Detailed Visual Cortical Responses Generated by Retinal Sheet Transplants in Rats with Severe Retinal Degeneration

Abstract

To combat retinal degeneration, healthy fetal retinal sheets have been successfully transplanted into both rodent models and humans, with synaptic connectivity between transplant and degenerated host retina having been confirmed. In rodent studies, transplants have been shown to restore responses to flashes of light in a region of the superior colliculus corresponding to the location of the transplant in the host retina. To determine the quality and detail of visual information provided by the transplant, visual responsivity was studied here at the level of visual cortex where higher visual perception is processed. For our model, we used the transgenic Rho-S334ter line-3 rat (both sexes), which loses photoreceptors at an early age and is effectively blind at postnatal day 30. These rats received fetal retinal sheet transplants in one eye between 24 and 40 d of age. Three to 10 months following surgery, visually responsive neurons were found in regions of primary visual cortex matching the transplanted region of the retina that were as highly selective as normal rat to stimulus orientation, size, contrast, and spatial and temporal frequencies. Conversely, we found that selective response properties were largely absent in nontransplanted line-3 rats. Our data show that fetal retinal sheet transplants can result in remarkably normal visual function in visual cortex of rats with a degenerated host retina and represents a critical step toward developing an effective remedy for the visually impaired human population.SIGNIFICANCE STATEMENT Age-related macular degeneration and retinitis pigmentosa lead to profound vision loss in millions of people worldwide. Many patients lose both retinal pigment epithelium and photoreceptors. Hence, there is a great demand for the development of efficient techniques that allow for long-term vision restoration. In this study, we transplanted dissected fetal retinal sheets, which can differentiate into photoreceptors and integrate with the host retina of rats with severe retinal degeneration. Remarkably, we show that transplants generated visual responses in cortex similar in quality to normal rats. Furthermore, transplants preserved connectivity within visual cortex and the retinal relay from the lateral geniculate nucleus to visual cortex, supporting their potential application in curing vision loss associated with retinal degeneration.

Keywords: neurophysiology; orientation selectivity; primary visual cortex; visual cortex; visual pathway; visual rehabilitation.

Figure 1.

Diagram of experimental setup. A retinal sheet transplant derived from fetal transgenic rats is carefully placed in the subretinal space between host-degenerated retina and retinal pigment epithelium using a custom-made surgical tool (Left). The transplant is placed close to the optic disc in the upper temporal area of the visual field (Right). The horizontal meridian (HM) bisects the upper and lower visual field representations.

Figure 2.

Retinal transplant can survive and integrate with degenerated host retina. Examples of retinal transplants verified in vivo by high-resolution OCT at 1 month after surgery are shown for Rats R15–13 (A, B) and R15–15 (D, E). A, D, B scans show the transplant placement in the subretinal space. Regions with partial lamination are clear in addition to photoreceptor rosettes. B, E, Fundus images represent the transplant placement nasal-dorsal to the optic disc. C, F, Examples are shown of BCIP staining for human placental alkaline phosphatase (dark blue to purple) labeling donor tissue in the subretinal space at 4.4 months (C) and 3.5 months (F) after surgery in Rats R15–13 and R15–15, respectively. Dark blue visible beyond the transplant represents cells that migrated from fetal tissue and continued to develop within the host retina.

Figure 3.

Retinal transplants generate new photoreceptors and rod bipolar cells. All images represent transplant at 3.5 months after surgery from Rat R15–15 and are oriented with ganglion cell side up and RPE side down. A, PKCα is a marker of rod bipolar cells and labels both the host (hPAP⁻, red) and donor (hPAP⁺, green) tissue. Donor bipolar cells (yellow) surround photoreceptor rosettes (white asterisks) and interact with photoreceptor terminals to form a putative outer plexiform layer. B, Magnification of boxed region in A with blue channel removed. Donor bipolar cells (yellow) have synaptic projections that innervate the host inner plexiform layer. Without these connections, the photoreceptor light response will not be detected in the brain. C, Rhodopsin (Rho) expression within donor-derived photoreceptor rosettes. Rho⁺ outer segments (red) are indicative of functional rods and critical for light response. D, Magnification of boxed region in C with blue channel removed. Rho is localized within rosettes and the photoreceptor cell bodies (green) surround the inward-pointing outer segments. E, Red-green (R/G) opsin (red) labels cone outer segments and is mostly found within donor rosettes. There are almost no host cones remaining. F, Magnification of boxed region in E with green channel removed to allow R/G opsin signal to be more clearly seen. Cones are generated at a lower frequency than rods. Only transplant cones have outer segments (arrowhead). Arrow indicates remaining host cone.

Figure 4.

Summary of single-unit recordings in V1. A, Percentage of visually responsive cells in tested animal groups. Bars represent the percentage of visually responsive cells. B, Drawing represents the position of V1 electrode tracks where visually responsive (open circles) or no visually responsive (filled circles) neurons were found relative to the optic disk representation of the transplanted eye in all cases. Top inset, Transplanted area relative to visual space and the location of the optic disk. C, For transplanted rats, the percentages of visually responsive and nonresponsive neurons are plotted over distance relative to the center of the transplant.

