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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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Evidence of cytoplasmic localization of melatonin receptors.(A) and (B) Double immunolabeling of rat retina cryostat sections using MT1/MT2 antibody and Alexa Fluor 555-conjugated WGA. In a vast majority of neurons in the INL and GCL, granular signals for either MT1 or MT2 are both closely circumscribed by the WGA-labeled cell membrane, suggesting the cytoplasmic staining. Scale bar = 10 μm. (C) and (D) A comparison of the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts (left two columns) and whole retinal homogenates (right two columns). The MT1 and MT2 antibodies both generated a band of appropriate size (40–45 kDa). Note that bands for membrane extracts (arrows) were much lighter than those for whole retinal homogenates.
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pone.0117967.g003: Evidence of cytoplasmic localization of melatonin receptors.(A) and (B) Double immunolabeling of rat retina cryostat sections using MT1/MT2 antibody and Alexa Fluor 555-conjugated WGA. In a vast majority of neurons in the INL and GCL, granular signals for either MT1 or MT2 are both closely circumscribed by the WGA-labeled cell membrane, suggesting the cytoplasmic staining. Scale bar = 10 μm. (C) and (D) A comparison of the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts (left two columns) and whole retinal homogenates (right two columns). The MT1 and MT2 antibodies both generated a band of appropriate size (40–45 kDa). Note that bands for membrane extracts (arrows) were much lighter than those for whole retinal homogenates.

Mentions: The cytoplasmic staining of both melatonin receptors was further confirmed by a double-staining control experiment using the MT1/MT2 antibody and fluorophore-conjugated WGA (a well-established cell membrane marker [47]). As shown in Fig. 3A1-A3 and B1-B3, in a vast majority of retinal neurons examined, the area circumscribed by WGA-positive signals was fully filled by the MT1/MT2 labeling, thus providing direct evidence for the cytoplasmic labeling for MT1/MT2 receptors. One may argue that staining for MT1/MT2 should be limited to the membrane given the fact that melatonin receptors are thought to be membrane receptors. It is likely that the cytoplasmic staining in the present work may be largely because the antibodies used preferentially recognize non-membrane-located melatonin receptors. This possibility was strengthened by making a comparison between the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts, and those using whole retinal homogenates (Fig. 3C and D). The bands obtained with retinal membrane extracts were found to be much weaker, as compared with those obtained with retinal homogenates.


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)

Evidence of cytoplasmic localization of melatonin receptors.(A) and (B) Double immunolabeling of rat retina cryostat sections using MT1/MT2 antibody and Alexa Fluor 555-conjugated WGA. In a vast majority of neurons in the INL and GCL, granular signals for either MT1 or MT2 are both closely circumscribed by the WGA-labeled cell membrane, suggesting the cytoplasmic staining. Scale bar = 10 μm. (C) and (D) A comparison of the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts (left two columns) and whole retinal homogenates (right two columns). The MT1 and MT2 antibodies both generated a band of appropriate size (40–45 kDa). Note that bands for membrane extracts (arrows) were much lighter than those for whole retinal homogenates.
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getmorefigures.php?uid=PMC4340921&req=5

pone.0117967.g003: Evidence of cytoplasmic localization of melatonin receptors.(A) and (B) Double immunolabeling of rat retina cryostat sections using MT1/MT2 antibody and Alexa Fluor 555-conjugated WGA. In a vast majority of neurons in the INL and GCL, granular signals for either MT1 or MT2 are both closely circumscribed by the WGA-labeled cell membrane, suggesting the cytoplasmic staining. Scale bar = 10 μm. (C) and (D) A comparison of the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts (left two columns) and whole retinal homogenates (right two columns). The MT1 and MT2 antibodies both generated a band of appropriate size (40–45 kDa). Note that bands for membrane extracts (arrows) were much lighter than those for whole retinal homogenates.
Mentions: The cytoplasmic staining of both melatonin receptors was further confirmed by a double-staining control experiment using the MT1/MT2 antibody and fluorophore-conjugated WGA (a well-established cell membrane marker [47]). As shown in Fig. 3A1-A3 and B1-B3, in a vast majority of retinal neurons examined, the area circumscribed by WGA-positive signals was fully filled by the MT1/MT2 labeling, thus providing direct evidence for the cytoplasmic labeling for MT1/MT2 receptors. One may argue that staining for MT1/MT2 should be limited to the membrane given the fact that melatonin receptors are thought to be membrane receptors. It is likely that the cytoplasmic staining in the present work may be largely because the antibodies used preferentially recognize non-membrane-located melatonin receptors. This possibility was strengthened by making a comparison between the results yielded by Western blot analysis for the MT1/MT2 antibodies using retinal plasma membrane extracts, and those using whole retinal homogenates (Fig. 3C and D). The bands obtained with retinal membrane extracts were found to be much weaker, as compared with those obtained with retinal homogenates.

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