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. Significance Statement: Retinitis Pigmentosa (RP) is an inherited degenerative disease that affects more than two million people worldwide. RP patients first lose peripheral and low-light vision due to the progressive death of their highly sensitive rod photoreceptors. Photoreceptor degeneration induces pathological noise within the retinal circuit, leading to dramatic structural changes that may hamper therapies to restore visual sensitivity. We discovered a pharmacological treatment that reduces pathological activity in a mouse model of RP without diminishing signaling in surviving circuitry. Partially replacing the neurotransmitter lost when photoreceptors die reduced noise in the retinal circuit without eliminating light sensitivity. This approach could limit the impact of the disease on retinal neurons and preserve the efficacy of subsequent restorative therapies.

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.

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.

Transient expression of a GABA receptor subunit during early development is critical for inhibitory synapse maturation and function.

Developing neural circuits, including GABAergic circuits, switch receptor types. But the role of early GABA receptor expression for establishment of functional inhibitory circuits remains unclear. Tracking the development of GABAergic synapses across axon terminals of retinal bipolar cells (BCs), we uncovered a crucial role of early GABAA receptor expression for the formation and function of presynaptic inhibitory synapses. Specifically, early α3-subunit-containing GABAA (GABAAα3) receptors are a key developmental organizer. Before eye opening, GABAAα3 gives way to GABAAα1 at individual BC presynaptic inhibitory synapses. The developmental downregulation of GABAAα3 is independent of GABAAα1 expression. Importantly, lack of early GABAAα3 impairs clustering of GABAAα1 and formation of functional GABAA synapses across mature BC terminals. This impacts the sensitivity of visual responses transmitted through the circuit. Lack of early GABAAα3 also perturbs aggregation of LRRTM4, the organizing protein at GABAergic synapses of rod BC terminals, and their arrangement of output ribbon synapses.

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.

Range, routing and kinetics of rod signaling in primate retina.

Stimulus- or context-dependent routing of neural signals through parallel pathways can permit flexible processing of diverse inputs. For example, work in mouse shows that rod photoreceptor signals are routed through several retinal pathways, each specialized for different light levels. This light-level-dependent routing of rod signals has been invoked to explain several human perceptual results, but it has not been tested in primate retina. Here, we show, surprisingly, that rod signals traverse the primate retina almost exclusively through a single pathway - the dedicated rod bipolar pathway. Identical experiments in mouse and primate reveal substantial differences in how rod signals traverse the retina. These results require reevaluating human perceptual results in terms of flexible computation within this single pathway. This includes a prominent speeding of rod signals with light level - which we show is inherited directly from the rod photoreceptors themselves rather than from different pathways with distinct kinetics.

Parallel Processing of Rod and Cone Signals: Retinal Function and Human Perception.

We know a good deal about the operation of the retina when either rod or cone photoreceptors provide the dominant input (i.e., under very dim or very bright conditions). However, we know much less about how the retina operates when rods and cones are coactive (i.e., under intermediate lighting conditions, such as dusk). Such mesopic conditions span 20-30% of the light levels over which vision operates and encompass many situations in which vision is essential (e.g., driving at night). These lighting conditions are challenging because rod and cone signals differ substantially: Rod responses are nearing saturation, while cone responses are weak and noisy. A rich history of perceptual studies guides our investigation of how the retina operates under mesopic conditions and in doing so provides a powerful opportunity to link general issues about parallel processing in neural circuits with computation and perception. We review some of the successes and challenges in understanding the retinal basis of perceptual rod-cone interactions.

Flexible Neural Hardware Supports Dynamic Computations in Retina.

The ability of the retina to adapt to changes in mean light intensity and contrast is well known. Classically, however, adaptation is thought to affect gain but not to change the visual modality encoded by a given type of retinal neuron. Recent findings reveal unexpected dynamic properties in mouse retinal neurons that challenge this view. Specifically, certain cell types change the visual modality they encode with variations in ambient illumination or following repetitive visual stimulation. These discoveries demonstrate that computations performed by retinal circuits with defined architecture can change with visual input. Moreover, they pose a major challenge for central circuits that must decode properties of the dynamic visual signal from retinal outputs.

Stimulation of functional neuronal regeneration from Müller glia in adult mice.

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.

A simple retinal mechanism contributes to perceptual interactions between rod- and cone-mediated responses in primates.

