Hierarchical retinal computations rely on hybrid chemical-electrical signaling.

Bipolar cells (BCs) are integral to the retinal circuits that extract diverse features from the visual environment. They bridge photoreceptors to ganglion cells, the source of retinal output. Understanding how such circuits encode visual features requires an accounting of the mechanisms that control glutamate release from bipolar cell axons. Here, we demonstrate orientation selectivity in a specific genetically identifiable type of mouse bipolar cell-type 5A (BC5A). Their synaptic terminals respond best when stimulated with vertical bars that are far larger than their dendritic fields. We provide evidence that this selectivity involves enhanced excitation for vertical stimuli that requires gap junctional coupling through connexin36. We also show that this orientation selectivity is detectable postsynaptically in direction-selective ganglion cells, which were not previously thought to be selective for orientation. Together, these results demonstrate how multiple features are extracted by a single hierarchical network, engaging distinct electrical and chemical synaptic pathways.

Gain control by sparse, ultra-slow glycinergic synapses.

In the retina, ON starburst amacrine cells (SACs) play a crucial role in the direction-selective circuit, but the sources of inhibition that shape their response properties remain unclear. Previous studies demonstrate that ∼95% of their inhibitory synapses are GABAergic, yet we find that the light-evoked inhibitory currents measured in SACs are predominantly glycinergic. Glycinergic inhibition is extremely slow, relying on non-canonical glycine receptors containing α4 subunits, and is driven by both the ON and OFF retinal pathways. These attributes enable glycine inputs to summate and effectively control the output gain of SACs, expanding the range over which they compute direction. Serial electron microscopic reconstructions reveal three specific types of ON and OFF narrow-field amacrine cells as the presumptive sources of glycinergic inhibition. Together, these results establish an unexpected role for specific glycinergic amacrine cells in the retinal computation of stimulus direction by SACs.

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.

Rapid multi-directed cholinergic transmission in the central nervous system.

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.

Retinal direction selectivity in the absence of asymmetric starburst amacrine cell responses.

In the mammalian retina, direction-selectivity is thought to originate in the dendrites of GABAergic/cholinergic starburst amacrine cells, where it is first observed. However, here we demonstrate that direction selectivity in downstream ganglion cells remains remarkably unaffected when starburst dendrites are rendered non-directional, using a novel strategy combining a conditional GABAA α2 receptor knockout mouse with optogenetics. We show that temporal asymmetries between excitation/inhibition, arising from the differential connectivity patterns of starburst cholinergic and GABAergic synapses to ganglion cells, form the basis for a parallel mechanism generating direction selectivity. We further demonstrate that these distinct mechanisms work in a coordinated way to refine direction selectivity as the stimulus crosses the ganglion cell's receptive field. Thus, precise spatiotemporal patterns of inhibition and excitation that determine directional responses in ganglion cells are shaped by two 'core' mechanisms, both arising from distinct specializations of the starburst network.

Cholinergic excitation complements glutamate in coding visual information in retinal ganglion cells.

KEY POINTS: Starburst amacrine cells release GABA and ACh. This study explores the coordinated function of starburst-mediated cholinergic excitation and GABAergic inhibition to bistratified retinal ganglion cells, predominantly direction-selective ganglion cells (DSGCs). In rat retina, under our recording conditions, starbursts were found to provide the major excitatory drive to a sub-population of ganglion cells whose dendrites co-stratify with starburst dendrites (putative DSGCs). In mouse retina, recordings from genetically identified DSGCs at physiological temperatures reveal that ACh inputs dominate the response to small spot-high contrast light stimuli, with preferential addition of bipolar cell input shifting the balance towards glutamate for larger spot stimuli In addition, starbursts also appear to gate glutamatergic excitation to DSGCs by postsynaptic and possibly presynaptic inhibitory processes ABSTRACT: Starburst amacrine cells release both GABA and ACh, allowing them to simultaneously mediate inhibition and excitation. However, the precise pre- and postsynaptic targets for ACh and GABA remain under intense investigation. Most previous studies have focused on starburst-mediated postsynaptic GABAergic inhibition and its role in the formation of directional selectivity in ganglion cells. However, the significance of postsynaptic cholinergic excitation is only beginning to be appreciated. Here, we found that light-evoked responses measured in bi-stratified rat ganglion cells with dendrites that co-fasciculate with ON and OFF starburst dendrites (putative direction-selective ganglion cells, DSGCs) were abolished by the application of nicotinic receptor antagonists, suggesting ACh could act as the primary source of excitation. Recording from genetically labelled DSGCs in mouse retina at physiological temperatures revealed that cholinergic synaptic inputs dominated the excitation for high contrast stimuli only when the size of the stimulus was small. Canonical glutamatergic inputs mediated by bipolar cells were prominent when GABA/glycine receptors were blocked or when larger spot stimuli were utilized. In mouse DSGCs, bipolar cell excitation could also be unmasked through the activation of mGluR2,3 receptors, which we show suppresses starburst output, suggesting that GABA from starbursts serves to inhibit bipolar cell signals in DSGCs. Taken together, these results suggest that starbursts amplify excitatory signals traversing the retina, endowing DSGCs with the ability to encode fine spatial information without compromising their ability to encode direction.

