Deafferented Adult Rod Bipolar Cells Create New Synapses with Photoreceptors to Restore Vision

Abstract

Upon degeneration of photoreceptors in the adult retina, interneurons, including bipolar cells, exhibit a plastic response leading to their aberrant rewiring. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of deafferented bipolar cells to establish functional synapses with photoreceptors is poorly understood. Here we use photocoagulation to selectively destroy photoreceptors in adult rabbits while preserving the inner retina. We find that rods and cones shift into the ablation zone over several weeks, reducing the blind spot at scotopic and photopic luminances. During recovery, rod and cone bipolar cells exhibit markedly different responses to deafferentation. Rod bipolar cells extend their dendrites to form new synapses with healthy photoreceptors outside the lesion, thereby restoring visual function in the deafferented retina. Secretagogin-positive cone bipolar cells did not exhibit such obvious dendritic restructuring. These findings are encouraging to the idea of photoreceptor reintroduction for vision restoration in patients blinded by retinal degeneration. At the same time, they draw attention to the postsynaptic side of photoreceptor reintroduction; various bipolar cell types, representing different visual pathways, vary in their response to the photoreceptor loss and in their consequent dendritic restructuring.SIGNIFICANCE STATEMENT Loss of photoreceptors during retinal degeneration results in permanent visual impairment. Strategies for vision restoration based on the reintroduction of photoreceptors inherently rely on the ability of the remaining retinal neurons to correctly synapse with new photoreceptors. We show that deafferented bipolar cells in the adult mammalian retina can reconnect to rods and cones and restore retinal sensitivity at scotopic and photopic luminances. Rod bipolar cells extend their dendrites to form new synapses with healthy rod photoreceptors. These findings support the idea that bipolar cells might be able to synapse with reintroduced photoreceptors, thereby restoring vision in patients blinded by retinal degeneration.

Keywords: dendrite restructuring; photoreceptor; plasticity; retina; retinal bipolar cell; synapse formation.

Figure 1.

Photoreceptor outer segments and their synapses shift into lesions over time and restore visual sensitivity in the rod and cone pathways. A, Rod and cone outer segments at the edges of a 3-d-old and 4-month-old, 100-μm-wide lesion. Dashed gray line indicates lesion center. Ribbons (in the OPL) are visible at the lesion edges. Lesion edges were defined as the solid white outlines, calculated with custom software. Both cone (circled) and rod ribbons are found inside the former lesion edges. B, Lesion width over time, as measured by lack of ribbon coverage (calculated from lesion outlines in A). Widths are reported for 100-, 200-, and 300-μm-wide × 1.5-mm-long laser scanned areas. Lines connect acute (3–16 d) and older (1, 2, or 4 month) lesions placed in the same rabbit. One of five rabbits did not show lesion closure (grayed out, Student's t test, p > 0.25). Data are mean ± SEM. C, Examples of visual sensitivity plots, obtained from spike-triggered average sensitivity profiles, in retinas with 300 μm lesions. Gaps in visual sensitivity in both the photopic and scotopic regime are much wider in 1-week-old lesions (white arrows) than in 4-month-old lesions (gray arrows). Pixel size in these maps is defined by the stimulus pixel size. All panels are scaled equally. D, Lesion width as measured by lack of visual sensitivity in the photopic regime over time. Data are mean ± SEM. E, Comparison of visual sensitivity lesion widths in the photopic and scotopic regime (n = 12 lesions).

Figure 2.

Bipolar cell dendritic trees change in response to photoreceptor loss. A, Dendritic tips of healthy rod bipolar cells (PKCα, green) share a rod spherule with another rod bipolar cell, which is visible as a doublet structure (see insets) of the glutamate receptors (mGluR6, magenta). Rod bipolar cells lose their dendritic tips and glutamate receptors within 3 d of photoreceptor loss. Dendrite simplification and pruning are evident in 14-day-old lesions. Fine processes are lost, leaving only major dendritic branches (see cell on the left). Some cells show signs of asymmetric pruning (see cell on the right); dendrites directed toward a lesion edge (up) are strengthened compared with the dendritic branches directed toward the lesion center (down). B, Dendrite pruning in rod bipolar cells of acute and older 200 μm lesions in the same rabbit. Data are mean ± SEM. C, Rod bipolar cells and glutamate receptors at the OPL of a 4-month-old 100 μm lesion. D, Inset from C. Dendritic tips are visible at the ends of the thickened rod bipolar cell dendrite (arrowheads). Dendritic tips colocalize with glutamate receptors and ribbons (CtBP2, cyan). E, Projection angle and length of thickened dendrites from rod bipolar cells left (blue) and right (red) of lesion center. Lesion orientation is vertical, shown with black line. F, Dendrites of secretagogin-positive cone bipolar cells in acute and older 200 μm lesions in the same rabbit. These lesions are the same lesions as those in B and pictured in Figure 3A. G, Cone bipolar cell (secretagogin, cyan) dendrites inside and outside a lesion. Small dendrites are still visible within the lesion, but their network appears abnormal.

