Scott F. Basinger

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.

Decorin and suramin inhibit ocular fibroblast collagen production

Abstract• 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 Tenons 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 μg/ml, suramin reduced the collagen production by more than 80%. Decorin, at a concentration of 100 μg/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 Tenons 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.

Phosphatidyl choline metabolism in the frog rod photoreceptor

Abstract Using an in vitro incubation system, phosphatidyl choline metabolism was studied in the frog rod photoreceptor using [3H]choline as a precursor of phosphatidyl choline incorporated into the rod outer segment disc membranes. Phosphatidyl choline incorporation was not affected when visual pigment synthesis was inhibited with puromycin, but was significantly inhibited by hemicholinium, EDTA, EGTA, dinitrophenol, oligomycin and cell lysis by detergent or sonication. The presence or absence of the pigment epithelium has no effect on phosphatidyl choline metabolism other than that of a competitive nature. While neither rhodopsin, phosphatidyl ethanolamine, or phosphatidyl inositol incorporation is affected by incubation in light, phosphatidyl choline incorporation is inhibited by both prior bleaching and light incubation. Changes in the phosphatidyl choline composition of disc membranes may be one of the initial steps in the process by which rod photoreceptor outer segments shed their tips.

Biosynthesis of Somatostatin-Like Immunoreactivity by Frog Retinas In Vitro

Abstract: 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 increases 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.

Cholecystokinin-like immunoreactivity in frog retina: Localization, characterization, and biosynthesis

We sought to localize and characterize cholecystokinin-like immunoreactivity (CCK-LI) in frog retinal tissues, CCK-LI was present in concentrations of 2.20 +/- 0.19 pmol/g wet weight (mean +/- SE, n = 6) in water extracts of frog retina and 0.23 +/- 0.04 pmol/g in acid extracts. The predominant form of CCK-LI co-eluted with the octapeptide of CCK on Sephadex G50 column chromatography. A minor form which eluted as a molecule larger than the 39 amino acid form of CCK was found only in the acid extracts. By immunohistochemistry, CCK-LI was localized to amacrine cells which give rise to processes ramifying in the distal 2/3 of the inner plexiform layer. Time dependent uptake of 35S-methionine into CCK-LI was observed in cultured frog retinas and this uptake was blocked with 0.2 mM cycloheximide. Our findings suggest that the retina may be a useful system for further characterization of the neural function of CCK-LI.

The effects of local anaesthetics on retinal function.

Isolated frog eyecups were incubated in Ringer containing local anaesthetics to study the effects of these drugs on dark-adaptation of the ERG. Relative to controls, dark-adaptation in eyecups treated with millimolar concentrations of MS-222, benzocaine, and procaine HCl was significantly inhibited during 10 to 120 min following the cessation of the adapting light. These drugs also prevented the recovery of the c-wave during dark-adaptation, resulting in ERG waveforms resembling those found in light-adapted eyecups. Measurements of rhodopsin in the retina were consistent with previous findings showing that rhodopsin regeneration in situ is inhibited by local anaesthetics. In vitro regeneration experiments in which bleached rod outer segment fragments were added to 11-cis retinal showed that preincubation of retinal with MS-222 in ethanol prevents rhodopsin regeneration. Evidence was obtained spectrophotometrically for the formation of a complex between MS-222 and 11-cis retinal with a gamma max of 512 nm. We propose that the formation of a Schiffs base between these two compounds blocks the recombination of rhodopsin, and in situ, leads to the inhibition of dark-adaptation.

Purification of Somatostatin from Frog Brain: Coisolation with Retinal Somatostatin-Like Immunoreactivity

Abstract: 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.

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.

CONTROL OF ROD SHEDDING IN THE FROG RETINA

In all vertebrate species examined thus far, rod outer segment shedding follows a cyclic pattern in which the outer segment tips are shed shortly after the onset of light. Work in the rat retina suggests that rod shedding may follow a circadian rhythm which is controlled by one or more circadian oscillators. Our results in the frog retina are significantly different in that: rod shedding can be driven by the onset of light or other environmental cues; shedding does not persist in constant darkness; shedding is unaffected in frogs with chronic unilateral or bilateral optic nerve section; and shedding will rapidly phase shift to the time of light onset on a wide variety of diurnal cycles. Thus, rod shedding in the frog retina does not appear to be a classical circadian rhythm.

[101] Rhodopsin biosynthesis in isolated retinas

Abstract The system described in this section has been successfully used to study rhodopsin biosynthesis in the isolated frog retina, and modifications can be made for the retinas of other species. 14 The system is particularly effective for studying the short-term events in visual pigment biosynthesis in that it allows the use of a variety of experimental manipulations not possible in vivo . Using a combination of autoradiography and biochemistry, this system has supported the in vivo 6,7 data that demonstrated that opsin is synthesized and partially glycosylated on the rough endoplasmic reticulum, further glycosylated in the Golgi, then transported to the base of the outer segment, where it is incorporated into the evaginating plasma membrane. 15 Further, the autoradiography shown in Fig. 4 demonstrates that this system supports rhodopsin synthesis and rod outer segment renewal for many hours. Column chromatography (Fig. 5) and/or SDS-disc gels (Fig. 6) of purified disk membranes demonstrate that rhodopsin synthesized in the isolated retina has all the normal characteristics of rhodopsin synthesized in vivo . However, the specific activity of purified visual pigment from isolated retinas is usually much higher, and, of course, the biosynthesis is under experimental control.