Direction-selective Retinal Ganglion Cells Research Articles

Differential Expression Analysis Identifies Candidate Synaptogenic Molecules for Wiring Direction-Selective Circuits in the Retina.

An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.

Retinal origin of orientation but not direction selective maps in the superior colliculus

Neurons in the mouse superior colliculus ("colliculus") are arranged in ordered spatial maps. While orientation-selective (OS) neurons form a concentric map aligned to the center of vision, direction-selective (DS) neurons are arranged in patches with changing preferences across the visual field. It remains unclear whether these maps are a consequence of feedforward input from the retina or local computations in the colliculus. To determine whether these maps originate in the retina, we mapped the local and global distribution of OS and DS retinal ganglion cell axon boutons using invivo two-photon calcium imaging. We found that OS boutons formed patches that matched the distribution of OS neurons within the colliculus. DS boutons displayed fewer regional specializations, better reflecting the organization of DS neurons in the retina. Both eyes convey similar orientation but different DS inputs to the colliculus, as shown in recordings from retinal explants. These data demonstrate that orientation and direction maps within the colliculus are independent, where orientation maps are likely inherited from the retina, but direction maps require additional computations.

GABAergic Inhibition Controls Receptive Field Size, Sensitivity, and Contrast Preference of Direction Selective Retinal Ganglion Cells Near the Threshold of Vision.

Information about motion is encoded by direction-selective retinal ganglion cells (DSGCs). These cells reliably transmit this information across a broad range of light levels, spanning moonlight to sunlight. Previous work indicates that adaptation to low light levels causes heterogeneous changes to the direction tuning of ON-OFF (oo)DSGCs and suggests that superior-preferring ON-OFF DSGCs (s-DSGCs) are biased toward detecting stimuli rather than precisely signaling direction. Using a large-scale multielectrode array, we measured the absolute sensitivity of ooDSGCs and found that s-DSGCs are 10-fold more sensitive to dim flashes of light than other ooDSGCs. We measured their receptive field (RF) sizes and found that s-DSGCs also have larger receptive fields than other ooDSGCs; however, the size difference does not fully explain the sensitivity difference. Using a conditional knock-out of gap junctions and pharmacological manipulations, we demonstrate that GABA-mediated inhibition contributes to the difference in absolute sensitivity and receptive field size at low light levels, while the connexin36-mediated gap junction coupling plays a minor role. We further show that under scotopic conditions, ooDSGCs exhibit only an ON response, but pharmacologically removing GABA-mediated inhibition unmasks an OFF response. These results reveal that GABAergic inhibition controls and differentially modulates the responses of ooDSGCs under scotopic conditions.

A circuit suppressing retinal drive to the optokinetic system during fast image motion

Optokinetic nystagmus (OKN) assists stabilization of the retinal image during head rotation. OKN is driven by ON direction selective retinal ganglion cells (ON DSGCs), which encode both the direction and speed of global retinal slip. The synaptic circuits responsible for the direction selectivity of ON DSGCs are well understood, but those sculpting their slow-speed preference remain enigmatic. Here, we probe this mechanism in mouse retina through patch clamp recordings, functional imaging, genetic manipulation, and electron microscopic reconstructions. We confirm earlier evidence that feedforward glycinergic inhibition is the main suppressor of ON DSGC responses to fast motion, and reveal the source for this inhibition—the VGluT3 amacrine cell, a dual neurotransmitter, excitatory/inhibitory interneuron. Together, our results identify a role for VGluT3 cells in limiting the speed range of OKN. More broadly, they suggest VGluT3 cells shape the response of many retinal cell types to fast motion, suppressing it in some while enhancing it in others.

Asymmetric retinal direction tuning predicts optokinetic eye movements across stimulus conditions.

Across species, the optokinetic reflex (OKR) stabilizes vision during self-motion. OKR occurs when ON direction-selective retinal ganglion cells (oDSGCs) detect slow, global image motion on the retina. How oDSGC activity is integrated centrally to generate behavior remains unknown. Here, we discover mechanisms that contribute to motion encoding in vertically tuned oDSGCs and leverage these findings to empirically define signal transformation between retinal output and vertical OKR behavior. We demonstrate that motion encoding in vertically tuned oDSGCs is contrast-sensitive and asymmetric for oDSGC types that prefer opposite directions. These phenomena arise from the interplay between spike threshold nonlinearities and differences in synaptic input weights, including shifts in the balance of excitation and inhibition. In behaving mice, these neurophysiological observations, along with a central subtraction of oDSGC outputs, accurately predict the trajectories of vertical OKR across stimulus conditions. Thus, asymmetric tuning across competing sensory channels can critically shape behavior.

