ARTICLE |
Correspondence to: Makoto Kaneda, Dept. of Physiology, Keio Univ. School of Medicine, Tokyo 160-8582, Japan. E-mail: mkaneda@physiol.med.keio.ac.jp
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Summary |
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In the retina, zinc is believed to be a modulator of synaptic transmission and a constituent of metalloenzymes. To determine whether the intracellular localization of zinc correlates with function, we examined the localization of endogenous zinc in the rat retina using the silver amplification method. By light microscopy, reaction products were detected in the pigment epithelial cells (PE), the inner segment of photoreceptors (IS), the outer nuclear layer (ONL) and the inner nuclear layer (INL), the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and the ganglion cell layer (GC). The heaviest accumulation of precipitate was observed in PE and IS, whereas only a little precipitate was found in GC. When the intracellular zinc was chelated with diethyldithiocarbamate, a small amount of precipitate was observed only in ONL. By electron microscopy, zinc was associated with three compartments. In OPL and IPL, zinc was associated with neural processes, while in PE, IS, INL, and GC it was associated with the Golgi apparatus. In ONL, zinc was associated with the nucleus. Zinc in the neural processes is believed to act as a modulator of synaptic transmission, and zinc associated with the Golgi apparatus is assumed to catalyze metalloenzyme reactions. (J Histochem Cytochem 49:8796, 2001)
Key Words: synaptic terminal, Golgi apparatus, silver amplification, pigment epithelial cell, electron microscopy
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Introduction |
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IN THE CENTRAL NERVOUS SYSTEM, zinc is an essential constituent of metalloenzymes and a modulator of synaptic transmission (
Because zinc modulates the activity of the amino acid receptors (
In this study we examined the precise intracellular localization of zinc in the rat retina with the silver amplification method by both light microscopy and electron microscopy. We found that zinc was localized in specific compartments: the neural processes of the outer plexiform layer and the inner plexiform layer, the Golgi apparatus of the pigment epithelial cells, the inner segment of photoreceptors, the cells of the inner nuclear layer and the ganglion cells, and the nucleus of photoreceptors. The presence of zinc in these specific compartments is believed to reflect the fact that zinc is involved in multiple functions in the retina. A portion of the present study has been published in abstract form (
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Materials and Methods |
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Fixation of Animals
Research protocols were approved by the Animal Care and Use Committee of RIKEN. For the silver amplification, we followed the methods described in detail by
Silver Amplification
Fixed retina was embedded in 3% agar and sectioned at 100 µm thickness with a microslicer (DTK-1500; Dosaka EM, Kyoto, Japan). Sections were washed thoroughly with distilled water before the silver amplification. The IntenSE M silver Enhancement kit (Amersham International; Little Chalfont, Bucks, UK) was used to intensify zinc signals (
After the silver amplification, retinas were postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hr at 4C, dehydrated with a graded ethanol series, and flat-embedded in epoxy resin (Araldyte CY212; TAAB, Aldermaston, UK). Semithin 2-µm-thick sections were cut for light microscopic observation. For electron microscopic observation, the retina was sectioned at 80-nm thickness with an ultramicrotome (Ultracut-UCT; Leica, Heidelberg, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate.
Light and Electron Microscopic Observation
To observe the distribution of silver precipitates under the microscope (Nikon, ECLIPS E800; Tokyo, Japan), no counterstaining was carried out. Photomicrographic data recorded on color reversal film (Fujichrome 100, Fuji Photofilm, Tokyo, Japan) were scanned and digitized through a film scanner (LS-4500; Nikon). Digitized images processed by commercially available software (Photoshop; Adobe, San Jose, CA) were printed on a color printer (Pictrography 3000; Fuji Photofilm).
Electron micrographs recorded on imaging plates through an LEO 912 electron microscope (LEO Electron Microscopy; Oberkochen, Germany) were scanned and digitized by an FDL 5000 imaging system (Fuji Photofilm). Digitized images were processed by the computer program "Image Gauge" and printed out on a Pictrography 3000 color printer.
All experiments were carried out on at least three different animals.
