Supplemental Information. Melanopsin-Encoded Response Properties. of Intrinsically Photosensitive. Retinal Ganglion Cells

Transcription

1 Neuron, Volume 90 Supplemental Information Melanopsin-Encoded Response Properties of Intrinsically Photosensitive Retinal Ganglion Cells Ludovic S. Mure, Megumi Hatori, Quansheng Zhu, James Demas, Irene M. Kim, Surendra K. Nayak, and Satchidananda Panda

2 Supplementary Information Supplementary Table Table S1 (related to Fig. 3): Altered melanopsin C-terminus tail results in altered response properties that translate in vivo. Light responses from the retina of rd;opn4 -/- mice expressing WT (Opn4 WT, n=57) and altered melanopsins; truncated (Opn4 380, n=33; Opn4 397 n=45), phosphonull mutations (Opn4 2A, n=103; Opn4 4A, n=100; Opn4 9A, n=125) and phosphomimetic mutation (Opn4 2D, n=61,): MANOVA. p values Latency (fig3 F, G) Duration (fig3 H, I) Opn ns * Opn4 2D 0.73 ns ** Opn4 2A ** * Opn4 4A *** *** Opn4 9A *** ns

3 Supplementary Figures

4 Figure S1 (related to Fig. 2). FLIPER/Ca 2+ release assays. Melanopsin photoresponse in CHO cells transduced with different mutants. (A) Average (in sec, +s.e.m.) and relative time for the peak response to return to 30% and absolute (F/F 0, +s.e.m.) and relative peak amplitude (Raw values are the average of the pooled individual data (n=20) while relative values are the mean of the normalized data per plates (n=3)). (B) Progressive S to A mutation of the phosphorylable region of melanopsin C terminus tail results in additive effects on both photoresponse duration and peak. duration peak Opn4 WT p value p value Opn4 397Δ ** ** Opn4 9A397Δ *** *** Opn4 9A *** *** Opn4 8A *** *** Opn4 7A *** *** Opn4 6A *** *** Opn4 5A ** *** Opn4 4A ns ** Opn4 3A 0.53 ns *** Opn4 2A 0.22 ns 0.17 ns Opn4 1A 0.41 ns 0.44 ns Opn4 3Ai 0.04 * ** Opn4 3Ac 0.69 ns *** (C) Melanopsin photoresponse in CHO cells transduced with different non-phosphorylable mutants. The same residues are affected but in different cluster combinations. Average (in sec, +s.e.m.) and relative time for the peak response to return to 30% and absolute (F/F 0, +s.e.m.) and relative peak amplitude (Raw values are the average of the pooled individual data (n=20) while relative values are the mean of the normalized data per plates (n=3)).

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6 Figure S2 (related to Fig. 3). Functional expression of melanopsin in mrgcs imparts sustained photosensitivity. (A) Restoration of circadian photoentrainment in rd;opn4 -/- mice upon AAV Opn4 WT administration in the retina (arrow). Daily wheel running activity over several days are shown. (a 1 ) Within few days of virus administration the daily activity pattern entrains to light:dark cycle and this entrainment is (a 2 ) stable over several weeks. (a 3 )As is known in WT mice under constant light, these mice also exhibit lengthening of circadian activity rhythm. (a 4 ) The AAV expression does not affect the intrinsic short period (<24 h) periodcity of the activity rhythm under constant darkness and the newly imparted photoentrainment property is intact even after several months of virus administration. (B) Lack of circadian photoentrainment in rd;opn4 Cre/Cre ;idtr mice with ablated iprgcs (red arrow representing DT administration), upon AAV Opn4 WT expression in the retina (blue arrow).. (C) Lack of circadian photoentrainment in rd;opn4 -/- mice upon AAV GFP expression in the retina. Expression of (D) AAV Opn4 4A or (E) AAV Opn4 2D restored circadian photoentrainment in rd;opn4 -/- mice upon viral mediated expression.

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8 Figure S3 (related to Fig. 3). (A) Diagram summarizing the different parameters of iprgcs electrophysiological response as recorded extracellularly with MEA. Comparaison of responses from Opn4 WT and Opn4 9A to 10ms stimulations recorded in cells responding to 100ms stimulations. (B) in rd;opn4 -/- mice injected with Opn4 WT and Opn4 9A (n=6 and 14) and (C) in rd;opn4 cre/cre mice injected with floxed Opn4 WT and Opn4 9A (n=16 and 18). Representative single units responses to 1s, 10s and 60s stimulations recorded from rd;opn4 -/- mice retinas transduced with Opn4 WT, Opn4 380, Opn4 397, Opn4 2A, Opn4 4A, Opn4 9A and Opn4 2D (D) or in rd mice compared to rd;opn4 cre/cre mice conditionally transduced with Opn4 WT, Opn4 397, Opn4 9A and Opn4 9A397 (E). (F, G) Light responses from the retina of rd;opn4 -/- mice expressing WT (Opn4 WT, n=57) and altered melanopsins; truncated (Opn4 380, n=33; Opn4 397 n=45), phosphonull mutations (Opn4 2A, n=103; Opn4 4A, n=100; Opn4 9A, n=125) and phosphomimetic mutation (Opn4 2D, n=61,): number of spikes. MANOVA: p value Opn4 397Δ ** Opn4 2A * Opn4 4A * Opn4 9A ns Opn4 2D 0.54 ns