An Old Neuron Learns New Tricks: Redefining Motion Processing in the Primate Retina.

Motion sensitivity requires the comparison of neural responses activated by nearby points in visual space. In this issue of Neuron, Manookin et al. (2018) find that in the primate retina, such comparisons are already manifest in second-order retinal bipolar cells, relying on lateral excitation mediated by gap junctions.

"Silent" NMDA Synapses Enhance Motion Sensitivity in a Mature Retinal Circuit.

Retinal direction-selective ganglion cells (DSGCs) have the remarkable ability to encode motion over a wide range of contrasts, relying on well-coordinated excitation and inhibition (E/I). E/I is orchestrated by a diverse set of glutamatergic bipolar cells that drive DSGCs directly, as well as indirectly through feedforward GABAergic/cholinergic signals mediated by starburst amacrine cells. Determining how direction-selective responses are generated across varied stimulus conditions requires understanding how glutamate, acetylcholine, and GABA signals are precisely coordinated. Here, we use a combination of paired patch-clamp recordings, serial EM, and large-scale multi-electrode array recordings to show that a single high-sensitivity source of glutamate is processed differentially by starbursts via AMPA receptors and DSGCs via NMDA receptors. We further demonstrate how this novel synaptic arrangement enables DSGCs to encode direction robustly near threshold contrasts. Together, these results reveal a space-efficient synaptic circuit model for direction computations, in which "silent" NMDA receptors play critical roles.

A Central Role for Mixed Acetylcholine/GABA Transmission in Direction Coding in the Retina.

A surprisingly large number of neurons throughout the brain are endowed with the ability to co-release both a fast excitatory and inhibitory transmitter. The computational benefits of dual transmitter release, however, remain poorly understood. Here, we address the role of co-transmission of acetylcholine (ACh) and GABA from starburst amacrine cells (SACs) to direction-selective ganglion cells (DSGCs). Using a combination of pharmacology, optogenetics, and linear regression methods, we estimated the spatiotemporal profiles of GABA, ACh, and glutamate receptor-mediated synaptic activity in DSGCs evoked by motion. We found that ACh initiates responses to motion in natural scenes or under low-contrast conditions. In contrast, classical glutamatergic pathways play a secondary role, amplifying cholinergic responses via NMDA receptor activation. Furthermore, under these conditions, the network of SACs differentially transmits ACh and GABA to DSGCs in a directional manner. Thus, mixed transmission plays a central role in shaping directional responses of DSGCs.

Origins of spontaneous activity in the degenerating retina.

Sensory deafferentation resulting from the loss of photoreceptors during retinal degeneration (rd) is often accompanied by a paradoxical increase in spontaneous activity throughout the visual system. Oscillatory discharges are apparent in retinal ganglion cells in several rodent models of rd, indicating that spontaneous activity can originate in the retina. Understanding the biophysical mechanisms underlying spontaneous retinal activity is interesting for two main reasons. First, it could lead to strategies that reduce spontaneous retinal activity, which could improve the performance of vision restoration strategies that aim to stimulate remnant retinal circuits in blind patients. Second, studying emergent network activity could offer general insights into how sensory systems remodel upon deafferentation. Here we provide an overview of the work describing spontaneous activity in the degenerating retina, and outline the current state of knowledge regarding the cellular and biophysical properties underlying spontaneous neural activity.

