On-Off Direction-selective Ganglion Cells Research Articles

A new role for excitation in the retinal direction-selective circuit.

A key feature of the receptive field of neurons in the visual system is their centre-surround antagonism, whereby the centre and the surround exhibit responses of opposite polarity. This organization is thought to enhance visual acuity, but whether and how such antagonism plays a role in more complex processing remains poorly understood. Here, we investigate the role of centre and surround receptive fields in retinal direction selectivity by exposing posterior-preferring On-Off direction-selective ganglion cells (pDSGCs) to adaptive light and recording their response to globally moving objects. We reveal that light adaptation leads to surround expansion in pDSGCs. The pDSGCs maintain their original directional tuning in the centre receptive field, but present the oppositely tuned response in their surround. Notably, although inhibition is the main substrate for retinal direction selectivity, we found that following light adaptation, both the centre- and surround-mediated responses originate from directionally tuned excitatory inputs. Multi-electrode array recordings show similar oppositely tuned responses in other DSGC subtypes. Together, these data attribute a new role for excitation in the direction-selective circuit. This excitation carries an antagonistic centre-surround property, possibly designed to sharpen the detection of motion direction in the retina. KEY POINTS: Receptive fields of direction-selective retinal ganglion cells expand asymmetrically following light adaptation. The increase in the surround receptive field generates a delayed spiking phase that is tuned to the null direction and is mediated by excitation. Following light adaptation, excitation rules the computation in the centre receptive field and is tuned to the preferred direction. GABAergic and glycinergic inputs modulate the null-tuned delayed response differentially. Null-tuned delayed spiking phases can be detected in all types of direction-selective retinal ganglion cells. Light adaptation exposes a hidden directional excitation in the circuit, which is tuned to opposite directions in the centre and surround receptive fields.

Cadherin-13 Maintains Retinotectal Synapses via Transneuronal Interactions.

Maintaining precise synaptic contacts between neuronal partners is critical to ensure the proper functioning of the mammalian central nervous system (CNS). Diverse cell recognition molecules, such as classic cadherins (Cdhs), are part of the molecular machinery mediating synaptic choices during development and synaptic maintenance. Yet, the principles governing neuron-neuron wiring across diverse CNS neuron types remain largely unknown. The retinotectal synapses, connections from the retinal ganglion cells (RGCs) to the superior collicular (SC) neurons, offer an ideal experimental system to reveal molecular logic underlying synaptic choices and formation. This is due to the retina's unidirectional and laminar-restricted projections to the SC and the large databases of presynaptic RGC subtypes and postsynaptic SC neuronal types. Here, we focused on determining the role of Type II Cdhs in wiring the retinotectal synapses. We surveyed Cdhs expression patterns at neuronal resolution and revealed that Cdh13 is enriched in the wide-field neurons in the superficial SC (sSC). In either the Cdh13 null mutant or selective adult deletion within the wide-field neurons, there is a significant reduction of spine densities in the distal dendrites of these neurons in both sexes. Additionally, Cdh13 removal from presynaptic RGCs reduced dendritic spines in the postsynaptic wide-field neurons. Cdh13-expressing RGCs use differential mechanisms than αRGCs and On-Off Direction-Selective Ganglion Cells (ooDSGCs) to form specific retinotectal synapses. The results revealed a selective transneuronal interaction mediated by Cdh13 to maintain proper retinotectal synapses in vivo.

Neural Circuits Underlying Multifeature Extraction in the Retina.

