Fig 1.
(A) A sketch of Opto-mGluR6 composed of the N-terminus (NT), transmembrane domains (TM1–TM7), and extracellular loops 1–3 (EL1–EL3) of melanopsin and the intracellular loops (IL) 2 and 3, and the C-terminus (CT) of mGluR6. Locations of the splice sites (connection sites between the melanopsin and mGluR6 peptides) are indicated (a–g). (B) Amino acid sequence alignment of mouse melanopsin, mouse mGluR6, and Opto-mGluR6; splice sites are indicated by arrows (see Supporting Information). Amino acid numbering is indicated to the right. Cages indicate amino acids that are either identical (black) or conserved (grey) on the basis of polarity and acidity. (C) Light response of a HEK293-GIRK cell transiently transfected with Opto-mGluR6 showing the Kir3.1/3.2 induced continuous hyperpolarization in response to 60-s illumination (black bar). (D) Example whole-cell current responses from the same HEK293-GIRK cell to 1-s voltage ramps between -150 mV and +60 mV in the dark and during light stimulation. The subtraction curve (black line) represents the light-induced GIRK current with the characteristic inward rectification of Kir3.1/3.2 channels. See S1 Table for a comprehensive overview of GIRK data.
Fig 2.
(A) Schematic of the rAAV expression cassette with internal tandem repeats (ITR), the mGluR6 enhancer element (GRM6), the sv40 eukaryotic promoter sequence (psv40), an internal ribosomal entry site (IRES), woodchuck posttranscriptional regulatory element (WPRE), and bovine growth hormone polyadenylation sequence (BGHpA). (B) Retinal whole mount transduced by intravitreal injection and stained with an antibody against TurboFP635. The green dots represent transfected ON-bipolar cells. Scale bar, 500 μm. (C) Confocal image of a traverse section through a C57BL/6 retina stained against the rod bipolar cell marker PKCα (green), anti-TurboFP635 (red), and DAPI (blue), showing expressing rod bipolar cells (arrows). Also see S1 Fig. Scale bar, 20 μm. (D) Raster plots of seven RGCs from rAAV_Opto-mGluR6–treated rd1 retinas showing light responses to 1-s-long blue light pulses (bright band), four traces per cell. Cells are numbered and separated by stippled lines. (E) Fluorescent micrograph of the ganglion cell (green) giving rise to the transient ON response from D (star), with the underlying TurboFP635-labeled bipolar cells shown in red. Scale bar, 40 μm.
Fig 3.
Immunocytochemistry of the rd1_Opto-mGluR6 retina.
(A) Double labeling against cytoplasmic TurboFP635 (red) and ChAT (green), serving as depth marker in the inner plexiform layer (IPL), subdividing ON and OFF sublaminae (white brackets). All TurboFP635-positive bipolar cells project their axons to the ON-sublamina of the IPL. Nuclei are labeled with DAPI (blue). (B) Staining against the N-terminal part of melanopsin labels both ipRGCs and Opto-mGluR6 in the perikarya of ON-bipolar cells (ON-BPs). (C) Double labeling of TurboFP635 (red) with the pan ON-bipolar cell marker Gαo (green) shows a 100% overlay, indicating that all ON-BPs express Opto-mGluR6_IRES_TurboFP635. Scale bars, 20 μm.
Fig 4.
Light responses in rd1_Opto-mGluR6 retinas.
(A) Despite expression of Opto-mGluR6 only in ON-bipolar cells, the RGCs have both ON and OFF receptive fields (left). RGCs of rd1 littermates did not respond to the same light stimulus (right). Light steps are indicated by bright underlays. (B) Example light responses of a sustained ON (left) and a transient ON (right) RGC that are not antagonized by 20-μM L-AP4 but are blocked by 10-μM CNQX, indicating that these light-ON responses arise from bipolar cells. Light step is indicated by broken vertical lines. Pharmacology performed on a total of six cells. (C) Average light intensity response curves of seven RGCs from transgenic Opto-mGluR6 retinas and six RGCs from rAAV-transduced Opto-mGluR6 retinas compared to published values for ChR2 [9,12]. The Opto-mGluR6 response saturated at light intensities required for ChR2 activation. The light-induced changes in spike rate were normalized to a maximum of 1 for each RGC (see S4 Fig for details).
Fig 5.
Example traces of direct patch-clamp recordings from bipolar cells demonstrating inverse response polarity of Opto-mGluR6 compared to photoreceptor-activation without changes in response kinetics.
