The retina's neurovascular unit: Müller glial sheaths and neuronal contacts.

The neurovascular unit (NVU), comprising vascular, glial and neural elements, supports the energetic demands of neural computation, but this aspect of the retina's trilaminar vessel network is poorly understood. Only the innermost vessel layer - the superficial vascular plexus (SVP) - is ensheathed by astrocytes, like brain capillaries, whereas glial ensheathment in other layers derives from radial Müller glia. Using serial electron microscopy reconstructions from mouse and primate retina, we find that Müller processes cover capillaries in a tessellating pattern, mirroring the tiled astrocytic endfeet wrapping brain capillaries. However, gaps in the Müller sheath, found mainly in the intermediate vascular plexus (IVP), permit different neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with a reduced or absent vascular basement lamina. Focal application of adenosine triphosphate (ATP) to the vitreal surface evoked Ca2+ signals in Müller sheaths in all three vascular layers. Pharmacological experiments confirmed that Müller sheaths express purinergic receptors that, when activated, trigger intracellular Ca2+ signals that are amplified by IP3-controlled intracellular Ca2+ stores. When rod photoreceptors die in a mouse model of retinitis pigmentosa (rd10), Müller sheaths dissociate from the deep vascular plexus (DVP) but are largely unchanged within the IVP or SVP. Thus, Müller glia interact with retinal vessels in a laminar, compartmentalized manner: glial sheathes are virtually complete in the SVP but fenestrated in the IVP, permitting direct neural-to-vascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.

A metabotropic glutamate receptor agonist enhances visual signal fidelity in a mouse model of retinitis pigmentosa.

Many inherited retinal diseases target photoreceptors, which transduce light into a neural signal that is processed by the downstream visual system. As photoreceptors degenerate, physiological and morphological changes to retinal synapses and circuitry reduce sensitivity and increase noise, degrading visual signal fidelity. Here, we pharmacologically targeted the first synapse in the retina in an effort to reduce circuit noise without sacrificing visual sensitivity. We tested a strategy to partially replace the neurotransmitter lost when photoreceptors die with an agonist of receptors that ON bipolars cells use to detect glutamate released from photoreceptors. In rd10 mice, which express a photoreceptor mutation that causes retinitis pigmentosa (RP), we found that a low dose of the mGluR6 agonist l-2-amino-4-phosphonobutyric acid (L-AP4) reduced pathological noise induced by photoreceptor degeneration. After making in vivo electroretinogram recordings in rd10 mice to characterize the developmental time course of visual signal degeneration, we examined effects of L-AP4 on sensitivity and circuit noise by recording in vitro light-evoked responses from individual retinal ganglion cells (RGCs). L-AP4 decreased circuit noise evident in RGC recordings without significantly reducing response amplitudes, an effect that persisted over the entire time course of rod photoreceptor degeneration. Subsequent in vitro recordings from rod bipolar cells (RBCs) showed that RBCs are more depolarized in rd10 retinas, likely contributing to downstream circuit noise and reduced synaptic gain, both of which appear to be ameliorated by hyperpolarizing RBCs with L-AP4. These beneficial effects may reduce pathological circuit remodeling and preserve the efficacy of therapies designed to restore vision.

Layers of inhibitory networks shape receptive field properties of AII amacrine cells.

In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways via different bipolar cells, enabling AIIs to operate at night and during the day. Previous work has examined luminance-dependent changes in AII gap junction connectivity, but less is known about how surrounding circuitry shapes AII receptive fields across light levels. Here, we report that moderate contrast stimuli elicit surround inhibition in AIIs under all but the dimmest visual conditions, due to actions of horizontal cells and at least two ACs that inhibit presynaptic bipolar cells. Under photopic (daylight) conditions, surround inhibition transforms AII response kinetics, which are inherited by downstream ganglion cells. Ablating neuronal nitric oxide synthase type-1 (nNOS-1) ACs removes AII surround inhibition under mesopic (dusk/dawn), but not photopic, conditions. Our findings demonstrate how multiple layers of neural circuitry interact to encode signals across a wide physiological range.

Sensory deprivation arrests cellular and synaptic development of the night-vision circuitry in the retina.

