Iron contributes to photoreceptor degeneration and Müller glia proliferation in the zebrafish light-treated retina.

Zebrafish possess the ability to completely regenerate the retina following injury, however little is understood about the damage signals that contribute to inducing Müller glia reprogramming and proliferation to regenerate lost neurons. Multiple studies demonstrated that iron contributes to various retinal injuries, however no link has been shown between iron and zebrafish retinal regeneration. Here we demonstrate that Müller glia exhibit transcriptional changes following injury to regulate iron levels within the retina, allowing for increased iron uptake and decreased export. The response of the zebrafish retina to intravitreal iron injection was then characterized, showing that ferrous, and not ferric, iron induces retinal cell death. Additionally, iron chelation resulted in decreased numbers of TUNEL-positive photoreceptors and fewer proliferating Müller glia. Despite the contribution of iron to retinal cell death, inhibition of ferroptosis did not significantly reduce cell death following light treatment. Finally, we demonstrate that both the anti-ferroptotic protein Glutathione peroxidase 4b and the Transferrin receptor 1b are required for Müller glia proliferation following light damage. Together these findings show that iron contributes to cell death in the light-damaged retina and is essential for inducing the Müller glia regeneration response.

PKM2 Is a Potential Diagnostic and Therapeutic Target for Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

The retinal ipRGC-preoptic circuit mediates the acute effect of light on sleep.

Light regulates daily sleep rhythms by a neural circuit that connects intrinsically photosensitive retinal ganglion cells (ipRGCs) to the circadian pacemaker, the suprachiasmatic nucleus. Light, however, also acutely affects sleep in a circadian-independent manner. The neural circuits involving the acute effect of light on sleep remain unknown. Here we uncovered a neural circuit that drives this acute light response, independent of the suprachiasmatic nucleus, but still through ipRGCs. We show that ipRGCs substantially innervate the preoptic area (POA) to mediate the acute light effect on sleep in mice. Consistently, activation of either the POA projecting ipRGCs or the light-responsive POA neurons increased non-rapid eye movement (NREM) sleep without influencing REM sleep. In addition, inhibition of the light-responsive POA neurons blocked the acute light effects on NREM sleep. The predominant light-responsive POA neurons that receive ipRGC input belong to the corticotropin-releasing hormone subpopulation. Remarkably, the light-responsive POA neurons are inhibitory and project to well-known wakefulness-promoting brain regions, such as the tuberomammillary nucleus and the lateral hypothalamus. Therefore, activation of the ipRGC-POA circuit inhibits arousal brain regions to drive light-induced NREM sleep. Our findings reveal a functional retina-brain circuit that is both necessary and sufficient for the acute effect of light on sleep.

Modeling of Retinal Degeneration.

We developed a model of retinal degeneration in rabbits based on exposure to light with a wavelength of 405 nm. This model allows reproducing structural and functional disorders in the central parts of the retina, including primarily degeneration of the outer layers of the retina (retinal pigment epithelium and layer of photoreceptor cells), and is designed to study the mechanisms of formation, progression and effectiveness of new drugs and methods of treatment of degenerative diseases of the retina.

Regulatory Impact of the C-Terminal Tail on Charge Transfer Pathways in Drosophila Cryptochrome.

Interconnected transcriptional and translational feedback loops are at the core of the molecular mechanism of the circadian clock. Such feedback loops are synchronized to external light entrainment by the blue light photoreceptor cryptochrome (CRY) that undergoes conformational changes upon light absorption by an unknown photoexcitation mechanism. Light-induced charge transfer (CT) reactions in Drosophila CRY (dCRY) are investigated by state-of-the-art simulations that reveal a complex, multi-redox site nature of CT dynamics on the microscopic level. The simulations consider redox-active chromophores of the tryptophan triad (Trp triad) and further account for pathways mediated by W314 and W422 residues proximate to the C-terminal tail (CTT), thus avoiding a pre-bias to specific W-mediated CT pathways. The conducted dissipative quantum dynamics simulations employ microscopically derived model Hamiltonians and display complex and ultrafast CT dynamics on the picosecond timescale, subtly balanced by the electrostatic environment of dCRY. In silicio point mutations provide a microscopic basis for rationalizing particular CT directionality and demonstrate the degree of electrostatic control realized by a discrete set of charged amino acid residues. The predicted participation of CT states in proximity to the CTT relates the directionality of CT reactions to the spatial vicinity of a linear interaction motif. The results stress the importance of CTT directional charge transfer in addition to charge transfer via the Trp triad and call for the use of full-length CRY models including the interactions of photolyase homology region (PHR) and CTT domains.