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Results |
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Light Microscopic Observation
Fig 1A shows the distribution of silver precipitates corresponding to zinc in retinal layers developed by the IntenSE M silver Enhancement kit. In the pigment epithelial cells and the inner segment of photoreceptors, precipitates accumulated heavily and were observed as a black band. No precipitate was observed between the pigment epithelial cells and the inner segment of photoreceptors. In the outer plexiform layer and the inner plexiform layer, precipitate was diffusely distributed. There was substantially less precipitate in the outer nuclear layer and the inner nuclear layer than in the outer plexiform layer and the inner plexiform layer. In the inner nuclear layer, most zinc was distributed near the marginal regions of the layer. Some precipitate was also observed in the ganglion cell layer.
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In the silver amplification method, the perfusion of sodium sulfide is necessary to precipitate free zinc and zinc weakly bound to protein as sulfide compounds in the tissue (
Electron Microscopic Observation
Pigment Epithelial Cells.
Silver grains corresponding to zinc were distributed in the cytoplasm of the pigment epithelial cells (Fig 2A). These silver grains were frequently associated with the Golgi apparatus. Silver grains were usually localized on one side of the lamellae (Fig 2B). Because the lamellae appeared to be straight, we could not identify whether silver grains were associated with the cis side or the trans side of the Golgi apparatus. There were very few silver grains not associated with the Golgi apparatus.
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Outer and Inner Segments of Photoreceptors. Silver grains were localized to the inner segment (Fig 2C), where the grains had accumulated in the myoid region. Most grains were associated with only one side of the Golgi apparatus (Fig 2D). No grains were found in the outer segment.
Outer Nuclear Layer. Many silver grains were associated with the nucleus of photoreceptors (Fig 3A). These nuclei were polygonal and had uniform density. Some of the grains were found in the internuclear space, but most of them were found in the processes of the Müller cells (Fig 3B). A few precipitates were observed in both the nucleus and the processes, even after DEDTC pretreatment.
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Outer Plexiform Layer.
On the scleral side, silver grains were found in the electron-lucent neural processes containing vesicles (Fig 3C). These electron-lucent neural processes had contact with electron-dense neural processes (Fig 3D), and an invagination of the electron-lucent processes into the electron-dense neural processes was also observed (Fig 3C). Because the electron-dense terminals were located near the scleral side and had a single synaptic ribbon (
Inner Nuclear Layer.
There are different types of cells in this layer. Cell bodies of the horizontal cells and the bipolar cells are located on the scleral side, whereas those of the amacrine cells are located on the vitreal side and often have an indented nucleus. Cell bodies of the Müller cells are also localized together with them, and often show a fusiform shape (
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Inner Plexiform Layer. Silver grains were frequently found in the neural processes (Fig 5A). They contacted both the electron-lucent (Fig 5B) and electron-dense processes (Fig 5C). In Fig 5B, the processes contain small clear vesicles and occasionally cored vesicles, and contact electron-lucent neural processes with sparse vesicles. This contact is believed to be a non-synaptic contact. In Fig 5C, the electron-lucent processes contain small clear vesicles and contact electron-dense neural processes with rich vesicles. This contact is believed to be a synaptic contact. In the present study, the ratio of two types of contacts was not analyzed quantitatively. The presence of synaptic ribbons was used to identify terminals of bipolar cells, where we could not find any silver grains (Fig 5A and Fig 5C).
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Ganglion Cell Layer. A few ganglion cells had silver grains in the cytoplasm. Asymmetrical distribution of the grains was obvious when they were found near the Golgi apparatus (Fig 5D).
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Discussion |
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In this study we employed silver amplification methods to localize zinc in the rat retina at the ultrastructural level. We found that zinc was localized in three distinct subcellular compartments, each of which was specific for each layer. Table 1 summarizes the subcellular localization of silver grains in the rat retina. These compartments were not detectable by light microscopic observation.
