Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition.

Photoreceptor degeneration is mediated by apoptosis in several animal models, although the underlying mechanisms are yet to be elucidated. We present here an apoptotic model based on a primary cell culture of tiger salamander photoreceptors, in which treatment with carbonyl cyanide m-chlorophenylhydrazone (cccp), a protonophore, induced apoptosis. Cells exposed to cccp showed condensed nuclei and displayed positive TdT-dUTP terminal nick-end labeling (TUNEL). In addition, 10-100 microM cccp rapidly induced a reduction of Delta psi(m) and > or = 30 microM cccp induced a significant leakage of calcein from mitochondria to cytosol and nucleus, indicating a change in mitochondrial inner membrane permeability. Cyclosporin A (CsA), a transition pore blocker, did not prevent the cccp-induced MPT or the cccp-evoked apoptotic cell death, suggesting that cccp-induced apoptotic process was mediated by a CsA-insensitive pathway. This cell model provides an in vitro tool for studying mechanisms of photoreceptor apoptosis in isolated photoreceptors and may provide clues to the etiology of retinal degeneration.

Decorin and suramin inhibit ocular fibroblast collagen production.

BACKGROUND: The process of ocular wound healing with respect to glaucomatous filtering procedures is of current interest. Delaying this response in patients could possibly lead to more favorable surgical results. So far, only highly toxic antimetabolites have come into frequent clinical use. The possible efficacy of other groups of substances such as growth factor inhibitors has not yet been examined in vitro. METHODS: We exposed Tenon's capsule fibroblasts in tissue culture to various concentrations of decorin and suramin. The dose responses of type I and type III collagen to these inhibitors were measured using an ELISA-type dot blot assay. Total cellular protein production was assayed by measuring the incorporation of tritiated leucine. RESULTS: At a concentration of 10 micrograms/ml, suramin reduced the collagen production by more than 80%. Decorin, at a concentration of 100 micrograms/ml, reduced type I collagen production by about 50% while type III collagen was reduced by 80%. At these concentrations, the total cellular protein production was not inhibited. CONCLUSIONS: Both suramin and decorin, which specifically inhibit the action of growth factors on target cells, reduce the production of collagen synthesis by Tenon's capsule fibroblasts. This is a specific effect, because total protein production is not influenced. This sets these substances apart from antimetabolites. Decorin and suramin may have clinical relevance in that they appear to interfere with ocular wound healing more specifically than the substances so far frequently used.

Noise and information in interferometric cross correlators.

We consider optical interferometric cross correlators based on broadband light sources. We derive the signal-to-noise ratio from basic principles and supply experimental evidence that corroborates the theoretical analysis. Noise sources are discussed, and the signal-to-noise ratio of our experimental system is measured.

Ascorbate stimulates type I and type III collagen in human Tenon's fibroblasts.

PURPOSE: To better understand wound healing after glaucoma filtration surgery by measuring the production of type I and type III collagen in cultured Tenon's fibroblasts and determine the effect of ascorbic acid on collagen subtype production. METHODS: An ELISA-type dot blot assay was used to directly measure the production of types I and III collagen by subconfluent cultures of fibroblasts from human Tenon's capsule. Because ascorbic acid is both high in aqueous humor and necessary for the production of collagen, we measured the dose response of type I and type III collagen production to ascorbic acid. RESULTS: Ascorbic acid stimulated an increase in collagen production that reached a maximum level at 100 micrograms/ml. This is approximately half of the ascorbic acid concentration found in human aqueous humor. Unlike previous reports, we found no toxic effects from ascorbic acid at concentrations as high as 250 micrograms/ml over a 24-hour period. The lack of toxicity may result from the use of serum-free media in the assay. CONCLUSIONS: This culture system will be useful for exploring factors that may alter collagen production and could potentially affect wound healing.

Effects of excitatory amino acids on phosphoinositide metabolism in frog retina.

