Development of the Binocular Circuit.

Seeing in three dimensions is a major property of the visual system in mammals. The circuit underlying this property begins in the retina, from which retinal ganglion cells (RGCs) extend to the same or opposite side of the brain. RGC axons decussate to form the optic chiasm, then grow to targets in the thalamus and midbrain, where they synapse with neurons that project to the visual cortex. Here we review the cellular and molecular mechanisms of RGC axonal growth cone guidance across or away from the midline via receptors to cues in the midline environment. We present new views on the specification of ipsi- and contralateral RGC subpopulations and factors implementing their organization in the optic tract and termination in subregions of their targets. Lastly, we describe the functional and behavioral aspects of binocular vision, focusing on the mouse, and discuss recent discoveries on the evolution of the binocular circuit.

The Retinal Ganglion Cell Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration Consortium.

Purpose: The Retinal Ganglion Cell (RGC) Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) consortium was founded in 2021 to help address the numerous scientific and clinical obstacles that impede development of vision-restorative treatments for patients with optic neuropathies. The goals of the RReSTORe consortium are: (1) to define and prioritize the most critical challenges and questions related to RGC regeneration; (2) to brainstorm innovative tools and experimental approaches to meet these challenges; and (3) to foster opportunities for collaborative scientific research among diverse investigators. Design and Participants: The RReSTORe consortium currently includes > 220 members spanning all career stages worldwide and is directed by an organizing committee comprised of 15 leading scientists and physician-scientists of diverse backgrounds. Methods: Herein, we describe the structure and organization of the RReSTORe consortium, its activities to date, and the perceived impact that the consortium has had on the field based on a survey of participants. Results: In addition to helping propel the field of regenerative medicine as applied to optic neuropathies, the RReSTORe consortium serves as a framework for developing large collaborative groups aimed at tackling audacious goals that may be expanded beyond ophthalmology and vision science. Conclusions: The development of innovative interventions capable of restoring vision for patients suffering from optic neuropathy would be transformative for the ophthalmology field, and may set the stage for functional restoration in other central nervous system disorders. By coordinating large-scale, international collaborations among scientists with diverse and complementary expertise, we are confident that the RReSTORe consortium will help to accelerate the field toward clinical translation. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium.

Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system's limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium's efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

Semaphorin-6D and Plexin-A1 Act in a Non-Cell-Autonomous Manner to Position and Target Retinal Ganglion Cell Axons.

Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of semaphorins to act both as signaling receptors and ligands yields a multitude of responses. Here, we describe a novel role for Semaphorin-6D (Sema6D) and Plexin-A1 in the positioning and targeting of retinogeniculate axons. In Plexin-A1 or Sema6D mutant mice of either sex, the optic tract courses through, rather than along, the border of the dorsal lateral geniculate nucleus (dLGN), and some retinal axons ectopically arborize adjacent and lateral to the optic tract rather than defasciculating and entering the target region. We find that Sema6D and Plexin-A1 act together in a dose-dependent manner, as the number of the ectopic retinal projections is altered in proportion to the level of Sema6D or Plexin-A1 expression. Moreover, using retinal in utero electroporation of Sema6D or Plexin-A1 shRNA, we show that Sema6D and Plexin-A1 are both required in retinal ganglion cells for axon positioning and targeting. Strikingly, nonelectroporated retinal ganglion cell axons also mistarget in the tract region, indicating that Sema6D and Plexin-A1 can act non-cell-autonomously, potentially through axon-axon interactions. These data provide novel evidence for a dose-dependent and non-cell-autonomous role for Sema6D and Plexin-A1 in retinal axon organization in the optic tract and dLGN.SIGNIFICANCE STATEMENT Before innervating their central brain targets, retinal ganglion cell axons fasciculate in the optic tract and then branch and arborize in their target areas. Upon deletion of the guidance molecules Plexin-A1 or Semaphorin-6D, the optic tract becomes disorganized near and extends within the dorsal lateral geniculate nucleus. In addition, some retinal axons form ectopic aggregates within the defasciculated tract. Sema6D and Plexin-A1 act together as a receptor-ligand pair in a dose-dependent manner, and non-cell-autonomously, to produce this developmental aberration. Such a phenotype highlights an underappreciated role for axon guidance molecules in tract cohesion and appropriate defasciculation near, and arborization within, targets.

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism.

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

SpineRacks and SpinalJ for efficient analysis of neurons in a 3D reference atlas of the mouse spinal cord.

Spatial analysis of spinal neurons is currently limited by a lack of tools for efficient preparation and imaging of the whole spinal cord (SC) and the absence of a 3D reference atlas. Here, we describe protocols for efficient sectioning of whole SC using SpineRacks and subsequent image registration, atlas mapping, and 3D analysis of cells and projections, using SpinalJ. Together, these tools enable high-throughput analyses of adult mouse SC and direct comparison of spatial information of neurons between animals and studies. For complete details on the use and execution of this protocol, please refer to Fiederling et al. (2021).

Tools for efficient analysis of neurons in a 3D reference atlas of whole mouse spinal cord.

To fill the prevailing gap in methodology for whole spinal cord (SC) analysis, we have (1) designed scaffolds (SpineRacks) that facilitate efficient and ordered cryo-sectioning of the entire SC in a single block, (2) constructed a 3D reference atlas of adult mouse SC, and (3) developed software (SpinalJ) to register images of sections and for standardized analysis of cells and projections in atlas space. We have verified mapping accuracies for known neurons and demonstrated the usefulness of this platform to reveal unknown neuronal distributions. Together, these tools provide high-throughput analyses of whole mouse SC and enable direct comparison of 3D spatial information between animals and studies.

Retinal Ganglion Cell Axon Wiring Establishing the Binocular Circuit.

Binocular vision depends on retinal ganglion cell (RGC) axon projection either to the same side or to the opposite side of the brain. In this article, we review the molecular mechanisms for decussation of RGC axons, with a focus on axon guidance signaling at the optic chiasm and ipsi- and contralateral axon organization in the optic tract prior to and during targeting. The spatial and temporal features of RGC neurogenesis that give rise to ipsilateral and contralateral identity are described. The albino visual system is highlighted as an apt comparative model for understanding RGC decussation, as albinos have a reduced ipsilateral projection and altered RGC neurogenesis associated with perturbed melanogenesis in the retinal pigment epithelium. Understanding the steps for RGC specification into ipsi- and contralateral subtypes will facilitate differentiation of stem cells into RGCs with proper navigational abilities for effective axon regeneration and correct targeting of higher-order visual centers.

Deaths associated with GA for dentistry 1948 - 2016: the evolution of a policy for general anaesthesia (GA) for dental treatment.

BACKGROUND: Following the inception of the National Health Service in 1948 dental treatment under General Anesthesia (GA) became easily available. An unexpected consequence of this was a disconcerting number of deaths associated with GA. Over the decades since 1948 there have been a number of specialist medical society, royal college, and government working parties deliberating on the appropriateness of GA being conducted in general dental practice and community dental practice. METHODS: The figures for the number of general anaesthetics per annum in England and Wales were obtained from the general dental services board, the community dental service, and records from hospital inpatient episodes. The number of deaths per annum were obtained from coroners' enquiries and dental protection societies. FINDINGS: Prior to 2001 there is a strong correlation between the number of GA's per annum and deaths. Since 2001, when the UK government directed that all GAs for dentistry must be administered in a hospital with Intensive Care facilities the number of deaths per annum has reduced to nil. INTERPRETATION: The change in the arrangements under which GA for dentistry are administered was coincident with improved training and knowledge of GA for dentistry. This has led to a cessation of deaths associated with GA for dentistry. The incidence rate is now estimated at less than 1 death per 3.5 million GAs.

Conversations with Ray Guillery on albinism: linking Siamese cat visual pathway connectivity to mouse retinal development.

In albinism of all species, perturbed melanin biosynthesis in the eye leads to foveal hypoplasia, retinal ganglion cell misrouting, and, consequently, altered binocular vision. Here, written before he died, Ray Guillery chronicles his discovery of the aberrant circuitry from eye to brain in the Siamese cat. Ray's characterization of visual pathway anomalies in this temperature sensitive mutation of tyrosinase and thus melanin synthesis in domestic cats opened the exploration of albinism and simultaneously, a genetic approach to the organization of neural circuitry. I follow this account with a remembrance of Ray's influence on my work. Beginning with my postdoc research with Ray on the cat visual pathway, through my own work on the mechanisms of retinal axon guidance in the developing mouse, Ray and I had a continuous and rich dialogue about the albino visual pathway. I will present the questions Ray posed and clues we have to date on the still-elusive link between eye pigment and the proper balance of ipsilateral and contralateral retinal ganglion cell projections to the brain.

Spatiotemporal distribution of glia in and around the developing mouse optic tract.

In the developing mouse optic tract, retinal ganglion cell (RGC) axon position is organized by topography and laterality (i.e., eye-specific or ipsi- and contralateral segregation). Our lab previously showed that ipsilaterally projecting RGCs are segregated to the lateral aspect of the developing optic tract and found that ipsilateral axons self-fasciculate to a greater extent than contralaterally projecting RGC axons in vitro. However, the full complement of axon-intrinsic and -extrinsic factors mediating eye-specific segregation in the tract remain poorly understood. Glia, which are known to express several guidance cues in the visual system and regulate the navigation of ipsilateral and contralateral RGC axons at the optic chiasm, are natural candidates for contributing to eye-specific pre-target axon organization. Here, we investigate the spatiotemporal expression patterns of both putative astrocytes (Aldh1l1+ cells) and microglia (Iba1+ cells) in the embryonic and neonatal optic tract. We quantified the localization of ipsilateral RGC axons to the lateral two-thirds of the optic tract and analyzed glia position and distribution relative to eye-specific axon organization. While our results indicate that glial segregation patterns do not strictly align with eye-specific RGC axon segregation in the tract, we identify distinct spatiotemporal organization of both Aldh1l1+ cells and microglia in and around the developing optic tract. These findings inform future research into molecular mechanisms of glial involvement in RGC axon growth and organization in the developing retinogeniculate pathway.

Distinct timing of neurogenesis of ipsilateral and contralateral retinal ganglion cells.

In higher vertebrates, the circuit formed by retinal ganglion cells (RGCs) projecting ipsilaterally (iRGCs) or contralaterally (cRGCs) to the brain permits binocular vision and depth perception. iRGCs and cRGCs differ in their position within the retina and in expression of transcription, guidance and activity-related factors. To parse whether these two populations also differ in the timing of their genesis, a feature of distinct neural subtypes and associated projections, we used newer birthdating methods and cell subtype specific markers to determine birthdate and cell cycle exit more precisely than previously. In the ventrotemporal (VT) retina, i- and cRGCs intermingle and neurogenesis in this zone lags behind RGC production in the rest of the retina where only cRGCs are positioned. In addition, within the VT retina, i- and cRGC populations are born at distinct times: neurogenesis of iRGCs surges at E13, and cRGCs arise as early as E14, not later in embryogenesis as reported. Moreover, in the ventral ciliary margin zone (CMZ), which contains progenitors that give rise to some iRGCs in ventral neural retina (Marcucci et al., 2016), cell cycle exit is slower than in other retinal regions in which progenitors give rise only to cRGCs. Further, when the cell cycle regulator Cyclin D2 is missing, cell cycle length in the CMZ is further reduced, mirroring the reduction of both i- and cRGCs in the Cyclin D2 mutant. These results strengthen the view that differential regulation of cell cycle dynamics at the progenitor level is associated with specific RGC fates and laterality of axonal projection.

Activation of Wnt signaling reduces ipsilaterally projecting retinal ganglion cells in pigmented retina.

In mammalian albinism, disrupted melanogenesis in the retinal pigment epithelium (RPE) is associated with fewer retinal ganglion cells (RGCs) projecting ipsilaterally to the brain, resulting in numerous abnormalities in the retina and visual pathway, especially binocular vision. To further understand the molecular link between disrupted RPE and a reduced ipsilateral RGC projection in albinism, we compared gene expression in the embryonic albino and pigmented mouse RPE. We found that the Wnt pathway, which directs peripheral retinal differentiation and, generally, cell proliferation, is dysregulated in the albino RPE. Wnt2b expression is expanded in the albino RPE compared with the pigmented RPE, and the expanded region adjoins the site of ipsilateral RGC neurogenesis and settling. Pharmacological activation of Wnt signaling in pigmented mice by lithium (Li+) treatment in vivo reduces the number of Zic2-positive RGCs, which are normally fated to project ipsilaterally, to numbers observed in the albino retina. These results implicate Wnt signaling from the RPE to neural retina as a potential factor in the regulation of ipsilateral RGC production, and thus the albino phenotype.

Alcohol enhances type 1 interferon-α production and mortality in young mice infected with Mycobacterium tuberculosis.

In the current study, we used a mouse model and human blood samples to determine the effects of chronic alcohol consumption on immune responses during Mycobacterium tuberculosis (Mtb) infection. Alcohol increased the mortality of young mice but not old mice with Mtb infection. CD11b+Ly6G+ cells are the major source of IFN-α in the lungs of Mtb-infected alcohol-fed young mice, and IFN-α enhances macrophage necroptosis in the lungs. Treatment with an anti-IFNAR-1 antibody enhanced the survival of Mtb-infected alcohol-fed young mice. In response to Mtb, peripheral blood mononuclear cells (PBMCs) from alcoholic young healthy individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α than those from non-alcoholic young healthy LTBI+ individuals and alcoholic and non-alcoholic old healthy LTBI+ individuals. Our study demonstrates that alcohol enhances IFN-α production by CD11b+Ly6G+ cells in the lungs of young Mtb-infected mice, which leads to macrophage necroptosis and increased mortality. Our findings also suggest that young alcoholic LTBI+ individuals have a higher risk of developing active TB infection.

Differing Strategies Despite Shared Lineages of Motor Neurons and Glia to Achieve Robust Development of an Adult Neuropil in Drosophila.

In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages is not understood. We show that two stem cells that produce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. We suggest that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution.

Eye-specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway.

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1-/- mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.

Live or Die? Depends on Who You Are.

In this issue of Neuron, Welsbie et al. (2017) and Norsworthy et al. (2017) implicate the transcription factor Sox11 as a key player after optic nerve injury-in DLK signaling of RGC cell death, and in RGC regeneration and survival but only in certain RGCs.

SoxC Transcription Factors Promote Contralateral Retinal Ganglion Cell Differentiation and Axon Guidance in the Mouse Visual System.

Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells in vitro and project ipsilaterally at the chiasm midline in vivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.

Ipsilateral and Contralateral Retinal Ganglion Cells Express Distinct Genes during Decussation at the Optic Chiasm.

The increasing availability of transcriptomic technologies within the last decade has facilitated high-throughput identification of gene expression differences that define distinct cell types as well as the molecular pathways that drive their specification. The retinal projection neurons, retinal ganglion cells (RGCs), can be categorized into distinct morphological and functional subtypes and by the laterality of their projections. Here, we present a method for purifying the sparse population of ipsilaterally projecting RGCs in mouse retina from their contralaterally projecting counterparts during embryonic development through rapid retrograde labeling followed by fluorescence-activated cell sorting. Through microarray analysis, we uncovered the distinct molecular signatures that define and distinguish ipsilateral and contralateral RGCs during the critical period of axonal outgrowth and decussation, with more than 300 genes differentially expressed within these two cell populations. Among the differentially expressed genes confirmed through in vivo expression validation, several genes that mark "immaturity" are expressed within postmitotic ipsilateral RGCs. Moreover, at least one complementary pair, Igf1 and Igfbp5, is upregulated in contralateral or ipsilateral RGCs, respectively, and may represent signaling pathways that determine ipsilateral versus contralateral RGC identity. Importantly, the cell cycle regulator cyclin D2 is highly expressed in peripheral ventral retina with a dynamic expression pattern that peaks during the period of ipsilateral RGC production. Thus, the molecular signatures of ipsilateral and contralateral RGCs and the mechanisms that regulate their differentiation are more diverse than previously expected.