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Zinc Stained by the Silver Amplification Method
The silver amplification method is a useful means of detecting free zinc and zinc weakly bound to protein because the soluble zinc is precipitated in the tissue as sulfide compounds (
We observed that reaction products were widely distributed throughout the retina except for the outer segment of photoreceptors. A similar distribution of reaction products has been reported using the neo-Timm method in the rat retina (
Zinc in the Neural Processes
In the present study, we found endogenous zinc in the neural processes of the outer plexiform layer and the inner plexiform layer. We observed many synaptic vesicles in these processes. In the hippocampus, zinc in the synaptic terminals (300 µM (
In the central nervous system, neural processes containing zinc are glutamatergic (
Zinc in the Golgi Apparatus
We observed that zinc was associated with one side of the Golgi apparatus in pigment epithelial cells. Melanin is the main source of zinc in pigment epithelial cells (
Zinc in the Outer Nuclear Layer
In the outer nuclear layer, silver precipitate was associated with the nucleus and some silver precipitate was insensitive to DEDTC pretreatment. Therefore, it is likely that free zinc or zinc weakly bound to protein exists in the nucleus. In the nucleus, however, zinc has been found at the structural site of some transcription factors (
The silver amplification method is a useful means of detecting zinc in the central nervous system (
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Acknowledgments |
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Supported by a BSI grant (No. 57911) from RIKEN and a grant-in-aid to M. Kaneda (No. 09680820) from the Ministry of Education, Science and Culture.
We thank Prof A. Kaneko for critical reading of our manuscript.
Received for publication May 4, 2000; accepted September 8, 2000.
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Literature Cited |
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Akagi T, Kaneda M, Ishii K, Suzuki W, Hashikawa T (1999) Localization of zinc in the rat retina. Soc Neurosci Abstr 25:135
Aniksztejn L, Charton G, Ben-Ari Y (1987) Selective release of endogenous zinc from the hippocampal mossy fibers in situ. Brain Res 404:58-64[Medline]
Assaf SY, Chung S-H (1984) Release of endogenous Zn2+ from brain tissue during activity. Nature 308:734-736[Medline]
Baylor DA, Fuortes MGF, O'Bryan PM (1971) Receptive fields of cones in the retina of the turtle. J Physiol (Lond) 214:265-294[Medline]
Coleman JE (1992) Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem 61:897-946[Medline]
Danscher G (1981) Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electronmicroscopy. Histochemistry 71:1-16[Medline]
Danscher G (1996) The autometallographic zinc-sulphide method. A new approach involving in vivo creation of nanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles. Histochem J 28:361-373[Medline]
Danscher G, Nørgaard JOR, Baatrup E (1987) Autometallography: tissue metals demonstrated by a silver enhancement kit. Histochemistry 86:465-469[Medline]
De Biasi S, Bendotti C (1998) A simplified procedure for the physical development of the sulphide silver method to reveal synaptic zinc in combination with immunocytochemistry at light and electron microscopy. J Neurosci Methods 79:87-96[Medline]
Dong C-J, Werblin FS (1994) Zinc downmodulates the GABAC receptor current in cone horizontal cells acutely isolated from the catfish retina. J Neurophysiol 73:916-919
Dowling JE (1987) The Retina: An Approachable Part of the Brain. Cambridge, MA, Belknap Press, Harvard University Press
Dunphy WG, Rothman JE (1985) Compartmental organization of the Golgi stack. Cell 42:13-21[Medline]
Farquhar MG (1985) Progress in unraveling pathways of Golgi traffic. Annu Rev Cell Biol 1:447-488
Frederickson CJ (1989) Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 31:145-238[Medline]
Frederickson CJ, Danscher G (1990) Zinc-containing neurons in hippocampus and related CNS structures. Prog Brain Res 83:71-84[Medline]
Han Y, Wu SM (1999) Modulation of glycine receptors in retinal ganglion cells by zinc. Proc Natl Acad Sci USA 96:3234-3238