The effects of excitatory amino acid receptor agonists on the hydrolysis of phosphoinositides were examined using frog retinal membranes prelabeled in vitro with either 32PO4 or [3H]inositol. Glutamate stimulated release of [3H]inositol phosphates (IPs) from the retinas and altered the 32P-labeling pattern of phosphatidylinositol (PI) cycle intermediates. This indicates that glutamate affects not only the hydrolysis of phosphoinositides but possibly other steps involved in the PI cycle. Among glutamate analogs, kainate (KA), quisqualate (QA), and, to a lesser extent, N-methyl-D-aspartate (NMDA) mimicked the glutamate effect, whereas L-2-amino-4-phosphonobutyrate (L-AP4) was not effective in causing either the accumulation of [3H]IPs or the alteration of the 32P-labeling pattern of PI cycle intermediates. Among QA specific agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), but not ibotenate (IBO) or trans-1-aminocyclopentane-1,3-dicarboxylate (ACPD) was active in stimulating IPs formation. KA effect on IPs formation may be due to indirect (polysynaptic) activation of receptor(s) other than L-AP4, IBO, or ACPD specific QA receptors. To avoid activating polysynaptic pathways, retinal synaptoneurosomes prelabeled with [3H]inositol were used to examine the hydrolysis of phosphoinositides. As in whole retinas, KA, carbachol (CARB), and NMDA stimulated the release of IPs while L-AP4 had minimal effect. Glycine (GLY) had no effect. Our results show CARB and KA to be the most effective in stimulating the production of IPs. Their effects were exerted directly through separate receptors and not through polysynaptic pathways. ACPD and IBO were the least effective in eliciting the release of IPs. Our studies provide evidence that ionotropic and not metabotropic glutamate receptors are involved in PI metabolism in the retina.

Pattern recognition of amino acid signatures in retinal neurons.

Pattern recognition of amino acid signals partitions the cells of the goldfish retina into nine statistically unique biochemical theme classes and permits a first-order chemical mapping of virtually all cellular space. Photoreceptors, bipolar cells, and ganglion cells display a set of unique, nominally glutamatergic type E1, E1+E2, and E4 signatures, respectively. All horizontal cells are assignable to a GABAergic gamma 2 class or a non-GABAergic class with a glutamate-rich E3 signature. The amacrine cell layer is largely a mixture of (1) a taurine-dominated T1 Müller's cell signature and (2) GABAergic gamma 1, glycinergic G1, and dual glycinergic/GABAergic G gamma 1 amacrine cell signatures. Several major conclusions emerge from this work. (1) Glutamatergic, GABAergic, and glycinergic neural signatures and glial signatures account for over 99% of the cellular space in the retina. (2) All known neurons in the goldfish retina are associated with a set of conventional nonpeptide neurotransmitters. (3) Multiple forms of metabolic profiles are associated with a single nominal neurotransmitter category. (4) Glutamate and aspartate contents exhibit overlapping distributions and are not adequate univariate probes for identifying cell classes. (5) Signatures can serve as quantitative measures of cell state.

Glycine stimulates calcium-independent release of 3H-GABA from isolated retinas of Xenopus laevis.

A perfusion system was used to monitor the release of [3H]-GABA from isolated retinas of Xenopus laevis. Measurable release was stimulated by glycine at concentrations as low as 200 microM. Glycine-stimulated release was blocked by strychnine, and was not reduced in "calcium-free" Ringer's solution (0 Ca2+/20 mM Mg2+). Glutamate also stimulated calcium-independent release, using concentrations as low as 100 microM. In contrast, release stimulated by 25 mM potassium was reduced by 80% in calcium-free medium. In most experiments, agonists were applied in six consecutive 4-min pulses separated by 10-min washes with Ringer's solution. Under these conditions, the release stimulated by 0.5 mM glutamate or 25 mM potassium decreased by at least 50% from the first to the second pulse, and then gradually decreased with successive applications. In contrast, the response to 0.5 mM glycine at first increased and then only gradually decreased with successive pulses. These patterns of response to different agonists were similar in calcium-free medium. Somatostatin (-14 or -28) also stimulated release, and this effect was inhibited by AOAA, an inhibitor of GABA degradation. In the presence of AOAA, somatostatin had little effect, except at high concentrations of somatostatin (5 microM), which increased both basal and glycine-stimulated release. In contrast to somatostatin, glycine-stimulated release was much larger in the presence of AOAA. Autoradiography was used to investigate which cell types released [3H]-GABA under our conditions. Autoradiograms showed that horizontal cells and a population of apparent "off" bipolar cells were well-labeled by [3H]-GABA high-affinity uptake. In addition, light labeling was seen over numerous amacrine cells. After application of glycine, glutamate, or potassium, there was a decrease in label density over horizontal cells.