The transcription factor Tbx5 regulates direction-selective retinal ganglion cell development and image stabilization

The diversity of visual input processed by the mammalian visual system requires the generation of many distinct retinal ganglion cell (RGC) types, each tuned to a particular feature. The molecular code needed to generate this cell-type diversity is poorly understood. Here, we focus on the molecules needed to specify one type of retinal cell: the upward-preferring ON direction-selective ganglion cell (up-oDSGC) of the mouse visual system. Single-cell transcriptomic profiling of up- and down-oDSGCs shows that the transcription factor Tbx5 is selectively expressed in up-oDSGCs. The loss of Tbx5 in up-oDSGCs results in a selective defect in the formation of up-oDSGCs and a corresponding inability to detect vertical motion. A downstream effector of Tbx5, Sfrp1, is also critical for vertical motion detection but not up-oDSGC formation. These results advance our understanding of the molecular mechanisms that specify a rare retinal cell type and show how disrupting this specification leads to a corresponding defect in neural circuitry and behavior.

Characterization of non-alpha retinal ganglion cell injury responses reveals a possible block to restoring ipRGC function

Visual impairment caused by retinal ganglion cell (RGC) axon damage or degeneration affects millions of individuals throughout the world. While some progress has been made in promoting long-distance RGC axon regrowth following injury, it remains unclear whether RGC axons can properly reconnect with their central targets to restore visual function. Additionally, the regenerative capacity of many RGC subtypes remains unknown in part due to a lack of available genetic tools. Here, we use a new mouse line, Sema6ACreERT2, that labels On direction-selective RGCs (oDSGCs) and characterize the survival and regenerative potential of these cells following optic nerve crush (ONC). In parallel, we use a previously characterized mouse line, Opn4CreERT2, to answer these same questions for M1 intrinsically photosensitive RGCs (ipRGCs). We find that both M1 ipRGCs and oDSGCs are resilient to injury but do not display long-distance axon regrowth following Lin28a overexpression. Unexpectedly, we found that M1 ipRGC, but not oDSGC, intraretinal axons exhibit ectopic branching and are misaligned near the optic disc between one- and three-weeks following injury. Additionally, we observe that numerous ectopic presynaptic specializations associate with misguided ipRGC intraretinal axons. Taken together, these results reveal insights into the injury response of M1 ipRGCs and oDSGCs, providing a foundation for future efforts seeking to restore visual system function following injury.

Conserved circuits for direction selectivity in the primate retina

The detection of motion direction is a fundamental visual function and a classic model for neural computation. In the non-primate retina, direction selectivity arises in starburst amacrine cell (SAC) dendrites, which provide selective inhibition to direction-selective retinal ganglion cells (dsRGCs). Although SACs are present in primates, their connectivity and the existence of dsRGCs remain open questions. Here, we present a connectomic reconstruction of the primate ON SAC circuit from a serial electron microscopy volume of the macaque central retina. We show that the structural basis for the SACs' ability to confer directional selectivity on postsynaptic neurons is conserved. SACs selectively target a candidate homolog to the mammalian ON-sustained dsRGCs that project to the accessory optic system (AOS) and contribute to gaze-stabilizing reflexes. These results indicate that the capacity to compute motion direction is present in the retina, which is earlier in the primate visual system than classically thought.

FLRT3 Marks Direction-Selective Retinal Ganglion Cells That Project to the Medial Terminal Nucleus.

The mammalian retina extracts a multitude of diverse features from the visual scene such as color, contrast, and direction of motion. These features are transmitted separately to the brain by more than 40 different retinal ganglion cell (RGC) subtypes. However, so far only a few genetic markers exist to fully characterize the different RGC subtypes. Here, we present a novel genetic Flrt3-CreERT2 knock-in mouse that labels a small subpopulation of RGCs. Using single-cell injection of fluorescent dyes in Flrt3 positive RGCs, we distinguished four morphological RGC subtypes. Anterograde tracings using a fluorescent Cre-dependent Adeno-associated virus (AAV) revealed that a subgroup of Flrt3 positive RGCs specifically project to the medial terminal nucleus (MTN), which is part of the accessory optic system (AOS) and is essential in driving reflex eye movements for retinal image stabilization. Functional characterization using ex vivo patch-clamp recordings showed that the MTN-projecting Flrt3 RGCs preferentially respond to downward motion in an ON-fashion. These neurons distribute in a regular pattern and most of them are bistratified at the level of the ON and OFF bands of cholinergic starburst amacrine cells where they express the known ON-OFF direction-selective RGC marker CART. Together, our results indicate that MTN-projecting Flrt3 RGCs represent a new functionally homogeneous AOS projecting direction-selective RGC subpopulation.