Specific wiring of distinct amacrine cells in the directionally selective retinal circuit permits independent coding of direction and size.

Local and global forms of inhibition controlling directionally selective ganglion cells (DSGCs) in the mammalian retina are well documented. It is established that local inhibition arising from GABAergic starburst amacrine cells (SACs) strongly contributes to direction selectivity. Here, we demonstrate that increasing ambient illumination leads to the recruitment of GABAergic wide-field amacrine cells (WACs) endowing the DS circuit with an additional feature: size selectivity. Using a combination of electrophysiology, pharmacology, and light/electron microscopy, we show that WACs predominantly contact presynaptic bipolar cells, which drive direct excitation and feedforward inhibition (through SACs) to DSGCs, thus maintaining the appropriate balance of inhibition/excitation required for generating DS. This circuit arrangement permits high-fidelity direction coding over a range of ambient light levels, over which size selectivity is adjusted. Together, these results provide novel insights into the anatomical and functional arrangement of multiple inhibitory interneurons within a single computational module in the retina.

Nonlinear dendritic integration of electrical and chemical synaptic inputs drives fine-scale correlations.

Throughout the CNS, gap junction-mediated electrical signals synchronize neural activity on millisecond timescales via cooperative interactions with chemical synapses. However, gap junction-mediated synchrony has rarely been studied in the context of varying spatiotemporal patterns of electrical and chemical synaptic activity. Thus, the mechanism underlying fine-scale synchrony and its relationship to neural coding remain unclear. We examined spike synchrony in pairs of genetically identified, electrically coupled ganglion cells in mouse retina. We found that coincident electrical and chemical synaptic inputs, but not electrical inputs alone, elicited synchronized dendritic spikes in subregions of coupled dendritic trees. The resulting nonlinear integration produced fine-scale synchrony in the cells' spike output, specifically for light stimuli driving input to the regions of dendritic overlap. In addition, the strength of synchrony varied inversely with spike rate. Together, these features may allow synchronized activity to encode information about the spatial distribution of light that is ambiguous on the basis of spike rate alone.

Post-receptor adaptation: lighting up the details.

The very first rays of the rising sun enrich our visual world with spectacular detail. A recent study reveals how retinal circuits downstream of photoreceptors 'functionally re-wire' to trade-off sensitivity for high spatial acuity during night-day transitions.

Dynamic tuning of electrical and chemical synaptic transmission in a network of motion coding retinal neurons.

Recently, we demonstrated that gap junction coupling in the population of superior coding ON-OFF directionally selective ganglion cells (DSGCs) genetically labeled in the Hb9::eGFP mouse retina allows the passage of lateral anticipatory signals that help track moving stimuli. Here, we examine the properties of gap junctions in the DSGC network, and address how interactions between electrical and chemical synapses and intrinsic membrane properties contribute to the dynamic tuning of lateral anticipatory signals. When DSGC subtypes coding all four cardinal directions were individually loaded with the gap junction-permeable tracer Neurobiotin, only superior coding DSGCs exhibited homologous coupling. Consistent with these anatomical findings, gap junction-dependent feedback spikelets were only observed in Hb9(+) DSGCs. Recordings from pairs of neighboring Hb9(+) DSGCs revealed that coupling was reciprocal, non-inactivating, and relatively weak, and provided a substrate for an extensive subthreshold excitatory receptive field around each cell. This subthreshold activity appeared to boost coincident light-driven chemical synaptic responses. However, during responses to moving stimuli, gap junction-mediated boosting appeared to be dynamically modulated such that upstream DSGCs primed downstream cells, but not vice versa, giving rise to highly skewed responses in individual cells. We show that the asymmetry in priming arises from a combination of spatially offset GABAergic inhibition and activity-dependent changes in intrinsic membrane properties of DSGCs. Thus, dynamic interactions between electrical and chemical synapses and intrinsic membrane properties allow the network of DSGCs to propagate anticipatory responses most effectively along their preferred direction without leading to runaway excitation.

Early remodeling of Müller cells in the rd/rd mouse model of retinal dystrophy.

