In the outer retina, G protein-coupled receptor (GPCR) signaling mediates phototransduction and synaptic transmission between photoreceptors and ON bipolar cells. In contrast, the functions of modulatory GPCR signaling networks in the inner retina are less well understood. We addressed this question by determining the consequences of augmenting modulatory Gi/o signaling driven by endogenous transmitters. This was done by analyzing the effects of genetically ablating the R7 RGS-binding protein (R7BP), a membrane-targeting protein and positive allosteric modulator of R7-RGS (regulator of the G protein signaling 7) family that deactivates Gi/oα subunits. We found that R7BP is expressed highly in starburst amacrine cells and retinal ganglion cells (RGCs). As indicated by electroretinography and multielectrode array recordings of adult retina, ablation of R7BP preserved outer retina function, but altered the firing rate and latency of ON RGCs driven by rods and cones but not rods alone. In developing retina, R7BP ablation increased the burst duration of glutamatergic waves whereas cholinergic waves were unaffected. This effect on glutamatergic waves did not result in impaired segregation of RGC projections to eye-specific domains of the dorsal lateral geniculate nucleus. R7BP knockout mice exhibited normal spatial contrast sensitivity and visual acuity as assessed by optomotor reflexes. Taken together these findings indicate that R7BP-dependent regulation of R7-RGS proteins shapes specific aspects of light-evoked and spontaneous activity of RGCs in mature and developing retina.

Activity is thought to guide the patterning of synaptic connections in the developing nervous system. Specifically, differences in the activity of converging inputs are thought to cause the elimination of synapses from less active inputs and increase connectivity with more active inputs. Here we present findings that challenge the generality of this notion and offer a new view of the role of activity in synapse development. To imbalance neurotransmission from different sets of inputs in vivo, we generated transgenic mice in which ON but not OFF types of bipolar cells in the retina express tetanus toxin (TeNT). During development, retinal ganglion cells (RGCs) select between ON and OFF bipolar cell inputs (ON or OFF RGCs) or establish a similar number of synapses with both on separate dendritic arborizations (ON-OFF RGCs). In TeNT retinas, ON RGCs correctly selected the silenced ON bipolar cell inputs over the transmitting OFF bipolar cells, but were connected with them through fewer synapses at maturity. Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination. Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones. The remaining active zones often recruited multiple, instead of single, synaptic ribbons. ON-OFF RGCs in TeNT mice maintained convergence of ON and OFF bipolar cells inputs and had fewer synapses on their ON arbor without changes to OFF arbor synapses. Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

The size of dendrite arbors shapes their function and differs vastly between neuron types. The signals that control dendritic arbor size remain obscure. Here, we find that in the retina, starburst amacrine cells (SACs) and rod bipolar cells (RBCs) express the homophilic cell-surface protein AMIGO2. In Amigo2 knockout (KO) mice, SAC and RBC dendrites expand while arbors of other retinal neurons remain stable. SAC dendrites are divided into a central input region and a peripheral output region that provides asymmetric inhibition to direction-selective ganglion cells (DSGCs). Input and output compartments scale precisely with increased arbor size in Amigo2 KO mice, and SAC dendrites maintain asymmetric connectivity with DSGCs. Increased coverage of SAC dendrites is accompanied by increased direction selectivity of DSGCs without changes to other ganglion cells. Our results identify AMIGO2 as a cell-type-specific dendritic scaling factor and link dendrite size and coverage to visual feature detection.

