Journal: PLoS ONE

Article Title: The organization of melanopsin-immunoreactive cells in microbat retina

doi: 10.1371/journal.pone.0190435

Figure Lengend Snippet: (A, B) The negative test. (C, D) The preabsorption test. These tests were performed to assess the specificity of the rabbit polyclonal melanopsin antibody in the R . ferrumequinum retina. Melanopsin-IR cells were not detected in the R . ferrumequinum retina. IR, immunoreactive; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar = 50 μm.

Article Snippet: Polyclonal rabbit anti-melanopsin (1:200, Thermo Scientific, Rockford, IL, USA) was used as the primary antibody.

Techniques:

Journal: Nature neuroscience

Article Title: Trans-Seq maps a selective mammalian retinotectal synapse instructed by Nephronectin

doi: 10.1038/s41593-022-01068-8

Figure Lengend Snippet: The numbers of fluorescence-positive neurons in the SGS from different mWGA-RFP configurations were counted. The percentages were normalized to the highest number as (100%) set by the mWGA-mCherry configuration, which possessed the highest efficiency. The anterograde transfer number was quantified by counting the number of RFP-positive neurons in acute SC slices on the contralateral side (right SC) without fluorescence amplification through immunostaining. We focused on the contralateral (right) SC, as 90% of RGCs project contralaterally31. We examined RGC coverage across the retina to ensure there was no retinotopy bias in SC analysis. mWGA-fusion protein configurations were compared under consistent conditions by delivering AAV2 expressing the fusion proteins into the left eyes (Fig. 1a). We used standard AAV Serotype-2 followed by 4 weeks post-injection (wpi) sampling, a commonly established window to examine RGC axonal projections and carry out the optogenetic measurement8. a, Several WGA-fusion proteins were compared side-by-side using an AAV-mediated in vivo screen for highly efficient anterograde transfer from the retina to the brain. b, Comparisons between N-terminal (mCherry-mWGA) and C-terminal fusion (mWGA-mCherry) transfer efficiencies were quantified. c, Transfer efficiencies of different RFP C-terminal fusions, including mWGA-mCherry, mWGA-Ruby3, mWGA-tdTmt, were quantified, n=4 animals for each condition. Statistics for b, two-sided Student’s t-test, ****, P<0.0001; c, one-way ANOVA test. ****, P<0.0001. e-f, (e) Additional samples showing live red-fluorescent labeling of the contralateral SC after acute brain slice preparation 4 weeks post-injection (wpi), indicating transsynaptic transfer onto the recipient neurons enriched in stratum griseum superficiale (SGS) and stratum opticum (SO), but not in stratum griseum intermedium (SGI). f, Magnified view of inset from e showing individual neurons labeled with bright red fluorescent protein from RGC anterograde transfer without signal amplification through immunostaining. The ability to detect native fluorescence is unique to mWGA-mCherry (mWmC) but absent in other mWGA-RFP configurations, such as mWGA-TdTomato shown here (g, h). Scale bars: (e, g, 5 mm; f, h, 50μm). i-l, Intrinsic electrophysiological properties of mCherry-positive recipient neurons (red, n=5 animals) and neighboring mCherry-negative neurons (black, n=4 animals) are similar as showed in Fig. 1m. i, Action potential amplitudes, j, sustained firing rates, k, resting membrane potentials, and l, EPSC frequencies are shown. These intrinsic properties were unperturbed by mWmC-transfer. N.S. not significant. two-sided Student’s t-test. m-p, Retinal vertical sections to show high mWmC coverage across major RGC types, including (m) Spp1 for αRGCs, (n) Cart for ooDSGCs, (o) Melanopsin for ipRGCs, and (p) Foxp2 for F-RGCs. Scale bar: 20 μm. INL: inner nuclear layer; GCL: ganglion cell layer. The percentages of each RGC subclasses were quantified in q, representing a similar fraction of RGC subclasses among all RGCs14, n=5 animals. f-i, Intraocular injections of mWmC lead to efficient monosynaptic transfer to connected neurons in multiple retino-recipient areas, including SC (t,u) and LGN (r, s). By contrast, the secondary relay neurons in V1 (u) or those in the lateral posterior nucleus of the thalamus (LP) (s) do not show mWmC transfer. Scale bars: (r, t, 2mm; s, u, 20mm). v, Immunostaining for RFP (mWmC) indicates high efficiency of anterograde transfer onto the recipient neurons in stratum griseum superficiale (SGS) and stratum opticum (SO). mWmC-positive cells are largely NeuN-positive. Dotted-yellow circles indicate mCherry and NeuN double-positive neurons from RGC anterograde transsynaptic transfer. w, mWmC-positive cells are largely GFAP-negative, which were quantified in x. n=5 animals for each condition. ****, p<0.0001, two-sided Student’s t-test. Scale bars: (v, w, 50μm). All data in this figure are presented as mean ± SEM.