Identification of long noncoding RNAs in injury-resilient and injury-susceptible mouse retinal ganglion cells

BackgroundEmerging evidence indicates that long noncoding RNAs (lncRNAs) are important regulators of various biological processes, and their expression can be altered following certain pathological conditions, including central nervous system injury. Retinal ganglion cells (RGCs), whose axons form the optic nerve, are a heterogeneous population of neurons with more than 40 molecularly distinct subtypes in mouse. While most RGCs, including the ON-OFF direction-selective RGCs (ooDSGCs), are vulnerable to axonal injury, a small population of RGCs, including the intrinsically photosensitive RGCs (ipRGCs), are more resilient.ResultsBy performing systematic analyses on RNA-sequencing data, here we identify lncRNAs that are expressed in ooDSGCs and ipRGCs with and without axonal injury. Our results reveal a repertoire of different classes of lncRNAs, including long intergenic noncoding RNAs and antisense ncRNAs that are differentially expressed between these RGC types. Strikingly, we also found dozens of lncRNAs whose expressions are altered markedly in response to axonal injury, some of which are expressed exclusively in either one of the types. Moreover, analyses into these lncRNAs unraveled their neighboring coding genes, many of which encode transcription factors and signaling molecules, suggesting that these lncRNAs may act in cis to regulate important biological processes in these neurons. Lastly, guilt-by-association analysis showed that lncRNAs are correlated with apoptosis associated genes, suggesting potential roles for these lncRNAs in RGC survival.ConclusionsOverall, the results of this study reveal RGC type-specific expression of lncRNAs and provide a foundation for future investigation of the function of lncRNAs in regulating neuronal type specification and survival.

Glycogen Synthase Kinase 3 Regulates the Genesis of Displaced Retinal Ganglion Cells3.

Glycogen synthase kinase 3 (GSK3) proteins (GSK3α and GSK3β) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphologic defects. A single wild-type allele of either Gsk3α or Gsk3β is able to rescue this phenotype. In this genetic context, all cell types are present in a functional retina. However, we unexpectedly detected a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele of Gsk3α is expressed. The excess of dRGCs leads to an increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs in Gsk3 mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the production of ganglion cells in the inner nuclear layer, which correspond to dRGCs, a rare and poorly characterized retinal cell type.

Dendrite Morphology Minimally Influences the Synaptic Distribution of Excitation and Inhibition in Retinal Direction-Selective Ganglion Cells.

Throughout the nervous system, the organization of excitatory and inhibitory synaptic inputs within a neuron’s receptive field shapes its output computation. In some cases, multiple motifs of synaptic organization can contribute to a single computation. Here, we compare two of these mechanisms performed by two morphologically distinct retinal direction-selective ganglion cells (DSGCs): directionally tuned inhibition and spatially offset inhibition. Using drifting stimuli, we found that DSGCs that have asymmetric dendrites exhibited stronger directionally tuned inhibition than symmetric DSGCs. Using stationary stimuli to map receptive fields, we found that DSGCs with both symmetric and asymmetric dendrites exhibited similar spatially offset inhibition. Interestingly, we observed that excitatory and inhibitory synapses for both cell types were locally correlated in strength. This result indicates that in the mouse retina, dendritic morphology influences the amount of tuned inhibition attained through asymmetric wiring but does not dictate the synaptic organization of excitation relative to inhibition.

Global Motion Processing by Populations of Direction-Selective Retinal Ganglion Cells.

Simple stimuli have been critical to understanding neural population codes in sensory systems. Yet it remains necessary to determine the extent to which this understanding generalizes to more complex conditions. To examine this problem, we measured how populations of direction-selective ganglion cells (DSGCs) from the retinas of male and female mice respond to a global motion stimulus with its direction and speed changing dynamically. We then examined the encoding and decoding of motion direction in both individual and populations of DSGCs. Individual cells integrated global motion over ∼200 ms, and responses were tuned to direction. However, responses were sparse and broadly tuned, which severely limited decoding performance from small DSGC populations. In contrast, larger populations compensated for response sparsity, enabling decoding with high temporal precision (<100 ms). At these timescales, correlated spiking was minimal and had little impact on decoding performance, unlike results obtained using simpler local motion stimuli decoded over longer timescales. We use these data to define different DSGC population decoding regimes that use or mitigate correlated spiking to achieve high-spatial versus high-temporal resolution.SIGNIFICANCE STATEMENT ON-OFF direction-selective ganglion cells (ooDSGCs) in the mammalian retina are typically thought to signal local motion to the brain. However, several recent studies suggest they may signal global motion. Here we analyze the fidelity of encoding and decoding global motion in a natural scene across large populations of ooDSGCs. We show that large populations of DSGCs are capable of signaling rapid changes in global motion.