Visual perception across a broad range of light levels is shaped by interactions between rod- and cone-mediated signals. Because responses of retinal ganglion cells, the output cells of the retina, depend on signals from both rod and cone photoreceptors, interactions occurring in retinal circuits provide an opportunity to link the mechanistic operation of parallel pathways and perception. Here we show that rod- and cone-mediated responses interact nonlinearly to control the responses of primate retinal ganglion cells; these nonlinear interactions, surprisingly, were asymmetric, with rod responses strongly suppressing subsequent cone responses but not vice-versa. Human psychophysical experiments revealed a similar perceptual asymmetry. Nonlinear interactions in the retinal output cells were well-predicted by linear summation of kinetically-distinct rod- and cone-mediated signals followed by a synaptic nonlinearity. These experiments thus reveal how a simple mechanism controlling interactions between parallel pathways shapes circuit output and perception.

Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.

Neuronal microcircuits, small, localized signaling motifs involving two or more neurons, underlie signal processing and computation in the brain. Compartmentalized signaling within a neuron may enable it to participate in multiple, independent microcircuits. Each A17 amacrine cell in the mammalian retina contains within its dendrites hundreds of synaptic feedback microcircuits that operate independently to modulate feedforward signaling in the inner retina. Each of these microcircuits comprises a small (<1 µm) synaptic varicosity that typically receives one excitatory synapse from a presynaptic rod bipolar cell (RBC) and returns two reciprocal inhibitory synapses back onto the same RBC terminal. Feedback inhibition from the A17 sculpts the feedforward signal from the RBC to the AII, a critical component of the circuitry mediating night vision. Here, we show that the two inhibitory synapses from the A17 to the RBC express kinetically distinct populations of GABA receptors: rapidly activating GABA(A)Rs are enriched at one synapse while more slowly activating GABA(C)Rs are enriched at the other. Anatomical and electrophysiological data suggest that macromolecular complexes of voltage-gated (Cav) channels and Ca(2+)-activated K(+) channels help to regulate GABA release from A17 varicosities and limit GABA(C)R activation under certain conditions. Finally, we find that selective elimination of A17-mediated feedback inhibition reduces the signal to noise ratio of responses to dim flashes recorded in the feedforward pathway (i.e., the AII amacrine cell). We conclude that A17-mediated feedback inhibition improves the signal to noise ratio of RBC-AII transmission near visual threshold, thereby improving visual sensitivity at night.

Cross-synaptic synchrony and transmission of signal and noise across the mouse retina.

Cross-synaptic synchrony--correlations in transmitter release across output synapses of a single neuron--is a key determinant of how signal and noise traverse neural circuits. The anatomical connectivity between rod bipolar and A17 amacrine cells in the mammalian retina, specifically that neighboring A17s often receive input from many of the same rod bipolar cells, provides a rare technical opportunity to measure cross-synaptic synchrony under physiological conditions. This approach reveals that synchronization of rod bipolar cell synapses is near perfect in the dark and decreases with increasing light level. Strong synaptic synchronization in the dark minimizes intrinsic synaptic noise and allows rod bipolar cells to faithfully transmit upstream signal and noise to downstream neurons. Desynchronization in steady light lowers the sensitivity of the rod bipolar output to upstream voltage fluctuations. This work reveals how cross-synaptic synchrony shapes retinal responses to physiological light inputs and, more generally, signaling in complex neural networks.

Specialized postsynaptic morphology enhances neurotransmitter dilution and high-frequency signaling at an auditory synapse.

Sensory processing in the auditory system requires that synapses, neurons, and circuits encode information with particularly high temporal and spectral precision. In the amphibian papillia, sound frequencies up to 1 kHz are encoded along a tonotopic array of hair cells and transmitted to afferent fibers via fast, repetitive synaptic transmission, thereby promoting phase locking between the presynaptic and postsynaptic cells. Here, we have combined serial section electron microscopy, paired electrophysiological recordings, and Monte Carlo diffusion simulations to examine novel mechanisms that facilitate fast synaptic transmission in the inner ear of frogs (Rana catesbeiana and Rana pipiens). Three-dimensional anatomical reconstructions reveal specialized spine-like contacts between individual afferent fibers and hair cells that are surrounded by large, open regions of extracellular space. Morphologically realistic diffusion simulations suggest that these local enlargements in extracellular space speed transmitter clearance and reduce spillover between neighboring synapses, thereby minimizing postsynaptic receptor desensitization and improving sensitivity during prolonged signal transmission. Additionally, evoked EPSCs in afferent fibers are unaffected by glutamate transporter blockade, suggesting that transmitter diffusion and dilution, and not uptake, play a primary role in speeding neurotransmission and ensuring fidelity at these synapses.