How sensory systems extract salient features from natural environments and organize them across neural pathways is unclear. Combining single-cell and population two-photon calcium imaging in mice, we discover that retinal ON bipolar cells (second-order neurons of the visual system) are divided into two blocks of four types. The two blocks distribute temporal and spatial information encoding, respectively. ON bipolar cell axons co-stratify within each block, but separate laminarly between them (upper block: diverse temporal, uniform spatial tuning; lower block: diverse spatial, uniform temporal tuning). ON bipolar cells extract temporal and spatial features similarly from artificial and naturalistic stimuli. In addition, they differ in sensitivity to coherent motion in naturalistic movies. Motion information is distributed across ON bipolar cells in the upper and the lower blocks, multiplexed with temporal and spatial contrast, independent features of natural scenes. Comparing the responses of different boutons within the same arbor, we find that axons of all ON bipolar cell types function as computational units. Thus, our results provide insights into the visual feature extraction from naturalistic stimuli and reveal how structural and functional organization cooperate to generate parallel ON pathways for temporal and spatial information in the mammalian retina.

To integrate information from different presynaptic cell types, dendrites receive distinct patterns of synapses from converging axons. How different afferents in vivo establish specific connectivity patterns with the same dendrite is poorly understood. Here, we examine the synaptic development of three glutamatergic bipolar cell types converging onto a common postsynaptic retinal ganglion cell. We find that after axons and dendrites target appropriate synaptic layers, patterns of connections among these neurons diverge through selective changes in the conversion of axo-dendritic appositions to synapses. This process is differentially regulated by neurotransmission, which is required for the shift from single to multisynaptic appositions of one bipolar cell type but not for maintenance and elimination, respectively, of connections from the other two types. Thus, synaptic specificity among converging excitatory inputs in the retina emerges via differential synaptic maturation of axo-dendritic appositions and is shaped by neurotransmission in a cell type-dependent manner.

Neurons receive synaptic inputs on extensive neurite arbors. How information is organized across arbors and how local processing in neurites contributes to circuit function is mostly unknown. Here, we used two-photon Ca2+ imaging to study visual processing in VGluT3-expressing amacrine cells (VG3-ACs) in the mouse retina. Contrast preferences (ON vs. OFF) varied across VG3-AC arbors depending on the laminar position of neurites, with ON responses preferring larger stimuli than OFF responses. Although arbors of neighboring cells overlap extensively, imaging population activity revealed continuous topographic maps of visual space in the VG3-AC plexus. All VG3-AC neurites responded strongly to object motion, but remained silent during global image motion. Thus, VG3-AC arbors limit vertical and lateral integration of contrast and location information, respectively. We propose that this local processing enables the dense VG3-AC plexus to contribute precise object motion signals to diverse targets without distorting target-specific contrast preferences and spatial receptive fields.

The spike trains of retinal ganglion cells (RGCs) are the only source of visual information to the brain. Here, we genetically identify an RGC type in mice that functions as a pixel encoder and increases firing to light increments (PixON-RGC). PixON-RGCs have medium-sized dendritic arbors and non-canonical center-surround receptive fields. From their receptive field center, PixON-RGCs receive only excitatory input, which encodes contrast and spatial information linearly. From their receptive field surround, PixON-RGCs receive only inhibitory input, which is temporally matched to the excitatory center input. As a result, the firing rate of PixON-RGCs linearly encodes local image contrast. Spatially offset (i.e., truly lateral) inhibition of PixON-RGCs arises from spiking GABAergic amacrine cells. The receptive field organization of PixON-RGCs is independent of stimulus wavelength (i.e., achromatic). PixON-RGCs project predominantly to the dorsal lateral geniculate nucleus (dLGN) of the thalamus and likely contribute to visual perception.

Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells.

In humans, midget and parasol ganglion cells account for most of the input from the eyes to the brain. Yet, how they encode visual information is unknown. Here, we perform large-scale multi-electrode array recordings from retinas of treatment-naive patients who underwent enucleation surgery for choroidal malignant melanomas. We identify robust differences in the function of midget and parasol ganglion cells, consistent asymmetries between their ON and OFF types (that signal light increments and decrements, respectively) and divergence in the function of human versus non-human primate retinas. Our computational analyses reveal that the receptive fields of human midget and parasol ganglion cells divide naturalistic movies into adjacent spatiotemporal frequency domains with equal stimulus power, while the asymmetric response functions of their ON and OFF types simultaneously maximize stimulus coverage and information transmission and minimize metabolic cost. Thus, midget and parasol ganglion cells in the human retina efficiently encode our visual environment.

