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Retinal prosthetics are one of the leading therapeutic strategies to restore lost vision in patients with retinitis pigmentosa and age-related macular degeneration. Much work has described patterns of spiking in retinal ganglion cells (RGCs) in response to electrical stimulation, but less work has examined the underlying retinal circuitry that is activated by electrical stimulation to drive these responses. Surprisingly, little is known about the role of inhibition in generating electrical responses or how inhibition might be altered during degeneration. Using whole-cell voltage-clamp recordings during subretinal electrical stimulation in the rd10 and wild-type ( wt ) retina, we found electrically evoked synaptic inputs differed between ON and OFF RGC populations, with ON cells receiving mostly excitation and OFF cells receiving mostly inhibition and very little excitation. We found that the inhibition of OFF bipolar cells limits excitation in OFF RGCs, and a majority of both pre- and postsynaptic inhibition in the OFF pathway arises from glycinergic amacrine cells, and the stimulation of the ON pathway contributes to inhibitory inputs to the RGC. We also show that this presynaptic inhibition in the OFF pathway is greater in the rd10 retina, compared with that in the wt retina., (Copyright © 2024 Carleton and Oesch.)

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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Tworig et al.)

VGluT3-expressing mouse retinal amacrine cells (VG3s) respond to small-object motion and connect to multiple types of bipolar cells (inputs) and retinal ganglion cells (RGCs, outputs). Because these input and output connections are intermixed on the same dendrites, making sense of VG3 circuitry requires comparing the distribution of synapses across their arbors to the subcellular flow of signals. Here, we combine subcellular calcium imaging and electron microscopic connectomic reconstruction to analyze how VG3s integrate and transmit visual information. VG3s receive inputs from all nearby bipolar cell types but exhibit a strong preference for the fast type 3a bipolar cells. By comparing input distributions to VG3 dendrite responses, we show that VG3 dendrites have a short functional length constant that likely depends on inhibitory shunting. This model predicts that RGCs that extend dendrites into the middle layers of the inner plexiform encounter VG3 dendrites whose responses vary according to the local bipolar cell response type., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

In mammals, starburst amacrine cells are centrally involved in motion vision and a new study in PLOS Biology, by Yan and colleagues finds that zebrafish have them, too. They coexist with a second pair of starburst-like neurons, but neither appears to be strongly motion selective., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yoshimatsu, Baden. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dendritic mechanisms driving input-output transformation in starburst amacrine cells (SACs) are not fully understood. Here, we combine two-photon subcellular voltage and calcium imaging and electrophysiological recording to determine the computational architecture of mouse SAC dendrites. We found that the perisomatic region integrates motion signals over the entire dendritic field, providing a low-pass-filtered global depolarization to dendrites. Dendrites integrate local synaptic inputs with this global signal in a direction-selective manner. Coincidental local synaptic inputs and the global motion signal in the outward motion direction generate local suprathreshold calcium transients. Moreover, metabotropic glutamate receptor 2 (mGluR2) signaling in SACs modulates the initiation of calcium transients in dendrites but not at the soma. In contrast, voltage-gated potassium channel 3 (Kv3) dampens fast voltage transients at the soma. Together, complementary mGluR2 and Kv3 signaling in different subcellular regions leads to dendritic compartmentalization and direction selectivity, highlighting the importance of these mechanisms in dendritic computation., (© 2024. The Author(s).)

In early sensory systems, cell-type diversity generally increases from the periphery into the brain, resulting in a greater heterogeneity of responses to the same stimuli. Surround suppression is a canonical visual computation that begins within the retina and is found at varying levels across retinal ganglion cell types. Our results show that heterogeneity in the level of surround suppression occurs subcellularly at bipolar cell synapses. Using single-cell electrophysiology and serial block-face scanning electron microscopy, we show that two retinal ganglion cell types exhibit very different levels of surround suppression even though they receive input from the same bipolar cell types. This divergence of the bipolar cell signal occurs through synapse-specific regulation by amacrine cells at the scale of tens of microns. These findings indicate that each synapse of a single bipolar cell can carry a unique visual signal, expanding the number of possible functional channels at the earliest stages of visual processing., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ , we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Lee et al.)