Experience regulates synapse formation and function across sensory circuits. How inhibitory synapses in the mammalian retina are sculpted by visual cues remains unclear. By use of a sensory deprivation paradigm, we find that visual cues regulate maturation of two GABA synapse types (GABAA and GABAC receptor synapses), localized across the axon terminals of rod bipolar cells (RBCs)-second-order retinal neurons integral to the night-vision circuit. Lack of visual cues causes GABAA synapses at RBC terminals to retain an immature receptor configuration with slower response profiles and prevents receptor recruitment at GABAC synapses. Additionally, the organizing protein for both these GABA synapses, LRRTM4, is not clustered at dark-reared RBC synapses. Ultrastructurally, the total number of ribbon-output/inhibitory-input synapses across RBC terminals remains unaltered by sensory deprivation, although ribbon synapse output sites are misarranged when the circuit develops without visual cues. Intrinsic electrophysiological properties of RBCs and expression of chloride transporters across RBC terminals are additionally altered by sensory deprivation. Introduction to normal 12-h light-dark housing conditions facilitates maturation of dark-reared RBC GABA synapses and restoration of intrinsic RBC properties, unveiling a new element of light-dependent retinal cellular and synaptic plasticity.

Sources of Calcium at Connexin 36 Gap Junctions in the Retina.

Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.

Unpaired mesh-to-image translation for 3D fluorescent microscopy images of neurons.

While Generative Adversarial Networks (GANs) can now reliably produce realistic images in a multitude of imaging domains, they are ill-equipped to model thin, stochastic textures present in many large 3D fluorescent microscopy (FM) images acquired in biological research. This is especially problematic in neuroscience where the lack of ground truth data impedes the development of automated image analysis algorithms for neurons and neural populations. We therefore propose an unpaired mesh-to-image translation methodology for generating volumetric FM images of neurons from paired ground truths. We start by learning unique FM styles efficiently through a Gramian-based discriminator. Then, we stylize 3D voxelized meshes of previously reconstructed neurons by successively generating slices. As a result, we effectively create a synthetic microscope and can acquire realistic FM images of neurons with control over the image content and imaging configurations. We demonstrate the feasibility of our architecture and its superior performance compared to state-of-the-art image translation architectures through a variety of texture-based metrics, unsupervised segmentation accuracy, and an expert opinion test. In this study, we use 2 synthetic FM datasets and 2 newly acquired FM datasets of retinal neurons.

Dendro-somatic synaptic inputs to ganglion cells contradict receptive field and connectivity conventions in the mammalian retina.

The morphology of retinal neurons strongly influences their physiological function. Ganglion cell (GC) dendrites ramify in distinct strata of the inner plexiform layer (IPL) so that GCs responding to light increments (ON) or decrements (OFF) receive appropriate excitatory inputs. This vertical stratification prescribes response polarity and ensures consistent connectivity between cell types, whereas the lateral extent of GC dendritic arbors typically dictates receptive field (RF) size. Here, we identify circuitry in mouse retina that contradicts these conventions. AII amacrine cells are interneurons understood to mediate "crossover" inhibition by relaying excitatory input from the ON layer to inhibitory outputs in the OFF layer. Ultrastructural and physiological analyses show, however, that some AIIs deliver powerful inhibition to OFF GC somas and proximal dendrites in the ON layer, rendering the inhibitory RFs of these GCs smaller than their dendritic arbors. This OFF pathway, avoiding entirely the OFF region of the IPL, challenges several tenets of retinal circuitry. These results also indicate that subcellular synaptic organization can vary within a single population of neurons according to their proximity to potential postsynaptic targets.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

A high-density narrow-field inhibitory retinal interneuron with direct coupling to Müller glia.

Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation and neurovascular control. Here, we report that in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Müller glia, leading us to suggest the name "Müller glia-coupled amacrine cell," or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus (dLGN) showed selective connections between MACs and a subpopulation of RGC types. Visually-evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.Significance Statement:Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.

A17 Amacrine Cells and Olfactory Granule Cells: Parallel Processors of Early Sensory Information.

Neurons typically receive synaptic input in their dendritic arbor, integrate inputs in their soma, and send output action potentials through their axon, following Cajal's law of dynamic polarization. Two notable exceptions are retinal amacrine cells and olfactory granule cells (GCs), which flout Cajal's edict by providing synaptic output from the same dendrites that collect synaptic input. Amacrine cells, a diverse cell class comprising >60 subtypes, employ various dendritic input/output strategies, but A17 amacrine cells (A17s) in particular share further interesting functional characteristics with GCs: both receive excitatory synaptic input from neurons in the primary glutamatergic pathway and return immediate, reciprocal feedback via GABAergic inhibitory synapses to the same synaptic terminals that provided input. Both neurons thereby process signals locally within their dendrites, shaping many parallels, signaling pathways independently. The similarities between A17s and GCs cast into relief striking differences that may indicate distinct processing roles within their respective circuits: First, they employ partially dissimilar molecular mechanisms to transform excitatory input into inhibitory output; second, GCs fire action potentials, whereas A17s do not. Third, GC signals may be influenced by cortical feedback, whereas the mammalian retina receives no such retrograde input. Finally, A17s constitute just one subtype within a diverse class that is specialized in a particular task, whereas the more homogeneous GCs may play more diverse signaling roles via multiple processing modes. Here, we review these analogies and distinctions between A17 amacrine cells and granule cells, hoping to gain further insight into the operating principles of these two sensory circuits.

Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake.

Genetically encoded fluorescent glutamate indicators (iGluSnFRs) enable neurotransmitter release and diffusion to be visualized in intact tissue. Synaptic iGluSnFR signal time courses vary widely depending on experimental conditions, often lasting 10-100 times longer than the extracellular lifetime of synaptically released glutamate estimated with uptake measurements. iGluSnFR signals typically also decay much more slowly than the unbinding kinetics of the indicator. To resolve these discrepancies, here we have modeled synaptic glutamate diffusion, uptake and iGluSnFR activation to identify factors influencing iGluSnFR signal waveforms. Simulations suggested that iGluSnFR competes with transporters to bind synaptically released glutamate, delaying glutamate uptake. Accordingly, synaptic transporter currents recorded from iGluSnFR-expressing astrocytes in mouse cortex were slower than those in control astrocytes. Simulations also suggested that iGluSnFR reduces free glutamate levels in extrasynaptic spaces, likely limiting extrasynaptic receptor activation. iGluSnFR and lower affinity variants, nonetheless, provide linear indications of vesicle release, underscoring their value for optical quantal analysis.

Ganglion Cells in Primate Retina Use Fuzzy Logic to Encode Complex Visual Receptive Fields.

In most neurons, all spikes look alike. However, in this issue of Neuron, Rhoades et al. (2019) describe a ganglion cell in primate retina that reports visual input to different regions of its receptive field with distinct action potential waveforms.

Synaptic inhibition tunes contrast computation in the retina.

Inhibition shapes activity and signal processing in neural networks through numerous mechanisms mediated by many different cell types. Here, we examined how one type of GABAergic interneuron in the retina, the A17 amacrine cell, influences visual information processing. Our results suggest that A17s, which make reciprocal feedback inhibitory synapses onto rod bipolar cell (RBC) synaptic terminals, extend the luminance range over which RBC synapses compute temporal contrast and enhance the reliability of contrast signals over this range. Inhibition from other amacrine cells does not influence these computational features. Although A17-mediated feedback is mediated by both GABAA and GABAC receptors, the latter plays the primary role in extending the range of contrast computation. These results identify specific functions for an inhibitory interneuron subtype, as well as specific synaptic receptors, in a behaviorally relevant neural computation.

How we see the forest and the trees.

Signaling pathways in the retina help us see spatial detail in our visual world.

Synaptic Transfer between Rod and Cone Pathways Mediated by AII Amacrine Cells in the Mouse Retina.

To understand computation in a neural circuit requires a complete synaptic connectivity map and a thorough grasp of the information-processing tasks performed by the circuit. Here, we dissect a microcircuit in the mouse retina in which scotopic visual information (i.e., single photon events, luminance, contrast) is encoded by rod bipolar cells (RBCs) and distributed to parallel ON and OFF cone bipolar cell (CBC) circuits via the AII amacrine cell, an inhibitory interneuron. Serial block-face electron microscopy (SBEM) reconstructions indicate that AIIs preferentially connect to one OFF CBC subtype (CBC2); paired whole-cell patch-clamp recordings demonstrate that, depending on the level of network activation, AIIs transmit distinct components of synaptic input from single RBCs to downstream ON and OFF CBCs. These findings highlight specific synaptic and circuit-level features that allow intermediate neurons (e.g., AIIs) within a microcircuit to filter and propagate information to downstream neurons.

Functional Compartmentalization within Starburst Amacrine Cell Dendrites in the Retina.