The medial entorhinal cortex (MEC) is hypothesized to function as a cognitive map for memory-guided navigation. How this map develops during learning and influences memory remains unclear. By imaging MEC calcium dynamics while mice successfully learned a novel virtual environment over ten days, we discovered that the dynamics gradually became more spatially consistent and then stabilized. Additionally, grid cells in the MEC not only exhibited improved spatial tuning consistency, but also maintained stable phase relationships, suggesting a network mechanism involving synaptic plasticity and rigid recurrent connectivity to shape grid cell activity during learning. Increased c-Fos expression in the MEC in novel environments further supports the induction of synaptic plasticity. Unsuccessful learning lacked these activity features, indicating that a consistent map is specific for effective spatial memory. Finally, optogenetically disrupting spatial consistency of the map impaired memory-guided navigation in a well-learned environment. Thus, we demonstrate that the establishment of a spatially consistent MEC map across learning both correlates with, and is necessary for, successful spatial memory.

Neuronal output is shaped by a balance of excitation and inhibition. How this balance is attained in the central nervous system during development is not well understood, and is complicated by the fact that, in vivo, GABAergic and glycinergic synaptogenesis precedes that of glutamatergic synapses. Here, we determined the distributions of inhibitory postsynaptic sites on the dendritic arbors of individual neurons, and compared their developmental patterns with that of excitatory postsynaptic sites. We focused on retinal ganglion cells (RGCs), the output neurons of the retina, which receive excitatory input from bipolar cells and inhibitory input from amacrine cells. To visualize and map inhibitory postsynaptic sites, we generated transgenic mice in which RGCs express fluorescently tagged Neuroligin 2 (YFP-NL2) under the control of the Thy1 promoter. By labeling RGC dendrites biolistically in YFP-NL2-expressing retinas, we were able to map the spatial distribution and thus densities of inhibitory postsynaptic sites on the dendritic arbors of individual large-field RGCs across ages.

Synaptic cell adhesion molecules (CAMs) promote synapse formation in the developing nervous system. To what extent they maintain and can restore connections in the mature nervous system is unknown. Furthermore, how synaptic CAMs affect the growth of synapse-bearing neurites is unclear. Here, we use adeno-associated viruses (AAVs) to delete, re-, and overexpress the synaptic CAM NGL2 in individual retinal horizontal cells. When we removed NGL2 from horizontal cells, their axons overgrew and formed fewer synapses, irrespective of whether Ngl2 was deleted during development or in mature circuits. When we re-expressed NGL2 in knockout mice, horizontal cell axon territories and synapse numbers were restored, even if AAVs were injected after phenotypes had developed. Finally, overexpression of NGL2 in wild-type horizontal cells elevated synapse numbers above normal levels. Thus, NGL2 promotes the formation, maintenance, and restoration of synapses in the developing and mature retina, and restricts axon growth throughout life.

Ether à go-go (Eag; KV10.1) voltage-gated K+ channels have been detected in cancer cell lines of diverse origin and shown to influence their rate of proliferation. The tricyclic antidepressant imipramine and the antihistamine astemizole inhibit the current through Eag1 channels and reduce the proliferation of cancer cells. Here we describe the mechanism by which both drugs block human Eag1 (hEag1) channels. Even if both drugs differ in their affinity for hEag1 channels (IC50s are approximately 2 microM for imipramine and approximately 200 nM for astemizole) and in their blocking kinetics, both drugs permeate the membrane and inhibit the hEag1 current by selectively binding to open channels. Furthermore, both drugs are weak bases and the IC50s depend on both internal an external pH, suggesting that both substances cross the membrane in their uncharged form and act from inside the cell in their charged forms. Accordingly, the block by imipramine is voltage dependent and antagonized by intracellular TEA, consistent with imipramine binding in its charged form to a site located close to the inner end of the selectivity filter. Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane. In contrast, the block by astemizole is voltage independent. However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site. The significance of these findings, in the context of structure-function of channels of the eag family is discussed.