Article Snippet: Antibodies used were as follows: rabbit and chicken anti-GFP (1:1000, Millipore; 1:500, Abcam); rabbit anti-RFP (1:500 Rockland); goat anti-WGA (1:500, Vector Labs); mouse anti-NeuN (1: 500, Millipore); mouse anti-GFAP (1:500, Sigma); rabbit anti-Satb1 (1:1000, Abcam); goat anti-choline acetyltransferase (ChAT) (1:500, Millipore); rabbit anti-Cart (1:2500, Phoenix Pharmaceuticals); rabbit anti-Melanopsin (1:5000, Thermo Scientific); guinea-pig anti-RBPMS (1:1000, PhosphoSolutions); goat anti-Osteopontin/Spp1 (1:500, R&D); rabbit anti-Nephronectin (1:100, produced in 68 ); goat anti-Itga8 (1:100, R&D); rabbit anti-Lamp1 (1:500, abcam); mouse anti-Brn3b/Pou4f2 (1:500, Santa Cruz Biotechnology).

Techniques: Fluorescence, Amplification, Immunostaining, Expressing, Injection, Sampling, In Vivo, Labeling, Slice Preparation

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: Table of primary antibodies used

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Sequencing

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: Melanopsin and PACAP immunoreactivity is found in inner and outer stratifying RGC’s. A-D. Outer (cell 1) and inner (cell 2) stratifying RGC’s expressing PACAP in magenta (A) and melanopsin identified using both a C-terminal antibody (B, green) and an N-terminal antibody (C, blue) and with the two antibodies shown merged (D). E. Extended view representing the same Z-stack of a total of 55 images of the cells shown in A-D presented in an XY (E), XZ (E1) and ZY (E2) plane to illustrate dendritic stratification. F. The same cells shown in A-E analysed for the localization of the dendritic processes using the Imaris filament tracer module (see Material and Methods). The majority of dendritic processes of cell 2 stratify in the most inner part of the inner plexiform layer (IPL)(magenta,F1-2F2). The dendrites of cell 1 stratify exclusively at the border of the INL/IPL (yellow, F1-2 F2) and its cell body is displaced to the INL (F1-2). Scale bars; 50 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Expressing