By analyzing light-evoked spike responses, cation currents (ΔI C ) and chloride currents (ΔI Cl ) of over 100 morphologically-identified retinal ganglion cells (GCs) in dark-adapted mouse retina, we found there are at least 14 functionally- and morphologically-distinct types of RGCs. These cells can be divided into 5 groups based on their patterns of spike response to whole field light steps (SRWFLS), a GC identification scheme commonly used in studies with extracellular recording techniques. We also found that all GCs in the mouse retina express strychnine-sensitive glycine receptors, and receive light-elicited chloride current (ΔI Cl ) accompanied by a conductance increase from narrow-field, glycinergic amacrine cells. As the dark membrane potential of RGC are near the chloride-equilibrium potential, mouse GCs' spike responses are mediated primarily by bipolar cells inputs, and modulated by "shunting inhibition" from narrow-field amacrine cells. Analysis of strychnine actions on light-evoked cation current ΔI C (bipolar cell inputs) in GCs suggests that narrow-field amacrine cells modulate GCs by sending ON-OFF crossover feedback signals to presynaptic bipolar cell axon terminals via sign-inverting glycinergic synapses, and the feedback signals are synergistic to the bipolar cell light responses. Therefore narrow-field amacrine cells enhance light-evoked bipolar cell inputs to GCs by presynaptic "synergistic addition", besides the abovementioned postsynaptic "shunting inhibition" in GCs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The vertebrate inner retina is driven by photoreceptors whose outputs are already pre-processed; in zebrafish, outer retinal circuits split "color" from "grayscale" information across four cone-photoreceptor types. It remains unclear how the inner retina processes incoming spectral information while also combining cone signals to shape grayscale functions. We address this question by imaging the light-driven responses of amacrine cells (ACs) and bipolar cells (BCs) in larval zebrafish in the presence and pharmacological absence of inner retinal inhibition. We find that ACs enhance opponency in some bipolar cells while at the same time suppressing pre-existing opponency in others, so that, depending on the retinal region, the net change in the number of color-opponent units is essentially zero. To achieve this "dynamic balance," ACs counteract intrinsic color opponency of BCs via the On channel. Consistent with these observations, Off-stratifying ACs are exclusively achromatic, while all color-opponent ACs stratify in the On sublamina., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stage II cholinergic retinal waves are one of the first instances of neural activity in the visual system as they are present at a developmental timepoint in which light-evoked activity remains largely undetectable. These waves of spontaneous neural activity sweeping across the developing retina are generated by starburst amacrine cells, depolarize retinal ganglion cells, and drive the refinement of retinofugal projections to numerous visual centers in the brain. Building from several well-established models, we assemble a spatial computational model of starburst amacrine cell-mediated wave generation and wave propagation that includes three significant advancements. First, we model the intrinsic spontaneous bursting of the starburst amacrine cells, including the slow afterhyperpolarization, which shapes the stochastic process of wave generation. Second, we establish a mechanism of wave propagation using reciprocal acetylcholine release, synchronizing the bursting activity of neighboring starburst amacrine cells. Third, we model the additional starburst amacrine cell release of GABA, changing the spatial propagation of retinal waves and in certain instances, the directional bias of the retinal wave front. In total, these advancements comprise a now more comprehensive model of wave generation, propagation, and direction bias., (© 2023. The Author(s).)

Midget and parasol ganglion cells (GCs) represent the major output channels from the primate eye to the brain. On-type midget and parasol GCs exhibit a higher background spike rate and thus can respond more linearly to contrast changes than their Off-type counterparts. Here, we show that a calcium-permeable AMPA receptor (CP-AMPAR) antagonist blocks background spiking and sustained light-evoked firing in On-type GCs while preserving transient light responses. These effects are selective for On-GCs and are occluded by a gap-junction blocker suggesting involvement of AII amacrine cells (AII-ACs). Direct recordings from AII-ACs, cobalt uptake experiments, and analyses of transcriptomic data confirm that CP-AMPARs are expressed by primate AII-ACs. Overall, our data demonstrate that under some background light levels, CP-AMPARs at the rod bipolar to AII-AC synapse drive sustained signaling in On-type GCs and thus contribute to the more linear contrast signaling of the primate On- versus Off-pathway., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system., (© 2022. The Author(s).)

