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Co-expression of two subtypes of melatonin receptor on rat M1-type intrinsically photosensitive retinal ganglion cells.

Sheng WL, Chen WY, Yang XL, Zhong YM, Weng SJ - PLoS ONE (2015)

Bottom Line: Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons.Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth.It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.

ABSTRACT
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

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Related in: MedlinePlus

Melanopsin immunostaining reveals a single subclass of ipRGCs in rat retina.(a1) Western blot analysis of whole rat retina homogenates for the melanopsin antibody revealed a single band of ~55 kDa. (a2) The immunoblot signals were completely eliminated when the primary antibody was omitted. MW scale (kDa) is shown on the left. (A-C) Photomicrographs captured from the same area of a whole-mount retinal preparation, but at different focal planes. (A) Five melanopsin-positive somata (arrow heads) were identified in the GCL, corresponding to the ‘conventionally placed’ cells. (B) A strongly-labeled plexus of immunoractive dendritic processes, which is located at the outer limit of the IPL. The soma of one ‘displaced’ cell (arrow heads) is in sharp focus in (C), which focuses on the INL. (D) A vertical section counterstained with DAPI (blue) to reveal cellular laminae of the inner retina, showing monotratified dendritic arborization (arrow heads) of the melanopsin-immunoreactive (red) cells at the boundary between the INL and IPL. (E) Representative examples demonstrating the morphological profiles of melanopsin-stained cells reconstructed from the whole-mount retinal preparations. Three conventionally located (upper) and two displaced cells (lower) are shown. The sparse dendritic branching pattern is similar among these cells, typical of M1-type ipRGCs. Arrows in the inset point to the apparent varicosity-like structures on the dendrites. Arrowheads indicate the axons. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar is 200 μm in (A-C, E) and 20 μm in (D).
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pone.0117967.g001: Melanopsin immunostaining reveals a single subclass of ipRGCs in rat retina.(a1) Western blot analysis of whole rat retina homogenates for the melanopsin antibody revealed a single band of ~55 kDa. (a2) The immunoblot signals were completely eliminated when the primary antibody was omitted. MW scale (kDa) is shown on the left. (A-C) Photomicrographs captured from the same area of a whole-mount retinal preparation, but at different focal planes. (A) Five melanopsin-positive somata (arrow heads) were identified in the GCL, corresponding to the ‘conventionally placed’ cells. (B) A strongly-labeled plexus of immunoractive dendritic processes, which is located at the outer limit of the IPL. The soma of one ‘displaced’ cell (arrow heads) is in sharp focus in (C), which focuses on the INL. (D) A vertical section counterstained with DAPI (blue) to reveal cellular laminae of the inner retina, showing monotratified dendritic arborization (arrow heads) of the melanopsin-immunoreactive (red) cells at the boundary between the INL and IPL. (E) Representative examples demonstrating the morphological profiles of melanopsin-stained cells reconstructed from the whole-mount retinal preparations. Three conventionally located (upper) and two displaced cells (lower) are shown. The sparse dendritic branching pattern is similar among these cells, typical of M1-type ipRGCs. Arrows in the inset point to the apparent varicosity-like structures on the dendrites. Arrowheads indicate the axons. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar is 200 μm in (A-C, E) and 20 μm in (D).

Mentions: In a recent work using a rabbit melanopsin antibody, three subclasses of rat ipRGCs were labeled [40]. We therefore first carried out a series of morphological analysis to determine which type (s) of rat ipRGCs could be stained by the goat antibody used. Even though this antibody has been reported to identify ipRGCs [36,37], we further examined the specificity of it using Western blot analysis. In rat retinal homogenates, the antibody against melanopsin recognized a single band at ~55 kDa (Fig. 1a1), which is comparable to that of vertebrate melanopsin [41,42]. No signal was detected when the antibody was omitted (Fig. 1a2). Fig. 1A is a photomicrograph taken with a fluorescence microscope, showing an array of immunofluorescent cell bodies at low density in the ganglion cell layer (GCL), in a flat-mounted retina labeled with this antibody. Meanwhile, a single plexus of melanopsin-positive dendritic processes, occupying the distal part of the inner plexiform layer (IPL), could be clearly seen (Fig. 1B), but there was no immunofluorescent dendritic network in the proximal part of the IPL. Occasionally, a very few number of somata, which may be those of displaced ipRGCs [4,38], appeared in the inner nuclear layer (INL) (Fig. 1C). Fig. 1D is the confocal micrograph of a retinal vertical section, showing a single layer of strongly labeled dendritic arbors located in the OFF sublamina in the IPL, which was immediately close to the IPL-INL border. The morphological features of the labeled cells, as shown in both the flat-mount and the section, were typical of the M1-type ipRGC, which is characterized by dendritic varicosities, as well as sparsely branched dendritic arbors monostratifying at the outmost of the IPL. It should be noted that all the cells labeled by this antibody showed similar morphological features, and they were morphologically distinct from M2~M5-type cells, which are either bistratified or mono-stratified in the ON sublamina of the IPL [43,44]. Based on a bulk of microphotographs taken along the Z-axis of the whole mount retina preparations, we reconstructed the dendritic profiles of 24 individual melanopsin-positive cells, chosen randomly from six retinas. Fig. 1E shows five cells with representative soma-dendritic structures. The major morphometric parameters of these cells, such as soma diameter (13.9±0.4 μm), dendritic field diameter (367.4±15.5 μm), primary dendrite number (2.7±0.1), terminal neurite tips number (11.5±1.0) and dendritic arbor branch point number (8.1±1.0), all correspond to those of previously reported “M1” cells or the subset of melanopsin-containing cells monostratified in the outermost of the IPL in rat [4,40,45,46] (Table 1).


