Injury to Cone Synapses by Retinal Detachment: Differences from Rod Synapses and Protection by ROCK Inhibition.

Attachment of a detached retina does not always restore vision to pre-injury levels, even if the attachment is anatomically successful. The problem is due in part to long-term damage to photoreceptor synapses. Previously, we reported on damage to rod synapses and synaptic protection using a Rho kinase (ROCK) inhibitor (AR13503) after retinal detachment (RD). This report documents the effects of detachment, reattachment, and protection by ROCK inhibition on cone synapses. Conventional confocal and stimulated emission depletion (STED) microscopy were used for morphological assessment and electroretinograms for functional analysis of an adult pig model of RD. RDs were examined 2 and 4 h after injury or two days later when spontaneous reattachment had occurred. Cone pedicles respond differently than rod spherules. They lose their synaptic ribbons, reduce invaginations, and change their shape. ROCK inhibition protects against these structural abnormalities whether the inhibitor is applied immediately or 2 h after the RD. Functional restoration of the photopic b-wave, indicating cone-bipolar neurotransmission, is also improved with ROCK inhibition. Successful protection of both rod and cone synapses with AR13503 suggests this drug will (1) be a useful adjunct to subretinal administration of gene or stem cell therapies and (2) improve recovery of the injured retina when treatment is delayed.

Coming of Age for the Photoreceptor Synapse.

Purpose: To discuss the potential contribution of rod and cone synapses to the loss of visual function in retinal injury and disease. Methods: The published literature and the authors' own work were reviewed. Results: Retinal detachment is used as a case study of rod spherule and cone pedicle plasticity after injury. Both rod and cone photoreceptors terminals are damaged after detachment although the structural changes observed are only partially overlapping. For second-order neurons, only those associated with rod spherules respond consistently to injury by remodeling. Examination of signaling pathways involved in plasticity of conventional synapses and in neural development has been and may continue to be productive in discovering novel therapeutic targets. Rho kinase (ROCK) inhibition is an example of therapy that may reduce synaptic damage by preserving normal synaptic structure of rod and cone cells. Conclusions: We hypothesize that synaptic damage contributes to poor visual restoration after otherwise successful anatomical repair of retinal detachment. A similar situation may exist for patients with degenerative retinal disease. Thus, synaptic structure and function should be routinely studied, as this information may disclose therapeutic strategies to mitigate visual loss.

ROCK inhibition reduces morphological and functional damage to rod synapses after retinal injury.

Retinal detachment (RD) causes damage, including disjunction, of the rod photoreceptor-bipolar synapse, which disrupts vision and may contribute to the poor visual recovery observed after retinal reattachment surgery. We created a model of iatrogenic RD in adult female pigs to study damage to the rod-bipolar synapse after injury and the ability of a highly specific Rho-kinase (ROCK) inhibitor to preserve synaptic structure and function. This model mimics procedures used in humans when viral vectors or cells are injected subretinally for treatment of retinal disease. Synaptic disjunction by retraction of rod spherules, quantified by image analysis of confocal sections, was present 2 h after detachment and remained 2 days later even though the retina had spontaneously reattached by then. Moreover, spherule retraction occurred in attached retina 1-2 cms from detached retina. Synaptic damage was significantly reduced by ROCK inhibition in detached retina whether injected subretinally or intravitreally. Dark-adapted full-field electroretinograms were recorded in reattached retinas to assess rod-specific function. Reduction in synaptic injury correlated with increases in rod-driven responses in drug-treated eyes. Thus, ROCK inhibition helps prevent synaptic damage and improves functional outcomes after retinal injury and may be a useful adjunctive treatment in iatrogenic RD and other retinal degenerative diseases.

Improving outcomes in retinal detachment: the potential role of rho-kinase inhibitors.

