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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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Expression of MT1 and MT2 receptors on rat M1 cells.(A1) Western blot analysis of retinal homogenates for the MT1 antibody revealed a single band at ~40 kDa. MW scale (kDa) is shown on the left. (A2) No band was detected when the MT1 antibody was pre-absorbed with the immunizing antigen. (B1-B3) and (C1-C3) Confocal fluorescence microphotographs of retinal sections, double labeled by melanopsin and MT1. The cytoplasm of a conventionally placed M1 cell (B1), and a displaced M1 cell (C1) are both MT1 immunoreactive. Note that, several faint MT1-immunoreactive strata could be seen in the IPL (B2 and C2). (D1) Nomarski image showing multiple layers of a retinal section. (D2) Same retinal section as in (D1), showing that no immunofluorescence labeling was present when the MT1 antibody was pre-absorbed with the immunizing antigen. (E1) Western blotting of retinal homogenates for the MT2 antibody revealed a single band at ~45 kDa, as expected for MT2. MW scale (kDa) is shown on the left. (E2) No band was seen when the MT2 antibody was pre-absorbed with the blocking peptide. (F1-F3) and (G1-G3) Confocal fluorescence microphotographs of retinal sections, showing colocalization of melanopsin and MT2 immunoreactivity. The cytoplasm of a conventionally placed M1 cell (F1) and a displaced M1 cell (G1) are both stained by MT2. (H1) Nomarski image showing retinal layers more clearly. (H2) Same retinal section as in (H1), which was treated with the immunofluorescence labeling procedure for MT2, but the MT2 antibody was pre-absorbed with the immunizing antigen. Double labeled cells are indicated by arrow heads. Scale bars = 20 μm.
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pone.0117967.g002: Expression of MT1 and MT2 receptors on rat M1 cells.(A1) Western blot analysis of retinal homogenates for the MT1 antibody revealed a single band at ~40 kDa. MW scale (kDa) is shown on the left. (A2) No band was detected when the MT1 antibody was pre-absorbed with the immunizing antigen. (B1-B3) and (C1-C3) Confocal fluorescence microphotographs of retinal sections, double labeled by melanopsin and MT1. The cytoplasm of a conventionally placed M1 cell (B1), and a displaced M1 cell (C1) are both MT1 immunoreactive. Note that, several faint MT1-immunoreactive strata could be seen in the IPL (B2 and C2). (D1) Nomarski image showing multiple layers of a retinal section. (D2) Same retinal section as in (D1), showing that no immunofluorescence labeling was present when the MT1 antibody was pre-absorbed with the immunizing antigen. (E1) Western blotting of retinal homogenates for the MT2 antibody revealed a single band at ~45 kDa, as expected for MT2. MW scale (kDa) is shown on the left. (E2) No band was seen when the MT2 antibody was pre-absorbed with the blocking peptide. (F1-F3) and (G1-G3) Confocal fluorescence microphotographs of retinal sections, showing colocalization of melanopsin and MT2 immunoreactivity. The cytoplasm of a conventionally placed M1 cell (F1) and a displaced M1 cell (G1) are both stained by MT2. (H1) Nomarski image showing retinal layers more clearly. (H2) Same retinal section as in (H1), which was treated with the immunofluorescence labeling procedure for MT2, but the MT2 antibody was pre-absorbed with the immunizing antigen. Double labeled cells are indicated by arrow heads. Scale bars = 20 μm.

Mentions: The specificity of the MT1 receptor antibody was assessed using Western blot analysis. In rat retinal homogenates, this antibody revealed a single predominant band at approximately 40 kDa (Fig. 2A1), corresponding to the known molecular weight of the mammalian MT1 receptor [23,35]. No such band was detected when the antibody was pre-absorbed with the immunogen peptide (Fig. 2A2). These data indicated that the protein recognized by the antibody might indeed be the MT1 receptor. Fig. 2B1-B2 show vertical cryostat sections of the rat retina, doubled labeled by antibodies against melanopsin and MT1. MT1 receptor immunoreactivity was clearly seen in many cells in the GCL, in addition to the strong labeling in the INL and diffuse staining in the IPL. Notably, most cells in the ONL and a few cells in the INL were MT1-negative (data not shown). Some of the MT1-positive cells in the GCL were also immunoreactive with melanopsin (Fig. 2B3). As clear from the merged image (Fig. 2B3), the labeling for MT1 appeared to be restricted to the cytoplasm. A few of melanopsin-positive M1 cells were displaced to the INL (Fig. 2C1), and these cells were also MT1-immunoreactive (Fig. 2C2, C3). When the antibody for MT1 was pre-absorbed with the blocking peptides (negative control), only low level of background was detected (Fig. 2D2), which was similar to the no primary antibody control (data not shown), confirming that the labeling was specific. The Nomarski image of Fig. 2D2 (Fig. 2D1) shows retinal layers more clearly.


