Some mammalian retinae lack FMRF-amide-like immunoreactive ... tion was cut, it was placed on a...
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May 1989 Vol. 30/5 Investigative Ophthalmology & Visual Science Articles Some Mammalian Retinae Lack FMRF-Amide-Like Immunoreacfive Efferents Anne C. Rusoff* and Anita E. Hendricksonf The retinae of a monkey, cat, rat and mouse were examined for FMRF-amide immunoreactivity, a marker for efferent fibers in the retinae of lower vertebrates. No FMRF-amide immunoreactive retinal fibers were found despite their presence in the rat CNS. Therefore, we conclude that efferent fibers, if present in mammalian retinae, are immunologically different from efferent fibers in lower vertebrates. Invest Ophthalmol Vis Sci 30:791-794, 1989 Centrifugal fibers that innervate the retina exist in lower vertebrates, including amphibians, 1 " 3 fish, 4 " 9 birds 10 " and reptiles' 2 " 15 as well as in cyclostomes. 16 These fibers (often called retinal efferents) have been demonstrated both physiologically and anatomically; they appear to have a variety of different sources in different species, and often have multiple sources in a given species, especially in fish. 7 - 816 In general, the centrifugal fibers terminate in the inner plexiform layer of the retina near the cell bodies of amacrine cells. 4 '"' 17 The function of these fibers is not known. The evidence for centrifugal innervation of the ret- ina is much more controversial in mammals. Cajal stated that he occasionally saw efferents terminating on amacrine cells in the dog retina. 17 Later attempts to demonstrate these fibers have met with varying degrees of success; for a review of earlier work see Itaya. 18 Recent anatomical evidence exists both for efferents 18 " 23 and against them. 24 In each of the species of frogs, fish and lizards that have been studied, some of the efferents to the retina have an internal antigen that reacts immunologically with antisera prepared against the molluscan car- dioexcitatory peptide, FMRF-amide (fish 25 ; frog 26 ' 27 ; lizard—Rusoff, unpublished observations). These From the 'Department of Biology, Montana State University, Bozeman, Montana, and the fDepartments of Biological Structure and Ophthalmology, University of Washington, Seattle, Washing- ton. Supported by NIH Grant EY-06495 to ACR and EY-04536 to AH. Submitted for publication: May 26, 1988, accepted October 20, 1988. Reprint requests: Dr. Anne C. Rusoff, Department of Biology, Montana State University, Bozeman, MT 59717. fibers are designated FMRF-amide immunoreactive (FMRF-ir). The role of this antigenic molecule in the vertebrate retina is not yet clear; however, some cells in the goldfish retina do change their response prop- erties after superfusion with FMRF-amide. 28 This result suggests that the antigenic molecule is either FMRF-amide or has a region whose configuration strongly resembles that of FMRF-amide. FMRF-amide-like peptides are also present in the mammalian central nervous system. 29 " 34 They have been studied extensively in the rat and can be demon- strated particularly in the hypothalamus, brainstem and spinal cord, using either immunocytoche- mistry 2931 or radioimmunoassay. 32 It is unlikely that the antigenic molecule is simply FMRF-amide 30 " 33 ; isolated mammalian peptides which cross-react with antiserum to FMRF-amide are all larger peptides, 33 but their carboxyl terminals all are very similar to FMRF-amide. The carboxyl end appears to be the region that the antiserum recognizes, and only this end is necessary to elicit a response, as FMRF-amide alone iontophoresed onto cells in the rat brainstem is excitatory. 34 Since FMRF immunoreactivity has been found in some retinal efferents in each of the lower vertebrates which has been examined and is present in the mam- malian central nervous system, we decided to search for efferents in mammalian retinae containing FMRF-amide immunoreactivity. Materials and Methods All procedures described herein conform to the ARVO Resolution on the Use of Animals in Re- search. We examined the retinae of five different 791 Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933377/ on 11/12/2018
Transcript of Some mammalian retinae lack FMRF-amide-like immunoreactive ... tion was cut, it was placed on a...