9 Figure S4 (related to Fig. 3 and 4): Altered melanopsin C-terminus tail results in altered response properties. Light responses of rd;opn4 -/- mice injected with WT (Opn4 WT, n=58 ) or truncated melanopsin; (Opn4 380, n=33). Average traces (A), response latency (B) and duration (C). Light responses of rd;opn4 cre/cre mice injected with floxed WT (Opn4 WT, n=54 ) or mutated/truncated melanopsin; (Opn4 9A397, n=29). average traces (D), response latency (E) and duration (F).

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11 Figure S5 (related to Fig. 4). Melanopsin immunostaining in the retina of rd;opn4 cre-/cre mice intravitreally transduced with AAV-DIO-Opn4 constructs. Retina of rd;opn4 -/- mice intravitreally injected with the indicated viruses were collected at least 4 weeks after virus injection, fixed, flat mounted and immunostained with polyclonal rabbit anti-opn4 antibodies raised against the N-terminus region of the protein, and imaged using a Zeiss LSM 780 scanning confocal microscope with a 20X objective following standard protocols (Brown et al., 2010; Lin et al., 2008). Maximum intensity projection images are shown.(b-e) Restoration of circadian photoentrainment in rd;opn4 cre/cre mice upon viral mediated expression of different floxed melanopsin mutants with altered phosphorylation sites or truncations (Opn4 WT, Opn4 397, Opn4 9A, Opn4 9A397 ). Electrophysiological responses measured from rd mice retinas or rd;opn4 cre/cre mice conditionally expressing Opn4 WT are virtually identical.: Responses latency (F, ANOVA p=0.1732, ns) and duration (G, ANOVA p=0.2447, ns) to increasing stimulations lengths or increasing irradiance (H, sum of squares F test, p=0.8252, ns). 480nm, photons/cm 2 /s, 100ms, 1, 10 and 60s in and 480nm, 60s, photons/cm 2 /s to photons/cm 2 /s), average values +/-SEM. (n=91 and 54 respectively for rd and rd;opn4 cre/cre injected with Opn4 WT mice and DRC n=35 and 19 respectively for rd and rd;opn4 cre/cre injected with Opn4 WT mice).

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13 Figure S6 (related to Fig. 4 and 5): While conditional expression of WT (Opn4 WT, n=54 ) and mutant melanopsins (Opn4 397, n=38; Opn4 9A, n=36; Opn4 9A397, n=29) restores photosensitivity in rd;opn4 cre/cre mice, the different groups display distinct response latencies (A) and durations (B) to 100ms, 1s, 10s and 60s stimulations. p values latency duration Opn ns *** Opn4 9A ns *** Opn4 9A ns ns (C) Dose response curve plotted for the discharge rate in responses to 1min stimulations of increasing irradiances in rd;opn4 cre/cre mice conditionally infected with melanopsins (Opn4 WT, n=35; Opn4 397, n=47; Opn4 9A, n=42).(manova, Opn4 9A vs Opn4 WT p=0.03, Opn4 397 vs Opn4 WT p=0.75). (D) Average pupil constriction recorded in rd;opn4 cre/cre mice conditionally infected with WT (Opn4 WT, n=4) and altered melanopsins; (Opn4 397 n=5, Opn4 9A, n=2 and Opn4 9A397 n=6) during the 60s light stimulation or during the 2min immediately following light OFF (persistence). 480nm, photons/cm 2 /s, average values +/-SEM. (E) Average pupil constriction recorded in response to successive 1s stimulation in rd;opn4 cre/cre mice conditionally infected with WT (Opn4 WT, n=4) and altered melanopsins; (Opn4 397 n=5, Opn4 9A, n=2). 480nm, photons/cm 2 /s, average values +/-SEM.

14 Figure S7 (related to Fig. 6). Deactivation speed of the mrgcs responding to increasing irradiance stimulations (at least 5/6) from retina transduced with either Opn4 WT (A, n=5) or Opn4 397 (B, n=10). (C and D) Deactivation of photoresponse from mrgcs expressing Opn4 WT or Opn4 397 following a 60sec light pulse. The 55sec-120sec portion of Fig 4A and 4B are shown. Last 5sec of 60sec illumination is shaded in cyan blue.