Expression of Chop2-GFP in Retinal Neurons In Vivo (A) rAAV-CAG-Chop2-GFP-WPRE expression cassette. CAG: a hybrid CMV enhancer/chickenβ-actin promoter. WPRE: woodchuck posttranscriptional regulatory element. BGHpA: a bovine growth hormone polyadenylation sequence. (B and C) Chop2-GFP fluorescence viewed in low (B) and high (C) magnifications from eyes two months after the viral vector injection. (D) Confocal images of a ganglion cell, which show a stacked image (left) and a single optical section image (right). (E) Chop2-GFP fluorescence in a horizontal cell, which shows GFP in soma, axon, and distal axon terminal. (F and G) Chop2-GFP fluorescence in amacrine cells (F) and a retinal bipolar cell (G). (H and I) Fluorescence image (H) and phase contrast image (I) taken from a retina 12 months after the injection of Chop2-GFP viral vectors. Images in (B-E) were taken from flat whole-mounts of rat retinas. Images in (F-I) were taken from vertical slice sections of rat retinas. Scale bar: 200μm in (B); 100μmin(C);15μmin(D);50μm in (E), H), and (I); 25μm in (F) and (G). ONL: outer nuclear layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.

Properties of Light-Evoked Currents of the ChR2-Expressing Retinal Neurons (A) Phase contrast image (left) and fluorescence image (right) of a GFP-positive retinal neuron dissociated from the viral vector injected eye. Scale bar: 25μm. (B) A recording of Chop2-GFP fluorescent retinal cell to light stimuli of wavelengths ranging from 420 to 580 nm. The light intensities were ranging from 1.0-1.6× 10¹⁸ photons cm^-2 s^-1. (C) A representative recording of the currents elicited by light stimuli at the wavelength of 460 nm with light intensities ranging from 2.2× 10¹⁵ to 1.8× 10¹⁸ photons cm^-2 s^-1. (D) Current traces after the onset of the light stimulation from (C) shown in the expanded time scale. The red trace shows the fitting of one current trace by an exponential function: I(_t =a₀ +a₁×(1-exp[-t/τ₁])+a₂×(exp[-t/τ₂]), in which τ₁ and τ₂ represent the activation and inactivation time constant, respectively. (E) Current traces after the termination of the light stimulation from (C) shown in the expanded time scale. The red trace shows the fitting of one current trace by a single exponential function: I_(t)=a₀+a₁×(1-exp[-t/τ), in which τ represent the deactivation time constant. (F) Light-intensity response curve. The data points were fitted with a single logistic function curve. (G and H) The relationships of light-intensity and activation time constant (G) and light-intensity and inactivation time constant (H) obtained from the fitting shown in (D). All recordings were made at the holding potential of -70 mV. The data points in (F)-(H) are shown as mean± SD (n = 7).

Properties of Light-Evoked Voltage Responses of ChR2-Expressing Retinal Neurons (A) A representative recordings from GFP-positive nonspiking neurons. The voltage responses were elicited by four incremental light stimuli at the wavelength of 460 nm with intensities ranging from 2.2× 10¹⁵ to 1.8× 10¹⁸ photons cm^-2 s^-1 in current clamp. The dotted line indicates the saturated potential level. (B) A representative recording from GFP-positive nonspiking neurons to repeat light stimulations. The light-evoked currents (top traces) and voltage responses (bottom traces) from a same cells were shown. Left panel shows the superimposition of the first (red) and second (black) traces in an expanded time scale. The dotted line indicates the sustained component of the currents (top) and plateau membrane potential (bottom). (C) A representative recording of GFP-positive spiking neurons to repeated light stimulations. The responses in (B) and (C) were evoked by light at the wavelength of 460 nm with the intensity of 1.8 × 10¹⁸ photons cm^-2 s^-1.

Expression and Light-Response Properties of ChR2 in Retinal Neurons ofrd1/rd1 Mice (A) Chop2-GFP fluorescence viewed in flat retinal whole-mount of a 15 month old mouse with the Chop2-GFP viral vector injection at 9 months of age. (B) Chop2-GFP fluorescence viewed in vertical section from the retina of a 6 month old mouse with the injection of Chop2-GFP viral vectors at 3 months of age. (C) Light microscope image of a semithin vertical retinal section from a 5 month old mouse (Chop2-GFP viral vectors injected at postnatal day 1). Scale bar: 50μm in (A) and 30μm in (B) and (C). (D-E) Representative recordings of transient spiking (D) and sustained spiking (E) GFP-positive neurons. The responses were elicited by light of four incremental intensities at the wavelength of 460 nm. The light intensity without neutral density (Log I = 0) was 3.6× 10¹⁷ photons cm^-2 s^-1. The currents were recorded at the holding potential of -70 mV. The superimposed second (black) and fourth (red) current and voltage traces are shown in the right panel in the expanded time scale. (F-I) The relationships of the amplitude of current (F), membrane depolarization (G), the number of spikes (H), and the time to the first spike peak (I) to light intensity. Recordings were made fromrd1/rd1 mice at≥4 months of age. The data points are shown as mean± SE (n = 6 in [F]-[H] and n = 4 in [I]).

Multielectrode Array Recordings of the ChR2-Expressing Retinas ofrd1/rd1 Mice (A) A sample recording of light-evoked spike activities from the retinas ofrd1/rd1 mice (ages≥4 months). The recording was made in the present of CNQX (25μM) and AP5 (25μM). Prominent light-evoked spike activity was observed in 49 out of 58 electrodes (electrode 15 was for grounding and electrode 34 was defective). (B) Sample light-evoked spikes recorded from a single electrode to three incremental light intensities. (C) The raster plots of 30 consecutive light-elicited spikes originated from a single neuron. (D) The averaged spike rate histograms. The light intensity without neutral density filters (Log I = 0) was 8.5× 10¹⁷ photons cm^-2 s^-1. The responses shown in (A) were elicited by a single light pulse without neutral density filters.

Central Projections of Chop2-GFP-Expressing Retinal Ganglion Cells and Visual-Evoked Potentials inrd1/rd1 Mice (A) GFP labeled terminal arbors of retinal ganglion cells in ventral lateral geniculate nucleus and dorsal lateral geniculate nucleus. (B) GFP-labeled terminal arbors of retinal ganglion cells in superior colliculus. OT: optical track; vLGN: ventral lateral geniculate nucleus; dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus. Scale bar: 200μm in (A), 100μm in (B). (C) VEPs recorded from a wild-type mouse. The responses were observed both to the wavelengths of 460 and 580 nm. (D) VEPs recorded from anrd1/rd1 mouse injected with Chop2-GFP viral vectors. The responses were elicited only by light at the wavelength of 460 nm but not at the wavelength of 580 nm. (E) No detectable VEPs were observed from rd1/rd1 mice injected with viral vectors carrying GFP alone. The light intensities measured at the corneal surface at the wavelengths of 460 and 580 nm were 5.5× 10¹⁶ and 5.2×10¹⁶ photons cm^-2 s^-1, respectively. (F) Plot of the amplitude of VEPs from rd1/rd1 mice injected with Chop2-GFP viral vectors to various light intensities at the wavelengths of 420, 460, 500, 520, and 540 nm. For each eye, the responses are normalized to the peak response obtained at 460 nm. The data are shown as mean± SD (n = 3 eyes). Spectral sensitivity at each wavelength was defined as the inverse of the interpolated light intensity to produce 40% of the normalized peak response, as indicated by the dot line. (G) The sensitivity data points are fitted by a vitamin-A₁-based visual pigment template with a peak wavelength of 461 nm.