Classic ON-OFF direction-selective ganglion cells (DSGCs) that encode the four cardinal directions were recently shown to also be orientation-selective. To clarify the mechanisms underlying orientation selectivity, we employed a variety of electrophysiological, optogenetic, and gene knock-out strategies to test the relative contributions of glutamate, GABA, and acetylcholine (ACh) input that are known to drive DSGCs, in male and female mouse retinas. Extracellular spike recordings revealed that DSGCs respond preferentially to either vertical or horizontal bars, those that are perpendicular to their preferred-null motion axes. By contrast, the glutamate input to all four DSGC types measured using whole-cell patch-clamp techniques was found to be tuned along the vertical axis. Tuned glutamatergic excitation was heavily reliant on type 5A bipolar cells, which appear to be electrically coupled via connexin 36 containing gap junctions to the vertically oriented processes of wide-field amacrine cells. Vertically tuned inputs are transformed by the GABAergic/cholinergic "starburst" amacrine cells (SACs), which are critical components of the direction-selective circuit, into distinct patterns of inhibition and excitation. Feed-forward SAC inhibition appears to "veto" preferred orientation glutamate excitation in dorsal/ventral (but not nasal/temporal) coding DSGCs "flipping" their orientation tuning by 90° and accounts for the apparent mismatch between glutamate input tuning and the DSGC's spiking response. Together, these results reveal how two distinct synaptic motifs interact to generate complex feature selectivity, shedding light on the intricate circuitry that underlies visual processing in the retina.

Asymmetric connections with starburst amacrine cells underlie the upward motion selectivity of J-type retinal ganglion cells.

Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons' stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity.

Functional convergence of on-off direction-selective ganglion cells in the visual thalamus

In the mouse visual system, multiple types of retinal ganglion cells (RGCs) each encode distinct features of the visual space. A clear understanding of how this information is parsed in their downstream target, the dorsal lateral geniculate nucleus (dLGN), remains elusive. Here, we characterized retinogeniculate connectivity in Cart-IRES2-Cre-D and BD-CreER2 mice, which labels subsets of on-off direction-selective ganglion cells (ooDSGCs) tuned to the vertical directions and to only ventral motion, respectively. Our immunohistochemical, electrophysiological, and optogenetic experiments reveal that only a small fraction (<15%) of thalamocortical (TC) neurons in the dLGN receives primary retinal drive from these subtypes of ooDSGCs. The majority of the functionally identifiable ooDSGC inputs in the dLGN are weak and converge together with inputs from other RGC types. Yet our modeling indicates that this mixing is not random: BD-CreER+ ooDSGC inputs converge less frequently with ooDSGCs tuned to the opposite direction than with non-CART-Cre+ RGC types. Taken together, these results indicate that convergence of distinct information lines in dLGN follows specific rules of organization.

Sodium Hyaluronate-Induced Ocular Hypertension in Rats Damages the Direction-Selective Circuit and Inner/Outer Retinal Plexiform Layers.

PurposeTo assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension.MethodsIntraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry.ResultsIOP was higher in HYA-treated eyes than in sham-operated eyes along the 10-week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina.ConclusionsSerial HYA treatment in the rat anterior eye chamber results in mild-to-moderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma.

Visual Stimulation Induces Distinct Forms of Sensitization of On-Off Direction-Selective Ganglion Cell Responses in the Dorsal and Ventral Retina.

Experience-dependent modulation of neuronal responses is a key attribute in sensory processing. In the mammalian retina, the On-Off direction-selective ganglion cell (DSGC) is well known for its robust direction selectivity. However, how the On-Off DSGC light responsiveness dynamically adjusts to the changing visual environment is underexplored. Here, we report that On-Off DSGCs tuned to posterior motion direction [i.e. posterior DSGCs (pDSGCs)] in mice of both sexes can be transiently sensitized by prior stimuli. Notably, distinct sensitization patterns are found in dorsal and ventral pDSGCs. Although responses of both dorsal and ventral pDSGCs to dark stimuli (Off responses) are sensitized, only dorsal cells show the sensitization of responses to bright stimuli (On responses). Visual stimulation to the dorsal retina potentiates a sustained excitatory input from Off bipolar cells, leading to tonic depolarization of pDSGCs. Such tonic depolarization propagates from the Off to the On dendritic arbor of the pDSGC to sensitize its On response. We also identified a previously overlooked feature of DSGC dendritic architecture that can support dendritic integration between On and Off dendritic layers bypassing the soma. By contrast, ventral pDSGCs lack a sensitized tonic depolarization and thus do not exhibit sensitization of their On responses. Our results highlight a topographic difference in Off bipolar cell inputs underlying divergent sensitization patterns of dorsal and ventral pDSGCs. Moreover, substantial crossovers between dendritic layers of On-Off DSGCs suggest an interactive dendritic algorithm for processing On and Off signals before they reach the soma.SIGNIFICANCE STATEMENT Visual neuronal responses are dynamically influenced by the prior visual experience. This form of plasticity reflects the efficient coding of the naturalistic environment by the visual system. We found that a class of retinal output neurons, On-Off direction-selective ganglion cells, transiently increase their responsiveness after visual stimulation. Cells located in dorsal and ventral retinas exhibit distinct sensitization patterns because of different adaptive properties of Off bipolar cell signaling. A previously overlooked dendritic morphologic feature of the On-Off direction-selective ganglion cell is implicated in the cross talk between On and Off pathways during sensitization. Together, these findings uncover a topographic difference in the adaptive encoding of upper and lower visual fields and the underlying neural mechanism in the dorsal and ventral retinas.