Figure 3.

Rod and cone bipolar cells within the same lesion do not restructure their dendrites in the same manner. A, Cone bipolar cells (secretagogin, cyan) within 200 μm lesions at 14 d, 1 month, 2 months, and 4 months. Rod bipolar cells (PKCα, green) in the same field of view as the cone bipolar cell images. Ribbons (CtBP2, magenta) in the rod bipolar images show lesion edges. B, Example vector sum calculations for a cone (secretagogin, cyan) and rod (PKCα, green) bipolar cell. Measured dendrite projection angles (dashed white lines slightly offset from dendrites) are placed in a unit circle (gray circle) and summed to create a dendritic projection vector (solid white line) for each cell. C, Dendritic projection vector sum analysis of the four lesions pictured in A. Dendritic projection vectors representing all dendrites from cone and rod bipolar cells left (blue) and right (red) of lesion center. Lesion orientation is vertical, shown with a black line. D, The directionality index of cone and rod bipolar cell dendrites inside or outside of a lesion. Rod bipolar cells (RBCs) inside 2- and 4-month-old lesions bias their dendrites to exit the lesion (Kolmogorov–Smirnov test, p < 0.01). Secretagogin-stained cone bipolar cells (CBCs) do not bias their dendrites to exit the lesion (p > 0.67).

Figure 4.

Rod bipolar cells actively search for new synaptic partners. A, The dendritic reach of healthy rod bipolar cell dendrites (blue) and thickened dendrites (red) in 100, 200, and 300 μm lesions. Data are mean ± SEM (shown above the two populations). p values from the Kolmogorov–Smirnov test are printed for each lesion size. B, Example rod bipolar cell (*) in a lesion with a 45 μm dendritic reach making synaptic contact outside the lesion, shown by an mGluR6 doublet located distally on the dendrite (arrow). C, Examples of cone pedicles (outlined by dashed shape) being approached, avoided, or ignored by a rod bipolar cell dendrite. Typically, rod bipolar cell dendritic tips (PKCα, green) terminate at rod spherules (mGluR6 doublets, bright magenta outside dashed shape). Occasionally, a rod bipolar cell dendritic tip terminates within a cone pedicle (arrow). D, Fraction of cone pedicles that are approached by a rod bipolar cell dendrite. Fractions are given as mean ± SEM. p values are calculated using unpooled binomial statistics.

Figure 5.

Sprouting, but not cell body movement, is reversed in recovering lesions. A, Cross sections of confocal z stacks of 2-week-, 1-month-, and 4-month-old lesions. Green represents rod bipolar cells (PKCα). Magenta represents ribbons (CtBP2). Cyan represents nuclei of the retinal neural cells labeled with DAPI. Rod bipolar cells have axons and axon terminals at all lesion ages. Dendritic sprouts (arrows) are common in 2-week- and 1-month-old lesions, but not present at 4 months. In the 4-month-old lesion, asterisks indicate two example cells that have moved vertically (up and down) out of their normal layer. Horizontal gray bars represent width of ONL missing (DAPI stained) photoreceptor cell bodies. Variation in retinal thickness between these cross sections results from variable squashing of the tissue during electrophysiological recording. B, The frequency of sprouting rod bipolar cells in 300 μm lesions (gray dots) over time. Data are mean ± SEM. p values (Student's t test). C, Axon terminals of rod bipolar cells within a 4-month-old lesion, viewed as a maximal z projection, as indicated by the dashed box in A.