid pool, probably mediated by a fatty acyl exchange reaction.