Glycine high-affinity uptake labels a subpopulation of somatostatin-like immunoreactive cells in the Rana pipiens retina.

Somatostatin-like immunoreactivity (Som-LI) and glycine high-affinity uptake have been characterized in the Rana pipiens retina. These labels are found in both the outer and inner plexiform layers (OPL and IPL), suggesting that interplexiform cells (IPCs) contain both Som and glycine in this retina. In double-label experiments these labels colocalize to an abundant population of cells in the mid-inner nuclear layer (INL), in the second or third cell layer distal from the IPL. These cells have medium sized spherical or oval somas, each with a single thin descending dendrite which ramifies in the distal IPL. Processes ascending from cells at this location were not visualized by immunocytochemistry, but could be seen by autoradiography of tissue processed for glycine high-affinity uptake. In autoradiographs apparent IPCs were the most intensely labeled cell type in this retina. Som-LI is also found in two types of probable amacrine cells in the proximal INL adjacent to the IPL, neither of which is labeled by glycine high-affinity uptake. One of these is rare (about 10 cells/mm2), and has a large pyriform soma with a thick dendrite that branches in the proximal IPL. The other type is more common (324 +/- 20 cells/mm2), has medium-sized spherical or horizontally elongated elliptical somas, and has multiple thin dendrites projecting into the distal IPL. In addition to the above cell types, faint Som-LI was seen in cells of the ganglion cell layer, possibly indicating the presence of somatostatinergic ganglion cells or displaced amacrine cells.

Somatostatin-like immunoreactivity and glycine high-affinity uptake colocalize to an interplexiform cell of the Xenopus laevis retina.

Antibodies directed against somatostatin have been used to label a population of interplexiform cells (IPCs) in the Xenopus laevis retina. These cells have spherical soma which lie in the inner nuclear layer (INL), adjacent to or one cell distal to the inner plexiform layer (IPL). Processes from these cells project throughout the IPL, with a fairly dense accumulation of labeled dendrites in the upper two-fifths of the IPL and a dense, narrow band of labeled dendrites adjacent to the ganglion cell layer. These cells also have finer processes, originating at the cell body, that traverse the INL and ramify in the outer plexiform layer (OPL). Double label experiments show that all of the cells that contain somatostatin-like immunoreactivity (SOM-LI) in the INL are also labeled by high-affinity uptake with 3H-glycine. Immunocytochemistry of retinal whole mounts shows that these cells are evenly distributed across the retina at a density of 542 +/- 65 cells/mm2. On the basis of the colocalization experiments and the morphological homogeneity of these cells, we suggest that they represent a single cell type. Interplexiform cell processes were further characterized by electron microscopy after immunocytochemistry or 3H-glycine autoradiography. In the IPL, IPC processes are seen to be postsynaptic at both ribbon and conventional synapses. This input is found almost entirely in the distal two-fifths of the IPL. Interplexiform cell processes are presynaptic to unlabeled processes in both the distal and proximal IPL. In the OPL, labeled processes are found near or contiguous with photoreceptor bases, and are often presynaptic to small-diameter processes. The postsynaptic processes have been identified as bipolar cell dendrites in six cases. Interplexiform cell processes may also contact horizontal cell processes in the OPL.