Distinct inhibitory pathways control velocity and directional tuning in the mouse retina

The sensory periphery is responsible for detecting ethologically relevant features of the external world, using compact, predominantly feedforward circuits. Visual motion is a particularly prevalent sensory feature, the presence of which can be a signal to enact diverse behaviors ranging from gaze stabilization reflexes to predator avoidance or prey capture. To understand how the retina constructs the distinct neural representations required for these behaviors, we investigated two circuits responsible for encoding different aspects of image motion: ON and ON-OFF direction-selective ganglion cells (DSGCs). Using a combination of two-photon targeted whole-cell electrophysiology, pharmacology, and conditional knockout mice, we show that distinct inhibitory pathways independently control tuning for motion velocity and motion direction in these two cell types. We further employ dynamic clamp and numerical modeling techniques to show that asymmetric inhibition provides a velocity-invariant mechanism of directional tuning, despite the strong velocity dependence of classical models of direction selectivity. We therefore demonstrate that invariant representations of motion features by inhibitory interneurons act as computational building blocks to construct distinct, behaviorally relevant signals at the earliest stages of the visual system.

Parallel processing in active dendrites during periods of intense spiking activity.

A neuron's ability to perform parallel computations throughout its dendritic arbor substantially improves its computational capacity. However, during natural patterns of activity, the degree to which computations remain compartmentalized, especially in neurons with active dendritic trees, is not clear. Here, we examine how the direction of moving objects is computed across the bistratified dendritic arbors of ON-OFF direction-selective ganglion cells (DSGCs) in the mouse retina. We find that although local synaptic signals propagate efficiently throughout their dendritic trees, direction-selective computations in one part of the dendritic arbor have little effect on those being made elsewhere. Independent dendritic processing allows DSGCs to compute the direction of moving objects multiple times as they traverse their receptive fields, enabling them to rapidly detect changes in motion direction on a sub-receptive-field basis. These results demonstrate that the parallel processing capacity of neurons can be maintained even during periods of intense synaptic activity.

Cholinergic feedback to bipolar cells contributes to motion detection in the mouse retina.

SUMMARYRetinal bipolar cells are second-order neurons that transmit basic features of the visual scene to postsynaptic partners. However, their contribution to motion detection has not been fully appreciated. Here, we demonstrate that cholinergic feedback from starburst amacrine cells (SACs) to certain presynaptic bipolar cells via alpha-7 nicotinic acetylcholine receptors (α7-nAChRs) promotes direction-selective signaling. Patch clamp recordings reveal that distinct bipolar cell types making synapses at proximal SAC dendrites also express α7-nAChRs, producing directionally skewed excitatory inputs. Asymmetric SAC excitation contributes to motion detection in On-Off direction-selective ganglion cells (On-Off DSGCs), predicted by computational modeling of SAC dendrites and supported by patch clamp recordings from On-Off DSGCs when bipolar cell α7-nAChRs is eliminated pharmacologically or by conditional knockout. Altogether, these results show that cholinergic feedback to bipolar cells enhances direction-selective signaling in postsynaptic SACs and DSGCs, illustrating how bipolar cells provide a scaffold for postsynaptic microcircuits to cooperatively enhance retinal motion detection.

