A,B KA-driven (25 μM) signaling in hrhoG mouse retinas in focal regions of cone survival. Patches of transformed cones are indicated by the blue triangles. (A) γ.B.E → rgb mapped and (B) greyscale AGB images show that KA-responsive bipolar cells are common near cones, demonstrating local persistence of iGluR expression. Amacrine and ganglion cell signals are also robust. All images are 0.3 mm wide.

Endogenous signaling phase 3 hrhoG mutant mouse retinas tracked by AGB permeation. Symbols have the same meanings as in Figure 1. (A) γ.B.E → rgb (GABA.AGB.glutamate) mapped and (B) greyscale AGB images images demonstrate that only amacrine and ganglion cells are endogenously active. The inner nuclear layer box contains only lightly labeled neurons, indicating no AGB permeation. Different, albeit modest amacrine (upward arrows) and ganglion cell (downward arrows) responses are consistent with intrinsic signaling. Signaling in the inner plexiform layer (ipl) is relatively uniform but diffuse, indicating remodeling of rod signaling layers. All images are 0.3 mm wide; onl, outer nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

A,B KA-driven (25 μM) signaling in wt mouse retinas. Symbols have the same meanings as in Figure 1. (A) G.B.E (glycine.AGB.glutamate) → rgb mapped and (B) greyscale AGB images show that many, but not all neurons are responsive. The inl box contains a mixture of responsive and unresponsive bipolar cells, suggesting selective visualization of the normal iGluR-driven OFF bipolar cell cohort. Most amacrine (upward arrows) and ganglion cells (downward arrows) have strong responses, whereas Müller cells are unresponsive (angled arrows). Signaling in the ipl is uniformly strong, consistent with the pervasive expression of AMPA receptors in all layers. C,D KA-driven (25 μM) signaling in hrhoG mouse retinas. (C) G.B.E → rgb mapped and (D) greyscale AGB images show that only amacrine and ganglion cells are responsive. The inl box contains only unresponsive bipolar cells. The circle contains a single weakly responsive cell. Amacrine and ganglion cell responses are weaker and more variable than wt. All images are 0.3 mm wide. E, Univariate AGB signal histograms comparing KA-activated wt and hrhoG retinal activity for specific cell classes. BC histogram (left): KA-activated AGB permeation signals of wt bipolar cells are segmented into responsive OFF (pale green) and non-responsive ON bipolar cells (pale blue and red striped). Nearly all hrhoG bipolar cells are below background, implying little or no iGluR-mediated signaling. Gly AC, γ AC and GC histograms: both wt and hrhoG amacrine and ganglion cell signals demonstrate broad response profiles, with most cells exhibiting vigorous responses in the wt animals and a broad dispersion in the mutants. Horizontal cells were identified only in wt retinas.

KA-driven (25 μM) signaling in (A) normal baboon and (B) human RP retina. Symbols have the same meanings as in Figure 1. (A) G.B.E (glycine.AGB.glutamate) → rgb mapped and (B) greyscale AGB images show that that many, but not all neurons are responsive. The onl ellipses illustrate (A) normal and (B) aberrant cone morphologies in the blue glutamate channel. The inl boxes contain mixtures of responsive and unresponsive bipolar cells, allowing selective visualization of the normal iGluR-driven OFF bipolar cell cohort for each condition. Most amacrine (upward arrows) and ganglion cells (downward arrows) have strong responses. Signaling in the ipl is uniformly strong, consistent with the pervasive expression of AMPA receptors in all layers. C,D Rhodopsin and LWS1 (red and green) cone opsin immunoreactivity in normal and dystrophic primate retinas. C Rhodopsin is expressed uniformly in rods (cyan) while most cones express LWS1 opsin immunoreactivity. Circled areas represent putative blue cones. D Remnant rhodopsin-containing (green, arrows) or LWS1 cone opsin-containing (red, circles) outer segments are rare and short. Most cones (blue) are highly modified and lack any detectable opsin immunoreactivity. E,F KA-driven (25 μM) signaling in normal and dystrophic primate retinas segmented by cluster analysis16 into ON rod (blue), ON cone (red) and active OFF cone bipolar cell (green) cohorts; horizontal cells are orange and Müller cells or unclassified cells are black. Image widths: A,B 0.6 mm; C,D 47 μm; E 120 μm; F 77 μm. G, Scaled univariate butterfly histograms of KA-activated bipolar cell signals for normal baboon (Papio anubis) and dystrophic human retinas. Left wing (PA wt): OFF bipolar cells (pale green) comprise 39% and non-responsive ON cone and rod bipolar cells (pale blue and red striped) comprise 71% of all BCs in normal perhiperal primate retina. Right wing (HS RP, H. sapiens RP): OFF bipolar cells (pale green) comprise 79% and non-responsive ON cone bipolar cells (pale red) comprise 21% of all bipolar cells in the dystrophic retina. Rod bipolar cells comprise less than 3% of the remnant cells