May 1989 Vol. 30/5
Investigative Ophthalmology
& Visual Science
Articles
Some Mammalian Retinae Lack FMRF-Amide-LikeImmunoreacfive Efferents
Anne C. Rusoff* and Anita E. Hendricksonf
The retinae of a monkey, cat, rat and mouse were examined for FMRF-amide immunoreactivity, amarker for efferent fibers in the retinae of lower vertebrates. No FMRF-amide immunoreactiveretinal fibers were found despite their presence in the rat CNS. Therefore, we conclude that efferentfibers, if present in mammalian retinae, are immunologically different from efferent fibers in lowervertebrates. Invest Ophthalmol Vis Sci 30:791-794, 1989
Centrifugal fibers that innervate the retina exist inlower vertebrates, including amphibians,1"3 fish,4"9
birds10" and reptiles'2"15 as well as in cyclostomes.16
These fibers (often called retinal efferents) have beendemonstrated both physiologically and anatomically;they appear to have a variety of different sources indifferent species, and often have multiple sources in agiven species, especially in fish.7-816 In general, thecentrifugal fibers terminate in the inner plexiformlayer of the retina near the cell bodies of amacrinecells.4'"'17 The function of these fibers is not known.
The evidence for centrifugal innervation of the ret-ina is much more controversial in mammals. Cajalstated that he occasionally saw efferents terminatingon amacrine cells in the dog retina.17 Later attemptsto demonstrate these fibers have met with varyingdegrees of success; for a review of earlier work seeItaya.18 Recent anatomical evidence exists both forefferents18"23 and against them.24
In each of the species of frogs, fish and lizards thathave been studied, some of the efferents to the retinahave an internal antigen that reacts immunologicallywith antisera prepared against the molluscan car-dioexcitatory peptide, FMRF-amide (fish25; frog26'27;lizard—Rusoff, unpublished observations). These
From the 'Department of Biology, Montana State University,Bozeman, Montana, and the fDepartments of Biological Structureand Ophthalmology, University of Washington, Seattle, Washing-ton.
Supported by NIH Grant EY-06495 to ACR and EY-04536 toAH.
Submitted for publication: May 26, 1988, accepted October 20,1988.
Reprint requests: Dr. Anne C. Rusoff, Department of Biology,Montana State University, Bozeman, MT 59717.
fibers are designated FMRF-amide immunoreactive(FMRF-ir). The role of this antigenic molecule in thevertebrate retina is not yet clear; however, some cellsin the goldfish retina do change their response prop-erties after superfusion with FMRF-amide.28 Thisresult suggests that the antigenic molecule is eitherFMRF-amide or has a region whose configurationstrongly resembles that of FMRF-amide.
FMRF-amide-like peptides are also present in themammalian central nervous system.29"34 They havebeen studied extensively in the rat and can be demon-strated particularly in the hypothalamus, brainstemand spinal cord, using either immunocytoche-mistry2931 or radioimmunoassay.32 It is unlikely thatthe antigenic molecule is simply FMRF-amide30"33;isolated mammalian peptides which cross-react withantiserum to FMRF-amide are all larger peptides,33
but their carboxyl terminals all are very similar toFMRF-amide. The carboxyl end appears to be theregion that the antiserum recognizes, and only thisend is necessary to elicit a response, as FMRF-amidealone iontophoresed onto cells in the rat brainstem isexcitatory.34
Since FMRF immunoreactivity has been found insome retinal efferents in each of the lower vertebrateswhich has been examined and is present in the mam-malian central nervous system, we decided to searchfor efferents in mammalian retinae containingFMRF-amide immunoreactivity.
Materials and Methods
All procedures described herein conform to theARVO Resolution on the Use of Animals in Re-search. We examined the retinae of five different
791
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792 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / May 1989 Vol. 30
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Fig. 1. (A) Photograph of a section of the retina of an adult rainbow cichlid fish taken midway between the optic disc and the peijipheralmargin of the retina. The arrow indicates an efferent axon near the amacrine cells in the inner nuclear layer. This axon is immunoreactive forFMRF-amide. Scale line indicates 25 *im. (B) Photograph of a section from the posterior pituitary of a rat. The arrows point to FMRF-ir cells.The arrowhead points to a cell which is not immunoreactive. Scale line indicates 10 ^m. (C) Photograph of a section from the dorsal retina ofa monkey taken midway between the optic disc and the peripheral margin of the retina. This section received the same treatment as the sectionshown in (A); however, no immunoreactive fibers are present. Scale line indicates 25 fun. GCL, ganglion cell layer; IPL, inner plexiform layer;INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (D) Photograph of a section from the retina of the same ratwhose pituitary is shown in (B). This section received the same treatment as the section shown in (B); however, no immunoreactive fibers arepresent. Scale line indicates 25 )im. Abbreviations are as in (C).