Amacrine cells, inhibitory interneurons of the retina, feature synaptic inputs and outputs in close proximity throughout their dendritic trees, making them notable exceptions to prototypical somato-dendritic integration with output transmitted via axonal action potentials. The extent of dendritic compartmentalization in amacrine cells with widely differing dendritic tree morphology, however, is largely unexplored. Combining compartmental modeling, dendritic Ca 2+ imaging, targeted microiontophoresis and multielectrode patch-clamp recording (voltage and current clamp, capacitance measurement of exocytosis), we investigated integration in the AII amacrine cell, a narrow-field electrically coupled interneuron that participates in multiple, distinct microcircuits. Physiological experiments were performed with in vitro slices prepared from retinas of both male and female rats. We found that the morphology of the AII enables simultaneous local and global integration of inputs targeted to different dendritic regions. Local integration occurs within spatially restricted dendritic subunits and narrow time windows and is largely unaffected by the strength of electrical coupling. In contrast, global integration across the dendritic tree occurs over longer time periods and is markedly influenced by the strength of electrical coupling. These integrative properties enable AII amacrines to combine local control of synaptic plasticity with location-independent global integration. Dynamic inhibitory control of dendritic subunits is likely to be of general importance for amacrine cells, including cells with small dendritic trees, as well as for inhibitory interneurons in other regions of the CNS. SIGNIFICANCE STATEMENT Our understanding of synaptic integration is based on the prototypical morphology of a neuron with multiple dendrites and a single axon at opposing ends of a cell body. Many neurons, notably retinal amacrine cells, are exceptions to this arrangement, and display input and output synapses interspersed along their dendritic branches. In the large dendritic trees of some amacrine cells, such arrangements can give rise to multiple computational subunits. Other amacrine cells, with small dendritic trees, have been assumed to operate as single computational units. Here, we report the surprising result that despite a small dendritic tree, the AII amacrine cell simultaneously performs local integration of synaptic inputs (over smaller dendritic subregions) and global integration across the entire cell., (Copyright © 2022 the authors.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Retinal direction-selectivity originates in starburst amacrine cells (SACs), which display a centrifugal preference, responding with greater depolarization to a stimulus expanding from soma to dendrites than to a collapsing stimulus. Various mechanisms were hypothesized to underlie SAC centrifugal preference, but dissociating them is experimentally challenging and the mechanisms remain debatable. To address this issue, we developed the Retinal Stimulation Modeling Environment (RSME), a multifaceted data-driven retinal model that encompasses detailed neuronal morphology and biophysical properties, retina-tailored connectivity scheme and visual input. Using a genetic algorithm, we demonstrated that spatiotemporally diverse excitatory inputs-sustained in the proximal and transient in the distal processes-are sufficient to generate experimentally validated centrifugal preference in a single SAC. Reversing these input kinetics did not produce any centrifugal-preferring SAC. We then explored the contribution of SAC-SAC inhibitory connections in establishing the centrifugal preference. SAC inhibitory network enhanced the centrifugal preference, but failed to generate it in its absence. Embedding a direction selective ganglion cell (DSGC) in a SAC network showed that the known SAC-DSGC asymmetric connectivity by itself produces direction selectivity. Still, this selectivity is sharpened in a centrifugal-preferring SAC network. Finally, we use RSME to demonstrate the contribution of SAC-SAC inhibitory connections in mediating direction selectivity and recapitulate recent experimental findings. Thus, using RSME, we obtained a mechanistic understanding of SACs' centrifugal preference and its contribution to direction selectivity., Competing Interests: The authors have declared that no competing interests exist.

The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8-10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Purpose: The neuronal ELAV-like proteins (nElavls; Elavl2, Elavl3, Elavl4) have been known to regulate neuronal differentiation, maintenance, and axonogenesis in the brain. However, the specific role of nElavls in retina remains unclear. Here, we attempted to identify the expression pattern of Elavl2 during retinogenesis and aimed to decipher the function of Elavl2 in the retina., Methods: We have used the Cre-loxP system to conditionally inactivate Elavl2 in order to examine its role in developing retina. Eyes were collected for histology, immunohistochemistry, and TUNEL analysis to identify the structure of retina, and examined by RNA sequencing to analyze the function and pathway enrichment of differentially expressed genes in transgenic mice. Moreover, the mechanism by which Elavl2 regulates the differentiation of amacrine cells (ACs) was explored by RNA immunoprecipitation assays. Finally, eyes were functionally assessed by whole-cell patch-clamp, electroretinography (ERG) and optomotor response., Results: Elavl2 was expressed in retinal progenitor cells and retinal ganglion cells (RGCs), ACs, and horizontal cells. Retina-specific ablation of Elavl2 led to the loss of ACs and the transcription factors involved in ACs differentiation were also downregulated. In addition, the spontaneous activities of RGCs were obviously increased in Elavl2-deficient mice. Meanwhile, the loss of ACs that induced by Elavl2 deficiency lead to a decrease in ERG responses and visual acuity., Conclusions: Elavl2 is an intrinsic factor that involved in the differentiation of ACs subtype during retinogenesis, and essential for maintaining the normal retinal function.

