Amacrine Cells Forming Gap Junctions With Intrinsically Photosensitive Retinal Ganglion Cells: ipRGC Types, Neuromodulator Contents, and Connexin Isoform.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with amacrine cells, (2) the neuromodulator contents of ipRGC-coupled amacrine cells, and (3) whether connexin36 (Cx36) contributes to ipRGC-amacrine coupling. Methods: Gap junction-permeable Neurobiotin tracer was injected into green fluorescent protein (GFP)-labeled ipRGCs in Opn4Cre/+; Z/EG mice to stain coupled amacrine cells, and immunohistochemistry was performed to reveal the neuromodulator contents of the Neurobiotin-stained amacrine cells. We also created Opn4Cre/+; Cx36flox/flox; Z/EG mice to knock out Cx36 in GFP-labeled ipRGCs and looked for changes in the number of ipRGC-coupled amacrine cells. Results: Seventy-three percent of ipRGCs, including all six types (M1-M6), were tracer-coupled with amacrine somas 5.7 to 16.5 µm in diameter but not with ganglion cells. Ninety-two percent of the ipRGC-coupled somas were in the ganglion cell layer and the rest in the inner nuclear layer. Some ipRGC-coupled amacrine cells were found to accumulate serotonin or to contain nitric oxide synthase or neuropeptide Y. Knocking out Cx36 in M2 and M4 dramatically reduced the number of coupled somas. Conclusions: Heterologous gap junction coupling with amacrine cells is widespread across mouse ipRGC types. ipRGC-coupled amacrine cells probably comprise multiple morphologic types and use multiple neuromodulators, suggesting that gap junctional ipRGC-to-amacrine signaling likely exerts diverse modulatory effects on retinal physiology. ipRGC-amacrine coupling is mediated partly, but not solely, by Cx36.

Prolonged Melanopsin-based Photoresponses Depend in Part on RPE65 and Cellular Retinaldehyde-binding Protein (CRALBP).

PURPOSE: Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and can signal light continuously for many hours. Melanopsin is excited when its chromophore 11-cis-retinal absorbs a photon and becomes all-trans-retinal, which must be reisomerized to 11-cis-retinal to regenerate photoexcitable melanopsin. Due to the great distance separating ipRGCs from the retinal pigment epithelium (RPE) whose retinoid cycle produces 11-cis-retinal, ipRGCs had been assumed to regenerate all melanopsin molecules autonomously. Surprisingly, we previously found that pharmacologically inhibiting the retinoid cycle rendered melanopsin-based responses to prolonged illumination less sustained, suggesting that the RPE may supply retinoids to help ipRGCs regenerate melanopsin during extended photostimulation. However, the specificity of those drugs is unclear. Here, we reexamined the role of the retinoid cycle, and tested whether the RPE-to-ipRGC transport of retinoids utilizes cellular retinaldehyde-binding protein (CRALBP), present throughout the RPE and Müller glia. METHODS: To measure melanopsin-mediated photoresponses in isolation, all animals were 8- to 12-month-old rod/cone-degenerate mice. We genetically knocked out RPE-specific 65 kDa protein (RPE65), a critical enzyme in the retinoid cycle. We also knocked out the CRALBP gene rlbp1 mainly in Foxg1-expressing Müller cells. We obtained multielectrode-array recordings from ipRGCs in a novel RPE-attached mouse retina preparation, and imaged pupillary light reflexes in vivo. RESULTS: Melanopsin-based ipRGC responses to prolonged light became less tonic in both knockout lines, and pupillary light reflexes were also less sustained in RPE65-knockout than control mice. CONCLUSIONS: These results confirm that ipRGCs rely partly on the retinoid cycle to continuously regenerate melanopsin during prolonged photostimulation, and suggest that CRALBP in Müller glia likely transports 11-cis-retinal from the RPE to ipRGCs - this is the first proposed functional role for CRALBP in the inner retina.

The Roles of Rods, Cones, and Melanopsin in Photoresponses of M4 Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs) and Optokinetic Visual Behavior.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate not only image-forming vision like other ganglion cells, but also non-image-forming physiological responses to light such as pupil constriction and circadian photoentrainment. All ipRGCs respond to light through their endogenous photopigment melanopsin as well as rod/cone-driven synaptic inputs. A major knowledge gap is how melanopsin, rods, and cones differentially drive ipRGC photoresponses and image-forming vision. We whole-cell-recorded from M4-type ipRGCs lacking melanopsin, rod input, or cone input to dissect the roles of each component in ipRGCs' responses to steady and temporally modulated (≥0.3 Hz) lights. We also used a behavioral assay to determine how the elimination of melanopsin, rod, or cone function impacts the optokinetic visual behavior of mice. Results showed that the initial, transient peak in an M4 cell's responses to 10-s light steps arises from rod and cone inputs. Both the sustainability and poststimulus persistence of these light-step responses depend only on rod and/or cone inputs, which is unexpected because these ipRGC photoresponse properties have often been attributed primarily to melanopsin. For temporally varying stimuli, the enhancement of response sustainedness involves melanopsin, whereas stimulus tracking is mediated by rod and cone inputs. Finally, the behavioral assay showed that while all three photoreceptive systems are nearly equally important for contrast sensitivity, only cones and rods contribute to spatial acuity.