Figure 5.

Example V1 neuron tuning curves in response to an array of visual stimuli. Left column, Cells from normal rats. Middle column, Transplanted rat cells. Right column, Cells from degenerated rats. Response profiles to drifting sign wave gratings presented at different orientations (A–C). OSI and HWHH values are included to facilitate comparisons. Response profiles are also shown for stimulus size (D–F), spatial frequency (G–I), temporal frequency (J–L), and contrast (M–O). Horizontal dashed lines indicate average spontaneous activity.

Figure 6.

Population comparisons of orientation response for normal, transplanted, and degenerated rats. The distribution (A,D), CDF (B,E), and average (C,F) OSI (A–C) and HWHH (D–F) of V1 neurons are plotted for three different rat groups. Population orientation tuning curves for all cells are shown for normal (G), transplant (L), and degenerated (Q) rats. Additional example orientation tuning curves for V1 cells from normal (H–K), transplanted (M–P), and degenerated rats (R–U). p values are given where significant differences between groups were found. A, D, p values apply to B and E as well.

Figure 7.

Population comparisons of size tuning for normal, transplanted, and degenerated rats. The distribution (A), CDF (B), and average (C) optimal size for V1 neurons are plotted for three different rat groups. Population size tuning curves for all cells are shown for normal (D), transplant (I), and degenerated (N) rats. Additional example size tuning curves for V1 cells from normal (E–H), transplanted (J–M), and degenerated rats (O–R). Conventions are as described in Figure 6.

Figure 8.

Population comparisons of preferred spatial frequency for normal, transplanted, and degenerated rats. The distribution (A), CDF (B), and average (C) preferred spatial frequencies for V1 neurons are plotted for three different rat groups. Population spatial frequency tuning curves for all cells are shown for normal (D), transplant (I), and degenerated (N) rats. Additional example spatial frequency tuning curves for V1 cells from normal (E–H), transplanted (J–M), and degenerated rats (O–R). Conventions are as described in Figure 6.

Figure 9.

Population comparisons of preferred temporal frequency for normal, transplanted, and degenerated rats. The distribution (A), CDF (B), and average (C) preferred temporal frequencies for V1 neurons are plotted for three different rat groups. Population temporal frequency tuning curves for all cells are shown for normal (D), transplant (I), and degenerated (N) rats. Additional example temporal frequency tuning curves for V1 cells from normal (E–H), transplanted (J–M), and degenerated rats (O–R). Conventions are as described in Figure 6.

Figure 10.

Population comparisons of contrast response for normal, transplanted, and degenerated rats. A, The distribution (A), CDF (B), and average (C) C50 for V1 neurons are plotted for three different rat groups. Population contrast response profiles for all cells are shown for normal (D), transplant (I), and degenerated (N) rats. Additional contrast response profiles for V1 cells from normal (E–H), transplanted (J–M), and degenerated rats (O–R). Conventions are as described in Figure 6.

Figure 11.

Response latency and spontaneous and maximum firing rates for normal, transplanted, and degenerated rat V1 neurons. Raster plots (A–C) and poststimulus time histograms (D–F) represent responses of example neurons from a normal (A, D), transplanted (B, E), and degenerated (C, F) rat. A full-screen bright stimulus was shown for 0.5 s beginning at Time 0. Vertical dashed line indicates response onset, based on a 10% increase over the average spontaneous firing rate. Spontaneous firing rate was determined over the 0.5 s before stimulus onset. Population responses over time are shown for normal (G), transplant (H), and degenerated (I) rats. Average response latency (J), spontaneous activity (K), and maximum firing rate (L) are shown for the V1 population of the three rat groups. Conventions are as described in Figure 6.

Figure 12.

V1 connectivity revealed by glycoprotein-deleted GFP encoding rabies virus in transplanted and degenerated rats. A, A V1 injection site in transplanted Rat R15–13 made into a region containing visually responsive neurons confirmed through recordings. B, A larger magnification of injection site shown in A. C, Labeled neurons found in the LGN from the V1 injection in A. D, A V1 injection site in degenerated Rat R15–08 that did not receive a retinal transplant. E, A larger magnification of the site shown in D. F, Labeled neurons found in the LGN from the injection shown (D). Scale bars, 500 μm.

Figure 13.

Distribution of V1 connections in cortex and thalamus for normal, transplanted, and degenerated rats. A, The number of neurons retrogradely infected locally (within 300 μm of the injection site) in V1 following injections of glycoprotein-deleted rabies virus were averaged across cases within the three rat groups. B, The percentage of infected neurons in the LGN, LP, higher visual cortex, and at long range in V1 (>300 μm from injection site) are shown for the three rat populations. For each case, percentages were derived by dividing the number of infected cells in each brain region by the number of cells found locally in V1. The same cases were used for A and B; normal (n = 4), transplant (n = 5), and degenerated (n = 4) rat groups. p values are given where significant differences between groups were found.