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.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH + ) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH + ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH + ACs. Optogenetic stimulation of local CRH + ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH + ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits. SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons., (Copyright © 2021 the authors.)

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax -mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax -KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population. SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina., (Copyright © 2021 the authors.)

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: QC, RS, XH, WW No competing interests declared, (© 2020, Chen et al.)

Correlated spontaneous activity in the developing retina (termed "retinal waves") plays an instructive role in refining neural circuits of the visual system. Depolarizing (ON) and hyperpolarizing (OFF) starburst amacrine cells (SACs) initiate and propagate cholinergic retinal waves. Where cholinergic retinal waves stop, SACs are thought to be driven by glutamatergic retinal waves initiated by ON-bipolar cells. However, the properties and function of cholinergic and glutamatergic waves in ON- and OFF-SACs still remain poorly understood. In the present work, we performed whole-cell patch-clamp recordings and Ca 2+ imaging from genetically labeled ON- and OFF-SACs in mouse flat-mount retinas. We found that both SAC subtypes exhibited spontaneous rhythmic depolarization during cholinergic and glutamatergic waves. Interestingly, ON-SACs had wave-induced action potentials (APs) in an age-dependent manner, but OFF-SACs did not. Simultaneous Ca 2+ imaging and patch-clamp recordings demonstrated that, during a cholinergic wave, APs of an ON-SAC appeared to promote the dendritic release of acetylcholine onto neighboring ON- and OFF-SACs, which enhances their Ca 2+ transients. These results advance the understanding of the cellular mechanisms underlying correlated spontaneous activity in the developing retina.

Our understanding of nervous system function is limited by our ability to identify and manipulate neuronal subtypes within intact circuits. We show that the Gbx2 CreERT2-IRES-EGFP mouse line labels two amacrine cell (AC) subtypes in the mouse retina that have distinct morphological, physiological, and molecular properties. Using a combination of RNA-seq, genetic labeling, and patch clamp recordings, we show that one subtype is GABAergic that receives excitatory input from On bipolar cells. The other population is a non-GABAergic, non-glycinergic (nGnG) AC subtype that lacks the expression of standard neurotransmitter markers. Gbx2 + nGnG ACs have smaller, asymmetric dendritic arbors that receive excitatory input from both On and Off bipolar cells. Gbx2 + nGnG ACs also exhibit spatially restricted tracer coupling to bipolar cells (BCs) through gap junctions. This study identifies a genetic tool for investigating the two distinct AC subtypes, and it provides a model for studying synaptic communication and visual circuit function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, there have been increasing indications that the endocannabinoid (eCB) system is involved in vision. Multiple research teams studied the cannabinoid receptor type 2 (CB2R) expression and function in the mouse retina. Here, we examined the consequence of CB2R modulation on visual acuity using genetic and pharmacologic tools. We found that Cnr2 knockout mice show an enhanced visual acuity, CB2R activation decreased visual acuity while CB2R blockade with the inverse agonist AM630 increased it. The inhibition of 2-arachidonylglycerol (2-AG) synthesis and degradation also greatly increased and decreased visual acuity, respectively. No differences were seen when the cannabinoid receptor type 1 (CB1R) was deleted, blocked or activated implying that CB2R exclusively mediates cannabinoid modulation of the visual acuity. We also investigated the role of cannabinoids in retinal function using electroretinography (ERG). We found that modulating 2-AG levels affected many ERG components, such as the a-wave and oscillatory potentials (OPs), suggesting an impact on cones and amacrine cells. Taken together, these results reveal that CB2R modulates visual acuity and that eCBs such as 2-AG can modulate both visual acuity and retinal sensitivity. Finally, these findings establish that CB2R is present in visual areas and regulates vision-related functions.

How astounding neuronal diversity arises from variable cell lineages in vertebrates remains mostly elusive. By in vivo lineage tracing of ∼1,000 single zebrafish retinal progenitors, we identified a repertoire of subtype-specific stereotyped neurogenic lineages. Remarkably, within these stereotyped lineages, GABAergic amacrine cells were born with photoreceptor cells, whereas glycinergic amacrine cells were born with OFF bipolar cells. More interestingly, post-mitotic differentiation blockage of GABAergic and glycinergic amacrine cells resulted in their respecification into photoreceptor and bipolar cells, respectively, suggesting lineage constraint in cell subtype specification. Using single-cell RNA-seq and ATAC-seq analyses, we further identified lineage-specific progenitors, each defined by specific transcription factors that exhibited characteristic chromatin accessibility dynamics. Finally, single pro-neural factors could specify different neuron types/subtypes in a lineage-dependent manner. Our findings reveal the importance of lineage context in defining neuronal subtypes and provide a demonstration of in vivo lineage-dependent induction of unique retinal neuron subtypes for treatment purposes., (© 2020 Wang et al.)