Dendrites in many neurons actively compute information. In retinal starburst amacrine cells, transformations from synaptic input to output occur within individual dendrites and mediate direction selectivity, but directional signal fidelity at individual synaptic outputs and correlated activity among neighboring outputs on starburst dendrites have not been examined systematically. Here, we record visually evoked calcium signals simultaneously at many individual synaptic outputs within single starburst amacrine cells in mouse retina. We measure visual receptive fields of individual output synapses and show that small groups of outputs are functionally compartmentalized within starburst dendrites, creating distinct computational units. Inhibition enhances compartmentalization and directional tuning of individual outputs but also decreases the signal-to-noise ratio. Simulations suggest, however, that the noise underlying output signal variability is well tolerated by postsynaptic direction-selective ganglion cells, which integrate convergent inputs to acquire reliable directional information.

Inhibitory Interneurons in the Retina: Types, Circuitry, and Function.

Visual signals in the vertebrate retina are shaped by feedback and feedforward inhibition in two synaptic layers. In one, horizontal cells establish fundamental center-surround receptive-field properties via morphologically and physiologically complex synapses with photoreceptors and bipolar cells. In the other, a panoply of amacrine cells imbue ganglion cell responses with spatiotemporally complex information about the visual world. Here, I review current ideas about horizontal cell signaling, considering the evidence for and against the leading, competing theories. I also discuss recent work that has begun to make sense of the remarkable morphological and physiological diversity of amacrine cells. These latter efforts have been aided tremendously by increasingly complete connectivity maps of inner retinal circuitry and new genetic tools that enable study of individual, sparsely expressed amacrine cell types.

Species-specific wiring for direction selectivity in the mammalian retina.

Directionally tuned signalling in starburst amacrine cell (SAC) dendrites lies at the heart of the circuit that detects the direction of moving stimuli in the mammalian retina. The relative contributions of intrinsic cellular properties and network connectivity to SAC direction selectivity remain unclear. Here we present a detailed connectomic reconstruction of SAC circuitry in mouse retina and describe two previously unknown features of synapse distributions along SAC dendrites: input and output synapses are segregated, with inputs restricted to proximal dendrites; and the distribution of inhibitory inputs is fundamentally different from that observed in rabbit retina. An anatomically constrained SAC network model suggests that SAC­SAC wiring differences between mouse and rabbit retina underlie distinct contributions of synaptic inhibition to velocity and contrast tuning and receptive field structure. In particular, the model indicates that mouse connectivity enables SACs to encode lower linear velocities that account for smaller eye diameter, thereby conserving angular velocity tuning. These predictions are confirmed with calcium imaging of mouse SAC dendrites responding to directional stimuli.

Retinal Circuitry Balances Contrast Tuning of Excitation and Inhibition to Enable Reliable Computation of Direction Selectivity.

UNLABELLED: Feedforward (FF) inhibition is a common motif in many neural networks. Typically, excitatory inputs drive both principal neurons and interneurons; the interneurons then inhibit the principal neurons, thereby regulating the strength and timing of the FF signal. The interneurons introduce a likely nonlinear processing step that could distort the excitation/inhibition (E/I) ratio in the principal neuron, potentially degrading the reliability of computation in the circuit. In the retina, FF inhibition is an essential feature of the circuitry underlying direction selectivity (DS): glutamatergic bipolar cells (BCs) provide excitatory input to direction-selective ganglion cells (DSGCs) and GABAergic starburst amacrine cells (SACs), and the SACs then provide FF inhibition onto DSGCs. Robust DS computation requires a consistent synaptic E/I ratio in the DSGC in various visual conditions. Here, we show in mouse retina that the E/I ratio is maintained in DSGCs over a wide stimulus contrast range due to compensatory mechanisms in the diverse population of presynaptic BCs. BC inputs to SACs exhibit higher contrast sensitivity, so that the subsequent nonlinear transformation in SACs reduces the contrast sensitivity of FF inhibition to match the sensitivity of direct excitatory inputs onto DSGCs. Measurements of light-evoked responses from individual BC synaptic terminals suggest that the distinct sensitivity of BC inputs reflects different contrast sensitivity between BC subtypes. Numerical simulations suggest that this network arrangement is crucial for reliable DS computation. SIGNIFICANCE STATEMENT: Properly balanced excitation and inhibition are essential for many neuronal computations across brain regions. Feedforward inhibition circuitry, in which a common excitatory source drives both the principal cell and an interneuron, is a typical mechanism by which neural networks maintain this balance. Feedforward circuits may become imbalanced at low stimulation levels, however, if the excitatory drive is too weak to overcome the activation threshold in the interneuron. Here we reveal how excitation and inhibition remain balanced in direction selective ganglion cells in the mouse retina over a wide visual stimulus range.

Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.