(A) Schematic of the DNA expression cassette delivered by AAV2/2 vector to the retina. A human rod opsin coding sequence (RHO) is driven by a hybrid CMV enhancer/chickenβ-actin (CAG) promoter. The sequence is flanked by inverted terminal repeats (ITRs) and stabilized by a polyadenylation signal sequence (polyA) and a woodchuck hepatitis posttranscriptional regulatory element (WPRE).

(B and C) Exemplar images of a section through an rd1 mouse retina >4 months after intravitreal delivery of vector in (A) in conjunction with glycosidic enzymes. Expression of human rod opsin in cells of the ganglion cell layer (GCL) and inner nuclear layer (INL) and processes in the inner plexiform layer (IPL) are revealed by staining with an α-hRho antibody (red) and counterstaining of nuclei with DAPI (blue) to aid orientation (B). A monochrome version of α-hRho antibody staining in (B) in which rod opsin expression appears in white is shown in (C). Calibration bar = 50 μm.

(D and E) Perievent rasters and associated perievent firing rate histograms (PSTHs) for eight representative single units isolated from multi-electrode array (MEA) recordings of rd1-CAG-RHO retinas without (D) and with (E) exogenous 9-cis-retinal. Each set of rasters depicts spiking activity for 20 sequential presentations of a 2-s white light flash (4 × 1014 rod photons/cm2/s; interstimulus interval 20 s) starting at time 0. PSTHs below depict mean firing rate in 100-ms epochs across all 20 repeats. In both conditions, units show increases in firing associated with light presentation (from 0 to 2 s), but these are most pronounced for the first few trials (lower traces in raster) in (D), indicating bleaching, while inclusion of 9-cis-retinal (E) renders them repeatable across many trials.

(F and G) Heatmap representations of mean firing rate across at least 20 presentations of 2-s stimulus (ON at time 0) for 104 units from 5 rd1-CAG-RHO mice (F) and six units from three control rd1-CAG-GFP mice (G) meeting an objective criterion of stimulus-associated change in firing. Color code represents normalized firing rate (−1 and 1 being minimum and maximum firing rate for that unit, respectively). Traces are ordered according to response latency.

(H) Population mean (±SEM) normalized firing rate profiles for rd1-CAG-RHO units grouped according to response latency (horizontal white lines in F delineate extent of clusters).

(I) Mean ± SEM normalized firing rate (mean firing rate from −2 s to 6 s was normalized to maximum and minimum, and the normalized pre-stimulus firing rate (−2 to 0 s) was then subtracted) for all light-responsive units exposed to 2-s pulses (starting at 0 s) at 4 × 1014, 4 × 1013, and 4 × 1012 rod photons/cm2/s.

(J and K) Distribution of response amplitudes (J; mean change in firing rate) and latencies (K; mean time at which mean firing rate first fell outside 2 SDs of baseline firing) for units in (F) responding with increases (excit’n) or decreases (inhib’n) in firing at 4 × 1014, 4 × 1013, and 4 × 1012 rod photons/cm2/s.

(L) Perievent rasters for three single units showing firing of three units across multiple repeats of a 2-s light pulse (4 × 1014 rod photons/cm2/s) without (above) and with (below; shaded in green) application of GABA receptor antagonists (TPMP 25 μM and picrotoxin 50 μM).