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Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel(1c) melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel(1c) but not Mel(1b) receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.
FIG. 1. Immunocytochemistry of X. laevis ciliary epithelium with melatonin receptor antibody. Fixed cryostat sections of
Xenopus eye were incubated with the Mel1c receptor antibody only or with antibody plus excess Mel1c peptide (control). The
primary antibody was labeled with a ¯uorescein-antibody conjugate, and counter stained with a blue nuclear dye (Hoechst).
(A) Schematic representation of the structure of the Xenopus eye, indicating the region where the ciliary body is located. The
rectangular inset represents the region examined in (B)±(F). (B) Retina and ciliary body stained with the Hoechst blue nuclear
dye. (C) Retina and ciliary body stained with the hematoxylin and eosin. (D) Same section as in (B) labelled with the Mel1c
melatonin receptor antibody. Speci®c immunolabeling is observed in the nonpigmented ciliary epithelium (CE), but not in the
pigmented ciliary epithelium (PCE: arrow) or iris. Immunoreactivity is also present in the retina. (E) Retina and ciliary body
incubated with the Mel1c receptor antibody that was preincubated with the antigen peptide, and counter stained with Hoechst
dye. No speci®c immunoreactivity is observed. (F) Merged images of (B) and (D) showing labelling with the Mel1c melatonin
receptor antibody, and Hoechst dye staining of the cell nuclei. Scale bar 100 mm. RPE, retinal pigment epithelium; OS,
photoreceptor outer segments; IPL, inner plexiform layer.
FIG. 2. Immunocytochemistry of X. laevis ciliary body
with melatonin receptor antibody preincubated with receptor
subtype peptides. Fixed cryostat sections of Xenopus
retina were incubated with the Mel1c receptor antibody only
or with antibody plus an excess of Mel1a , Mel1b , or Mel1c
peptide. The primary antibody was labelled with a
¯uorescein-antibody conjugate, and the tissue was counter
stained with Hoechst blue nuclear dye. (A) Incubation with
the Mel1c antibody only shows speci®c immunolabeling in
the non-pigmented ciliary epithelium (CE). (B) Antibody
pre-incubation with Mel1a homologous region peptide does
not block any immunoreactivity. (C) Antibody pre-incubation
with Mel1b homologous region peptide signi®cantly
reduces immunoreactivity in the retina and CE. (D)
Antibody pre-incubation with Mel1c antigen peptide blocks
all immunoreactivity. Scale bar ˆ 100 mm. RPE, retinal
pigment epithelium; OS, photoreceptor outer segments; IPL,
inner plexiform layer; PCE, pigmented ciliary epithelium.
FIG. 3. Immunocytochemistry and laser scanning confocal
microscopy of X. laevis ciliary epithelium with
melatonin receptor antibody. Fixed cryostat sections of
Xenopus eye were incubated with the Mel1c receptor
antibody, which was labelled with a ¯uorescein-antibody
conjugate, and the tissue was counter stained with a blue
nuclear dye (Hoechst). (A±C) The basolateral surfaces
(arrows) of the nonpigmented ciliary epithelium (CE) are
immunoreactive. The apical surface of the CE and the
pigmented ciliary epithelium (PCE) are not immunoreactive.
Scale bar  50 mm.
FIG. 4. Melatonin receptor subtype Mel1c and Mel1b
receptor in situ hybridization in sections of Xenopus ciliary
epithelium. (A) Xenopus ciliary epithelium section hybridized
with Xenopus Mel1c melatonin receptor antisense riboprobe,
and stained with toluidine blue. A diffuse band of
hybridization (represented by black silver grains) is located
over cells of the nonpigmented ciliary epithelium (CE). (B)
Tissue section treated with a sense (control) riboprobe,
demonstrates the level of non-speci®c background hybridization.
No speci®c hybridization is observed in the CE. (C)
Tissue section treated with the Mel1b antisense riboprobe
does not demonstrate speci®c hybridization (some pigment
granules from the PCE appear over the CE). Scale
bar  100 mm. PCE, pigmented ciliary epithelium.