Object motion sensitive (OMS) W3-retinal ganglion cells (W3-RGCs) in mice respond to local movements in a visual scene but remain silent during self-generated global image motion. The excitatory inputs that drive responses of W3-RGCs to local motion were recently characterized, but which inhibitory neurons suppress W3-RGCs' responses to global motion, how these neurons encode motion information, and how their connections are organized along the excitatory circuit axis remains unknown. Here, we find that a genetically identified amacrine cell (AC) type, TH2-AC, exhibits fast responses to global motion and slow responses to local motion. Optogenetic stimulation shows that TH2-ACs provide strong GABAA receptor-mediated input to W3-RGCs but only weak input to upstream excitatory neurons. Cell-type-specific silencing reveals that temporally coded inhibition from TH2-ACs cancels W3-RGC spike responses to global but not local motion stimuli and, thus, controls the feature selectivity of OMS signals sent to the brain.

Predators use vision to hunt, and hunting success is one of evolution's main selection pressures. However, how viewing strategies and visual systems are adapted to predation is unclear. Tracking predator-prey interactions of mice and crickets in 3D, we find that mice trace crickets with their binocular visual fields and that monocular mice are poor hunters. Mammalian binocular vision requires ipsi- and contralateral projections of retinal ganglion cells (RGCs) to the brain. Large-scale single-cell recordings and morphological reconstructions reveal that only a small subset (9 of 40+) of RGC types in the ventrotemporal mouse retina innervate ipsilateral brain areas (ipsi-RGCs). Selective ablation of ipsi-RGCs (<2% of RGCs) in the adult retina drastically reduces the hunting success of mice. Stimuli based on ethological observations indicate that five ipsi-RGC types reliably signal prey. Thus, viewing strategies align with a spatially restricted and cell-type-specific set of ipsi-RGCs that supports binocular vision to guide predation.

Dendrite and axon arbor sizes are critical to neuronal function and vary widely between different neuron types. The relative dendrite and axon sizes of synaptic partners control signal convergence and divergence in neural circuits. The developmental mechanisms that determine cell-type-specific dendrite and axon size and match synaptic partners' arbor territories remain obscure. Here, we discover that retinal horizontal cells express the leucine-rich repeat domain cell adhesion molecule AMIGO1. Horizontal cells provide pathway-specific feedback to photoreceptors-horizontal cell axons to rods and horizontal cell dendrites to cones. AMIGO1 selectively expands the size of horizontal cell axons. When Amigo1 is deleted in all or individual horizontal cells of either sex, their axon arbors shrink. By contrast, horizontal cell dendrites and synapse formation of horizontal cell axons and dendrites are unaffected by AMIGO1 removal. The dendrites of rod bipolar cells, which do not express AMIGO1, shrink in parallel with horizontal cell axons in Amigo1 knockout (Amigo1 KO) mice. This territory matching maintains the function of the rod bipolar pathway, preserving bipolar cell responses and retinal output signals in Amigo1 KO mice. We previously identified AMIGO2 as a scaling factor that constrains retinal neurite arbors. Our current results identify AMIGO1 as a scaling factor that expands retinal neurite arbors and reveal territory matching as a novel homeostatic mechanism. Territory matching interacts with other homeostatic mechanisms to stabilize the development of the rod bipolar pathway, which mediates vision near the threshold.SIGNIFICANCE STATEMENT Neurons send and receive signals through branched axonal and dendritic arbors. The size of these arbors is critical to the function of a neuron. Axons and dendrites grow during development and are stable at maturity. The mechanisms that determine axon and dendrite size are not well understood. Here, we identify a cell surface protein, AMIGO1, that selectively promotes axon growth of horizontal cells, a retinal interneuron. Removal of AMIGO1 reduces the size of horizontal cell axons without affecting the size of their dendrites or the ability of both arbors to form connections. The changes in horizontal cell axons are matched by changes in synaptic partner dendrites to stabilize retinal function. This identifies territory matching as a novel homeostatic plasticity mechanism.

Understanding a sensory system implies the ability to predict responses to a variety of inputs from a common model. In the retina, this includes predicting how the integration of signals across visual space shapes the outputs of retinal ganglion cells. Existing models of this process generalize poorly to predict responses to new stimuli. This failure arises in part from properties of the ganglion cell response that are not well captured by standard receptive-field mapping techniques: nonlinear spatial integration and fine-scale heterogeneities in spatial sampling. Here we characterize a ganglion cell's spatial receptive field using a mechanistic model based on measurements of the physiological properties and connectivity of only the primary excitatory circuitry of the retina. The resulting simplified circuit model successfully predicts ganglion-cell responses to a variety of spatial patterns and thus provides a direct correspondence between circuit connectivity and retinal output.

Mitochondria move throughout neuronal dendrites and localize to sites of energy demand. The prevailing view of dendritic mitochondria as highly motile organelles whose distribution is continually adjusted by neuronal activity via Ca(2+)-dependent arrests is based on observations in cultured neurons exposed to artificial stimuli. Here, we analyze the movements of mitochondria in ganglion cell dendrites in the intact retina. We find that whereas during development 30% of mitochondria are motile at any time, as dendrites mature, mitochondria all but stop moving and localize stably to synapses and branch points. Neither spontaneous nor sensory-evoked activity and Ca(2+) transients alter motility of dendritic mitochondria; and pathological hyperactivity in a mouse model of retinal degeneration elevates rather than reduces motility. Thus, our findings indicate that dendritic mitochondria reach stable positions during a critical developmental period of high motility, and challenge current views about the role of activity in regulating mitochondrial transport in dendrites.

Neurons that release more than one transmitter exist throughout the CNS. Yet, how these neurons deploy multiple transmitters and shape the function of specific circuits is not well understood. VGluT3-expressing amacrine cells (VG3-ACs) provide glutamatergic input to ganglion cells activated by contrast and motion. Using optogenetics, we find that VG3-ACs provide selective glycinergic input to a retinal ganglion cell type suppressed by contrast and motion (SbC-RGCs). Firing of SbC-RGCs is suppressed at light ON and OFF over a broad range of stimulus sizes. Anatomical circuit reconstructions reveal that VG3-ACs form inhibitory synapses preferentially on processes that link ON and OFF arbors of SbC-RGC dendrites. Removal of VG3-ACs from mature circuits reduces inhibition and attenuates spike suppression of SbC-RGCs in a contrast- and size-selective manner, illustrating the modularity of retinal circuits. VG3-ACs thus use dual transmitters in a target-specific manner and shape suppressive contrast responses in the retina by glycinergic transmission.

Convergent input from different presynaptic partners shapes the responses of postsynaptic neurons. Whether developing postsynaptic neurons establish connections with each presynaptic partner independently or balance inputs to attain specific responses is unclear. Retinal ganglion cells (RGCs) receive convergent input from bipolar cell types with different contrast responses and temporal tuning. Here, using optogenetic activation and pharmacogenetic silencing, we found that type 6 bipolar (B6) cells dominate excitatory input to ONα-RGCs. We generated mice in which B6 cells were selectively removed from developing circuits (B6-DTA). In B6-DTA mice, ONα-RGCs adjusted connectivity with other bipolar cells in a cell-type-specific manner. They recruited new partners, increased synapses with some existing partners, and maintained constant input from others. Patch-clamp recordings revealed that anatomical rewiring precisely preserved contrast and temporal frequency response functions of ONα-RGCs, indicating that homeostatic plasticity shapes cell-type-specific wiring in the developing retina to stabilize visual information sent to the brain.