PURPOSE OF REVIEW: Retinal detachment initiates a series of events that lead to degenerative changes in retinal synaptic architecture as well as the well-known phenomena of gliosis and photoreceptor apoptosis. Retinal reattachment does not always result in complete visual recovery, even if the fovea is not directly involved in the detachment. Rho-kinase (ROCK) inhibitors may mitigate some of these deleterious changes including disruption of synaptic architecture, photoreceptor apoptosis, and initiation of the epithelial-mesenchymal transition that characterizes proliferative vitreoretinopathy (PVR). This review focuses on the use of ROCK inhibitors to modulate synaptic disjunction. RECENT FINDINGS: ROCK inhibition prevents retinal detachment-induced photoreceptor synaptic terminal retraction (i.e., synaptic disjunction), thereby diminishing the damage of the first synapse in the visual pathway. ROCK inhibition also reduces retinal detachment-induced photoreceptor apoptosis and suppresses PVR progression in preclinical models. SUMMARY: Inhibition of ROCK may help to optimize visual recovery after retinal detachment surgery or iatrogenic detachments during cell transplantation or viral subretinal injection and might play a role in reducing the risk of PVR after retinal detachment surgery.

Actin Dynamics, Regulated by RhoA-LIMK-Cofilin Signaling, Mediates Rod Photoreceptor Axonal Retraction After Retinal Injury.

Purpose: Retraction of the axon terminals of rod photoreceptors after retinal detachment breaks the first synapse in the visual pathway, resulting in visual impairment. Previous work showed that the mechanism of axonal retraction involves RhoA signaling and its downstream effector LIM Kinase (LIMK) activation. We examined the response of the downstream component cofilin, a direct binding protein of actin filaments, as well as the regulation by RhoA-LIMK-Cofilin signaling of actin assembly/disassembly, in the presynaptic ribbon terminal of injured rod cells. Methods: Injury was produced by retinal detachment or rod cell isolation. Detached porcine retina was probed for levels and localization of phosphorylated cofilin with Western blots and confocal microscopy, whereas rod cell cultures of dissociated salamander retina were examined for filamentous actin assembly/disassembly with a barbed end assay and phalloidin staining. Results: A detachment increased phosphorylation of cofilin in retinal explants; phosphorylation occurred in rod terminals in sections of detached retina. Isolation of rod cells resulted in axon retraction accompanied by an increase in actin barbed ends and a decrease in net filament labeling. All changes were significantly reduced by either Rho kinase (ROCK) or LIMK inhibition, using Y27632 or BMS-5, respectively. Cytochalasin D also reduced retraction and stabilized filaments in isolated rod cells. Conclusions: These results indicate that actin depolymerization via activation of RhoA downstream kinases and cofilin contributes to axon retraction. Preventing depolymerization, in addition to actomyosin contraction, may stabilize ribbon synapses after trauma.

Concise Review: Update on Retinal Pigment Epithelium Transplantation for Age-Related Macular Degeneration.

Retinal cell therapy can have the objectives of rescue (i.e., modulation of metabolic abnormalities primarily for sight preservation) as well as replacement (i.e., replace cells lost due to injury or disease for sight restoration as well as preservation). The first clinical trials of retinal pigment epithelium (RPE) transplantation for vision-threatening complications of age-related macular degeneration (AMD) have begun with some preliminary signs of success (e.g., improvement in vision in some patients, anatomic evidence of transplant-host integration with some evidence of host photoreceptor recovery, long-term survival of autologous induced pluripotent stem cell-derived RPE transplants without immune suppression) as well as limitations (e.g., limited RPE suspension survival in the AMD eye, limited tolerance for long-term systemic immune suppression in elderly patients, suggestion of uncontrolled cell proliferation in the vitreous cavity). RPE survival on aged and AMD Bruch's membrane can be improved with chemical treatment, which may enhance the efficacy of RPE suspension transplants in AMD patients. Retinal detachment, currently used to deliver transplanted RPE cells to the subretinal space, induces disjunction of the first synapse in the visual pathway: the photoreceptor-bipolar synapse. This synaptic change occurs even in areas of attached retina near the locus of detachment. Synaptic disjunction and photoreceptor apoptosis associated with retinal detachment can be reduced with Rho kinase inhibitors. Addition of Rho kinase inhibitors may improve retinal function and photoreceptor survival after subretinal delivery of cells either in suspension or on scaffolds.