Vitamin A aldehyde-taurine adduct and the visual cycle.

Visual pigment consists of opsin covalently linked to the vitamin A-derived chromophore, 11-cis-retinaldehyde. Photon absorption causes the chromophore to isomerize from the 11-cis- to all-trans-retinal configuration. Continued light sensitivity necessitates the regeneration of 11-cis-retinal via a series of enzyme-catalyzed steps within the visual cycle. During this process, vitamin A aldehyde is shepherded within photoreceptors and retinal pigment epithelial cells to facilitate retinoid trafficking, to prevent nonspecific reactivity, and to conserve the 11-cis configuration. Here we show that redundancy in this system is provided by a protonated Schiff base adduct of retinaldehyde and taurine (A1-taurine, A1T) that forms reversibly by nonenzymatic reaction. A1T was present as 9-cis, 11-cis, 13-cis, and all-trans isomers, and the total levels were higher in neural retina than in retinal pigment epithelium (RPE). A1T was also more abundant under conditions in which 11-cis-retinaldehyde was higher; this included black versus albino mice, dark-adapted versus light-adapted mice, and mice carrying the Rpe65-Leu450 versus Rpe65-450Met variant. Taurine levels paralleled these differences in A1T. Moreover, A1T was substantially reduced in mice deficient in the Rpe65 isomerase and in mice deficient in cellular retinaldehyde-binding protein; in these models the production of 11-cis-retinal is compromised. A1T is an amphiphilic small molecule that may represent a mechanism for escorting retinaldehyde. The transient Schiff base conjugate that the primary amine of taurine forms with retinaldehyde would readily hydrolyze to release the retinoid and thus may embody a pool of 11-cis-retinal that can be marshalled in photoreceptor cells.

Shihu Yeguang Pill protects against bright light-induced photoreceptor degeneration in part through suppressing photoreceptor apoptosis.

Photoreceptor cells are first-order retinal neurons that directly contribute to the formation of vision. Photoreceptor degeneration is the primary cause of vision impairment during the course of retinopathies such as retinitis pigmentosa and age-related macular degeneration, for which photoreceptor-targeted therapies are currently unavailable. Shihu Yeguang Pill (SYP), a classic formula in traditional Chinese medicine, has a long histology of clinical application for the treatment of a wide range of retinopathies in China. However, whether SYP is pharmacological effective at protecting photoreceptor cells is unclear. The current study thus directly addressed the pharmacological implications of SYP in photoreceptor degeneration in a mouse model characterized by bright light-induced retinal degeneration. Non-invasive full-retinal assessment was carried out to evaluate the effect of SYP on the retinal structure and function through optical coherence tomography and electroretinography, respectively. In addition, photoreceptor apoptosis, second-order neuron impairment and reactive changes in retinal microglial and müller cells, hallmark pathologies associated with photoreceptor degeneration, were assessed using immunohistochemistry and real-time PCR analyses. The results showed that SYP treatment attenuated bright light-induced impairment of the retinal structure and function. Moreover, SYP treatment suppressed photoreceptor apoptosis, alleviated the impairment of bipolar and horizontal cells and mitigated the reactive changes of müller and microglial cells in the bright light-exposed retinas. Real-time PCR analyses showed that dysregulated expression of pro-apoptotic c-fos and c-jun and anti-apoptotic bcl-2 as well as proinflammatory TNF-α in the bright light-exposed retinas was partially normalized as a result of SYP treatment. In summary, the work here demonstrates for the first time that SYP treatment protects the retinas from developing bright light-induced photoreceptor degeneration and associated alterations in second-order neurons and glial cells. The findings here thus provide experimental evidence to better support the mechanism-guided clinical application of SYP in the treatment of related retinal degenerative diseases.

Changes in the Inner Retinal Cells after Intense and Constant Light Exposure in Sprague-Dawley Rats.

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Light Stress-Induced Increase of Sphingosine 1-Phosphate in Photoreceptors and Its Relevance to Retinal Degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

The 40S Ribosomal Protein S6 Response to Blue Light by Interaction with SjAUREO in Saccharina japonica.

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 × 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO.

Gene Therapy Rescues Retinal Degeneration in Receptor Expression-Enhancing Protein 6 Mutant Mice.