Satb1 expression in retinal ganglion cells of marmosets, macaques, and humans.

Recent advances in single-cell RNA sequencing have enabled the molecular distinction of ganglion cell populations in mammalian retinas. Here we used antibodies against the transcription factor special AT-rich binding protein 1 (Satb1, a protein which is expressed by on-off direction-selective ganglion cells in mouse retina) to study Satb1 expression in the retina of marmosets (Callithrix jacchus), macaques (Macaca fascicularis), and humans. In all species, Satb1 was exclusively expressed in retinal ganglion cells. The Satb1 cells made up ∼2% of the ganglion cell population in the central retina of all species, rising to a maximum ∼7% in peripheral marmoset retina. Intracellular injections in marmoset and macaque retinas revealed that most Satb1 expressing ganglion cells are widefield ganglion cells. In marmoset, Satb1 cells have a densely branching dendritic tree and include broad and narrow thorny, recursive bistratified, and parasol cells, all of which show some costratification with the outer or inner cholinergic amacrine cells. The recursive bistratified cells showed the strongest costratification but did not show extensive cofasciculation as reported for on-off direction-selective ganglion cells in rabbit and rodent retinas. In macaque, Satb1 was not expressed in recursive bistratified cells, but in large sparsely branching cells. Our findings further support the idea that the expression of transcription factors in retinal ganglion cells is not conserved across Old World (human and macaque) and New World (marmoset) primates and provides a further step to link a molecular marker with specific cell types.

Spatially displaced excitation contributes to the encoding of interrupted motion by a retinal direction-selective circuit.

Spatially distributed excitation and inhibition collectively shape a visual neuron's receptive field (RF) properties. In the direction-selective circuit of the mammalian retina, the role of strong null-direction inhibition of On-Off direction-selective ganglion cells (On-Off DSGCs) on their direction selectivity is well-studied. However, how excitatory inputs influence the On-Off DSGC's visual response is underexplored. Here, we report that On-Off DSGCs have a spatially displaced glutamatergic receptive field along their horizontal preferred-null motion axes. This displaced receptive field contributes to DSGC null-direction spiking during interrupted motion trajectories. Theoretical analyses indicate that population responses during interrupted motion may help populations of On-Off DSGCs signal the spatial location of moving objects in complex, naturalistic visual environments. Our study highlights that the direction-selective circuit exploits separate sets of mechanisms under different stimulus conditions, and these mechanisms may help encode multiple visual features.

Preserving inhibition with a disinhibitory microcircuit in the retina.

Previously, we found that in the mammalian retina, inhibitory inputs onto starburst amacrine cells (SACs) are required for robust direction selectivity of On-Off direction-selective ganglion cells (On-Off DSGCs) against noisy backgrounds (Chen et al., 2016). However, the source of the inhibitory inputs to SACs and how this inhibition confers noise resilience of DSGCs are unknown. Here, we show that when visual noise is present in the background, the motion-evoked inhibition to an On-Off DSGC is preserved by a disinhibitory motif consisting of a serially connected network of neighboring SACs presynaptic to the DSGC. This preservation of inhibition by a disinhibitory motif arises from the interaction between visually evoked network dynamics and short-term synaptic plasticity at the SAC-DSGC synapse. Although the disinhibitory microcircuit is well studied for its disinhibitory function in brain circuits, our results highlight the algorithmic flexibility of this motif beyond disinhibition due to the mutual influence between network and synaptic plasticity mechanisms.

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.

Early visual motion experience shapes the gap junction connections among direction selective ganglion cells.