Early Visual Motion Experience Improves Retinal Encoding of Motion Directions.

Altered sensory experience in early life often leads to altered response properties of the sensory neurons. This process is mostly thought to happen in the brain, not in the sensory organs. We show that in the mouse retina of both sexes, exposed to a motion-dominated visual environment from eye-opening, the ON-OFF direction selective ganglion cells (ooDSGCs) develop significantly stronger direction encoding ability for motion in all directions. This improvement occurs independent of the motion direction used for training. We demonstrated that this enhanced ability to encode motion direction is mainly attributed to increased response reliability of ooDSGCs. Closer examination revealed that the excitatory inputs from the ON bipolar pathway showed enhanced response reliability after the motion experience training, while other synaptic inputs remain relatively unchanged. Our results demonstrate that retina adapts to the visual environment during neonatal development.SIGNIFICANCE STATEMENT We found that retina, as the first stage of visual sensation, can also be affected by experience dependent plasticity during development. Exposure to a motion enriched visual environment immediately after eye-opening greatly improves motion direction encoding by direction selective retinal ganglion cells (RGCs). These results motivate future studies aimed at understanding how visual experience shapes the retinal circuits and the response properties of retinal neurons.

Thrombospondin-1 Promotes Circuit-Specific Synapse Formation Via β1-Integrin

Glial cells regulate synaptic connectivity during development, but whether they selectively instruct the formation of specific synaptic circuits is not known. Here we show that the major perisynaptic glia of the retina, the Muller glia (MG), control the proper establishment of the direction-selective (DS) circuit by a synaptogenic protein, Thrombospondin 1 (TSP1). We found that TSP1 promotes excitatory synapse formation specifically in on-off Direction-Selective retinal Ganglion Cells (ooDSGCs). Lack of TSP1 leads to reduced synapse formation within the inner plexiform sublayers containing DS-circuit, resulting in deficits of ooDSGC function. Even though pan-TSP receptor, α2δ-1, interaction is required for TSP1-induced synapse formation, the ooDSGC-subtype specificity of TSP1 is conferred by a second neuronal TSP1 receptor, β1-Integrin. Furthermore, conditional deletion of β1-Integrin in ooDSGCs results in diminished excitatory synapse formation without disturbing laminar organization showing that MG-secreted TSP1 controls circuit-specific synapse formation via β1-Integrin.

Effect of ocular hypertension on the pattern of retinal ganglion cell subtype loss in a mouse model of early-onset glaucoma

Glaucoma is a neurodegenerative disease with elevated intraocular pressure as one of the major risk factors. Glaucoma leads to irreversible loss of vision and its progression involves optic nerve head cupping, axonal degeneration, retinal ganglion cell (RGC) loss, and visual field defects. Despite its high global prevalence, glaucoma still remains a major neurodegenerative disease. Introduction of mouse models of experimental glaucoma has become integral to glaucoma research due to well-studied genetics as well as ease of manipulations. Many established inherent and inducible mouse models of glaucoma are used to study the molecular and physiological progression of the disease. One such model of spontaneous mutation is the nee model, which is caused by mutation of the Sh3pxd2b gene. In both humans and mice, mutations disrupting function of the SH3PXD2B adaptor protein cause a developmental syndrome including secondary congenital glaucoma. The purpose of this study was to characterize the early onset nee glaucoma phenotype on the C57BL/6J background and to evaluate the pattern of RGC loss and axonal degeneration in specific RGC subtypes. We found that the B6.Sh3pxd2bnee mutant animals exhibit glaucoma phenotypes of elevated intraocular pressure, RGC loss and axonal degeneration. Moreover, the non-image forming RGCs survived longer than the On-Off direction selective RGCs (DSGC), and the axonal death in these RGCs was independent of their respective RGC subtype. In conclusion, through this study we characterized an experimental model of early onset glaucoma on a C57BL/6J background exhibiting key glaucoma phenotypes. In addition, we describe that RGC death has subtype-specific sensitivities and follows a specific pattern of cell death under glaucomatous conditions.

