ARTICLE |
Correspondence to: Egemen Savaskan, Dept. of Psychiatry, University of Basel, Wilhelm Klein-Str.27, CH-4025 Basel, Switzerland. E-mail: esavaskan@datacomm.ch
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Summary |
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Melatonin is synthesized in the pineal gland and retina during the night. Retinal melatonin is believed to be involved in local cellular modulation and in regulation of light-induced entrainment of circadian rhythms. The present study provides the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT1) in human retina of aged subjects. Ganglion, amacrine, and photoreceptor cells expressed MT1. In addition, MT1 immunoreactivity was localized to cell processes in the inner plexiform layer and to central vessels of the retina, as well as to retinal vessels but not to ciliary or choroidal vessels. These results support a variety of cellular and vascular effects of melatonin in the human retina. Preliminary evidence from patients with Alzheimer's disease (AD) revealed increased MT1 immunoreactivity in ganglion and amacrine cells, as well as in vessels. In AD cases photoreceptor cells were degenerated and showed low MT1 expression.
(J Histochem Cytochem 50:519525, 2002)
Key Words: melatonin MT1 receptor, retina, ganglion cell, photoreceptor, amacrine cell, vessels, Alzheimer's disease
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Introduction |
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The hormone melatonin, secreted from the pineal gland at night and suppressed by light during the day, provides a circadian and seasonal signal to the organism. The circadian rhythm is generated by the suprachiasmatic nucleus (SCN). Light information to the SCN is transduced via the retina, itself containing a circadian pacemaker (
In mammals, the specific actions of melatonin are mediated by two different subtypes of G-protein-coupled receptors, the melatonin 1a (MT1) and melatonin 1b (MT2) receptors (
The aim of this study was to provide immunohistochemical evidence, the first we are aware of, for the distribution of MT1 in human retina and in the ocular vascular system. In addition, eyes of two AD patients were included in the study as a preliminary investigation of possible alterations in the disease process.
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Materials and Methods |
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Human Tissue
Paraffin-embedded human eyes fixed in 4% paraformaldehyde were obtained from seven subjects without neurological or psychiatric disease (six women and one man, mean age 83.1 ± 9.8 years and two male AD cases, 83 and 84 years old. The mean postmortem delay was 21 hr and 31 min ± 9 hr and 10 min) for controls and 16 hr and 15 min for AD cases (Table 1). The diagnosis of AD was made with clinical evaluation and confirmed by postmortem neuropathological examination.The cause of death in most cases was heart failure or pneumonia, and pancreatitis for one control case (Table 1). The sample collection was approved by the Ethics Committee criteria and according to the Helsinki Declaration of 1975. Tissue samples were cut in the sagittal plane and 4-µm-thick consecutive sections were made with a microtome.
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Immunohistochemistry
The sections were mounted on coated glass slides and deparaffinized. Endogenous peroxidase activity was blocked by bathing the sections in 80% methanol, 0.6% H2O2 for 20 min at room temperature (RT). After several washes in PBS and preincubation in PBS with 3% blocking serum for 30 min the sections were reacted with primary antibody overnight at RT. The affinity-purified polyclonal antibody used to specifically detect MT1 was developed against a peptide (peptide 536) corresponding to a sequence found in the C-terminus of the receptor and the antibody recognition of native MT1 has been ascertained (
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Results |
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The cytoarchitectural classification regarding cells, different layers, and vascularization in this section follows detailed descriptions by
Cellular Structures
Different cells within the retina displayed specific MT1 immunoreactivity (Fig 1A1D), whereas adjacent control sections omitting the primary antibody revealed no immune reaction (Fig 1E and Fig 1F). The majority of ganglion cells were immunoreactive for MT1 in their somata and dendritic processes, both in controls and in AD cases (Fig 1A and Fig 1C). Staining was homogeneously distributed in the perinuclear area. In five control cases (Table 1), single MT1-immunoreactive amacrine cells were located in the inner nuclear layer immediately adjacent to the inner plexiform layer. Only a minor subset of amacrine cells were stained. In all cases there was a slight granular MT1 labeling in the inner plexiform layer located on cell processes. The outer plexiform layer and the horizontal cells were not immunolabeled. Photoreceptor cells revealed distinct MT1 immunoreactivity in their inner segments (Fig 1B). Cell somata and outer segments were excluded. All photoreceptor cells were stained so that MT1 extended as an immunoreactive band throughout the photoreceptor cell layer.