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: Melanopsin and PACAP immunoreactivity is found in inner and outer stratifying RGC’s. A. Three RGCs numbered 1-3 expressing both melanopsin (visualized using the N-terminal directed anti-melanopsin antibody) and PACAP, and one RGC stained only for PACAP (arrows), shown in an extended view representing a Z-stack of a total of 50 images presented in an XY (A), XZ (A1) and ZY (A2) plane. Co-localization of melanopsin and PACAP appears yellow. Note the weak expression of both melanopsin and PACAP in cell no. 2. Inner and outer stratifying dendritic processes are shown in the XZ (A1) and YZ (A2) planes. B. Focus is in the inner IPL near the GCL border, showing PACAP (green) and melanopsin (magenta) labelling of cells1 and.2 (same cells as shown in A). Arrow indicates the same PACAP labelled cell indicated by the arrow in A. Nuclei from other ganglion cells are stained with DAPI (blue). C. Focus is in the outer IPL near the INL border showing PACAP (green) and melanopsin (magenta) labelling of cell3 (same cell as shown in A). The cell body is displaced to the inner nuclear layer (INL). Co-localization of melanopsin and PACAP appears yellow. Nuclei from other cells in the INL stained with DAPI (blue). D-E. Extended view of the Z-stack of 50 images from A showing the melanopsin cells before (D) and after (E) being analysed for the localization of dendritic processes using the Imaris filament tracer module (see Materials and Methods). F. Focus as in B after Imaris filament tracer analysis showing cell 1 (yellow) and cell2 (green). The majority of dendrites of cell 1 are located in the most inner part of the IPL. Cell2 expressed low levels of melanopsin and the weakly stained proximal dendrites are also in the inner IPL. The axon of cell3 (magenta) can be seen descending through the IPL towards the GCL. G. Focus as in C after Imaris filament tracer analysis showing the cell body and outer stratifying dendrites of cell3 (magenta). Several dendrites from cell 1 (yellow) can be seen ascending to the outer IPL. H. Summary of the traced cells shown in E-G. Panel H1shows an extended view in the XZ plane and panel H2 shows an extended view in the YZ plane. The dendrites of cell3 are located exclusively in the outer IPL. GCL; ganglion cell layer, INL; inner nuclear layer. Scale bars; 50 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Expressing, Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: PACAP is found in melanopsin immunoreactive RGC’s of the macaque monkey. A. Melanopsin immunoreactivity(magenta) (visualized using the C-terminal directed anti-melanopsin antibody) shown in a flat-mount preparation of the Macaca mulatta (MM1) retina. The image represents a montage of 4 images each representing a Z-stack of 35 sections covering the depth required to ensure that both inner and outer stratifying melanopsin processes are visible. The melanopsin cells are numbered 1-9. Note the weak staining of two inner stratifying cells (2 and 9). B. PACAP(green) staining of the same piece of retina as shown in A with cells numbered as in A (1-9). Note the low level of PACAP found in cell 9.C.Extended view representing the same Z-stack of a total of 35 images of the cells shown in A-B presented in an XY (C), XZ (C1) and ZY (C2) plane to illustrate dendritic stratification. Melanopsin (magenta) and PACAP (green) co-stored in the same RGCs numbered 1-9. Scale bars: 80 μm. GCL; ganglion cell layer, INL; inner nuclear cell layer.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: Retinal innervation of the macaque rostral (A-C), rostral-mid (D-F), mid (G-I) and caudal (J-L) SCN. The SCN was identified by immunostaining for VIP (C, F, I, L, blue). Retinal projections were visualized by cholera toxin subunit B immunoreactivity (B, E, H, K, magenta) and the melanopsin projections were demonstrated by staining for PACAP (A, D, G, J, green) Boxed area in G and H are shown at higher magnification in Figure 5. Oc; optic chiasm. Scale bar; 100 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Immunostaining, Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: Retinal innervation of the macaque mid and caudal SCN. A-D. Higher magnification view of retinal projections representing the melanopsin ipRGCs in the mid SCN shown in boxed area in Figure 4G H, demonstrating co-localization between cholera toxin subunit B (B; CtB; magenta) and PACAP (A; green) immunoreactivity as visualized in C (yellow)and by white colour representing 100 % overlap in D (calculated using the co-localization plug-in in Fiji). Note the PACAP fibres not co-storing CtB may originate from the contralateral eye and to a minor extent from the brain. E-H. Same area of the caudal SCN shown in Figure 4J-K. Co-localization between PACAP (E; green) and CtB (F; magenta) was very sparse, as visualized in G (yellow) and by the few white dots representing 100 % overlap in H (calculated as in D). Oc; optic chiasm. Scale bars: A-D; 20 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques:

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: PACAP containing retinal projections representing the melanopsin ipRGCs innervating the olivary pretectal nucleus (PON).A. Retinal projections visualized by CtB immunostaining in the PON. The image consists of 3×3 tiled confocal images stitched together for better overview. B. Same section as in A co-stained for PACAP showing that PACAP was found in both retinal projections and in cell bodies located within the PON as seen at higher magnification (C-E) and ultrahigh (F-H) magnification representing the area indicated by the square in E. Co-localization between CtB and PACAP containing retinal projections is shown in white (determined using the colocalization module in ImageJ, see Material and Methods). Scale bars: A-B; 100 μm, C-E; 50 μm, F-H; 10 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Immunostaining, Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: PACAP containing retinal projections representing the melanopsin ipRGCs innervating the nucleus of the optic tract (NOT).A. Retinal projections visualized by CtB immunostaining in the NOT. The image consists of 4×4 tiled confocal images stitched together for better overview. B. Same section as in A co-stained for PACAP showing that PACAP was found in both retinal and non-retinal projections located within the NOT. C-E. High magnification images of the area indicated by the asterisks in A and B. Co-localization between CtB and PACAP containing retinal projections is shown in white in E (determined using the colocalization module in ImageJ, see Material and Methods). Scale bars: A-B; 200 μm, C-E; 20 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Immunostaining, Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: PACAP containing retinal projections representing the melanopsin ipRGCs innervating the brachium of the superior colliculus (BSC).A. Retinal projections visualized by CtB immunostaining in the BSC. The image consists of 3×3 tiled confocal images stitched together for better overview. B-C. Same section as in A co-stained for PACAP (B) and showing that PACAP was found in both retinal and non-retinal projections located within the BSC (C). D-F. Higher magnification images of the area indicated by the boxes in A-C.G-I. Ultrahigh magnification of the area indicated by the asterisks in D-F. Co-localization between CtB and PACAP containing retinal projections is shown in white in C, F, and I (determined using the colocalization module in ImageJ, see Material and Methods). Scale bars: A-C; 100 μm, D-F; 50 μm, G-I; 20 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Immunostaining, Staining