Hereditary retinal dystrophy is clinically defined as a broad group of chronic and progressive disorders that affect visual function by causing photoreceptor degeneration. Previously, we identified mutations in the gene encoding receptor expression-enhancing protein 6 (REEP6), in individuals with autosomal recessive retinitis pigmentosa (RP), the most common form of inherited retinal dystrophy. One individual was molecularly diagnosed with biallelic REEP6 mutations, a missense mutation over a frameshift mutation. In this study, we generated Reep6 compound heterozygous mice, Reep6L135P/-, which mimic the patient genotype and recapitulate the early-onset retinal degeneration phenotypes observed in the individual with RP. To determine the feasibility of rescuing the Reep6 mutant phenotype via gene replacement therapy, we delivered Reep6.1, the mouse retina-specific isoform of REEP6, to photoreceptors of Reep6 mutant mice on postnatal day 20. Evaluation of the therapeutic effects 2 months posttreatment showed improvements in the photoresponse as well as preservation of photoreceptor cells. Importantly, guanylyl cyclase 1 (GC1) expression was also restored to the outer segment after treatment. Furthermore, rAAV8-Reep6.1 single treatment in Reep6 mutant mice 1 year postinjection showed significant improvements in retinal function and morphology, suggesting that the treatment is effective even after a prolonged period. Findings from this study show that gene replacement therapy in the retina with rAAV overexpressing Reep6 is effective, preserving photoreceptor function in Reep6 mutant mice. These findings provide evidence that rAAV8-based gene therapy can prolong survival of photoreceptors in vivo and can be potentially used as a therapeutic modality for treatment of patients with RP.

Light modulates oscillatory alpha activity in the occipital cortex of totally visually blind individuals with intact non-image-forming photoreception.

The discovery of intrinsically photosensitive retinal ganglion cells (ipRGCs) marked a major shift in our understanding of how light information is processed by the mammalian brain. These ipRGCs influence multiple functions not directly related to image formation such as circadian resetting and entrainment, pupil constriction, enhancement of alertness, as well as the modulation of cognition. More recently, it was demonstrated that ipRGCs may also contribute to basic visual functions. The impact of ipRGCs on visual function, independently of image forming photoreceptors, remains difficult to isolate, however, particularly in humans. We previously showed that exposure to intense monochromatic blue light (465 nm) induced non-conscious light perception in a forced choice task in three rare totally visually blind individuals without detectable rod and cone function, but who retained non-image-forming responses to light, very likely via ipRGCs. The neural foundation of such light perception in the absence of conscious vision is unknown, however. In this study, we characterized the brain activity of these three participants using electroencephalography (EEG), and demonstrate that unconsciously perceived light triggers an early and reliable transient desynchronization (i.e. decreased power) of the alpha EEG rhythm (8-14 Hz) over the occipital cortex. These results provide compelling insight into how ipRGC may contribute to transient changes in ongoing brain activity. They suggest that occipital alpha rhythm synchrony, which is typically linked to the visual system, is modulated by ipRGCs photoreception; a process that may contribute to the non-conscious light perception in those blind individuals.

Bisretinoids mediate light sensitivity resulting in photoreceptor cell degeneration in mice lacking the receptor tyrosine kinase Mer.

The receptor tyrosine kinase Mer is expressed by retinal pigment epithelial (RPE) cells and participates in photoreceptor outer-segment phagocytosis, a process enabling membrane renewal. Mutations in the gene encoding MERTK cause blinding retinitis pigmentosa in humans. Targeted Mertk disruption in mice causes defective RPE-mediated phagocytosis of the outer segments, leading to deposition of autofluorescent debris at the RPE-photoreceptor cell interface, followed by photoreceptor cell degeneration. Here, we show that retinaldehyde adducts (bisretinoid fluorophores) that form in photoreceptor outer segments occupy the unphagocytosed outer-segment debris that accumulates in Mertk-/- mice. Bisretinoids measured by HPLC were elevated in Mertk-/- mice compared with WT animals. Bisretinoids were also more abundant in albino Mertk-/- mice expressing leucine at position 450 of the isomerase RPE65 (Rpe65-Leu450) rather than the variant methionine (Rpe65-450Met) that yields lower bisretinoid levels. In Royal College of Surgeons rats having dysfunctional Mertk, bisretinoids were higher than in WT rats. Intensities of in vivo fundus autofluorescence were higher in Mertk-/- mice than in WT mice and peaked earlier in albino Mertk-/-/Rpe65-Leu450 mice than in albino Mertk-/-/Rpe65-450Met mice. Of note, the rate of photoreceptor cell degeneration was more rapid in albino Mertk-/- mice exposed to higher levels of intraocular light (albino versus pigmented mice) and in mice carrying Rpe65-Leu450 than in Rpe65-450Met mice, revealing a link between bisretinoid accumulation and light-mediated acceleration of photoreceptor cell degeneration. In conclusion, the light sensitivity of photoreceptor cell degeneration arising from Mertk deficiency is consistent with the known phototoxicity of bisretinoids.

Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.

In response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hr of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hr once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

Asymmetric activation mechanism of a homodimeric red light-regulated photoreceptor.

Organisms adapt to environmental cues using diverse signaling networks. In order to sense and integrate light for regulating various biological functions, photoreceptor proteins have evolved in a modular way. This modularity is targeted in the development of optogenetic tools enabling the control of cellular events with high spatiotemporal precision. However, the limited understanding of signaling mechanisms impedes the rational design of innovative photoreceptor-effector couples. Here, we reveal molecular details of signal transduction in phytochrome-regulated diguanylyl cyclases. Asymmetric structural changes of the full-length homodimer result in a functional heterodimer featuring two different photoactivation states. Structural changes around the cofactors result in a quasi-translational rearrangement of the distant coiled-coil sensor-effector linker. Eventually, this regulates enzymatic activity by modulating the dimer interface of the output domains. Considering the importance of phytochrome heterodimerization in plant signaling, our mechanistic details of asymmetric photoactivation in a bacterial system reveal novel aspects of the evolutionary adaptation of phytochromes.

Expression of CCL2 and its receptor in activation and migration of microglia and monocytes induced by photoreceptor apoptosis.

Purpose: To explore the effect of the CCL2 and CCR2 system on the activation and migration of microglia and monocytes in light-induced photoreceptor apoptosis. Methods: At 1 day, 3 days, 7 days, and 14 days after light exposure, OX42 and ED1 immunostaining were used to label the activation and migration of microglia and monocytes. Double immunostaining of CCL2 with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), OX42, or glial fibrillary acidic protein (GFAP) was applied to explore the relationships among CCL2, apoptotic photoreceptors, activated microglia and monocytes, and macroglial cells (Müller cells and astrocytes). Real-time PCR was used to evaluate the mRNA levels of retinal CCL2 and CCR2 and the proinflammatory factors interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha. Results: Real-time PCR analyses showed that CCL2 and CCR2 expression gradually increased after light exposure and peaked at 3 days, coinciding with the infiltration of OX42-positive cells and the expression of IL-1 beta and TNF-alpha in the outer retina. Double immunostaining of CCL2 with TUNEL revealed that CCL2 was expressed robustly in about 30% of the apoptotic photoreceptors at the early stage. As degeneration progressed, immunostaining of CCL2 with OX42 showed that activated and migrated microglia and monocytes expressed CCL2. At the late stage, Müller cells became the main source of CCL2, which was illustrated by CCL2 immunostaining with GFAP. Conclusions: Light exposure led to apoptosis of photoreceptors, which expressed CCL2, accelerating an inflammation-mediated cascade by activating and attracting microglia and monocytes and promoting their secretion of CCL2 in the injured position.

Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine.

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Mapping physiological inputs from multiple photoreceptor systems to dopaminergic amacrine cells in the mouse retina.

In the vertebrate retina, dopamine is synthesized and released by a specialized type of amacrine cell, the dopaminergic amacrine cell (DAC). DAC activity is stimulated by rods, cones, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells upon illumination. However, the relative contributions of these three photoreceptor systems to the DAC light-induced response are unknown. Here we found that rods excite dark-adapted DACs across a wide range of stimulation intensities, primarily through connexin-36-dependent rod pathways. Similar rod-driven responses were observed in both ventral and dorsal DACs. We further found that in the dorsal retina, M-cones and melanopsin contribute to dark-adapted DAC responses with a similar threshold intensity. In the ventral retina, however, the threshold intensity for M-cone-driven responses was two log units greater than that observed in dorsal DACs, and melanopsin-driven responses were almost undetectable. We also examined the DAC response to prolonged adapting light and found such responses to be mediated by rods under dim lighting conditions, rods/M-cones/melanopsin under intermediate lighting conditions, and cones and melanopsin under bright lighting conditions. Our results elucidate the relative contributions of the three photoreceptor systems to DACs under different lighting conditions, furthering our understanding of the role these cells play in the visual system.