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The two AD cases showed the following differences in the distribution of MT1 immunoreactivity. First, the staining intensity and the numbers of MT1-immunoreactive cells were distinctly increased in ganglion cells and inner plexiform cell layers (Fig 1C). Second, both AD cases revealed well-stained amacrine cells, and the number of these MT1-immunoreactive cells was twice that in controls (Fig 1C). There was also increased staining intensity within the inner plexiform layer. A decreased MT1 staining in the highly degenerated photoreceptor cell layer could be observed (Fig 1D). Therefore, in AD cases only single cells were immunoreactive for MT1 so that there was no continuous band of immunoreactivity throughout the entire photoreceptor cell layer as in controls (Fig 1B and Fig D).
Ocular Vessels
In all control eyes a distinctly positive MT1 immunoreactivity in the adventitia of central retinal arteries and veins could be observed (Fig 2A and Fig 2B). The same staining pattern was found even in the small retinal vessels located in the ganglion cell layer and inner plexiform layer (Fig 2E). Both ciliary and choroidal vessels were devoid of MT1 immunoreactivity (Fig 2F). In AD, increased MT1 staining was detected in central arteries and veins and in retinal vessels, whereas ciliary and choroidal vessels were not stained (Fig 2D and Fig 2E).
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Discussion |
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The present study has demonstrated the localization of MT1 in cellular structures of the retina and in ocular vessels of aged humans, and has provided preliminary evidence for alterations in retinal MT1 expresssion in AD patients. After previous demonstration of melatonin, its synthesizing enzymes (
Several types of retinal cells displayed MT1 immunoreactivity. The localization of MT1 to ganglion and amacrine cells is in accordance with previous reports in rodent retina (
Another contrast to rodents (
Although only two AD cases were investigated, the MT1 alterations were notable. The increase of MT1 immunoreactivity in ganglion and amacrine cells may indicate receptor upregulation, possibly as a regulative response to declining nocturnal melatonin levels in elderly subjects and in AD (
Free radicals are generated in mitochondria owing to their high respiratory activity (
In addition to cellular structures, central and retinal vessels were also immunoreactive for MT1 in their adventitia. Melatonin is a potent vasoactive substance regulating cerebrovascular responsiveness and blood flow in brain (
Taken together, the results provide evidence that melatonin may be involved in different cellular and vascular processes in the human retina via MT1. The evaluation of a larger number of cases will be necessary to confirm the striking alterations in AD. Understanding the effects of melatonin in retina as well as brain may be important to combat neurodegeneration and circadian disturbances in AD.
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Acknowledgments |
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Supported by grants from the CNRS, the Université Paris VII, and the Association pour la Recherche sur le Cancer (ARC 5513) to LB and RJ.
We thank Prof Ch. E. Remé (University of Zürich, Switzerland) for her invaluable advice.
Received for publication August 21, 2001; accepted November 21, 2001.
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Literature Cited |
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Brydon L, Roka F, Petit L, De Coppet P, Tissot M, Barrett P, Morgan PJ, Nanoff C, Strosberg AD, Jockers R (1999) Dual signaling of human Mel1a melatonin receptors via Gi2, Gi3 and Gq/11 proteins. Mol Endocrinol 13:2025-2038
Bubenik GA, Brown GM, Uhlir I, Grota LJ (1974) Immunohistological localization of N-acetylindolalkylamines in pineal gland, retina and cerebellum. Brain Res 81:233-242[Medline]
De la Torre JC, Stefano GB (2000) Evidence that Alzheimer's disease is a microvascular disorder: the role of constitutive nitric oxide. Brain Res Rev 34:119-136[Medline]