Journal: The Journal of comparative neurology

Article Title: Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

doi: 10.1002/cne.23588

Figure Lengend Snippet: PACAP containing retinal projections representing the melanopsin ipRGCs innervating the superior colliculus (SC). A-B. Retinal projections visualized by CtB immunostaining in the ipsi and contralateral SC. The image consists of 2×8 tiled confocal images stitched together for better overview. C-D. Same section as in A-B co-stained for PACAP showing that PACAP was found in cell bodies of deeper layers of the SC (indicated by arrows). High magnification demonstrates PACAP in retinal projections of both the ipsi-(E,F,G) and contralateral side (H-M). Co-localization between CtB and PACAP containing retinal projections is shown in white in G, J and M (determined using the colocalization module in ImageJ, see Material and Methods). Scale bars: A D; 500 μm, E-G; 50 μm, H-M; 20 μm.

Article Snippet: Subsequently, the retinas received another treatment in antigen retrieval solution of 1.5 h at 80 °C when using the C-terminal anti-melanopsin antibody (see below) or no antigen retrieval when using the N-terminal anti-melanopsin antibody (see below)(DAKO ChemMate, code No. S2367 in distilled water, pH 9).

Techniques: Immunostaining, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Functional characterisation of naturally occurring mutations in human melanopsin

doi: 10.1007/s00018-018-2813-0

Figure Lengend Snippet: OPN4 genetic variants selected for in vitro screening. Location of 96 naturally occurring non-synonymous amino acid substitutions (thick black outline) in the human OPN4 protein, of which 16 (red) were screened using immunocytochemistry and calcium imaging of melanopsin-driven light responses. Location of variants not screened in vitro (yellow) and the K340A control mutation (black) is also shown. Secondary structure based on homology with bovine rhodopsin

Article Snippet: A rabbit polyclonal anti-human melanopsin antibody (H-300, Santa Cruz Biotechnology, 1:400) was incubated for 1 h at room temperature.

Techniques: In Vitro, Immunocytochemistry, Imaging, Control, Mutagenesis

Journal: Cellular and Molecular Life Sciences

Article Title: Functional characterisation of naturally occurring mutations in human melanopsin

doi: 10.1007/s00018-018-2813-0

Figure Lengend Snippet: Melanopsin variants show normal membrane localisation. Heterologous expression of pcDNA3.1 OPN4 WT and OPN4 variants in HEK293T cells labelled with anti-OPN4 antibody (red) and DAPI nuclear stain (blue). Scale bar 10 µm

Article Snippet: A rabbit polyclonal anti-human melanopsin antibody (H-300, Santa Cruz Biotechnology, 1:400) was incubated for 1 h at room temperature.

Techniques: Membrane, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Functional characterisation of naturally occurring mutations in human melanopsin

doi: 10.1007/s00018-018-2813-0

Figure Lengend Snippet: Melanopsin variants show abnormal intracellular calcium responses to light. Intracellular calcium levels of HEK293T cells transiently transfected with pcDNA3.1 OPN4 WT and OPN4 variants were monitored using the fluorescent calcium indicator Fluo4-AM. Melanopsin-driven light responses were triggered by the first light exposure used for fluorescent imaging (485 ± 6 nm). a Mean response amplitude (maximum Δ F / F 0 ) for OPN4 WT and each OPN4 variant tested. Dashed grey line shows OPN4 WT response. b , c Traces showing the kinetics of intracellular calcium responses recorded from b non-functional OPN4 variants (red) and c OPN4 variants with significantly attenuated (blue) or elevated (green) intracellular calcium responses compared to OPN4 WT (black). N = 6 biological replicates for all groups except OPN4 WT ( N = 24). Asterisk indicates significant Dunnett’s post hoc test ( p > 0.05) compared to OPN4 WT. NTC is no transfection control. Error bars show standard error of mean. Where error bars are smaller than symbol, error bars are not shown

Article Snippet: A rabbit polyclonal anti-human melanopsin antibody (H-300, Santa Cruz Biotechnology, 1:400) was incubated for 1 h at room temperature.

Techniques: Transfection, Imaging, Variant Assay, Functional Assay, Control