FMRFamide-immunoreactive retinopetal fibers in the frog, Rana pipiens: demonstration by lesion and immunocytochemical techniques.

Retinopetal fibers, immunoreactive to a molluscan cardioexcitatory-like peptide (FMRFamide-ir), were examined in Rana pipiens with the use of immunocytochemical and lesion techniques. In intact frogs, FMRFamide-ir retinopetal fibers were found in the optic nerve, optic nerve head, and nerve fiber, ganglion cell and inner plexiform layers of the retina. Presumptive monostratified amacrine cells were also labeled. As observed in flat-mounted retinas, the retinopetal fibers radiated from the optic disc toward the peripheral retina, branched many times along their course and were more prevalent in the dorsal retina. Crushing the optic nerve eliminated retinopetal fibers from all regions except the cerebral stump of the optic nerve, indicating that this projection was of central origin. Bilateral prechiasmatic lesions completely eliminated retinopetal fibers from both retinas, indicating that the fibers arose from the rostral forebrain. Within the rostral brain, FMRFamide-ir perikarya were found in olfactory bulb, diagonal band, medial septum, anterior commissure area, and two regions of the posterior preoptic area. Olfactory bulbectomy and midforebrain lesions equally reduced the numbers of these fibers in the retina, implicating the nervus terminalis as a possible source for some of the retinopetal projection. These data will serve as a foundation for future studies on the function of retinopetal fibers in the frog retina.

Monensin stimulates glycerolipid incorporation into rod outer segment membranes.

Monensin is an ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells. In this study, we examined the effects of monensin on the incorporation of newly synthesized glycerolipids into retinal rod outer segment (ROS) membranes. Frog retinas were incubated in the presence or absence of monensin (50 nM) with either [1,2,3-3H]glycerol or [9,10-3H]palmitic acid as radiolabeled substrate. Total lipids were extracted from retinas and ROS membranes and resolved into individual phospholipid classes and neutral lipids by thin-layer chromatography. In the presence of monensin, the specific activity of ROS phospholipids was increased about 2-fold with [3H]glycerol and nearly 3-fold with [3H]palmitate as substrates relative to controls. In contrast, the specific activity of total retinal lipids, the relative incorporation of label into ROS and retinal phospholipids, and the total lipid phosphorous content of ROS membranes and retinas were not significantly different from control values. These data suggest that the enhanced labeling of ROS phospholipids in the presence of monensin was due to altered intracellular routing of lipids rather than increased glycerolipid synthesis. Under the same conditions, total retinal protein synthesis was about 90% of control, but light microscopic autoradiography indicated that newly synthesized proteins were not transported to the ROS for assembly into disc membranes. Thus, newly synthesized glycerolipids can be delivered to the ROS by a mechanism which is independent of protein transport to that cellular compartment.

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types.

Oviduct tissue slices were incubated with [3H]-leucine or [3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [3H]-mannose incorporation (greater than 90% inhibition) but a minimal effect on [3H]-leucine incorporation (less than 10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower Mr when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of somatostatin from frog brain: coisolation with retinal somatostatin-like immunoreactivity.

Somatostatin-like immunoreactivity (SLI) was purified from frog brain and retina, and the structure of the brain peptide was determined. Frog brain (101 g) and retinal (45 g) tissues were extracted with 3% acetic acid, yielding 9.6 and 0.44 nmol of SLI, respectively. SLI was further purified by chromatography on a somatostatin immunoaffinity column followed by sequential application to reverse-phase C-18 HPLC columns. The brain and retinal peptides, purified roughly 100,000-fold with net yields of 7.5 and 2.3%, respectively, appeared identical in the final steps of purification. The amino acid sequence of brain SLI, as determined by a gas-phase automated Edman degradation technique, was as follows: Ala-Gly-(Cys)-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-(Cys). Our data indicate that despite structural variations in somatostatins of other lower vertebrates, the amino acid sequence of frog brain and, by deduction, retinal SLI is identical to that of somatostatin tetradecapeptide. These findings support the physiological relevance of studies directed at elucidating the neurotransmitter function of somatostatin using the well-established models of frog brain and retina.

Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.

Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [14C]leucine was not significantly affected. In a companion single-label incubation, TM blocked the incorporation of [3H]leucine into rod outer segment (ROS) membrane Cl3CCOOH-precipitable material by 95% relative to controls. When retinas were labeled with [35S]methionine, fluorograms of NaDodSO4/polyacrylamide gels from control retinas and ROS membranes exhibited a heavily labeled component (apparent Mr approximately 37,000) which had the electrophoretic and antigenic properties of opsin, the rod visual pigment apoglycoprotein. TM-treated retinas exhibited a substantially reduced labeling of the Mr 37,000 component and incorporation of label into a component (apparent Mr approximately 32,000) not found in control retinas, which exhibited the electrophoretic and antigenic behavior of nonglycosylated opsin. ROS membranes isolated from TM-treated retinas contained neither the Mr 37,000 nor the Mr 32,000 radiolabeled species. Light-microscope autoradiograms of retinas incubated with [3H]leucine in the absence of TM exhibited bands of silver grains at the base of ROS, indicative of new membrane assembly. However, no such bands were observed in autoradiograms of TM-treated retinas. These results suggest that glycosylation of opsin is required for its incorporation into ROS membranes.

Birefringent periodicities in amphibian rod outer segments.

Birefringence variations, seen as regularly spaced altering light and dark rings (bands), have been observed along the length of unfixed, freshly isolated rod outer segments (ROS) of both Rana pipiens and Xenopus laevis. In our hands, the spatial frequency of the banding pattern is from 1.3-1.6u/band in Rana pipiens and 1.8-2.2u/band in Xenopus laevis ROS, both corresponding closely to determinations we made in the same animals of the quantity of new ROS disks added each day. To further probe this correlation, Xenopus laevis were maintained at 16 degrees C to lower the disk renewal rate. A similar correspondence was found, with the banding pattern and renewal rate both at 0.8-1.Ou/day. Further experiments involving Xenopus laevis placed on altered lighting cycles have suggested the existence of two normally superimposed periodicities. The more intense component is driven by the environmental light cycle, and thus may be regarded as diurnal. The less intense component is seen infrequently, suggesting lability, and apparently follows a 24-hour period in both constant light and darkness.

The effects of monensin on transport of membrane components in the frog retinal photoreceptor. I. Light microscopic autoradiography and biochemical analysis.

We have explored the use of the Na+-H+ ionophore monensin as a potential tool for the investigation of membrane assembly and transport in retinal photoreceptors. Autoradiographic analysis of frog retinas incubated with [3H]leucine in the presence of monensin revealed a lack of concentrated silver grains ("bands") at the base of the rod outer segments, in contrast to controls. This is indicative of a pronounced monensin-induced decrease in disc membrane assembly. Biochemical analyses of whole retinas and isolated rod outer segment membranes showed that protein synthesis (including opsin synthesis) was not significantly inhibited under these conditions, whereas passage of membrane protein to the rod outer segment was blocked. Glycerolipid synthesis was not significantly affected by monensin. The results suggest that membrane proteins (e.g., opsin) destined for incorporation into the rod outer segment must pass through the Golgi apparatus and demonstrate the potential utility of monensin for inhibiting aspects of marcomolecule transport in photoreceptors.

Frog rod outer segment shedding in vitro: histologic and electrophysiologic observations.