Demontis GC, Longoni B, Gargini C, Cervetto L (1997) The energetic cost of photoreception in retinal rods of mammals. Arch Ital Biol 135:95-109[Medline]
Dubocovich ML (1983) Melatonin is a potent modulator of dopamine release in the retina. Nature 306:782-784[Medline]
Dubocovich ML, Masana MI, Benloucif S (2000) Molecular pharmacology and function of melatonin receptor subtypes. In Olcese J, ed. Melatonin after Four Decades. New York, Kluwer Academic/Plenum Publishers, 181-190
Ferrari E, Arcaini A, Gornati R, Pelanconi L, Cravello L, Fioravanti M, Solerte SB, Magri F (2000) Pineal and pituitary-adrenocortical function in physiological aging and in senile dementia. Exp Gerontol 35:1239-1250[Medline]
Freedman MS, Lucas RJ, Soni B, Von Schantz M, MuÚoz M, DavidGray Z, Foster R (1999) Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors. Science 284:502-504
Fujieda H, Hamadanizadeh SA, Wankiewicz E, Pang SF, Brown GM (1999) Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin. Neuroscience 93:793-799[Medline]
Fujieda H, Scher J, Hamadanizadeh SA, Wankiewicz E, Pang SF, Brown GM (2000) Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina. Vis Neurosci 17:63-70[Medline]
Herzog ED, Block GD (1999) Keeping an eye on retinal clocks. Chronobiol Int 16:229-247[Medline]
Hsu S-C, Molday RS (1994) Glucose metabolism in photoreceptor outer segments. J Biol Chem 269:17954-17959
Iuvone PM, Gan J (1995) Functional interaction of melatonin receptors and D1 dopamine receptors in cultured chick retinal neurons. J Neurosci 15:2179-2185[Abstract]
Lucas RJ, Freedman MS, MuÚoz M, GarciaFernández J-M, Foster RG (1999) Regulation of the mammalian pineal by non-rod, non-cone, ocular receptors. Science 284:505-507
Marchiafava PL, Longoni B (1999) Melatonin as an antioxidant in retinal photoreceptors. J Pineal Res 26:184-189[Medline]
Pappolla MA, Chyan Y-J, Poeggeler B, Frangione B, Wilson G, Ghiso J, Reiter RJ (2000) An assessment of the antioxidant and the antiamyloidogenic properties of melatonin: implications for Alzheimer's disease. J Neural Transm 107:203-231
Pierce ME, Besharse JC (1985) Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length. J Gen Physiol 86:671-689[Abstract]
Régriny O, Delagrange P, Scalbert E, Atkinson J, LartaudIdjouadienne I (1998) Melatonin improves cerebral circulation security margins in rats. Am J Physiol 275:H139-144
Remé CE, Hafezi F, Reinboth J, Clausen M (1996) Light damage revisited: converging evidence, diverging views? Graefes Arch Clin Exp Ophthalmol 234:2-11[Medline]
Savaskan E, Olivieri G, Brydon L, Jockers R, Kräuchi K, WirzJustice A, MüllerSpahn F (2001) Cerebrovascular melatonin MT1-receptor alterations in patients with Alzheimer's disease. Neurosci Lett 308:9-12[Medline]
Savaskan E, Olivieri G, Meier F, Brydon L, Jockers R, Ravid R, Wirz-Justice A, Müller-Spahn F (in press) Increased melatonin 1a-receptor immunoreactivity in the hippocampus of Alzheimer's disease patients. J Pineal Res
Spencer WH (1996) Ophthalmic Pathology. 4th ed Philadelphia, WB Saunders
Sugawara T, Sieving PA, Iuvone PM, Bush RA (1998) The melatonin antagonist luzindole protects retinal photoreceptors from light damage in rat. Invest Ophthalmol Vis Sci 39:2458-2465[Abstract]
Swaab DF, Fliers E, Partiman TS (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res 342:37-44[Medline]
Terman M, Remé CE, WirzJustice A (1991) The visual input stage of the mammalian circadian pacemaking system: II. The effect of light and drugs on retinal function. J Biol Rhythms 6:31-48[Medline]
Tosini G (2000) Melatonin circadian rhythm in the retina of mammals. Chronobiol Int 17:599-612[Medline]
VivienRoels B, Pévet P, Dubois MP, Arendt J, Brown GM (1981) Immunohistochemical evidence for the presence of melatonin in the pineal gland, the retina and the Harderian gland. Cell Tissue Res 217:105-115[Medline]
White MP, Fisher LJ (1989) Effects of exogenous melatonin on circadian disc shedding in the albino rat retina. Vis Res 29:167-179[Medline]
Wiechmann AF, Hollyfield JG (1989) HIOMT-like immunoreactivity in the vertebrate retina: a species comparison. Exp Eye Res 49:1079-1095[Medline]
Wiechmann AF, Yang XL, Wu SM, Hollyfield JG (1988) Melatonin enhances horizontal cell sensitivity in salamander retina. Brain Res 453:377-380[Medline]