Antibiotics Reduce Retinal Cell Survival In Vitro.

Antibiotics such as gentamicin (an aminoglycoside) and penicillin (a beta-lactam antibiotic) are routinely used in retinal cell and explant cultures. In many cases, these in vitro systems are testing parameters regarding photoreceptor transplantation or preparing cells for transplantation. In vivo, milligram doses of gentamicin are neurotoxic to the retina. However, little is known about the effects of antibiotics to retina in vitro and whether smaller doses of gentamicin are toxic to retinal cells. To test toxicity, retinal cells were dissociated from tiger salamander, placed in culture, and treated with either 20 µg/ml gentamicin, 100 µg/ml streptomycin, 100 U/ml antibiotic/antimycotic, 0.25 µg/ml amphotericin B, or 100 U/ml penicillin G. All dosages were within manufacturer's recommended levels. Control cultures had defined medium only. Cells were fixed at 2 h or 7 days. Three criteria were used to assess toxicity: (1) survival of retinal neurons, (2) neuritic growth of photoreceptors assessed by the development of presynaptic varicosities, and (3) survival and morphology of Mueller cells. Rod cells were immunolabeled for rod opsin, Mueller cells for glial fibrillary acidic protein, and varicosities for synaptophysin. Neuronal cell density was reduced with all pharmacological treatments. The number of presynaptic varicosities was also significantly lower in both rod and cone photoreceptors in treated compared to control cultures; further, rods were more sensitive to gentamicin than cones. Penicillin G (100 U/ml) was overall the least inhibitory and amphotericin B the most toxic of all the agents to photoreceptors. Mueller cell survival was reduced with all treatments; reduced survival was accompanied by the appearance of proportionally fewer stellate and more rounded glial morphologies. These findings suggest that even microgram doses of antibiotic and antimycotic drugs can be neurotoxic to retinal cells and reduce neuritic regeneration in cell culture systems.

Sema3A Reduces Sprouting of Adult Rod Photoreceptors In Vitro.

Purpose: Rod photoreceptor terminals respond to retinal injury with retraction and sprouting. Since the guidance cue Semaphorin3A (Sema3A) is observed in the retina after injury, we asked whether Sema3A contributes to structural plasticity in rod photoreceptors. Methods: We used Western blots and alkaline phosphatase (AP)-tagged neuropilin-1 (NPN-1) to detect the expression of Sema3A in an organotypic model of porcine retinal detachment. We then examined Sema3A binding to cultured salamander rod photoreceptors using AP-tagged Sema3A. For functional analysis, we used a microspritzer to apply a gradient of Sema3A-Fc to isolated salamander rod photoreceptors over 24 hours. Results: Sema3A protein was biochemically detected in porcine retinal explants in the retina 7, 24, and 72 hours after detachment. In sections, NPN-1 receptor was bound to the inner and outer retina. For isolated rod photoreceptors, Sema3A localized to synaptic terminals and to neuritic processes after 1 week in vitro. In microspritzed rod photoreceptors, process initiation occurred away from high concentrations of Sema3A. Sema3A significantly decreased the number of processes formed by rod photoreceptors although the average length of processes was not affected. The cellular orientation of rod photoreceptors relative to the microspritzer also significantly changed over time; this effect was reduced with the Sema3A inhibitor, xanthofulvin. Conclusion: Sema3A is expressed in the retina after detachment, binds to rod photoreceptors, affects cell orientation, and reduces photoreceptor process initiation in vitro. Our results suggest that Sema3A contributes to axonal retraction in retinal injury, whereas rod neuritic sprouting and regenerative synaptogenesis may require a reduction in semaphorin signaling.

Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment.