Purpose: Disruption in blood supply to active retinal circuits is the earliest hallmark of diabetic retinopathy (DR) and has been primarily attributed to vascular deficiency. However, accumulating evidence supports an early role for a disrupted neuronal function in blood flow impairment. Here, we tested the hypothesis that selectively stimulating cholinergic neurons could restore neurovascular signaling to preserve the capillary circulation in DR., Methods: We used wild type (wt) and choline acetyltransferase promoter (ChAT)-channelrhodopsin-2 (ChR2) mice expressing ChR2 exclusively in cholinergic cells. Mice were made diabetic by streptozotocin (STZ) injections. Two to 3 months after the last STZ injection, the rate of capillary blood flow was measured in vivo within each retinal vascular layer using high speed two-photon imaging. Measurements were done at baseline and following ChR2-driven activation of retinal cholinergic interneurons, the sole source of the vasodilating neurotransmitter acetylcholine. After recordings, retinas were collected and assessed for physiological and structural features., Results: In retinal explants from ChAT-ChR2 mice, we found that channelrhodopsin2 was selectively expressed in all cholinergic amacrine cells. Its direct activation by blue light led to dilation of adjacent retinal capillaries. In living diabetic ChAT-ChR2 animals, basal capillary blood flow was significantly higher than in diabetic mice without channelrhodopsin. However, optogenetic stimulation with blue light did not result in flickering light-induced functional hyperemia, suggesting a necessity for a concerted neurovascular interaction., Conclusions: These findings provide direct support to the utility and efficacy of an optogenetic approach for targeting selective retinal circuits to treat DR and its complications.

Background: Retinal circuitry provides a fundamental window to neural networks, featuring widely investigated visual phenomena ranging from direction selectivity to fast detection of approaching motion. As the divide between experimental and theoretical visual neuroscience is fading, neuronal modeling has proven to be important for retinal research. In neuronal modeling a delicate balance is maintained between bio-plausibility and model tractability, giving rise to myriad modeling frameworks. One biologically detailed framework for neuro modeling is NeuroConstruct, which facilitates the creation, visualization and analysis of neural networks in 3D., Results: Here, we extended NeuroConstruct to support the generation of structured visual stimuli, to feature different synaptic dynamics, to allow for heterogeneous synapse distribution and to enable rule-based synaptic connectivity between cell populations. We utilized this framework to demonstrate a simulation of a dense plexus of biologically realistic and morphologically detailed starburst amacrine cells. The amacrine cells were connected to a ganglion cell and stimulated with expanding and collapsing rings of light., Conclusions: This framework provides a powerful toolset for the investigation of the yet elusive underlying mechanisms of retinal computations such as direction selectivity. Particularly, we showcased the way NeuroConstruct can be extended to support advanced field-specific neuro-modeling.

Night vision in mammals depends fundamentally on rod photoreceptors and the well-studied rod bipolar (RB) cell pathway. The central neuron in this pathway, the AII amacrine cell (AC), exhibits a spatially tuned receptive field, composed of an excitatory center and an inhibitory surround, that propagates to ganglion cells, the retina's projection neurons. The circuitry underlying the surround of the AII, however, remains unresolved. Here, we combined structural, functional and optogenetic analyses of the mouse retina to discover that surround inhibition of the AII depends primarily on a single interneuron type, the NOS-1 AC: a multistratified, axon-bearing GABAergic cell, with dendrites in both ON and OFF synaptic layers, but with a pure ON (depolarizing) response to light. Our study demonstrates generally that novel neural circuits can be identified from targeted connectomic analyses and specifically that the NOS-1 AC mediates long-range inhibition during night vision and is a major element of the RB pathway., Competing Interests: SP, EL, JK, NR, PG, PR, NJ, HL, IK, KB, JD, JS No competing interests declared, (© 2020, Park et al.)

An antagonistic center-surround receptive field is a key feature in sensory processing, but how it contributes to specific computations such as direction selectivity is often unknown. Retinal On-starburst amacrine cells (SACs), which mediate direction selectivity in direction-selective ganglion cells (DSGCs), exhibit antagonistic receptive field organization: depolarizing to light increments and decrements in their center and surround, respectively. We find that a repetitive stimulation exhausts SAC center and enhances its surround and use it to study how center-surround responses contribute to direction selectivity. Center, but not surround, activation induces direction-selective responses in SACs. Nevertheless, both SAC center and surround elicited direction-selective responses in DSGCs, but to opposite directions. Physiological and modeling data suggest that the opposing direction selectivity can result from inverted temporal balance between excitation and inhibition in DSGCs, implying that SAC's response timing dictates direction selectivity. Our findings reveal antagonistic center-surround mechanisms for direction selectivity and demonstrate how context-dependent receptive field reorganization enables flexible computations., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Amacrine cells are a diverse population of interneurons in the retina that play a critical role in extracting complex features of the visual world and shaping the receptive fields of retinal output neurons (ganglion cells). While much of the computational power of amacrine cells is believed to arise from the immense mutual interactions among amacrine cells themselves, the intricate circuitry and functions of amacrine-amacrine interactions are poorly understood in general. Here we report a specific interamacrine pathway from a small-field, glutamate-glycine dual-transmitter amacrine cell (vGluT3) to a wide-field polyaxonal amacrine cell (PAS4/5). Distal tips of vGluT3 cell dendrites made selective glycinergic (but not glutamatergic) synapses onto PAS4/5 dendrites to provide a center-inhibitory, surround-disinhibitory drive that helps PAS4/5 cells build a suppressed-by-contrast (sbc) receptive field, which is a unique and fundamental trigger feature previously found only in a small population of ganglion cells. The finding of this trigger feature in a circuit upstream to ganglion cells suggests that the sbc form of visual computation occurs more widely in the retina than previously believed and shapes visual processing in multiple downstream circuits in multiple ways. We also identified two different subpopulations of PAS4/5 cells based on their differential connectivity with vGluT3 cells and their distinct receptive-field and luminance-encoding characteristics. Moreover, our results revealed a form of crosstalk between small-field and large-field amacrine cell circuits, which provides a mechanism for feature-specific local (<150 µm) control of global (>1 mm) retinal activity., Competing Interests: The authors declare no competing interest.

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals., Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices., Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs., Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal "b" wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the "a" wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber., Competing Interests: The authors declare no financial conflicts of interest.

Down syndrome cell adhesion molecules ( dscam and dscaml1 ) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), whereas abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders. SIGNIFICANCE STATEMENT Dscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders., (Copyright © 2020 the authors.)

Exocytotic release of neurotransmitter can be quantified by electrophysiological recording from postsynaptic neurons. Alternatively, fusion of synaptic vesicles with the cell membrane can be measured as increased capacitance by recording directly from a presynaptic neuron. The "Sine + DC" technique is based on recording from an unbranched cell, represented by an electrically equivalent RC-circuit. It is challenging to extend such measurements to branching neurons where exocytosis occurs at a distance from a somatic recording electrode. The AII amacrine is an important inhibitory interneuron of the mammalian retina and there is evidence that exocytosis at presynaptic lobular dendrites increases the capacitance. Here, we combined electrophysiological recording and computer simulations with realistic compartmental models to explore capacitance measurements of rat AII amacrine cells. First, we verified the ability of the "Sine + DC" technique to detect depolarization-evoked exocytosis in physiological recordings. Next, we used compartmental modeling to demonstrate that capacitance measurements can detect increased membrane surface area at lobular dendrites. However, the accuracy declines for lobular dendrites located further from the soma due to frequency-dependent signal attenuation. For sine wave frequencies ≥1 kHz, the magnitude of the total releasable pool of synaptic vesicles will be significantly underestimated. Reducing the sine wave frequency increases overall accuracy, but when the frequency is sufficiently low that exocytosis can be detected with high accuracy from all lobular dendrites (~100 Hz), strong electrical coupling between AII amacrines compromises the measurements. These results need to be taken into account in studies with capacitance measurements from these and other electrically coupled neurons., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Despite robust effects on immature neurons, growth factors minimally promote axon regeneration in the adult central nervous system (CNS). Attempting to improve growth-factor responsiveness in mature neurons by dedifferentiation, we overexpressed Lin28 in the retina. Lin28-treated retinas responded to insulin-like growth factor-1 (IGF1) by initiating retinal ganglion cell (RGC) axon regeneration after axotomy. Surprisingly, this effect was cell non-autonomous. Lin28 expression was required only in amacrine cells, inhibitory neurons that innervate RGCs. Ultimately, we found that optic-nerve crush pathologically upregulated activity in amacrine cells, which reduced RGC electrical activity and suppressed growth-factor signaling. Silencing amacrine cells or pharmacologically blocking inhibitory neurotransmission also induced IGF1 competence. Remarkably, RGCs regenerating across these manipulations localized IGF1 receptor to their primary cilia, which maintained their signaling competence and regenerative ability. Thus, our results reveal a circuit-based mechanism that regulates CNS axon regeneration and implicate primary cilia as a regenerative signaling hub., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.

Retinal waves, the spontaneous patterned neural activities propagating among developing retinal ganglion cells (RGCs), instruct the activity-dependent refinement of visuotopic maps. Although it is known that the wave is initiated successively by amacrine cells and bipolar cells, the behavior and function of glia in retinal waves remain unclear. Using multiple in vivo methods in larval zebrafish, we found that Müller glial cells (MGCs) display wave-like spontaneous activities, which start at MGC processes within the inner plexiform layer, vertically spread to their somata and endfeet, and horizontally propagate into neighboring MGCs. MGC waves depend on glutamatergic signaling derived from bipolar cells. Moreover, MGCs express both glia-specific glutamate transporters and the AMPA subtype of glutamate receptors. The AMPA receptors mediate MGC calcium activities during retinal waves, whereas the glutamate transporters modulate the occurrence of retinal waves. Thus, MGCs can sense and regulate retinal waves via AMPA receptors and glutamate transporters, respectively., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sholl analysis has been an important technique in dendritic anatomy for more than 60 years. The Sholl intersection profile is obtained by counting the number of dendritic branches at a given distance from the soma and is a key measure of dendritic complexity; it has applications from evaluating the changes in structure induced by pathologies to estimating the expected number of anatomical synaptic contacts. We find that the Sholl intersection profiles of most neurons can be reproduced from three basic, functional measures: the domain spanned by the dendritic arbor, the total length of the dendrite, and the angular distribution of how far dendritic segments deviate from a direct path to the soma (i.e., the root angle distribution). The first two measures are determined by axon location and hence microcircuit structure; the third arises from optimal wiring and represents a branching statistic estimating the need for conduction speed in a neuron., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

A neuron is conventionally regarded as a single processing unit. It receives input from one or several presynaptic cells, transforms these signals, and transmits one output signal to its postsynaptic partners. Exceptions exist: amacrine cells in the mammalian retina [1-3] or interneurons in the locust mesothoracic ganglion [4] are thought to represent many electrically isolated microcircuits within one neuron. An extreme case of such an amacrine cell has recently been described in the Drosophila visual system. This cell, called CT1, reaches into two neuropils of the optic lobe, where it visits each of 700 repetitive columns, thereby covering the whole visual field [5, 6]. Due to its unusual morphology, CT1 has been suspected to perform local computations [6, 7], but this has never been proven. Using 2-photon calcium imaging and visual stimulation, we find highly compartmentalized retinotopic response properties in neighboring terminals of CT1, with each terminal acting as an independent functional unit. Model simulations demonstrate that this extreme case of compartmentalization is at the biophysical limit of neural computation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During adaptation from dim to bright environments, changes in retinal signaling are mediated, in part, by dopamine. Dopamine is released with light and can modulate retinal receptive fields, neuronal coupling, inhibitory receptors, and rod pathway inhibition. However, it is unclear how dopamine affects inner retinal inhibition to cone bipolar cells, which relay visual information from photoreceptors to ganglion cells and are important signal processing sites. We tested the hypothesis that dopamine (D)1 receptor activation is sufficient to elicit light-adapted inhibitory changes. Local light-evoked inhibition and spontaneous activity were measured from OFF cone bipolar cells in dark-adapted mouse retinas while stimulating D1 receptors, which are located on bipolar, horizontal, and inhibitory amacrine cells. The D1 agonist SKF38393 reduced local inhibitory light-evoked response magnitude and increased response transience, which mimicked changes measured with light adaptation. D1-mediated reductions in local inhibition were more pronounced for glycinergic than GABAergic inputs, comparable with light adaptation. The effects of D1 receptors on light-evoked input were similar to the effects on spontaneous input. D1 receptor activation primarily decreased glycinergic spontaneous current frequency, similar to light adaptation, suggesting mainly a presynaptic amacrine cell site of action. These results expand the role of dopamine to include signal modulation of cone bipolar cell local inhibition. In this role, D1 receptor activation, acting primarily through glycinergic amacrine cells, may be an important mechanism for the light-adapted reduction in OFF bipolar cell inhibition since the actions are similar and dopamine is released during light adaptation. NEW & NOTEWORTHY Retinal adaptation to different luminance conditions requires the adjustment of local circuits for accurate signaling of visual scenes. Understanding mechanisms behind luminance adaptation at different retinal levels is important for understanding how the retina functions in a dynamic environment. In the mouse, we show that dopamine pathways reduce inner retinal inhibition similar to increased background luminance, suggesting the two are linked and highlighting a possible mechanism for light adaptation at an early retinal processing center.

Retinal dopamine is released by a specialized subset of amacrine cells in response to light and has a potent influence on how the retina responds to, and encodes, visual information. Here, we address the critical question of which retinal photoreceptor is responsible for coordinating the release of this neuromodulator. Although all three photoreceptor classes-rods, cones, and melanopsin-containing retinal ganglion cells (mRGCs)-have been shown to provide electrophysiological inputs to dopaminergic amacrine cells (DACs), we show here that the release of dopamine is defined only by rod photoreceptors. Remarkably, this rod signal coordinates both a suppressive signal at low intensities and drives dopamine release at very bright light intensities. These data further reveal that dopamine release does not necessarily correlate with electrophysiological activity of DACs and add to a growing body of evidence that rods define aspects of retinal function at very bright light levels., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Patterned spontaneous activity periodically displays in developing retinas termed retinal waves, essential for visual circuit refinement. In neonatal rodents, retinal waves initiate in starburst amacrine cells (SACs), propagating across retinal ganglion cells (RGCs), further through visual centers. Although these waves are shown temporally synchronized with transiently high PKA activity, the downstream PKA target important for regulating the transmission from SACs remains unidentified. A t-SNARE, synaptosome-associated protein of 25 kDa (SNAP-25/SN25), serves as a PKA substrate, implying a potential role of SN25 in regulating retinal development. Here, we examined whether SN25 in SACs could regulate wave properties and retinogeniculate projection during development. In developing SACs, overexpression of wild-type SN25b, but not the PKA-phosphodeficient mutant (SN25b-T138A), decreased the frequency and spatial correlation of wave-associated calcium transients. Overexpressing SN25b, but not SN25b-T138A, in SACs dampened spontaneous, wave-associated, postsynaptic currents in RGCs and decreased the SAC release upon augmenting the cAMP-PKA signaling. These results suggest that SN25b overexpression may inhibit the strength of transmission from SACs via PKA-mediated phosphorylation at T138. Moreover, knockdown of endogenous SN25b increased the frequency of wave-associated calcium transients, supporting the role of SN25 in restraining wave periodicity. Finally, the eye-specific segregation of retinogeniculate projection was impaired by in vivo overexpression of SN25b, but not SN25b-T138A, in SACs. These results suggest that SN25 in developing SACs dampens the spatiotemporal properties of retinal waves and limits visual circuit refinement by phosphorylation at T138. Therefore, SN25 in SACs plays a profound role in regulating visual circuit refinement., Competing Interests: The authors declare no conflict of interest.

During early development, waves of activity propagate across the retina and play a key role in the proper wiring of the early visual system. During a particular phase of the retina development (stage II) these waves are triggered by a transient network of neurons, called Starburst Amacrine Cells (SACs), showing a bursting activity which disappears upon further maturation. The underlying mechanisms of the spontaneous bursting and the transient excitability of immature SACs are not completely clear yet. While several models have attempted to reproduce retinal waves, none of them is able to mimic the rhythmic autonomous bursting of individual SACs and reveal how these cells change their intrinsic properties during development. Here, we introduce a mathematical model, grounded on biophysics, which enables us to reproduce the bursting activity of SACs and to propose a plausible, generic and robust, mechanism that generates it. The core parameters controlling repetitive firing are fast depolarizing V-gated calcium channels and hyperpolarizing V-gated potassium channels. The quiescent phase of bursting is controlled by a slow after hyperpolarization (sAHP), mediated by calcium-dependent potassium channels. Based on a bifurcation analysis we show how biophysical parameters, regulating calcium and potassium activity, control the spontaneously occurring fast oscillatory activity followed by long refractory periods in individual SACs. We make a testable experimental prediction on the role of voltage-dependent potassium channels on the excitability properties of SACs and on the evolution of this excitability along development. We also propose an explanation on how SACs can exhibit a large variability in their bursting periods, as observed experimentally within a SACs network as well as across different species, yet based on a simple, unique, mechanism. As we discuss, these observations at the cellular level have a deep impact on the retinal waves description.