Figure 1.
Generation of Thy-1.2 ChR2V transgenic rat.
Schematic drawing of DNA fragment injected into rat oocytes. (A) The cDNA coding channelrhodopsin-2 (ChR2) tagged with Venus was inserted at XhoI site of the modified mouse Thy-1.2 expression cassette. A linearized DNA fragment (7.5 kb) prepared by digestion with EcoRI and PvuI restriction enzymes was injected into rat oocytes. (B) Examples of PCR analysis of genomic DNA from transgenic founder rats injected with the transgene shown in A. Genomic DNAs from the injected DNA fragment (lane 1), a transgenic founder (lane 2) and a non transgenic founder (lane 3) were amplified by PCR. DNA bands at 173 bp and 324 bp correspond to amplified DNA fragments for the transgene (ChR2-Venus, ChR2V) and the T cell receptor gene as an internal control, respectively.
Figure 2.
Microphotographs showing ChR2V expression in the inner retinal layers of each transgenic line.
(A–D) The retinal organization of each transgenic line (A, W-TChR2V1; B, W-TChR2V4; C, W-TChR2V5; D, W-TChR2V7) showed normal features in the Nomarskii images (left). Fluorescence microphotography revealed various expression patterns in retinal slices (middle) and flat-mounted retinas (right). (E) Z-axis scan (pitch: 0.35 µm) images collected from a flat-mounted retina of a ChR2V+/− rat (line W-TChR2V4) showed that the ChR2V fluorescence (green) was coexpressed with fluorogold transported retrograde from the superior colliculus (blue).
Figure 3.
Direct photoactivation of ChR2V-expressing RGCs.
(A) The blue light-emitting diode (LED)-evoked membrane currents and potentials were recorded under whole-cell recording from one of the ChR2V-expressing RGCs. (B) The photocurrents and their dependency in the LED power density. (C–D) The membrane potential responses of a ChR2V-expressing RGC to LED pulses of 100-ms (C) or 10-ms (D) duration.
Figure 4.
Morphological evidences of the photoreceptor degeneration in either ChR2V−/− or ChR2V+/− rats.
(A, B) Normal retinal architecture was observed in each rat before photoreceptor degeneration. (C, D) After continuous light exposure (3000 lux for 7 days). Sections are from the superior and inferior regions at a distance of 0.24 mm from the optic nerve. Note the absence of the outer nuclear layer. (E, F)The severe degeneration extended to the whole retina. (G)Cryo-section of a retina from a ChR2V+/− rat. ChR2V expression was observed in the inner layer. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Figure 5.
Electrophysiological evidence of photoreceptor degeneration.
(A) Typical waveforms of electroretinogram (ERG) responses evoked by either blue or red light-emitting diode (LED) flash (duration: 10 ms; light intensity: 1000, 300, and 100 lux, top to bottom). (B)The ERG amplitudes (left, a-wave; middle, b-wave) and the latency of the a-wave (right) in response to the blue LED flash. Note that both amplitudes were significantly diminished in the ChR2V+/− rats compared to the ChR2V−/− rats without any differences in a-wave latency. (C) The b-wave amplitudes (left) and the latency (right) in response to the red LED flash. (D)Typical ERG waveforms evoked by either blue (upper traces) or red (lower traces) LED flash in the ChR2V−/− (left) and +/− (right) rats after continuous light (3000 lux) exposure for 7 days. (E)The b-wave amplitudes recorded from the rats after continuous light exposure. Error bars represent standard deviation (n = 8, **: P<0.01, unpaired t-test).
Figure 6.
The visually evoked potentials recorded from either the ChR2V−/− or ChR2V+/− rats before photoreceptor degeneration.
(A)Sample waveforms evoked by the blue or red LED flash. (B) The amplitude- (left) and the latency- (right) stimulus intensity relationships of VEPs evoked by the blue LED flash. (C) Similar to (A), but the responses are to the red LED flashes. Error bars represent standard deviation (n = 8, *: P = 0.04, **: P<0.01, unpaired t-test).
Figure 7.
The VEPs after photoreceptor degeneration.
(A)Sample waveforms evoked by blue or red LED flash. (B) The amplitude- (left) and the latency- (right) stimulus intensity relationships of VEPs evoked by the blue LED flash. (C) The summary of VEP responses to the red LED flashes. Error bars represent standard deviation (n = 8, *: P<0.05, **: P<0.01, unpaired t-test).
Figure 8.
Optomotor response of the ChR2V−/− and ChR2V+/− rats.
(A) The experimental design for the evaluation of optomotor response. The moving vertical stripes were displayed on the computer monitors so that the brightness-darkness contrast followed a sine-wave function of variable amplitude and spatial frequency. (B)The contrast sensitivity-spatial frequency relationship of the ChR2V−/− and ChR2V+/− rats before photoreceptor degeneration. (C) The contrast sensitivity-spatial frequency relationship of the ChR2V+/− rats after photoreceptor degeneration. LD = Light damaged. Error bars represent standard error of the mean (n = 8, *: P<0.05, Mann-Whitney U-test).