PURPOSE: Retinal detachment disrupts the rod-bipolar synapse in the outer plexiform layer by retraction of rod axons. We showed that breakage is due to RhoA activation whereas inhibition of Rho kinase (ROCK), using Y27632, reduces synaptic damage. We test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment. METHODS: Detachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). In some animals, fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours. RESULTS: Subretinal injection of fasudil (10 mM) reduced retraction of rod spherules by 51.3% compared to control detachments (n = 3 pigs, P = 0.002). Intravitreal injection of 10 mM fasudil, a more clinically feasible route of administration, also reduced retraction (28.7%, n = 5, P < 0.05). Controls had no photoreceptor degeneration at 2 hours, but by 4 hours apoptosis was evident. Fasudil 10 mM reduced pyknotic nuclei by 55.7% (n = 4, P < 0.001). Phosphorylation of cofilin and myosin light chain, downstream effectors of ROCK, was decreased with 30 µM fasudil (n = 8-10 explants, P < 0.05). CONCLUSIONS: Inhibition of ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment. TRANSLATIONAL RELEVANCE: These results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.

A Versatile Method of Patterning Proteins and Cells.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Lim kinase, a bi-functional effector in injury-induced structural plasticity of synapses.

The structural plasticity of synaptic terminals contributes to normal nervous system function but also to neural degeneration, in the form of terminal retraction, and regeneration, due to process growth. Synaptic morphological change is mediated through the actin cytoskeleton, which is enriched in axonal and dendritic terminals. Whereas the three RhoGTPases, RhoA, Cdc42 and Rac, function as upstream signaling nodes sensitive to extracellular stimuli, LIMK-cofilin activity serves as a common downstream effector to up-regulate actin turnover, which is necessary for both polymerization and depolymerization. The dual effects of LIMK activity make LIMK a potential target of therapeutic intervention for injury-induced synaptic plasticity, as LIMK inhibition can stabilize actin cytoskeleton and preserve existing structure. This therapeutic benefit of LIMK inhibition has been demonstrated in animal models of injury-induced axon retraction and neuritic sprouting by rod photoreceptors. A better understanding of the regulation of LIMK-cofilin activity and the interaction with the microtubular cytoskeleton may open new ways to promote synaptic regeneration that can benefit neuronal degenerative disease.

RhoA Signaling and Synaptic Damage Occur Within Hours in a Live Pig Model of CNS Injury, Retinal Detachment.

PURPOSE: The RhoA pathway is activated after retinal injury. However, the time of onset and consequences of activation are unknown in vivo. Based on in vitro studies we focused on a period 2 hours after retinal detachment, in pig, an animal whose retina is holangiotic and contains cones. METHODS: Under anesthesia, retinal detachments were created by subretinal injection of a balanced salt solution. Two hours later, animals were sacrificed and enucleated for GTPase activity assays and quantitative Western blot and confocal microscopy analyses. RESULTS: RhoA activity with detachment was increased 1.5-fold compared to that in normal eyes or in eyes that had undergone vitrectomy only. Increased phosphorylation of myosin light chain, a RhoA effector, also occurred. By 2 hours, rod cells had retracted their terminals toward their cell bodies, disrupting the photoreceptor-to-bipolar synapse and producing significant numbers of spherules with SV2 immunolabel in the outer nuclear layer of the retina. In eyes with detachment, distant retina that remained attached also showed significant increases in RhoA activity and synaptic disjunction. Increases in RAC1 activity and glial fibrillary acidic protein (GFAP) were not specific for detachment, and sprouting of bipolar dendrites, reported for longer detachments, was not seen. The RhoA kinase inhibitor Y27632 significantly reduced axonal retraction by rod cells. CONCLUSIONS: Activation of the RhoA pathway occurs quickly after injury and promotes synaptic damage that can be controlled by RhoA kinase inhibition. We suggest that retinal detachment joins the list of central nervous system injuries, such as stroke and spinal cord injury, that should be considered for rapid therapeutic intervention.

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury.

