Van Der List DA, Chapman B (2011) Targeted ablation of intrinsically photosensitive melanopsin expressing retinal ganglion cells early in development alters retinal morphology within the inner plexiform layer of mice. Neuroscience 2011 Abstracts 232.12. Society for Neuroscience, Washington, DC.

Summary: It has been demonstrated in adult mice, that eliminating a small subset of retinal ganglion cells expressing the photopigment melanopsin (ip-RGCs) with an immunotoxin alters the effects of light on circadian rhythms. The immunotoxin was made by conjugating the melanopsin antibody with ribosome-inactivating protein, saporin. It has also been observed that the ablation of ip-RGCs in adult mice did not alter retinal morphology. Specifically, it was found that dendrites arising from starburst amacrine cells retained their position within the inner plexiform layer (IPL) suggesting no reorganization within this synaptic layer (Goz et al. 2008). In this study, we used the same melanopsin immunotoxin (Mx) (Advanced Targeting Systems) to perform intravitreal injections into mice at postnatal day one. The animals were sacrificed at P26 and the retina fixed in 4%PFA, frozen transverse sections were then immunostained with antibodies against melanopsin, choline acetyl transferase (ChAT), calreinin, calbindin, PKC and Kv4.2. In control retinae, melanosin antibody stained ip-RGC cell bodies and dendrites stratifying in both On and Off layers of the IPL, whereas retinae treated with Mx shows a loss of melanopsin-containing cell bodies and dendrites. In control retinae, ChAT stains starburst amacrine cells with cell bodies in the RGC and INL layers and two distinct bands in the IPL. In Mx treated retinae, most starburst amacrine cells appear to be eliminated along with melanopsin RGCs. Interestingly, if there is a hint of residual melanopsin expressing dendrites remaining, there is also a ChAT expressing cell body and a hint of dendrites in the synaptic layer. In control retinae, calretinin and calbindin antibodies stain a subset of RGCs and amacrine cells and show a characteristic three-layered pattern of dendrites in the IPL. In Mx treated retinae, the calretinin and calbindin layers within the IPL are altered showing an absent or more diffuse labeling pattern in the ON and OFF bands. Antibodies against PKC (staining rod bipolar cells) and Kv4.2 (stains a subset of retinal ganglion cells) do not show an altered staining pattern. These findings suggest that the initial stratification and structural development of synaptic layers in the IPL are altered by Mx treatment.

Related Products: Melanopsin-SAP (Cat. #IT-44)

Chen S, Badea T, Hattar S (2011) Photoentrainment and pupillary light reflex are mediated by distinct populations of ipRGCs. Nature 476:92-95. doi: 10.1038/nature10206 PMID: 21765429

Summary: Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment Melanopsin and regulate a wide array of light dependent physiological processes. Genetic ablation of ipRGCs eliminates circadian photoentrainment and severely disrupts the pupillary light reflex (PLR). Scientists showed that ipRGCs consist of distinct subpopulation that differentially express the Brn3b transcription factor, and can be functionally distinguished. Brn3b-negative M1 ipRGCs innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, whereas Brn3b-positive ipRGCs innervate all other known brain targets. Selective ablation of Brn3b-postive ipRGCs severly disrupts the PLR, but does not impair circadian photoentrainment. The scientists concluded that molecularly distinct subpopulations of M1 ipRGCs, which are morphologically and electrophysiologically similar, innervate different brain regions to execute light-induced functions. A dilution of 1:1000 of Anti-Melanopsin (Cat. #AB-N38) was used for immunohistochemical analysis of retina sections.

Related Products: Melanopsin Rabbit Polyclonal (Cat. #AB-N38)

Do MTH, Yau K (2010) Intrinsically photosensitive retinal ganglion cells. Physiol Rev 90(4):1547-1581. doi: 10.1152/physrev.00013.2010

Summary: This review presents recent data that has established the importance of intrinsically photosensitive retinal ganglion cells (ipRPG) in nonimage visual functions. The use of melanopsin-SAP (Cat. #IT-44) in both mice and rats is discussed. It is of note that depletion of ipRPG’s using melanopsin-SAP resulted in deficits in communication to nonimage regions of the brain, but image vision appeared normal.

Related Products: Melanopsin-SAP (Cat. #IT-44)

Berson DM, Castrucci AM, Provencio I (2010) Morphology and mosaics of melanopsin-expressing retinal ganglion cell types in mice. J Comp Neurol 518(13):2405-2422. doi: 10.1002/cne.22381 PMID: 20503419

Objective: To provide a fuller description of murine cell types expressing melanopsin, their contribution to the plexuses of melanopsin dendrites, and mosaics formed by each type.

