Preservation of vision after CaMKII-mediated protection of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.

Brn3b regulates the formation of fear-related midbrain circuits and defensive responses to visual threat.

Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior.

The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3.

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (Dnm1-3), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas.

In zebrafish, Müller glia (MG) are a source of retinal stem cells that can replenish damaged retinal neurons and restore vision1. In mammals, however, MG do not spontaneously re-enter the cell cycle to generate a population of stem or progenitor cells that differentiate into retinal neurons. Nevertheless, the regenerative machinery may exist in the mammalian retina, as retinal injury can stimulate MG proliferation followed by limited neurogenesis2-7. Therefore, there is still a fundamental question regarding whether MG-derived regeneration can be exploited to restore vision in mammalian retinas. Gene transfer of ß-catenin stimulates MG proliferation in the absence of injury in mouse retinas8. Here we report that following gene transfer of ß-catenin, cell-cycle-reactivated MG can be reprogrammed to generate rod photoreceptors by subsequent gene transfer of transcription factors essential for rod cell fate specification and determination. MG-derived rods restored visual responses in Gnat1rd17Gnat2cpfl3 double mutant mice, a model of congenital blindness9,10, throughout the visual pathway from the retina to the primary visual cortex. Together, our results provide evidence of vision restoration after de novo MG-derived genesis of rod photoreceptors in mammalian retinas.

Photoreceptive Ganglion Cells Drive Circuits for Local Inhibition in the Mouse Retina.

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.

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

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

Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry.

Inhibitory interneurons sculpt the outputs of excitatory circuits to expand the dynamic range of information processing. In mammalian retina, >30 types of amacrine cells provide lateral inhibition to vertical, excitatory bipolar cell circuits, but functional roles for only a few amacrine cells are well established. Here, we elucidate the function of corticotropin-releasing hormone (CRH)-expressing amacrine cells labeled in Cre-transgenic mice of either sex. CRH cells costratify with the ON alpha ganglion cell, a neuron highly sensitive to positive contrast. Electrophysiological and optogenetic analyses demonstrate that two CRH types (CRH-1 and CRH-3) make GABAergic synapses with ON alpha cells. CRH-1 cells signal via graded membrane potential changes, whereas CRH-3 cells fire action potentials. Both types show sustained ON-type responses to positive contrast over a range of stimulus conditions. Optogenetic control of transmission at CRH-1 synapses demonstrates that these synapses are tuned to low temporal frequencies, maintaining GABA release during fast hyperpolarizations during brief periods of negative contrast. CRH amacrine cell output is suppressed by prolonged negative contrast, when ON alpha ganglion cells continue to receive inhibitory input from converging OFF-pathway amacrine cells; the converging ON- and OFF-pathway inhibition balances tonic excitatory drive to ON alpha cells. Previously, it was demonstrated that CRH-1 cells inhibit firing by suppressed-by-contrast (SbC) ganglion cells during positive contrast. Therefore, divergent outputs of CRH-1 cells inhibit two ganglion cell types with opposite responses to positive contrast. The opposing responses of ON alpha and SbC ganglion cells are explained by differing excitation/inhibition balance in the two circuits.SIGNIFICANCE STATEMENT A goal of neuroscience research is to explain the function of neural circuits at the level of specific cell types. Here, we studied the function of specific types of inhibitory interneurons, corticotropin-releasing hormone (CRH) amacrine cells, in the mouse retina. Genetic tools were used to identify and manipulate CRH cells, which make GABAergic synapses with a well studied ganglion cell type, the ON alpha cell. CRH cells converge with other types of amacrine cells to tonically inhibit ON alpha cells and balance their high level of excitation. CRH cells diverge to different types of ganglion cell, the unique properties of which depend on their balance of excitation and inhibition.

Function and Circuitry of VIP+ Interneurons in the Mouse Retina.

Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30-40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP+) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP+ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP+ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP+ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT: The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and communicate with a complex network of interneurons. These interneurons drive the responses of ganglion cells, which form the optic nerve and transmit visual information to the brain. Presently, we lack information about many of the retina's inhibitory amacrine interneurons. In this study, we used genetically modified mice to study the light responses and intercellular connections of specific amacrine cell types. The results show diversity in the shape and function of the studied amacrine cells and elucidate their connections with specific types of ganglion cell. The findings advance our understanding of the cellular basis for retinal function.