Gap junction connections between neurons play critical roles in the development of the nervous system. However, studies on the sensory experience–driven plasticity during the critical period rarely examine the involvement of gap junction connections. ON-OFF direction selective ganglion cells (ooDSGCs) in the mouse retina that prefer upward motion are connected by gap junctions throughout development. Here, we show that after exposing the mice to a visual environment dominated by upward motion from eye-opening to puberty, ooDSGCs that respond preferentially to upward motion show enhanced spike synchronization, while downward motion training has the opposite effect. The effect is long-term, persisting at least three months after the training. Correlated activity during training is tightly linked to this effect: Cells trained by stimuli that promote higher levels of activity correlation show stronger gap junction connection after the training, while stimuli that produce very low activity correlation leave the cells with much weaker gap junction connections afterwards. Direct investigation of the gap junction connections among upward motion–preferring ooDSGCs show that both the percentage of electrically coupled ooDSGCs and the strength of the coupling are affected by visual motion training. Our results demonstrate that in the retina, one of the peripheral sensory systems, gap junction connections can be shaped by experience during development.

Neural mechanisms of contextual modulation in the retinal direction selective circuit

Contextual modulation of neuronal responses by surrounding environments is a fundamental attribute of sensory processing. In the mammalian retina, responses of On–Off direction selective ganglion cells (DSGCs) are modulated by motion contexts. However, the underlying mechanisms are unknown. Here, we show that posterior-preferring DSGCs (pDSGCs) are sensitive to discontinuities of moving contours owing to contextually modulated cholinergic excitation from starburst amacrine cells (SACs). Using a combination of synapse-specific genetic manipulations, patch clamp electrophysiology and connectomic analysis, we identified distinct circuit motifs upstream of On and Off SACs that are required for the contextual modulation of pDSGC activity for bright and dark contrasts. Furthermore, our results reveal a class of wide-field amacrine cells (WACs) with straight, unbranching dendrites that function as “continuity detectors” of moving contours. Therefore, divergent circuit motifs in the On and Off pathways extend the information encoding of On-Off DSGCs beyond their direction selectivity during complex stimuli.

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.

Gap Junctions Contribute to Differential Light Adaptation across Direction-Selective Retinal Ganglion Cells

Direction-selective ganglion cells (DSGCs) deliver signals from the retina to multiple brain areas to indicate the presence and direction of motion. Delivering reliable signals in response to motion is critical across light levels. Here we determine how populations of DSGCs adapt to changes in light level, from moonlight to daylight. Using large-scale measurements of neural activity, we demonstrate that the population of DSGCs switches encoding strategies across light levels. Specifically, the direction tuning of superior (upward)-preferring ON-OFF DSGCs becomes broader at low light levels, whereas other DSGCs exhibit stable tuning. Using a conditional knockout of gap junctions, we show that this differential adaptation among superior-preferring ON-OFF DSGCs is caused by connexin36-mediated electrical coupling and differences in effective GABAergic inhibition. Furthermore, this adaptation strategy is beneficial for balancing motion detection and direction estimation at the lower signal-to-noise ratio encountered at night. These results provide insights into how light adaptation impacts motion encoding in the retina.

A Role for Mouse Primary Visual Cortex in Motion Perception

Visual motion is an ethologically important stimulus throughout the animal kingdom. In primates, motion perception relies on specific higher-order cortical regions. Although mouse primary visual cortex (V1) and higher-order visual areas show direction-selective (DS) responses, their role in motion perception remains unknown. Here, we tested whether V1 is involved in motion perception in mice. We developed a head-fixed discrimination task in which mice must report their perceived direction of motion from random dot kinematograms (RDKs). After training, mice made around 90% correct choices for stimuli with high coherence and performed significantly above chance for 16% coherent RDKs. Accuracy increased with both stimulus duration and visual field coverage of the stimulus, suggesting that mice in this task integrate motion information in time and space. Retinal recordings showed that thalamically projecting On-Off DS ganglion cells display DS responses when stimulated with RDKs. Two-photon calcium imaging revealed that neurons in layer (L) 2/3 of V1 display strong DS tuning in response to this stimulus. Thus, RDKs engage motion-sensitive retinal circuits as well as downstream visual cortical areas. Contralateral V1 activity played a key role in this motion direction discrimination task because its reversible inactivation with muscimol led to a significant reduction in performance. Neurometric-psychometric comparisons showed that an ideal observer could solve the task with the information encoded in DS L2/3 neurons. Motion discrimination of RDKs presents a powerful behavioral tool for dissecting the role of retino-forebrain circuits in motion processing.