PURPOSE: Rod photoreceptors retract their axon terminals and develop neuritic sprouts in response to retinal detachment and reattachment, respectively. This study examines the role of LIM kinase (LIMK), a component of RhoA and Rac pathways, in the presynaptic structural remodeling of rod photoreceptors. METHODS: Phosphorylated LIMK (p-LIMK), the active form of LIMK, was examined in salamander retina with Western blot and confocal microscopy. Axon length within the first 7 hours and process growth after 3 days of culture were assessed in isolated rod photoreceptors treated with inhibitors of upstream regulators ROCK and p21-activated kinase (Pak) (Y27632 and IPA-3) and a direct LIMK inhibitor (BMS-5). Porcine retinal explants were also treated with BMS-5 and analyzed 24 hours after detachment. Because Ca2+ influx contributes to axonal retraction, L-type channels were blocked in some experiments with nicardipine. RESULTS: Phosphorylated LIMK is present in rod terminals during retraction and in newly formed processes. Axonal retraction over 7 hours was significantly reduced by inhibition of LIMK or its regulators, ROCK and Pak. Process growth was reduced by LIMK or Pak inhibition especially at the basal (axon-bearing) region of the rod cells. Combining Ca2+ channel and LIMK inhibition had no additional effect on retraction but did further inhibit sprouting after 3 days. In detached porcine retina, LIMK inhibition reduced rod axonal retraction and improved retinal morphology. CONCLUSIONS: Thus structural remodeling, in the form of either axonal retraction or neuritic growth, requires LIMK activity. LIM kinase inhibition may have therapeutic potential for reducing pathologic rod terminal plasticity after retinal injury.

Position along the nasal/temporal plane affects synaptic development by adult photoreceptors, revealed by micropatterning.

In retinal degeneration, death of photoreceptors causes blindness. Repair of the retina by transplanting photoreceptors has resulted in limited functional connectivity between transplanted and host neurons. We hypothesize that absence of appropriate biological cues, specifically positional (retinotopographic) cues, reduces synaptogenesis. Here we use micropatterning to test whether regional origin affects the early synaptic development of photoreceptors. Right and left retinas from salamanders were first labelled with dextran tetramethyl-rhodamine and fluorescein, respectively, bisected into nasal (N)/temporal (T) or dorsal (D)/ventral (V) halves, individually dissociated, mixed, and cultured for 1 week. Origin of cells was identified by the fluorescent label. Interactions between photoreceptors and neighboring (target) cells were assessed by the number of neuritic contacts with a presynaptic swelling (varicosity). Randomly-plated photoreceptors showed no preference for cellular origin, likely due to multiple potential interactions available to each cell. To reduce cell-cell interactions, culture substrate was patterned using a microfluidic device with 10 µm-wide channels separated by 200 µm, thus allowing only 1-2 targets per photoreceptor. In patterned cultures, 36.89% of N rod cells contacted T targets but only 27.42% of N rod cells contacted N targets; similarly 35.05% of T rod cells contacted N cells but only 17.08% contacted T cells. Thus, opposite regions were more permissive of contact. However, neither cone nor D/V rod cells showed preferences for positional origin of targets. In conclusion, micropatterning demonstrated that neuritic differentiation by rod cells depends on retinotopographic cues along the nasal/temporal plane, suggesting that transplanting rod cells of known positional origin will increase transplant success.

Vacuum-assisted fluid flow in microchannels to pattern substrates and cells.

Substrate and cell patterning are widely used techniques in cell biology to study cell-to-cell and cell-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This paper describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. Our method builds upon a previous vacuum-assisted method used for micromolding (Jeon et al 1999 Adv. Mater 11 946) and successfully patterned collagen-I, fibronectin and Sal-1 substrates on glass and polystyrene surfaces, filling microchannels with lengths up to 120 mm and covering areas up to 13 × 10 mm(2). Vacuum-patterned substrates were subsequently used to culture mammalian PC12 and fibroblast cells and amphibian neurons. Cells were also patterned directly by injecting cell suspensions into microchannels using vacuum. Fibroblast and neuronal cells patterned using vacuum showed normal growth and minimal cell death indicating no adverse effects of vacuum on cells. Our method fills reversibly sealed PDMS microchannels. This enables the user to remove the PDMS microchannel cast and access the patterned biomaterial or cells for further experimental purposes. Overall, this is a straightforward technique that has broad applicability for cell biology.