Co-expression of two subtypes of melatonin receptor on rat M1-type intrinsically photosensitive retinal ganglion cells.

Sheng WL, Chen WY, Yang XL, Zhong YM, Weng SJ - PLoS ONE (2015)

Melanopsin immunostaining reveals a single subclass of ipRGCs in rat retina.(a1) Western blot analysis of whole rat retina homogenates for the melanopsin antibody revealed a single band of ~55 kDa. (a2) The immunoblot signals were completely eliminated when the primary antibody was omitted. MW scale (kDa) is shown on the left. (A-C) Photomicrographs captured from the same area of a whole-mount retinal preparation, but at different focal planes. (A) Five melanopsin-positive somata (arrow heads) were identified in the GCL, corresponding to the ‘conventionally placed’ cells. (B) A strongly-labeled plexus of immunoractive dendritic processes, which is located at the outer limit of the IPL. The soma of one ‘displaced’ cell (arrow heads) is in sharp focus in (C), which focuses on the INL. (D) A vertical section counterstained with DAPI (blue) to reveal cellular laminae of the inner retina, showing monotratified dendritic arborization (arrow heads) of the melanopsin-immunoreactive (red) cells at the boundary between the INL and IPL. (E) Representative examples demonstrating the morphological profiles of melanopsin-stained cells reconstructed from the whole-mount retinal preparations. Three conventionally located (upper) and two displaced cells (lower) are shown. The sparse dendritic branching pattern is similar among these cells, typical of M1-type ipRGCs. Arrows in the inset point to the apparent varicosity-like structures on the dendrites. Arrowheads indicate the axons. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar is 200 μm in (A-C, E) and 20 μm in (D).
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Related In: Results  -  Collection

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pone.0117967.g001: Melanopsin immunostaining reveals a single subclass of ipRGCs in rat retina.(a1) Western blot analysis of whole rat retina homogenates for the melanopsin antibody revealed a single band of ~55 kDa. (a2) The immunoblot signals were completely eliminated when the primary antibody was omitted. MW scale (kDa) is shown on the left. (A-C) Photomicrographs captured from the same area of a whole-mount retinal preparation, but at different focal planes. (A) Five melanopsin-positive somata (arrow heads) were identified in the GCL, corresponding to the ‘conventionally placed’ cells. (B) A strongly-labeled plexus of immunoractive dendritic processes, which is located at the outer limit of the IPL. The soma of one ‘displaced’ cell (arrow heads) is in sharp focus in (C), which focuses on the INL. (D) A vertical section counterstained with DAPI (blue) to reveal cellular laminae of the inner retina, showing monotratified dendritic arborization (arrow heads) of the melanopsin-immunoreactive (red) cells at the boundary between the INL and IPL. (E) Representative examples demonstrating the morphological profiles of melanopsin-stained cells reconstructed from the whole-mount retinal preparations. Three conventionally located (upper) and two displaced cells (lower) are shown. The sparse dendritic branching pattern is similar among these cells, typical of M1-type ipRGCs. Arrows in the inset point to the apparent varicosity-like structures on the dendrites. Arrowheads indicate the axons. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar is 200 μm in (A-C, E) and 20 μm in (D).
Mentions: In a recent work using a rabbit melanopsin antibody, three subclasses of rat ipRGCs were labeled [40]. We therefore first carried out a series of morphological analysis to determine which type (s) of rat ipRGCs could be stained by the goat antibody used. Even though this antibody has been reported to identify ipRGCs [36,37], we further examined the specificity of it using Western blot analysis. In rat retinal homogenates, the antibody against melanopsin recognized a single band at ~55 kDa (Fig. 1a1), which is comparable to that of vertebrate melanopsin [41,42]. No signal was detected when the antibody was omitted (Fig. 1a2). Fig. 1A is a photomicrograph taken with a fluorescence microscope, showing an array of immunofluorescent cell bodies at low density in the ganglion cell layer (GCL), in a flat-mounted retina labeled with this antibody. Meanwhile, a single plexus of melanopsin-positive dendritic processes, occupying the distal part of the inner plexiform layer (IPL), could be clearly seen (Fig. 1B), but there was no immunofluorescent dendritic network in the proximal part of the IPL. Occasionally, a very few number of somata, which may be those of displaced ipRGCs [4,38], appeared in the inner nuclear layer (INL) (Fig. 1C). Fig. 1D is the confocal micrograph of a retinal vertical section, showing a single layer of strongly labeled dendritic arbors located in the OFF sublamina in the IPL, which was immediately close to the IPL-INL border. The morphological features of the labeled cells, as shown in both the flat-mount and the section, were typical of the M1-type ipRGC, which is characterized by dendritic varicosities, as well as sparsely branched dendritic arbors monostratifying at the outmost of the IPL. It should be noted that all the cells labeled by this antibody showed similar morphological features, and they were morphologically distinct from M2~M5-type cells, which are either bistratified or mono-stratified in the ON sublamina of the IPL [43,44]. Based on a bulk of microphotographs taken along the Z-axis of the whole mount retina preparations, we reconstructed the dendritic profiles of 24 individual melanopsin-positive cells, chosen randomly from six retinas. Fig. 1E shows five cells with representative soma-dendritic structures. The major morphometric parameters of these cells, such as soma diameter (13.9±0.4 μm), dendritic field diameter (367.4±15.5 μm), primary dendrite number (2.7±0.1), terminal neurite tips number (11.5±1.0) and dendritic arbor branch point number (8.1±1.0), all correspond to those of previously reported “M1” cells or the subset of melanopsin-containing cells monostratified in the outermost of the IPL in rat [4,40,45,46] (Table 1).

Bottom Line: Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons.Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth.It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.

ABSTRACT
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

Show MeSH
Related in: MedlinePlus