Han M-H, Yang X-L (1999) Zn2+ differentially modulates kinetics of GABAC vs GABAA receptors in carp retinal bipolar cells. Neuroreport 10:2593-2597[Medline]
Hearing VJ, Tsukamoto K (1991) Enzymatic control of pigmentation in mammals. FASEB J 5:2902-2909
Howell GA, Welch MG, Frederickson CJ (1984) Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308:736-738[Medline]
Kaneda M, Andrásfalvy B, Kaneko (2000) A modulation by Zn2+ of GABA responses in bipolar cells of the mouse retina. Vis Neurosci 17:273-281[Medline]
Kaneda M, Mochizuki M, Aoki K, Kaneko A (1997) Modulation of GABAC response by Ca2+ and other divalent cations in horizontal cells of the catfish retina. J Gen Physiol 110:741-747
Kaneko A, Tachibana M (1986) Effects of -aminobutyric acid on isolated cone photoreceptors of the turtle retina. J Physiol (Lond) 373:443-461[Abstract]
LeureDupree AE, Bridges CDB (1982) Changes in retinal morphology and vitamin A metabolism as a consequence of decreased zinc availability. Retina 2:294-302[Medline]
Miceli MV, Tate DJ, Jr, Alcock NW, Newsome DA (1999) Zinc-deficiency and oxidative stress in the retina of pigmented rats. Invest Ophthalmol Vis Sci 40:1238-1244[Abstract]
Newsome DA, Oliver PD, Deupree DM, Miceli MV, Diamond JG (1992) Zinc uptake by primate retinal pigment epithelium and choroid. Curr Eye Res 11:213-217[Medline]
PérezClausell J, Danscher G (1985) Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study. Brain Res 337:91-98[Medline]
PérezClausell J, Danscher G (1986) Release of zinc sulphide accumulations into synaptic clefts after in vivo injection of sodium sulphide. Brain Res 362:358-361[Medline]
Piccolino M, Gerschenfeld HM (1980) Characteristics and ionic processes involved in the feedback spikes of turtle cones. Proc R Soc Lond 206:439-463. [B][Medline]
Qian H, Li L, Chappell RL, Ripps H (1997) GABA receptors of bipolar cells from the skate retina: actions of zinc on GABA-mediated membrane currents. J Neurophysiol 78:2402-2412
Qian H, Malchow RP, Chappell RL, Ripps H (1996) Zinc enhances ionic currents induced in skate Muller (glial) cells by the inhibitory neurotransmitter GABA. Proc R Soc Lond 263:791-796. [B][Medline]
Rodieck RW (1973) The Vertebrate Retina: Principles of Structure and Function. San Francisco, WH Freeman
Samuelson DA, Smith P, Ulshafer RJ, Hendricks DG, Whitley RD, Hendricks H, Leone NC (1993) X-ray microanalysis of ocular melanin in pigs maintained on normal and low zinc diets. Exp Eye Res 56:63-70[Medline]
Schraermeyer U, Heimann K (1999) Current understanding on the role of retinal pigment epithelium and its pigmentation. Pigment Cell Res 12:219-236[Medline]
Schraermeyer U, Stieve H (1994) A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle. Cell Tissue Res 276:273-279[Medline]
Solano F, MartinezLiarte JH, JiménezCervantes C, GarcíaBorrón JC, Lozano JA (1994) Dopachrome tautomerase is a zinc-containing enzyme. Biochem Biophys Res Commun 204:1243-1250[Medline]
Szél A, Röhlich P (1992) Two cone types of rat retina detected by anti-visual pigment antibodies. Exp Eye Res 55:47-52[Medline]
Tachibana M, Kaneko A (1984) -Aminobutyric acid acts at axon terminals of turtle photoreceptors: difference in sensitivity among cell types. Proc Natl Acad Sci USA 81:7961-7964[Abstract]
Tate DJ, Miceli MV, Newsome DA, Alcock NA, Oliver PD (1995) Influence of zinc on selected cellular functions of cultured human retinal pigment epithelium. Curr Eye Res 14:897-903[Medline]
Ugarte M, Osborne NN (1998) The localization of endogenous zinc and the in vitro effect of exogenous zinc on the GABA immunoreactivity and formation of reactive oxygen species in the retina. Gen Pharmacol 30:297-303[Medline]
Westbrook GL, Mayer ML (1987) Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neurons. Nature 328:640-643[Medline]
Wu SM (1991) Input-output relations of the feedback synapse between horizontal cells and cones in the tiger salamander retina. J Neurophysiol 65:1197-1206
Wu SM (1992) Feedback connections and operation of the outer plexiform layer of the retina. Curr Opin Neurobiol 2:462-468[Medline]
Wu SM, Qiao X, Noebels JY, Yang XL (1993) Localization and modulatory actions of zinc in vertebrate retina. Vis Res 33:2611-2616[Medline]