We studied the anatomical remodeling and gliosis of retinal Müller cells in the rd/rd mouse model of photoreceptor degeneration. A computational calculation of glutamine synthetase immunoreactivity was developed so we could specifically quantify changes in Müller cell anatomy between control mice (C57Bl/6) and the dystrophic strain. We found no change in the number of Müller cell somata between mice strains, indicating no cell proliferation as a function of development and degeneration. The retinal area occupied by the total Müller cell body (soma and processes) was significantly less in the rd/rd mouse retina compared with control mice. When only the outer retina was considered, we found rd/rd Müller cell processes were dramatically reduced during the cone phase of photoreceptor degeneration. However, at older ages an increase in Müller cell processes was seen. Conversely, glial fibrillary acidic protein (GFAP) expression showed a significant increase during cone degeneration followed by a reduction in older ages. Müller cell electrophysiology, particularly K(+) currents and membrane potential, was similar between rd/rd and control Müller cells during cone degeneration. Together, these results show that glial remodeling in the rd/rd retina follows separate phases-an initial conservative glial response involving the loss of Müller cells processes, hyperexpression of GFAP, and preservation of normal electrophysiology followed by an active growth of Müller cell processes, glial seal formation, and attenuation of GFAP expression after complete photoreceptor loss.

Lag normalization in an electrically coupled neural network.

Moving objects can cover large distances while they are processed by the eye, usually resulting in a spatially lagged retinal response. We identified a network of electrically coupled motion-coding neurons in mouse retina that act collectively to register the leading edges of moving objects at a nearly constant spatial location, regardless of their velocity. These results reveal a previously unknown neurophysiological substrate for lag normalization in the visual system.

Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels.

In the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10­20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (I(h)), voltage-gated Na(+) channels and gap junctions in mediating such oscillatory activity. Blocking I(h) (1 mm Cs(+)) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na(+) channels (1 µm TTX) abolished oscillatory activity in the AII amacrine-ON cone bipolar cell network. Voltage-gated Na(+) channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 µm meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na(+) channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 µm NBQX and 50 µm l-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine-ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na(+) channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na(+) channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity.

Vsx1 regulates terminal differentiation of type 7 ON bipolar cells.

Although retinal bipolar cells represent a morphologically well defined population of retinal interneurons, very little is known about the developmental mechanisms that regulate their processing. Furthermore, the identity of specific bipolar cell types that function in distinct visual circuits remains poorly understood. Here, we show that the homeobox gene Vsx1 is expressed in Type 7 ON bipolar cells. In the absence of Vsx1, Type 7 bipolar cells exhibit proper morphological specification but show defects in terminal gene expression. Vsx1 is required for the repression of bipolar cell-specific markers, including Calcium-binding protein 5 and Chx10. This contrasts its genetic requirement as an activator of gene expression in OFF bipolar cells. To assess possible ON signaling defects in Vsx1-null mice, we recorded specifically from ON-OFF directionally selective ganglion cells (DSGCs), which cofasciculate with Type 7 bipolar cell terminals. Vsx1-null ON-OFF DSGCs received more sustained excitatory synaptic input, possibly due to Type 7 bipolar cell defects. Interestingly, in Vsx1-null mice, the directionally selective circuit is functional but compromised. Together, these findings indicate that Vsx1 regulates terminal gene expression in Type 7 bipolar cells and is necessary for proper ON visual signaling within a directionally selective circuit.

Parallel mechanisms encode direction in the retina.

In the retina, presynaptic inhibitory mechanisms that shape directionally selective (DS) responses in output ganglion cells are well established. However, the nature of inhibition-independent forms of directional selectivity remains poorly defined. Here, we describe a genetically specified set of ON-OFF DS ganglion cells (DSGCs) that code anterior motion. This entire population of DSGCs exhibits asymmetric dendritic arborizations that orientate toward the preferred direction. We demonstrate that morphological asymmetries along with nonlinear dendritic conductances generate a centrifugal (soma-to-dendrite) preference that does not critically depend upon, but works in parallel with the GABAergic circuitry. We also show that in symmetrical DSGCs, such dendritic DS mechanisms are aligned with, or are in opposition to, the inhibitory DS circuitry in distinct dendritic subfields where they differentially interact to promote or weaken directional preferences. Thus, pre- and postsynaptic DS mechanisms interact uniquely in distinct ganglion cell populations, enabling efficient DS coding under diverse conditions.