The Atoh7 remote enhancer provides transcriptional robustness during retinal ganglion cell development.

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo.

BACKGROUND: The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. METHODS: We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. RESULTS: Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. CONCLUSIONS: The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Monocyte infiltration rather than microglia proliferation dominates the early immune response to rapid photoreceptor degeneration.

BACKGROUND: Activation of resident microglia accompanies every known form of neurodegeneration, but the involvement of peripheral monocytes that extravasate and rapidly transform into microglia-like macrophages within the central nervous system during degeneration is far less clear. METHODS: Using a combination of in vivo ocular imaging, flow cytometry, and immunohistochemistry, we investigated the response of infiltrating cells in a light-inducible mouse model of photoreceptor degeneration. RESULTS: Within 24 h, resident microglia became activated and began migrating to the site of degeneration. Retinal expression of CCL2 increased just prior to a transient period of CCR2+ cell extravasation from the retinal vasculature. Proliferation of microglia and monocytes occurred concurrently; however, there was no indication of proliferation in either population until 72-96 h after neurodegeneration began. Eliminating CCL2-CCR2 signaling blocked monocyte recruitment, but did not alter the extent of retinal degeneration. CONCLUSIONS: These results demonstrate that the immune response to photoreceptor degeneration includes both resident microglia and monocytes, even at very early times. Surprisingly, preventing monocyte infiltration did not block neurodegeneration, suggesting that in this model, degeneration is limited by cell clearance from other phagocytes or by the timing of intrinsic cell death programs. These results show monocyte involvement is not limited to disease states that overwhelm or deplete the resident microglial population and that interventions focused on modulating the peripheral immune system are not universally beneficial for staving off degeneration.

Loss of cone function without degeneration in a novel Gnat2 knock-out mouse.

Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod-1 s-1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130.

BACKGROUND: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice. METHODS: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor ß (gp-130) was injected intravitreally immediately after RD. RESULTS: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade. CONCLUSIONS: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

New Developments in Murine Imaging for Assessing Photoreceptor Degeneration In Vivo.

Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs' membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al., Vis Res 102:71-79, 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.

Adaptive optics scanning laser ophthalmoscopy and optical coherence tomography (AO-SLO-OCT) system for in vivo mouse retina imaging.

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are imaging technologies invented in the 1980s that have revolutionized the field of in vivo retinal diagnostics and are now commonly used in ophthalmology clinics as well as in vision science research. Adaptive optics (AO) technology enables high-fidelity correction of ocular aberrations, resulting in improved resolution and sensitivity for both SLO and OCT systems. The potential of gathering multi-modal cellular-resolution information in a single instrument is of great interest to the ophthalmic imaging community. Although similar instruments have been developed for imaging the human retina, developing such a system for mice will benefit basic science research and should help with further dissemination of AO technology. Here, we present our work integrating OCT into an existing mouse retinal AO-SLO system, resulting in a multi-modal AO-enhanced imaging system of the living mouse eye. The new system allows either independent or simultaneous data acquisition of AO-SLO and AO-OCT, depending on the requirements of specific scientific experiments. The system allows a data acquisition speed of 200 kHz A-scans/pixel rate for OCT and SLO, respectively. It offers ∼6 µm axial resolution for AO-OCT and a ∼1 µm lateral resolution for AO-SLO-OCT imaging.

Loss of the K+ channel Kv2.1 greatly reduces outward dark current and causes ionic dysregulation and degeneration in rod photoreceptors.