Cell specific post-translational processing of pikachurin, a protein involved in retinal synaptogenesis.

Pikachurin is a recently identified, highly conserved, extracellular matrix-like protein. Murine pikachurin has 1,017 amino acids (~110 kDa), can bind to α-dystroglycan, and has been found to localize mainly in the synaptic cleft of photoreceptor ribbon synapses. Its knockout selectively disrupts synaptogenesis between photoreceptor and bipolar cells. To further characterize this synaptic protein, we used an antibody raised against the N-terminal of murine pikachurin on Western blots of mammalian and amphibian retinas and found, unexpectedly, that a low weight ~60-kDa band was the predominant signal for endogenous pikachurin. This band was predicted to be an N-terminal product of post-translational cleavage of pikachurin. A similar sized protein was also detected in human Y79 retinoblastoma cells, a cell line with characteristics of photoreceptor cells. In Y79 cells, endogenous pikachurin immunofluorescence was found on the cell surface of living cells. The expression of the N-fragment was not significantly affected by dystroglycan overexpression in spite of the biochemical evidence for pikachurin-α-dystroglycan binding. The presence of a corresponding endogenous C-fragment was not determined because of the lack of a suitable antibody. However, a protein of ~65 kDa was detected in Y79 cells expressing recombinant pikachurin with a C-terminal tag. In contrast, in QBI-HEK 293A cells, whose endogenous pikachurin protein level is negligible, recombinant pikachurin did not appear to be cleaved. Instead pikachurin was found either intact or as dimers. Finally, whole and N- and C-fragments of recombinant pikachurin were present in the conditioned media of Y79 cells indicating the secretion of pikachurin. The site of cleavage, however, was not conclusively determined. Our data suggest the existence of post-translational cleavage of pikachurin protein as well as the extracellular localization of cleaved protein specifically by retinal cells. The functions of the pikachurin N- and C-fragments in the photoreceptor ribbon synapse are unknown.

Mislocalized opsin and cAMP signaling: a mechanism for sprouting by rod cells in retinal degeneration.

PURPOSE: In human retinal degeneration, rod photoreceptors reactively sprout neurites. The mechanism is unknown in part because of the paucity of animal models displaying this feature of human pathology. We tested the role of cAMP and opsin in sprouting by tiger salamander rod cells, photoreceptors that can produce reactive growth. METHODS: In vitro systems of isolated photoreceptor cells and intact neural retina were used. cAMP signaling was manipulated with nucleotide analogues, enzyme stimulators, agonists for adenosine and dopamine receptors, and the opsin agonist, ß-ionone. Levels of cAMP were determined by radioimmunoassay, and protein levels by Western blot and quantitative immunocytochemistry. Neuritic growth was assayed by image analysis and conventional and confocal microscopy. RESULTS: cAMP analogues and stimulation of adenylyl cyclase (AC) directly or through G-protein-coupled receptors resulted in significant increases in neuritic growth of isolated rod, but not cone, cells. The signaling pathway included protein kinase A (PKA) and phosphorylation of the transcription factor cAMP response element-binding protein (pCREB). Opsin, a G-linked receptor, is present throughout the plasmalemma of isolated cells; its activation also induced sprouting. In neural retina, rod sprouting was significantly increased by ß-ionone with concomitant increases in cAMP, pCREB, and synaptic proteins. Notably, opsin stimulated sprouting only when mislocalized to the plasmalemma of the rod cell body. CONCLUSIONS: cAMP causes neuritic sprouting in rod, but not cone, cells through the AC-PKA-CREB pathway known to be associated with synaptic plasticity. We propose that in retinal disease, mislocalized rod opsin gains access to cAMP signaling, which leads to neuritic sprouting.