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)

Expression of MT1 and MT2 receptors on rat M1 cells.(A1) Western blot analysis of retinal homogenates for the MT1 antibody revealed a single band at ~40 kDa. MW scale (kDa) is shown on the left. (A2) No band was detected when the MT1 antibody was pre-absorbed with the immunizing antigen. (B1-B3) and (C1-C3) Confocal fluorescence microphotographs of retinal sections, double labeled by melanopsin and MT1. The cytoplasm of a conventionally placed M1 cell (B1), and a displaced M1 cell (C1) are both MT1 immunoreactive. Note that, several faint MT1-immunoreactive strata could be seen in the IPL (B2 and C2). (D1) Nomarski image showing multiple layers of a retinal section. (D2) Same retinal section as in (D1), showing that no immunofluorescence labeling was present when the MT1 antibody was pre-absorbed with the immunizing antigen. (E1) Western blotting of retinal homogenates for the MT2 antibody revealed a single band at ~45 kDa, as expected for MT2. MW scale (kDa) is shown on the left. (E2) No band was seen when the MT2 antibody was pre-absorbed with the blocking peptide. (F1-F3) and (G1-G3) Confocal fluorescence microphotographs of retinal sections, showing colocalization of melanopsin and MT2 immunoreactivity. The cytoplasm of a conventionally placed M1 cell (F1) and a displaced M1 cell (G1) are both stained by MT2. (H1) Nomarski image showing retinal layers more clearly. (H2) Same retinal section as in (H1), which was treated with the immunofluorescence labeling procedure for MT2, but the MT2 antibody was pre-absorbed with the immunizing antigen. Double labeled cells are indicated by arrow heads. Scale bars = 20 μm.
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pone.0117967.g002: Expression of MT1 and MT2 receptors on rat M1 cells.(A1) Western blot analysis of retinal homogenates for the MT1 antibody revealed a single band at ~40 kDa. MW scale (kDa) is shown on the left. (A2) No band was detected when the MT1 antibody was pre-absorbed with the immunizing antigen. (B1-B3) and (C1-C3) Confocal fluorescence microphotographs of retinal sections, double labeled by melanopsin and MT1. The cytoplasm of a conventionally placed M1 cell (B1), and a displaced M1 cell (C1) are both MT1 immunoreactive. Note that, several faint MT1-immunoreactive strata could be seen in the IPL (B2 and C2). (D1) Nomarski image showing multiple layers of a retinal section. (D2) Same retinal section as in (D1), showing that no immunofluorescence labeling was present when the MT1 antibody was pre-absorbed with the immunizing antigen. (E1) Western blotting of retinal homogenates for the MT2 antibody revealed a single band at ~45 kDa, as expected for MT2. MW scale (kDa) is shown on the left. (E2) No band was seen when the MT2 antibody was pre-absorbed with the blocking peptide. (F1-F3) and (G1-G3) Confocal fluorescence microphotographs of retinal sections, showing colocalization of melanopsin and MT2 immunoreactivity. The cytoplasm of a conventionally placed M1 cell (F1) and a displaced M1 cell (G1) are both stained by MT2. (H1) Nomarski image showing retinal layers more clearly. (H2) Same retinal section as in (H1), which was treated with the immunofluorescence labeling procedure for MT2, but the MT2 antibody was pre-absorbed with the immunizing antigen. Double labeled cells are indicated by arrow heads. Scale bars = 20 μm.
Mentions: The specificity of the MT1 receptor antibody was assessed using Western blot analysis. In rat retinal homogenates, this antibody revealed a single predominant band at approximately 40 kDa (Fig. 2A1), corresponding to the known molecular weight of the mammalian MT1 receptor [23,35]. No such band was detected when the antibody was pre-absorbed with the immunogen peptide (Fig. 2A2). These data indicated that the protein recognized by the antibody might indeed be the MT1 receptor. Fig. 2B1-B2 show vertical cryostat sections of the rat retina, doubled labeled by antibodies against melanopsin and MT1. MT1 receptor immunoreactivity was clearly seen in many cells in the GCL, in addition to the strong labeling in the INL and diffuse staining in the IPL. Notably, most cells in the ONL and a few cells in the INL were MT1-negative (data not shown). Some of the MT1-positive cells in the GCL were also immunoreactive with melanopsin (Fig. 2B3). As clear from the merged image (Fig. 2B3), the labeling for MT1 appeared to be restricted to the cytoplasm. A few of melanopsin-positive M1 cells were displaced to the INL (Fig. 2C1), and these cells were also MT1-immunoreactive (Fig. 2C2, C3). When the antibody for MT1 was pre-absorbed with the blocking peptides (negative control), only low level of background was detected (Fig. 2D2), which was similar to the no primary antibody control (data not shown), confirming that the labeling was specific. The Nomarski image of Fig. 2D2 (Fig. 2D1) shows retinal layers more clearly.

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