Signaling and bipolar cell reprogramming. A Rod bipolar cell remodeling in cone-sparing RP. Normal “phase 0” rod bipolar cells bypass cone synapses to contact rods. In phase 1–2 RP, rod stress and death trigger dendrite retraction and in persistent phase 2 cone survival, some rod bipolar cells successfully make ectopic contacts with cones. B The cone synaptic ribbon zone viewed as a flattened 2D structure with central glutamate release from a ribbon site, laterally displaced dendrites of target cells, and a gradient of extracellular glutamate. Ectopic dendrites from rod ON bipolar cells (black) likely access too little glutamate to activate the mGluR6 (m) transduction cascade. Radially decreasing glutamate levels around the ribbon release site are effected by diffusion and transporter (black serrated border) losses. Dendrites near the ribbon expressing mGluR6 (cone ON BC) or AMPA receptors (HC) access perhaps a 30–90x higher glutamate level than peripheral dendrites (OFF BC) expressing more sensitive KA receptors (see Devries et al.23). Enhanced KA receptor expression by reprogrammed rod BCs could render them responsive once again. C The canonical high-sensitivity scotopic pathway of the mammalian retina. Rods drive rod ON bipolar cells via sign-inverting (−) mGluR6 synapses and cones drive OFF bipolar cells mostly via sign-conserving (+) KA receptors and ON bipolar cells via sign-inverting mGluR6 synapses. Cone bipolar cells alone directly drive ganglion cells with sign-conserving AMPA receptors, creating distinct cone OFF (filled cells) and cone ON (open cells) channels that culminate in ganglion cell spiking to light decrements and increments respectively. In scotopic conditions, cones are silent and rods capture cone bipolar cell channels via an intercalary neuron, a glycinergic rod amacrine cell that receives sign-conserving input directly from rod bipolar cells and distributes those signals to ON cone bipolar cell terminals via sign-conserving gap junctions or OFF cone bipolar cell terminals via sign-inverting glycinergic synapses. Tracing the hyperpolarizing rod response to a flash of light reveals that this network preserves the polarity of ON and OFF channel signaling. D In cone-sparing RP, survivor rod bipolar cells attempt to contact cones. If such cells reprogram by expressing iGluRs, a collision network emerges, corrupting cone pathway signaling driven by remnant functional cones.

Endogenous glutamatergic signaling in wt and mutant mouse retinas under mesopic light conditions tracked by AGB permeation. High resolution images of boxed regions are available at http://prometheus.med.utah.edu/~marclab/mss/IOVS.html). A,B Endogenous signaling in wt mouse retinas. (A) γ.B.E → rgb (GABA.AGB.glutamate) mapped and (B) greyscale AGB images show that nearly all neurons are active and display AGB permeation. The box in the inner nuclear layer (inl) contains AGB-rich cells, indicating that all bipolar cell classes are responsive. Different strengths of amacrine cell (upward arrows) and ganglion cell (downward arrows) responses are consistent with normal retinal activity patterns. Signaling in the inner plexiform layer (ipl) is relatively uniform with strong bands in the cone OFF (black dot), cone ON (yellow dot) and rod ON (yellow aster) layers. C,D Endogenous signaling in rdcl mouse retinas. (C) γ.B.E → rgb mapped and (D) greyscale AGB images demonstrate that only amacrine and ganglion cells are endogenously active. The inl box contains only unlabeled neurons, indicating no AGB permeation. Different strengths of amacrine cell (upward arrows) and ganglion cell (downward arrows) responses imply variation in endogenous activity. Signaling in the ipl is strong in the cone OFF and ON layers, but very weak in the rod ON layer. All images are 0.3 mm wide; onl, outer nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer. E, Univariate AGB butterfly histograms comparing endogenous wt and rdcl retinal activity for specific cell classes: BCs bipolar cells, GCs ganglion cells, gly ACs glycinergic amacrine cells and γ ACs GABAergic amacrine cells. The ordinate on each of the plots represents AGB signal strength as an 8-bit grey pixel value. The abscissas for each plot are normalized probability densities (P_N), allowing direct comparison of wt (left wing) and mutant (right wing) cell class response patterns. Each histogram represents over 100–500 adjacent cells, except for horizontal and ganglion cell histograms, representing 50–100 adjacent cells. BC Histogram (left): the wt Müller cell signal profile (M, white histogram) defines the background (bkgd) level, shaded across all histograms. Endogenous AGB permeation signals of wt bipolar cells are segmented by cluster analysis13 into ON cone BCs (pale red) and all other BCs (blue-green striped) and are significantly above background. All rdcl bipolar cells are below background, implying little or no endogenous signaling. GC Histogram: both wt and rdcl GC signals are broadly dispersed with stronger compression to high responses in wt animals. Gly AC Histogram: wt glycinergic amacrine cells yield a broad pseudo-unimodal response, while rdcl ACs generate a clearly bimodal response with a large fraction of nearly unresponsive cells, many of which project to the rod-driven layer of the inner plexiform layer *. γ AC Histogram (right): wt GABAergic amacrine cells also yield a broad pseudo-unimodal response, while rdcl amacrine cells generate a bimodal response, similar to glycinergic amacrine cells and with similar rod dependencies *. All horizontal cells (HCs) are uniformly highly responsive in wt retinas, but none were identified in the rdcl retina. F, Profiles of in vivo signaling in the inner plexiform layer of wt (left) and rdcl (right) mice. Absolute AGB PV signals at each level of the inner plexiform layer were plotted for total inner plexiform layer neurites (black trace), GABAergic neurites (orange dotted trace) and glycinergic neurites (blue diamond trace) as described in Methods. The rod bipolar cell terminal layer is indicated by the shaded box.