Vertebrate retinal photoreceptors signal light by suppressing a circulating "dark current" that maintains their relative depolarization in the dark. This dark current is composed of an inward current through CNG channels and NCKX transporters in the outer segment that is balanced by outward current exiting principally from the inner segment. It has been hypothesized that Kv2.1 channels carry a predominant fraction of the outward current in rods. We examined this hypothesis by comparing whole cell, suction electrode, and electroretinographic recordings from Kv2.1 knockout (Kv2.1-/-) and wild-type (WT) mouse rods. Single cell recordings revealed flash responses with unusual kinetics, and reduced dark currents that were quantitatively consistent with the measured depolarization of the membrane resting potential in the dark. A two-compartment (outer and inner segment) physiological model based on known ionic mechanisms revealed that the abnormal Kv2.1-/- rod photoresponses arise principally from the voltage dependencies of the known conductances and the NCKX exchanger, and a highly elevated fraction of inward current carried by Ca2+ through CNG channels due to the aberrant depolarization. Kv2.1-/- rods had shorter outer segments than WT and dysmorphic mitochondria in their inner segments. Optical coherence tomography of knockout animals demonstrated a slow photoreceptor degeneration over a period of 6 mo. Overall, these findings reveal that Kv2.1 channels carry 70-80% of the non-NKX outward dark current of the mouse rod, and that the depolarization caused by the loss of Kv2.1 results in elevated Ca2+ influx through CNG channels and elevated free intracellular Ca2+, leading to progressive degeneration.

Microglia Activation and Inflammation During the Death of Mammalian Photoreceptors.

Photoreceptors are highly specialized sensory neurons with unique metabolic and physiological requirements. These requirements are partially met by Müller glia and cells of the retinal pigment epithelium (RPE), which provide essential metabolites, phagocytose waste, and control the composition of the surrounding microenvironment. A third vital supporting cell type, the retinal microglia, can provide photoreceptors with neurotrophic support or exacerbate neuroinflammation and hasten neuronal cell death. Understanding the physiological requirements for photoreceptor homeostasis and the factors that drive microglia to best promote photoreceptor survival has important implications for the treatment and prevention of blinding degenerative diseases like retinitis pigmentosa and age-related macular degeneration.

Molecular profiling of resident and infiltrating mononuclear phagocytes during rapid adult retinal degeneration using single-cell RNA sequencing.

Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2+ leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b+CD45+ cells. Single-cell mRNA sequencing of the entire CD11b+CD45+ population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.

Temporal speckle-averaging of optical coherence tomography volumes for in-vivo cellular resolution neuronal and vascular retinal imaging.

It has been recently demonstrated that structures corresponding to the cell bodies of highly transparent cells in the retinal ganglion cell layer could be visualized noninvasively in the living human eye by optical coherence tomography (OCT) via temporal averaging. Inspired by this development, we explored the application of volumetric temporal averaging in mice, which are important models for studying human retinal diseases and therapeutic interventions. A general framework of temporal speckle-averaging (TSA) of OCT and optical coherence tomography angiography (OCTA) is presented and applied to mouse retinal volumetric data. Based on the image analysis, the eyes of mice under anesthesia exhibit only minor motions, corresponding to lateral displacements of a few micrometers and rotations of a fraction of 1 deg. Moreover, due to reduced eye movements under anesthesia, there is a negligible amount of motion artifacts within the volumes that need to be corrected to achieve volume coregistration. In addition, the relatively good optical quality of the mouse ocular media allows for cellular-resolution imaging without adaptive optics (AO), greatly simplifying the experimental system, making the proposed framework feasible for large studies. The TSA OCT and TSA OCTA results provide rich information about new structures previously not visualized in living mice with non-AO-OCT. The mechanism of TSA relies on improving signal-to-noise ratio as well as efficient suppression of speckle contrast due to temporal decorrelation of the speckle patterns, enabling full utilization of the high volumetric resolution offered by OCT and OCTA.

Aperture phase modulation with adaptive optics: a novel approach for speckle reduction and structure extraction in optical coherence tomography.

Speckle is an inevitable consequence of the use of coherent light in imaging and acts as noise that corrupts image formation in most applications. Optical coherence tomographic imaging, as a technique employing coherence time gating, suffers from speckle. We present here a novel method of suppressing speckle noise intrinsically compatible with adaptive optics (AO) for confocal coherent imaging: modulation of the phase in the system pupil aperture with a segmented deformable mirror (DM) to introduce minor perturbations in the point spread function. This approach creates uncorrelated speckle patterns in a series of images, enabling averaging to suppress speckle noise while maintaining structural detail. A method is presented that efficiently determines the optimal range of modulation of DM segments relative to their AO-optimized position so that speckle noise is reduced while image resolution and signal strength are preserved. The method is active and independent of sample properties. Its effectiveness and efficiency are quantified and demonstrated by both ex vivo non-biological and in vivo biological applications.

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.