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No. 5 MAMMALIAN RETINAE LACK FMRF-AMIDE-IR EFFERENT5 / Rusoff and Hendrickson 793
mammals: a primate, Macaca nemistrina, a domesticcat, a mouse with a pigmented coat and eyes(C57B1/6), an albino mouse (Balb/cByJ), and an al-bino rat (Holtzman). The primate eye was fixed byperfusion with 4% paraformaldehyde containing ly-sine and periodate. The rat was perfused with 4%paraformaldehyde in 0.1 M phosphate buffer with 3%sucrose; its retinae and brain were then further fixedby immersion in fixative. The other mammalian re-tinae and retinae from rainbow cichlid fish (Herotila-pia multispinosa) used as positive controls were fixedby immersion in the same fixative. All tissues werethen washed in phosphate buffer and cryo-protectedin 30% sucrose in phosphate buffer. They were thenimmersed in O.C.T. compound (Tissue-tek, Naper-ville, IL), frozen and sectioned at 10 or 20 ^m in aFrigocut cryostat (Reichert, West Germany). The en-tire retina of each mouse eye and one rat eye wasused; the portion of the cat retina centered on theoptic disc and fully surrounding it was used; and twoportions of the primate retina were used—that tissueperipheral to the fovea and that immediately superiorto the fovea, extending from the optic disc to theperipheral margin. The fish retinae and pituitaryfrom the rat brain that were used as tissue controlswere sectioned in an identical manner. As each sec-tion was cut, it was placed on a gelatin-subbed slide.The slides were stored in the refrigerator for 24 hr orless before being brought up to room temperature toreact the tissue.
The slides were then bathed in each of the solutionsrequired to reveal FMRF-amide immunoreactivityin the tissue, using the peroxidase-antiperoxidaseprocedure of Sternberger.35 All washes were phos-phate-buffered saline (PBS) plus 0.3% Triton-X 100,unless otherwise stated. Sections were incubated inwash plus 10% DMSO for 30 min, washed, incubatedin normal goat serum for 1 hr, and incubated over-night in the antibody to FMRF-amide (Immunonu-clear, Stillwater, MN and/or Cambridge Research Bi-ologicals, New York, NY) at room temperature. Theantibodies from the two sources were usually mixed,with that from Immunonuclear used at a dilution of1:1000 and that from Cambridge used at 1:5000. Thismixture yielded optimal staining versus backgroundon the fish retinae run with each mammalian retinaas positive controls. Negative control slides were in-cubated with normal rabbit serum at a dilution of1:1000. Slides were then washed, incubated with goatanti-rabbit serum for 1 hr, washed, incubated withperoxidase-anti-peroxidase complex (DAKO, Car-pinteria, CA) for 1 hr, washed, washed in 0.1 Mphosphate buffer, and reacted to reveal the peroxi-dase using diaminobenzidine as the chromogen andglucose-glucose oxidase as the hydrogen peroxidesource.36 The tissue was counterstained with cresyl
violet, dehydrated, cleared with xylene and cover-slipped with permount.
Results
All fish retinae run as positive controls containedFMRF-amide immunoreactive fibers crossing theinner plexiform layer and terminating near the ama-crine cell bodies in the inner nuclear layer (Fig. 1 A).As reported by others,30 the posterior, but not theanterior, pituitary of the rat brain contained numer-ous FMRF-ir cells and fibers. Figure IB shows twocells in the posterior pituitary densely filled with thereaction product (arrows). A neighboring cell that isnot immunoreactive is indicated by the arrowheadfor comparison. However, over 100 sections fromeach mammalian retina were examined and none ofthem contained any FMRF-ir fibers. Figure 1C and Dshow representative sections from the primate and ratretinae, respectively, in which no immunoreactivefibers are visible. Portions of the optic nerve headwere included in some of the sections from the ret-inae of the mice and the cat and no immunoreactivefibers were seen in these optic nerves either.
Discussion
We conclude that centrifugal fibers, if present inthe primate, cat, rat or mouse retinae, do not containdetectable levels of FMRF-amide immunoreactivity.If there are FMRF-amide-like peptides in these re-tinae, they are present either in extremely smallamounts or have different cross-reactivities from theFMRF-amide-like peptides found both in the retinaeof cold-blooded vertebrates and in the mammalianCNS. These results do not show that there are nocentrifugal fibers in these retinae. However, our abil-ity to easily demonstrate FMRF-ir cells and fibers inrat CNS and FMRF-ir efferents in the retinae of lowervertebrates but not in these mammalian retinaestrongly suggests that any efferent fibers present inthe mammalian retina differ in their internal chemi-cals from one major group of retinal efferents presentin most cold-blooded vertebrates.
Key words: mammals, retina, efferents, FMRF-amide im-munoreactivity
Acknowledgments
We thank Drs. S. Ewald, J. Starkey and J. McMillan forproviding the mouse and cat eyes used in this study. Themonkey eye was from the Tissue Program of the RegionalPrimate Research Center, University of Washington.
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