A subset of ipRGCs regulates both maturation of the circadian clock and segregation of retinogeniculate projections in mice.

The visual system consists of two major subsystems, image-forming circuits that drive conscious vision and non-image-forming circuits for behaviors such as circadian photoentrainment. While historically considered non-overlapping, recent evidence has uncovered crosstalk between these subsystems. Here, we investigated shared developmental mechanisms. We revealed an unprecedented role for light in the maturation of the circadian clock and discovered that intrinsically photosensitive retinal ganglion cells (ipRGCs) are critical for this refinement process. In addition, ipRGCs regulate retinal waves independent of light, and developmental ablation of a subset of ipRGCs disrupts eye-specific segregation of retinogeniculate projections. Specifically, a subset of ipRGCs, comprising ~200 cells and which project intraretinally and to circadian centers in the brain, are sufficient to mediate both of these developmental processes. Thus, this subset of ipRGCs constitute a shared node in the neural networks that mediate light-dependent maturation of the circadian clock and light-independent refinement of retinogeniculate projections.

M1 ipRGCs Influence Visual Function through Retrograde Signaling in the Retina.

UNLABELLED: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs, with five subtypes named M1-M5) are a unique subclass of RGCs with axons that project directly to many brain nuclei involved in non-image-forming functions such as circadian photoentrainment and the pupillary light reflex. Recent evidence suggests that melanopsin-based signals also influence image-forming visual function, including light adaptation, but the mechanisms involved are unclear. Intriguingly, a small population of M1 ipRGCs have intraretinal axon collaterals that project toward the outer retina. Using genetic mouse models, we provide three lines of evidence showing that these axon collaterals make connections with upstream dopaminergic amacrine cells (DACs): (1) ipRGC signaling to DACs is blocked by tetrodotoxin both in vitro and in vivo, indicating that ipRGC-to-DAC transmission requires voltage-gated Na(+) channels; (2) this transmission is partly dependent on N-type Ca(2+) channels, which are possibly expressed in the axon collateral terminals of ipRGCs; and (3) fluorescence microscopy reveals that ipRGC axon collaterals make putative presynaptic contact with DACs. We further demonstrate that elimination of M1 ipRGCs attenuates light adaptation, as evidenced by an impaired electroretinogram b-wave from cones, whereas a dopamine receptor agonist can potentiate the cone-driven b-wave of retinas lacking M1 ipRGCs. Together, the results strongly suggest that ipRGCs transmit luminance signals retrogradely to the outer retina through the dopaminergic system and in turn influence retinal light adaptation. SIGNIFICANCE STATEMENT: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a third class of retinal photoreceptors that are known to mediate physiological responses such as circadian photoentrainment. However, investigation into whether and how ipRGCs contribute to vision has just begun. Here, we provide convergent anatomical and physiological evidence that axon collaterals of ipRGCs constitute a centrifugal pathway to DACs, conveying melanopsin-based signals from the innermost retina to the outer retina. We further demonstrate that retrograde signals likely influence visual processing because elimination of axon collateral-bearing ipRGCs impairs light adaptation by limiting dopamine-dependent facilitation of the cone pathway. Our findings strongly support the hypothesis that retrograde melanopsin-based signaling influences visual function locally within the retina, a notion that refutes the dogma that RGCs only provide physiological signals to the brain.

Apoptosis regulates ipRGC spacing necessary for rods and cones to drive circadian photoentrainment.

The retina consists of ordered arrays of individual types of neurons for processing vision. Here, we show that such order is necessary for intrinsically photosensitive retinal ganglion cells (ipRGCs) to function as irradiance detectors. We found that during development, ipRGCs undergo proximity-dependent Bax-mediated apoptosis. Bax mutant mice exhibit disrupted ipRGC spacing and dendritic stratification with an increase in abnormally localized synapses. ipRGCs are the sole conduit for light input to circadian photoentrainment, and either their melanopsin-based photosensitivity or ability to relay rod/cone input is sufficient for circadian photoentrainment. Remarkably, the disrupted ipRGC spacing does not affect melanopsin-based circadian photoentrainment but severely impairs rod/cone-driven photoentrainment. We demonstrate reduced rod/cone-driven cFos activation and electrophysiological responses in ipRGCs, suggesting that impaired synaptic input to ipRGCs underlies the photoentrainment deficits. Thus, for irradiance detection, developmental apoptosis is necessary for the spacing and connectivity of ipRGCs that underlie their functioning within a neural network.

Intrinsically photosensitive retinal ganglion cells are the primary but not exclusive circuit for light aversion.

Photoallodynia (photophobia) occurs when normal levels of light cause pain ranging from uncomfortable to debilitating. The only current treatment for photoallodynia is light avoidance. The first step to understanding the mechanisms of photoallodynia is to develop reliable animal behavioral tests of light aversion and identify the photoreceptors required to initiate this response. A reliable light/dark box behavioral assay was developed that measures light aversion independently from anxiety, allowing direct testing of one endophenotype of photoallodynia in mice. Mice lacking intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit reduced aversion to bright light, suggesting these cells are the primary circuit for light aversion. Mice treated with exogenous µ opiate receptor agonists exhibited dramatically enhanced light aversion, which was not dependent on ipRGCs, suggesting an alternative pathway for light is engaged. Morphine enhances retinal electrophysiological responses to light but only at low levels. This suggests that for the dramatic light aversion observed, opiates also sensitize central brain regions of photoallodynia. Taken together, our results suggest that light aversion has at least two dissociable mechanisms by which light causes specific allodynia behaviors: a primary ipRGC-based circuit, and a secondary ipRGC-independent circuit that is unmasked by morphine sensitization. These models will be useful in delineating upstream light sensory pathways and downstream avoidance pathways that apply to photoallodynia.

Development of melanopsin-based irradiance detecting circuitry.

BACKGROUND: Most retinal ganglion cells (RGCs) convey contrast and motion information to visual brain centers. Approximately 2% of RGCs are intrinsically photosensitive (ipRGCs), express melanopsin and are necessary for light to modulate specific physiological processes in mice. The ipRGCs directly target the suprachiasmatic nucleus (SCN) to photoentrain circadian rhythms, and the olivary pretectal nucleus (OPN) to mediate the pupillary light response. How and when this ipRGC circuitry develops is unknown. RESULTS: Here, we show that some ipRGCs follow a delayed developmental time course relative to other image-forming RGCs. Specifically, ipRGC neurogenesis extends beyond that of other RGCs, and ipRGCs begin innervating the SCN at postnatal ages, unlike most RGCs, which innervate their image-forming targets embryonically. Moreover, the appearance of ipRGC axons in the OPN coincides precisely with the onset of the pupillary light response. CONCLUSIONS: Some ipRGCs differ not only functionally but also developmentally from RGCs that mediate pattern-forming vision.

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition.

BACKGROUND: Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. RESULTS: We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. CONCLUSION: These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS.