Evaluating neural crest cell migration in a Col4a1 mutant mouse model of ocular anterior segment dysgenesis.

The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome - a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.

The Interplay between Neurotransmitters and Calcium Dynamics in Retinal Synapses during Development, Health, and Disease.

The intricate functionality of the vertebrate retina relies on the interplay between neurotransmitter activity and calcium (Ca2+) dynamics, offering important insights into developmental processes, physiological functioning, and disease progression. Neurotransmitters orchestrate cellular processes to shape the behavior of the retina under diverse circumstances. Despite research to elucidate the roles of individual neurotransmitters in the visual system, there remains a gap in our understanding of the holistic integration of their interplay with Ca2+ dynamics in the broader context of neuronal development, health, and disease. To address this gap, the present review explores the mechanisms used by the neurotransmitters glutamate, gamma-aminobutyric acid (GABA), glycine, dopamine, and acetylcholine (ACh) and their interplay with Ca2+ dynamics. This conceptual outline is intended to inform and guide future research, underpinning novel therapeutic avenues for retinal-associated disorders.

Large-scale survey of excitatory synapses reveals sublamina-specific and asymmetric synapse disassembly in a neurodegenerative circuit.

In the nervous system, parallel circuits are organized in part by the lamina-specific compartmentalization of synaptic connections. In sensory systems such as mammalian retina, degenerating third-order neurons remodel their local presynaptic connectivity with second-order neurons. To determine whether there are sublamina-specific perturbations after injury of adult retinal ganglion cells, we comprehensively analyzed excitatory synapses across the inner plexiform layer (IPL) where bipolar cells connect to ganglion cells. Here, we show that pre- and postsynaptic component loss occurs throughout the IPL in a sublamina-dependent fashion after transient intraocular pressure elevation. Partnered synaptic components are lost as neurodegeneration progresses, while unpartnered synaptic components remain stable. Furthermore, presynaptic components are either lost first or simultaneously with the postsynaptic component. Our results demonstrate that this degenerating neural circuit exhibits differential vulnerability of excitatory synapses depending on IPL depth, highlighting the ordered disassembly of synapses that is specific to laminar compartments of the retina.

Rejection of inappropriate synaptic partners in mouse retina mediated by transcellular FLRT2-UNC5 signaling.

During nervous system development, neurons choose synaptic partners with remarkable specificity; however, the cell-cell recognition mechanisms governing rejection of inappropriate partners remain enigmatic. Here, we show that mouse retinal neurons avoid inappropriate partners by using the FLRT2-uncoordinated-5 (UNC5) receptor-ligand system. Within the inner plexiform layer (IPL), FLRT2 is expressed by direction-selective (DS) circuit neurons, whereas UNC5C/D are expressed by non-DS neurons projecting to adjacent IPL sublayers. In vivo gain- and loss-of-function experiments demonstrate that FLRT2-UNC5 binding eliminates growing DS dendrites that have strayed from the DS circuit IPL sublayers. Abrogation of FLRT2-UNC5 binding allows mistargeted arbors to persist, elaborate, and acquire synapses from inappropriate partners. Conversely, UNC5C misexpression within DS circuit sublayers inhibits dendrite growth and drives arbors into adjacent sublayers. Mechanistically, UNC5s promote dendrite elimination by interfering with FLRT2-mediated adhesion. Based on their broad expression, FLRT-UNC5 recognition is poised to exert widespread effects upon synaptic partner choices across the nervous system.

Inhibition, but not excitation, recovers from partial cone loss with greater spatiotemporal integration, synapse density, and frequency.

Neural circuits function in the face of changing inputs, either caused by normal variation in stimuli or by cell death. To maintain their ability to perform essential computations with partial inputs, neural circuits make modifications. Here, we study the retinal circuit's responses to changes in light stimuli or in photoreceptor inputs by inducing partial cone death in the mature mouse retina. Can the retina withstand or recover from input loss? We find that the excitatory pathways exhibit functional loss commensurate with cone death and with some aspects predicted by partial light stimulation. However, inhibitory pathways recover functionally from lost input by increasing spatiotemporal integration in a way that is not recapitulated by partially stimulating the control retina. Anatomically, inhibitory synapses are upregulated on secondary bipolar cells and output ganglion cells. These findings demonstrate the greater capacity for inhibition, compared with excitation, to modify spatiotemporal processing with fewer cone inputs.

Impact of Photoreceptor Loss on Retinal Circuitry.

Our sense of sight relies on photoreceptors, which transduce photons into the nervous system's electrochemical interpretation of the visual world. These precious photoreceptors can be disrupted by disease, injury, and aging. Once photoreceptors start to die, but before blindness occurs, the remaining retinal circuitry can withstand, mask, or exacerbate the photoreceptor deficit and potentially be receptive to newfound therapies for vision restoration. To maximize the retina's receptivity to therapy, one must understand the conditions that influence the state of the remaining retina. In this review, we provide an overview of the retina's structure and function in health and disease. We analyze a collection of observations on photoreceptor disruption and generate a predictive model to identify parameters that influence the retina's response. Finally, we speculate on whether the retina, with its remarkable capacity to function over light levels spanning nine orders of magnitude, uses these same adaptational mechanisms to withstand and perhaps mask photoreceptor loss.

Disassembly and rewiring of a mature converging excitatory circuit following injury.

Specificity and timing of synapse disassembly in the CNS are essential to learning how individual circuits react to neurodegeneration of the postsynaptic neuron. In sensory systems such as the mammalian retina, synaptic connections of second-order neurons are known to remodel and reconnect in the face of sensory cell loss. Here we analyzed whether degenerating third-order neurons can remodel their local presynaptic connectivity. We injured adult retinal ganglion cells by transiently elevating intraocular pressure. We show that loss of presynaptic structures occurs before postsynaptic density proteins and accounts for impaired transmission from presynaptic neurons, despite no evidence of presynaptic cell loss, axon terminal shrinkage, or reduced functional input. Loss of synapses is biased among converging presynaptic neuron types, with preferential loss of the major excitatory cone-driven partner and increased connectivity with rod-driven presynaptic partners, demonstrating that this adult neural circuit is capable of structural plasticity while undergoing neurodegeneration.

Asymmetric Functional Impairment of ON and OFF Retinal Pathways in Glaucoma.

Purpose: To investigate ON-pathway versus OFF-pathway dysfunction in glaucoma using handheld electroretinography (ERG) with a temporally modulated sinusoidal flicker stimulus. Design: Cross-sectional study. Participants: Fifty-nine participants accounting for 104 eyes, comprised of 19 control eyes, 26 glaucoma suspect eyes, and 59 glaucoma eyes. Methods: Participants underwent portable ERG testing, which included the photopic flash, photopic flicker, photopic negative response stimulus, ON-OFF stimulus, and a custom-written sinusoidal flicker stimulus that was modulated from 50 to 0.3 Hz. Main Outcome and Measures: The ERG response amplitudes were measured by the handheld ERG. For the custom-written sinusoidal flicker stimulus, we derived and compared the log10 first harmonic frequency response amplitudes. Patient discomfort and fatigue after ERG testing were rated on a scale from 1 to 5. Results: Baseline demographics were not significantly different between groups, except for ocular characteristics. Analysis was performed adjusting for participant age, sex, race, and dilation status, and the sinusoidal frequency responses were stratified at 10 Hz because higher frequencies are associated with the OFF-pathway, whereas lower frequencies are associated with the ON-pathway. After stratification, glaucoma eyes showed an adjusted decrease of 32.1% at frequencies of more than 10 Hz (95% confidence interval [CI], -51.8% to -4.1%; P = 0.03). For 10 Hz stimulus frequencies or less, an adjusted 11.5% reduction was found (95% CI, -39.5% to 29.1%; P = 0.50). Glaucoma suspect eyes did show a decreased response, but this was not significant at either frequency range. When comparing handheld ERG with traditional visual field assessments, participants found the handheld ERG to result in much less discomfort and fatigue. Conclusions: Our finding that glaucoma participants showed greater decreases in ERG response at higher frequencies supports the hypothesis that the OFF-pathway may be more vulnerable in human glaucoma. Using a handheld ERG device with a sinusoidal flicker stimulus may provide an objective assessment of visual function in glaucoma.

Probing ON and OFF Retinal Pathways in Glaucoma Using Electroretinography.

Glaucoma is a progressive neurodegenerative disease involving damage and eventually death of retinal ganglion cells (RGCs) that comprise the optic nerve. This review summarizes current understanding of specific RGC type vulnerability in glaucoma and how electroretinography (ERG) may provide an objective measure of these functional perturbations. There is building evidence to suggest that ON RGCs, which respond to light increments, may be more resilient to elevated intraocular pressure and glaucoma, whereas OFF RGCs, which respond to light decrements, may be more susceptible. ERG experiments in nonhuman primates and mice have also shown that the ON- and OFF-pathways can be separated using a variety of techniques such as pattern ERG and the photopic negative response. Another ERG paradigm of interest to separate the ON and OFF responses is a flicker stimulus at varying temporal frequencies. Response to lower temporal frequencies is associated with the ON-pathway, and ERG response to higher frequencies is associated with the OFF-pathway. In mice, experimental glaucoma models have shown greater decreases in ERG response at higher frequencies, suggesting that the OFF-pathway is more susceptible. We also summarize current clinical ERG protocols used for glaucoma and discuss innovations for developing new types of stimuli that can further separate the ON- and OFF-pathways. Applying these novel paradigms that distinguish ON- and OFF-pathways may ultimately improve glaucoma diagnostics and monitoring of glaucoma progression. Translational Relevance: Based on our current understanding of specific RGC type vulnerability in glaucoma, we explore how ERG may provide an objective measure of ON- versus OFF-pathway functional perturbations.

A dual role for Cav1.4 Ca2+ channels in the molecular and structural organization of the rod photoreceptor synapse.

Synapses are fundamental information processing units that rely on voltage-gated Ca2+ (Cav) channels to trigger Ca2+-dependent neurotransmitter release. Cav channels also play Ca2+-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Cav1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Cav1.4, we report that the Cav1.4 protein, but not its Ca2+ conductance, is required for the molecular assembly of rod synapses; however, Cav1.4 Ca2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Cav channels serve both as organizers of synaptic building blocks and as sources of Ca2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system.

Mature Retina Compensates Functionally for Partial Loss of Rod Photoreceptors.

Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit's output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.

Partial Cone Loss Triggers Synapse-Specific Remodeling and Spatial Receptive Field Rearrangements in a Mature Retinal Circuit.

Resilience of neural circuits has been observed in the persistence of function despite neuronal loss. In vision, acuity and sensitivity can be retained after 50% loss of cones. While neurons in the cortex can remodel after input loss, the contributions of cell-type-specific circuits to resilience are unknown. Here, we study the effects of partial cone loss in mature mouse retina where cell types and connections are known. At first-order synapses, bipolar cell dendrites remodel and synaptic proteins diminish at sites of input loss. Sites of remaining inputs preserve synaptic proteins. Second-order synapses between bipolar and ganglion cells remain stable. Functionally, ganglion cell spatio-temporal receptive fields retain center-surround structure following partial cone loss. We find evidence for slower temporal filters and expanded receptive field surrounds, derived mainly from inhibitory inputs. Surround expansion is absent in partially stimulated control retina. Results demonstrate functional resilience to input loss beyond pre-existing mechanisms in control retina.

Who's lost first? Susceptibility of retinal ganglion cell types in experimental glaucoma.

The purpose of this article is to summarize our current knowledge about the susceptibility of specific retinal ganglion cell (RGC) types in experimental glaucoma, and to delineate the initial morphological and functional alterations that occur in response to intraocular pressure (IOP) elevation. There has been debate in the field as to whether RGCs with large somata and axons are more vulnerable, with definitive conclusions still in progress because of the wide diversity of RGC types. Indeed, it is now estimated that there are greater than 30 different RGC types, and while we do not yet understand the complete details, we discuss a growing body of work that supports the selective vulnerability hypothesis of specific RGC types in experimental glaucoma. Specifically, structural and functional degeneration of various RGC types have been examined across different rodent models of experimental glaucoma (acute vs. chronic) and different strains, and an emerging consensus is that OFF RGCs appear to be more vulnerable to IOP elevation compared to ON RGCs. Understanding the mechanisms by which this selective vulnerability manifests across different RGC types should lead to novel and improved strategies for neuroprotection and neuroregeneration in glaucoma.

Selective Vulnerability of Specific Retinal Ganglion Cell Types and Synapses after Transient Ocular Hypertension.

UNLABELLED: Key issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs. Among the α-like RGCs studied, αOFF-transient RGCs exhibited higher rates of cell death, with corresponding reductions in dendritic area, dendritic complexity, and synapse density. Functionally, OFF-transient RGCs displayed decreases in spontaneous activity and receptive field size. In contrast, neither αOFF-sustained nor αON-sustained RGCs displayed decreases in light responses, although they did exhibit a decrease in excitatory postsynaptic sites, suggesting that synapse loss may be one of the earliest signs of degeneration. Interestingly, presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer, corroborating the hypothesis that RGCs with dendrites stratifying in the OFF sublamina may be damaged early. Indeed, OFF arbors of ON-OFF RGCs lose complexity more rapidly than ON arbors. Our results reveal type-specific differences in RGC responses to injury with a selective vulnerability of αOFF-transient RGCs, and furthermore, an increased susceptibility of synapses in the OFF sublamina. The selective vulnerability of specific RGC types offers new avenues for the design of more sensitive functional tests and targeted neuroprotection. SIGNIFICANCE STATEMENT: Conflicting reports regarding the selective vulnerability of specific retinal ganglion cell (RGC) types in glaucoma exist. We examine, for the first time, the effects of transient intraocular pressure elevation on the structure and function of various RGC types. Among the α-like RGCs studied, αOFF-transient RGCs are the most vulnerable to transient transient intraocular pressure elevation as measured by rates of cell death, morphologic alterations in dendrites and synapses, and physiological dysfunction. Specifically, we found that presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer. Our results suggest selective vulnerability both of specific types of RGCs and of specific inner plexiform layer sublaminae, opening new avenues for identifying novel diagnostic and treatment targets in glaucoma.

Glutamatergic Monopolar Interneurons Provide a Novel Pathway of Excitation in the Mouse Retina.

Excitatory and inhibitory neurons in the CNS are distinguished by several features, including morphology, transmitter content, and synapse architecture [1]. Such distinctions are exemplified in the vertebrate retina. Retinal bipolar cells are polarized glutamatergic neurons receiving direct photoreceptor input, whereas amacrine cells are usually monopolar inhibitory interneurons with synapses almost exclusively in the inner retina [2]. Bipolar but not amacrine cell synapses have presynaptic ribbon-like structures at their transmitter release sites. We identified a monopolar interneuron in the mouse retina that resembles amacrine cells morphologically but is glutamatergic and, unexpectedly, makes ribbon synapses. These glutamatergic monopolar interneurons (GluMIs) do not receive direct photoreceptor input, and their light responses are strongly shaped by both ON and OFF pathway-derived inhibitory input. GluMIs contact and make almost as many synapses as type 2 OFF bipolar cells onto OFF-sustained A-type (AOFF-S) retinal ganglion cells (RGCs). However, GluMIs and type 2 OFF bipolar cells possess functionally distinct light-driven responses and may therefore mediate separate components of the excitatory synaptic input to AOFF-S RGCs. The identification of GluMIs thus unveils a novel cellular component of excitatory circuits in the vertebrate retina, underscoring the complexity in defining cell types even in this well-characterized region of the CNS.

Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes.

The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit.

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels.

Illuminating the multifaceted roles of neurotransmission in shaping neuronal circuitry.

Across the nervous system, neurons form highly stereotypic patterns of synaptic connections that are designed to serve specific functions. Mature wiring patterns are often attained upon the refinement of early, less precise connectivity. Much work has led to the prevailing view that many developing circuits are sculpted by activity-dependent competition among converging afferents, which results in the elimination of unwanted synapses and the maintenance and strengthening of desired connections. Studies of the vertebrate retina, however, have recently revealed that activity can play a role in shaping developing circuits without engaging competition among converging inputs that differ in their activity levels. Such neurotransmission-mediated processes can produce stereotypic wiring patterns by promoting selective synapse formation rather than elimination. We discuss how the influence of transmission may also be limited by circuit design and further highlight the importance of transmission beyond development in maintaining wiring specificity and synaptic organization of neural circuits.

Functional architecture of the retina: development and disease.

Structure and function are highly correlated in the vertebrate retina, a sensory tissue that is organized into cell layers with microcircuits working in parallel and together to encode visual information. All vertebrate retinas share a fundamental plan, comprising five major neuronal cell classes with cell body distributions and connectivity arranged in stereotypic patterns. Conserved features in retinal design have enabled detailed analysis and comparisons of structure, connectivity and function across species. Each species, however, can adopt structural and/or functional retinal specializations, implementing variations to the basic design in order to satisfy unique requirements in visual function. Recent advances in molecular tools, imaging and electrophysiological approaches have greatly facilitated identification of the cellular and molecular mechanisms that establish the fundamental organization of the retina and the specializations of its microcircuits during development. Here, we review advances in our understanding of how these mechanisms act to shape structure and function at the single cell level, to coordinate the assembly of cell populations, and to define their specific circuitry. We also highlight how structure is rearranged and function is disrupted in disease, and discuss current approaches to re-establish the intricate functional architecture of the retina.

Interplay of cell-autonomous and nonautonomous mechanisms tailors synaptic connectivity of converging axons in vivo.

Neurons receive input from diverse afferents but form stereotypic connections with each axon type to execute their precise functions. Developmental mechanisms that specify the connectivity of individual axons across populations of converging afferents are not well-understood. Here, we untangled the contributions of activity-dependent and independent interactions that regulate the connectivity of afferents providing major and minor input onto a neuron. Individual transmission-deficient retinal bipolar cells (BCs) reduced synapses with retinal ganglion cells (RGCs), but active BCs of the same type sharing the dendrite surprisingly did not compensate for this loss. Genetic ablation of some BC neighbors resulted in increased synaptogenesis by the remaining axons in a transmission-independent manner. Presence, but not transmission, of the major BC input also dissuades wiring with the minor input and with synaptically compatible but functionally mismatched afferents. Cell-autonomous, activity-dependent and nonautonomous, activity-independent mechanisms thus together tailor connectivity of individual axons among converging inner retinal afferents.