We recorded from 21 bipolar cells in dark-adapted Pde6b+_Opto-mGluR6 retinal slices before (black traces) and after (grey traces) the photoreceptor input was eliminated by bleaching and application of L-AP4. (A,B) Bipolar cell responses to a long (2 s) light pulse. The native ON-bipolar cell hyperpolarized to a light-OFF stimulus (step down at 0.5 s) but depolarized to the same stimulus under direct Opto-mGluR6 activation (A). Conversely, the native OFF-bipolar cell responded with hyperpolarization to a light-ON stimulus (step up at 0.5 s) but depolarized to the same stimulus under Opto-mGluR6 activation (B). (D,E) ON-bipolar cell responses to short (50 ms) light stimuli. Opto-mGluR6-mediated responses have an identical time course to the photoreceptor-mediated response under inversion of response polarity. Opto-mGluR6 reliably encodes multiple short light pulses (D).
Fig 6.
Opto-mGluR6 flips the polarity of light responses in RGCs.
(A) Example spike trains recorded from an ON-Alpha RGC in an isolated Pde6b+_Opto-mGluR6 retina in response to a series of light steps (yellow underlay, 5.4 x 1016 photons cm-2 s-1, 100% contrast, yellow highlights), before (top trace) and after (bottom trace) the photoreceptor cells were bleached. (B) The same experiment from panel A performed on an OFF-Alpha cell. After recording, cells were labeled with neurobiotin (red). Panels (C,D) show flat views of the cells recorded from in A and B, respectively. (E,F) show transverse views of the marked areas in C and D, respectively. Colabeling with ChAT (green) confirms the identity of the ON- and OFF-Alpha cells on the basis of their dendritic stratification depth. Scale bars, 50 μm. (G) Spike-time histograms comparing the response kinetics of OFF-Alpha cells from C57BL/6 retinas (black) with that of ON-Alpha cells from bleached Pde6b+_Opto-mGluR6 retinas (red). Each trace shows the average of eight traces recorded from four cells from four retinas, bin width 20 ms.
Fig 7.
ERGs of rd1_Opto-mGluR6 mice show an inverse, electronegative b-wave.
(A) Negative deflection in response to a strobe flash measured in rd1_Opto-mGluR mice. (B) Blind rd1 littermates had no detectable ERG. (C,D) The ERGs of seeing Pde6b+ mice to a low-intensity (C) and high-intensity (D) strobe flash. ERGs of nonsaturating light intensities (C) lack oscillatory potentials and show slowed b-wave kinetics compared to ERGs of saturating light intensities (D). (E,F) Pharmacology on the electronegative ERG depicted in (A), before (grey traces) and after (broken traces) intravitreal injection of inhibitors. Application of DL-APV and L-AP4 slowed the response kinetics (E), and additional application of CNQX increased the ERG amplitude (F). The black trace in (E) shows the ERG of the control (left) eye after the inhibitor mix has been injected into the contralateral (right) eye; no change in the ERG is visible in this internal control.
Fig 8.
rd1_Opto-mGluR6 mice show significantly higher V1 activation compared to rd1 littermates both at p180 and p282.
(A,B) Quantification of V1 activation of the right and left primary visual cortex (V1) from rd1 (grey, p180: n = 5; p282: n = 4) and rd1_Opto-mGluR6 mice (blue, p180 n = 3; p282, n = 4), plotted separately for the two ages (A) or as a function of age (B) for V1 activation of the left (squares) and right (triangles) hemispheres (mean ± SEM, ANOVA, ***p < 0.001). (C) Typical examples of binocular V1 activity maps of rd1_Opto-mGluR6 mice and rd1 littermates. The grey-scale coded response magnitude maps (upper rows) are illustrated as fractional change in reflection x 10-4. Retinotopic maps (lower rows) are color-coded according to the scheme on the left. Scale bar, 1 mm.
Fig 9.
Visual performance determined in behavioral experiments.
(A) rd1_Opto_mGluR6 mice (n = 11) had optokinetic reflexes with a peak spatial acuity of 0.17 ± 0.04 cyc/deg, rd1 littermates (n = 8) did not respond to any frequency, and seeing Pde6b+ mice (n = 6) responded with a peak acuity of 0.35 ± 0.05 cyc/deg. Stimulus intensity 5.6 × 1013 photons cm-2 s-1. (B) The total swim distance to a hidden platform in a water maze marked by an overhead blue light-emitting diode (LED). From training day 2, rd1_Opto-mGluR6 mice (n = 6) learned to find the platform significantly faster than their blind rd1 littermates (n = 5) but not significantly slower than Pde6b+ mice (n = 3). Stimulus intensity 1 × 1016 photons cm-2 s-1. (C) Sensitivity of behavioral vision. rd1_Opto-mGluR6 mice were trained for 6 d at high and low light intensities (left) and subsequently tested for their visual performance at three test intensities (right). Nonsaturating training light intensities did not compromise learning, and the performance at the three test intensities did not differ significantly. Error bars indicate SEM in B and C. (D,E) Example paths swam by rd1_Opto-mGluR6 and rd1 mice, respectively, on training day 10.