Rod outer segment shedding in the frog, Rana pipiens, has been studied using an in vitro eyecup method. Control experiments have shown that shedding responses in vitro are comparable to those in vivo and, like the situation in vivo, shedding in isolated eyecups requires a dark period followed by light onset. We found an initial, rapid and light-evoked component of the shedding response to be critically dependent upon bicarbonate concentration, supporting the initial discovery of a bicarbonate requirement for Xenopus rod shedding by Besharse et al. In Rana, in vitro shedding occurs in the presence of 20 mM aspartate, suggesting that functional integrity of the inner retina is not a prerequisite for rod shedding. Additionally, shedding was found to be suppressed completely in the presence of the local anesthetic MS-222 and the phosphodiesterase inhibitor IBMX. In the case of IBMX, electrophysiologic recording indicated changes in photoreceptor sensitivity in the presence of the drug. Such changes may play a role in the observed inhibition of shedding.

Simple sugars inhibit rod outer segment disc shedding by the frog retina.

To investigate the hypothesis that sugar molecules might act as markers for ROS disc phagocytosis, frog eyecups were incubated in a normally permissive medium for ROS disc shedding with and without added simple sugars. L-fucose, alpha-methyl-D-mannopyranoside and D-mannose all significantly reduced the numbers of packets of ROS discs found in the retinal pigment epithelium. D-fucose, L-mannose, D-fructose, D-galactose, D-glucose and sucrose were without significant effect at the same concentration. Ultrastructural examination of the retinas indicates that the sugars were effective on the disc shedding process rather than on phagocytosis of already shed disc packets.

Retinomotor pigment migration in the teleost retinal pigment epithelium. II. Cyclic-3',5'-adenosine monophosphate induction of dark-adaptive movement in vitro.

The retinal pigment epithelium (RPE) and photoreceptors of teleosts exhibit dramatic examples of cell motility (called retinomotor movements) in response to diurnal changes in lighting conditions. In darkness the pigment granules of the RPE migrate to the scleral base of the RPE cell and cone photoreceptors elongate. In the light these movements are reversed; pigment granules disperse into the long apical projections of the RPE cell and cones contract. It is reported here that treatments that elevate cytoplasmic cyclic AMP induce dark-adaptive movements (pigment aggregation and cone elongation) in light-adapted retinas cultured in the light. Treatments designed to elevate cGMP had no effect. In dose-response studies with the cAMP analog, dibutyryl cyclic AMP (dbcAMP), we found that the RPE pigment did not exhibit intermediate states of aggregation with increasing concentrations of dbcAMP but instead changed abruptly from the fully light-adapted to the fully dark-adapted retinomotor positions between 10 microM and 50 microM exogenous dbcAMP concentrations. Cones, on the other hand, elongated to intermediate extents in proportion to increasing dbcAMP concentration between 10 microM and 500 microM. These observations suggest that cytoplasmic cAMP plays a role in regulating retinomotor position in both RPE and cones.

Biosynthesis of somatostatin-like immunoreactivity by frog retinas in vitro.

Somatostatin-like immunoreactivity has been localized to a wide variety of central nervous system neurons, including the retina. We utilized the unique advantages the retina provides for in vitro studies of nerves to examine the biosynthesis of somatostatin. Extracts of frog retinas pulse-labeled with [35S]cysteine for various time periods revealed uptake of radioactivity into material adsorbable by anti-somatostatin antibody linked to affinity beads. This uptake increased in a curvilinear fashion for 4 h and was inhibited by cycloheximide (0.2 mM) or by boiling the retinas prior to labeling. Pulse-chase experiments revealed that affinity-adsorbable radioactivity from retinal extracts decreased with time of incubation in chase medium; 89% of this decrease could be accounted for by increased in the affinity-adsorbable radioactivity of the chase medium. Chromatography of the retinal extracts on Sephadex G50 (superfine) revealed four elution peaks, whereas only one peak, co-eluted with somatostatin-14, could be identified in the medium. Chromatographic elution patterns of affinity-adsorbable radioactivity from extracts of pulse-labeled retinas incubated in chase medium for various times showed a gradual shift of radioactivity from the earlier-eluting peaks to the later ones. These studies indicate that biosynthesis of somatostatin occurs in frog retinas in vitro. The retina may be a useful model for further study of peptidergic neurons.