The synaptic and circuit mechanisms underlying a change in spatial encoding in the retina.

Components of neural circuits are often repurposed so that the same biological hardware can be used for distinct computations. This flexibility in circuit operation is required to account for the changes in sensory computations that accompany changes in input signals. Yet we know little about how such changes in circuit operation are implemented. Here we show that a single retinal ganglion cell performs a different computation in dim light--averaging contrast within its receptive field--than in brighter light, when the cell becomes sensitive to fine spatial detail. This computational change depends on interactions between two parallel circuits that control the ganglion cell's excitatory synaptic inputs. Specifically, steady-state interactions through dendro-axonal gap junctions control rectification of the synapses providing excitatory input to the ganglion cell. These findings provide a clear example of how a simple synaptic mechanism can repurpose a neural circuit to perform diverse computations.

Amacrine cell-mediated input to bipolar cells: variations on a common mechanistic theme.

Feedback is a ubiquitous feature of neural circuits in the mammalian central nervous system (CNS). Analogous to pure electronic circuits, neuronal feedback provides either a positive or negative influence on the output of upstream components/neurons. Although the particulars (i.e., connectivity, physiological encoding/processing/signaling) of circuits in higher areas of the brain are often unclear, the inner retina proves an excellent model for studying both the anatomy and physiology of feedback circuits within the functional context of visual processing. Inner retinal feedback to bipolar cells is almost entirely mediated by a single class of interneurons, the amacrine cells. Although this might sound like a simple circuit arrangement with an equally simple function, anatomical, molecular, and functional evidence suggest that amacrine cells represent an extremely diverse class of CNS interneurons that contribute to a variety of retinal processes. In this review, I classify the amacrine cells according to their anatomical output synapses and target cell(s) (i.e., bipolar cells, ganglion cells, and/or amacrine cells) and discuss specifically our current understandings of amacrine cell-mediated feedback and output to bipolar cells on the synaptic, cellular, and circuit levels, while drawing connections to visual processing.

Genetic targeting and physiological features of VGLUT3+ amacrine cells.

Amacrine cells constitute a diverse class of interneurons that contribute to visual signal processing in the inner retina, but surprisingly, little is known about the physiology of most amacrine cell subtypes. Here, we have taken advantage of the sparse expression of vesicular glutamate transporter 3 (VGLUT3) in the mammalian retina to target the expression of yellow fluorescent protein (YFP) to a unique population of amacrine cells using a new transgenic mouse line. Electrophysiological recordings made from YFP-positive (VGLUT3+) amacrine cells provide the first functional data regarding the active membrane properties and synaptic connections of this recently identified cell type. We found that VGLUT3+ amacrine cells receive direct synaptic input from bipolar cells via both N-methyl-d-aspartate receptors (NMDARs) and non-NMDARs. Voltage-gated sodium channels amplified these excitatory inputs but repetitive spiking was never observed. VGLUT3+ amacrine cells responded transiently to both light increments (ON response) and decrements (OFF response); ON responses consisted exclusively of inhibitory inputs, while OFF responses comprised both excitatory and inhibitory components, although the inhibitory conductance was larger in amplitude and longer in time course. The physiological properties and anatomical features of the VGLUT3+ amacrine cells suggest that this bistratified interneuron may play a role in disinhibitory signaling and/or crossover inhibition between parallel pathways in the retina.

Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration.

Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.

Retinal parallel processors: more than 100 independent microcircuits operate within a single interneuron.

Most neurons are highly polarized cells with branched dendrites that receive and integrate synaptic inputs and extensive axons that deliver action potential output to distant targets. By contrast, amacrine cells, a diverse class of inhibitory interneurons in the inner retina, collect input and distribute output within the same neuritic network. The extent to which most amacrine cells integrate synaptic information and distribute their output is poorly understood. Here, we show that single A17 amacrine cells provide reciprocal feedback inhibition to presynaptic bipolar cells via hundreds of independent microcircuits operating in parallel. The A17 uses specialized morphological features, biophysical properties, and synaptic mechanisms to isolate feedback microcircuits and maximize its capacity to handle many independent processes. This example of a neuron employing distributed parallel processing rather than spatial integration provides insights into how unconventional neuronal morphology and physiology can maximize network function while minimizing wiring cost.