Summary: M1 cells, corresponding to the originally described ganglion-cell photoreceptors, occupy the ganglion cell or inner nuclear layers. M2 cells ramify in the inner third of the IPL. Rare bistratified cells deploy terminal dendrites within both melanopsin-immunoreactive plexuses within the ON sublayer.

Usage: Immunohistochemistry: The tissue was incubated for 24 hr at 4°C in unpurified primary antiserum diluted 1:2500. Immunoperoxidase labeling of flat mount tissue was incubated for 3 days in the unpurified primary antiserum diluted 1:2500.

Related Products: Melanopsin Rabbit Polyclonal (Cat. #AB-N38)

Gonzalez-Menendez I, Contreras F, Cernuda-Cernuda R, Provencio I, Garcia-Fernandez JM (2010) Postnatal development and functional adaptations of the melanopsin photoreceptive system in the albino mouse retina. Invest Ophthalmol Vis Sci 51(9):4840-4847. doi: 10.1167/iovs.10-5253 PMID: 20435589

Summary: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) adjust the circadian pacemaker of mammals by detecting light. The authors tracked the development of ipRGCs in postnatal mice under varying light conditions. Immunohistochemistry for these experiments was done using an anti-mouse melanopsin polyclonal antibody (Cat. #AB-N38). Alteration of the standard light/dark cycle clearly affected the development of ipRGCs.

Related Products: Melanopsin Rabbit Polyclonal (Cat. #AB-N38)

Q: We’re interested in trying out your melanopsin antibody (Cat. #AB-N38) using immunohistochemistry in mouse retina. Do you have a recommended protocol?

A:  This protocol has been utilized successfully with anti-melanopsin.

Panda S. et al. 2002. Melanopsin (Opn4) requirement for normal light- induced circadian phase shifting. Science 298(5601):2213-2216.

Related: Anti-Melanopsin (Cat. #AB-N38), Anti-Melanopsin, affinity-purified (Cat. #AB-N39)

Ingham ES, Gunhan E, Fuller PM, Fuller CA (2009) Immunotoxin-induced ablation of melanopsin retinal ganglion cells in a non-murine mammalian model. J Comp Neurol 516:125-140. doi: 10.1002/cne.22103

Related Products: Melanopsin-SAP (Cat. #IT-44)

Göz D, Studholme K, Lappi DA, Rollag MD, Provencio I, Morin LP (2008) Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms. PLoS ONE 3(9):e3153. doi: 10.1371/journal.pone.0003153 PMID: 18773079

Summary: Retinal ganglion cells expressing melanopsin photopigment are thought to be involved in non-image forming visual responses to light. The authors had a custom conjugate made between saporin and an anti-melanopsin antibody. A 400-ng injection of the melanopsin-SAP conjugate into the eye of a mouse resulted in a 57% loss of the targeted cells. Rabbit IgG-SAP (Cat. #IT-35) was used as a control. The data indicates that melanopsin-containing cells are involved in the response to certain non-image forming visual input.

Related Products: Melanopsin-SAP (Cat. #IT-44), Melanopsin Rabbit Polyclonal (Cat. #AB-N38), Melanopsin Rabbit Polyclonal, affinity-purified (Cat. #AB-N39), Rabbit IgG-SAP (Cat. #IT-35)

Panda S, Sato TK, Castrucci AM, Rollag MD, DeGrip WJ, Hogenesch JB, Provencio I, Kay SA (2002) Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298(5601):2213-2216. doi: 10.1126/science.1076848 PMID: 12481141

Objective: To investigate the role of melanopsin in circadian photoentrainment in mammals.

Summary: The study focused on understanding how melanopsin, an opsin-based photopigment, affects the circadian rhythms in mammals. By generating melanopsin-null mice and observing their response to light-induced circadian phase shifting, the research demonstrated that while these mice maintain some entrainment to light/dark cycles, their phase resetting in response to monochromatic light is severely attenuated. This highlights melanopsin’s critical role in circadian photoentrainment, alongside other mechanisms that contribute to this process.

Usage: Anti-Melanopsin (AB-N38) was incubated with tissue for 24 hr at 4°C at a 1:2,500 dilution in a TBS incubating buffer containing 1% bovine serum albumin, 0.25% carrageenan lambda and 0.003% Triton X-100.

Related Products: Melanopsin Rabbit Polyclonal (Cat. #AB-N38), Melanopsin Rabbit Polyclonal, affinity-purified (Cat. #AB-N39)

Provencio I, Rollag MD, Castrucci AM (2002) Photoreceptive net in the mammalian retina. Nature 415:493. doi: 10.1038/415493a PMID: 11823848

Related Products: Melanopsin Rabbit Polyclonal (Cat. #AB-N38)