Neuronal Architecture of a Visual Center that Processes Optic Flow

Animals use global image motion cues to actively stabilize their position by compensatory movements. Neurons in the zebrafish pretectum distinguish different optic flow patterns, e.g., rotation and translation, to drive appropriate behaviors. Combining functional imaging and morphological reconstruction of single cells, we revealed critical neuroanatomical features of this sensorimotor transformation. Terminals of direction-selective retinal ganglion cells (DS-RGCs) are located within the pretectal retinal arborization field 5 (AF5), where they meet dendrites of pretectal neurons with simple tuning to monocular optic flow. Translation-selective neurons, which respond selectively to optic flow in the same direction for both eyes, are intermingled with these simple cells but do not receive inputs from DS-RGCs. Mutually exclusive populations of pretectal projection neurons innervate either the reticular formation or the cerebellum, which in turn control motor responses. We posit that local computations in a defined pretectal circuit transform optic flow signals into neural commands driving optomotor behavior. VIDEO ABSTRACT.

Activity Correlations between Direction-Selective Retinal Ganglion Cells Synergistically Enhance Motion Decoding from Complex Visual Scenes

SummaryNeurons in sensory systems are often tuned to particular stimulus features. During complex naturalistic stimulation, however, multiple features may simultaneously affect neuronal responses, which complicates the readout of individual features. To investigate feature representation under complex stimulation, we studied how direction-selective ganglion cells in salamander retina respond to texture motion where direction, velocity, and spatial pattern inside the receptive field continuously change. We found that the cells preserve their direction preference under this stimulation, yet their direction encoding becomes ambiguous due to simultaneous activation by luminance changes. The ambiguities can be resolved by considering populations of direction-selective cells with different preferred directions. This gives rise to synergistic motion decoding, yielding more information from the population than the summed information from single-cell responses. Strong positive response correlations between cells with different preferred directions amplify this synergy. Our results show how correlated population activity can enhance feature extraction in complex visual scenes.

Putative targets of direction-selective retinal ganglion cells in the tectum opticum of cyprinid fish

Responses of direction selective (DS) units of retinal and tectal origin were recorded extracellularly from the tectum opticum (TO) of immobilized fish. The data were collected from three cyprinid species – goldfish, carp and roach. Responses of the retinal DS ganglion cells (GCs) were recorded from their axon terminals in the superficial layers of TO. According to their preferred directions DS GCs, characterized by small receptive fields (3−8°), can be divided in three distinct groups, each group containing ON and OFF subtypes approximately in equal quantity. Conversely, direction-selective tectal neurons (DS TNs), recorded at two different tectal levels deeper than the zone of retinal DS afferents, are characterized by large receptive fields (up to 60°) and are indifferent to any sign of contrast i.e. can be considered as ON-OFF type units. Fish DS TNs unlike the retinal DS GCs, select four preferred directions. Three types of tectal DS units prefer practically the same directions as those already selected on the retinal level – caudo-rostral, dorso-ventral and ventro-dorsal. The fact that three preferred directions of DS GCs and DS TNs coincide allows us to assume that three types of DS GCs are input neurons for corresponding types of DS TNs. The fourth group of DS TNs has the emergent rostro-caudal preference not explicitly present in any of the DS GC inputs. These units are recorded in deep TO layers exclusively. Receptive fields of these DS neurons could be entirely formed on the tectal level. Possible interrelations between retinal and tectal DS units are discussed.

GABA release selectively regulates synapse development at distinct inputs on direction-selective retinal ganglion cells

Synaptic inhibition controls a neuron's output via functionally distinct inputs at two subcellular compartments, the cell body and the dendrites. It is unclear whether the assembly of these distinct inhibitory inputs can be regulated independently by neurotransmission. In the mammalian retina, γ-aminobutyric acid (GABA) release from starburst amacrine cells (SACs) onto the dendrites of on-off direction-selective ganglion cells (ooDSGCs) is essential for directionally selective responses. We found that ooDSGCs also receive GABAergic input on their somata from other amacrine cells (ACs), including ACs containing the vasoactive intestinal peptide (VIP). When net GABAergic transmission is reduced, somatic, but not dendritic, GABAA receptor clusters on the ooDSGC increased in number and size. Correlative fluorescence imaging and serial electron microscopy revealed that these enlarged somatic receptor clusters are localized to synapses. By contrast, selectively blocking vesicular GABA release from either SACs or VIP ACs did not alter dendritic or somatic receptor distributions on the ooDSGCs, showing that neither SAC nor VIP AC GABA release alone is required for the development of inhibitory synapses in ooDSGCs. Furthermore, a reduction in net GABAergic transmission, but not a selective reduction from SACs, increased excitatory drive onto ooDSGCs. This increased excitation may drive a homeostatic increase in ooDSGC somatic GABAA receptors. Differential regulation of GABAA receptors on the ooDSGC's soma and dendrites could facilitate homeostatic control of the ooDSGC's output while enabling the assembly of the GABAergic connectivity underlying direction selectivity to be indifferent to altered transmission.