An optimized fluorescent probe for visualizing glutamate neurotransmission.

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Developmental changes in NMDA receptor subunit composition at ON and OFF bipolar cell synapses onto direction-selective retinal ganglion cells.

In the developing mouse retina, spontaneous and light-driven activity shapes bipolar→ganglion cell glutamatergic synapse formation, beginning around the time of eye-opening (P12-P14) and extending through the first postnatal month. During this time, glutamate release can spill outside the synaptic cleft and possibly stimulate extrasynaptic NMDA-type glutamate receptors (NMDARs) on ganglion cells. Furthermore, the role of NMDARs during development may differ between ON and OFF bipolar synapses as in mature retina, where ON synapses reportedly include extrasynaptic NMDARs with GluN2B subunits. To better understand the function of glutamatergic synapses during development, we made whole-cell recordings of NMDAR-mediated responses, in vitro, from two types of genetically identified direction-selective ganglion cells (dsGCs): TRHR (thyrotropin-releasing hormone receptor) and Drd4 (dopamine receptor 4). Both dsGC types responded to puffed NMDA between P7 and P28; and both types exhibited robust light-evoked NMDAR-mediated responses at P14 and P28 that were quantified by conductance analysis during nicotinic and GABA(A) receptor blockade. For a given cell type and at a given age, ON and OFF bipolar cell inputs evoked similar NMDAR-mediated responses, suggesting that ON-versus-OFF differences in mature retina do not apply to the cell types or ages studied here. At P14, puff- and light-evoked NMDAR-mediated responses in both dsGCs were partially blocked by the GluN2B antagonist ifenprodil, whereas at P28 only TRHR cells remained ifenprodil-sensitive. NMDARs contribute at both ON and OFF bipolar cell synapses during a period of robust activity-dependent synaptic development, with declining GluN2B involvement over time in specific ganglion cell types.

Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion cells lack direction tuning.

Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina.

Alpha/Y-type retinal ganglion cells encode visual information with a receptive field composed of nonlinear subunits. This nonlinear subunit structure enhances sensitivity to patterns composed of high spatial frequencies. The Y-cell's subunits are the presynaptic bipolar cells, but the mechanism for the nonlinearity remains incompletely understood. We investigated the synaptic basis of the subunit nonlinearity by combining whole-cell recording of mouse Y-type ganglion cells with two-photon fluorescence imaging of a glutamate sensor (iGluSnFR) expressed on their dendrites and throughout the inner plexiform layer. A control experiment designed to assess iGluSnFR's dynamic range showed that fluorescence responses from Y-cell dendrites increased proportionally with simultaneously recorded excitatory current. Spatial resolution was sufficient to readily resolve independent release at intermingled ON and OFF bipolar terminals. iGluSnFR responses at Y-cell dendrites showed strong surround inhibition, reflecting receptive field properties of presynaptic release sites. Responses to spatial patterns located the origin of the Y-cell nonlinearity to the bipolar cell output, after the stage of spatial integration. The underlying mechanism differed between OFF and ON pathways: OFF synapses showed transient release and strong rectification, whereas ON synapses showed relatively sustained release and weak rectification. At ON synapses, the combination of fast release onset with slower release offset explained the nonlinear response of the postsynaptic ganglion cell. Imaging throughout the inner plexiform layer, we found transient, rectified release at the central-most levels, with increasingly sustained release near the borders. By visualizing glutamate release in real time, iGluSnFR provides a powerful tool for characterizing glutamate synapses in intact neural circuits.

Transsynaptic tracing with vesicular stomatitis virus reveals novel retinal circuitry.

The use of neurotropic viruses as transsynaptic tracers was first described in the 1960s, but only recently have such viruses gained popularity as a method for labeling neural circuits. The development of retrograde monosynaptic tracing vectors has enabled visualization of the presynaptic sources onto defined sets of postsynaptic neurons. Here, we describe the first application of a novel viral tracer, based on vesicular stomatitis virus (VSV), which directs retrograde transsynaptic viral spread between defined cell types. We use this virus in the mouse retina to show connectivity between starburst amacrine cells (SACs) and their known synaptic partners, direction-selective retinal ganglion cells, as well as to discover previously unknown connectivity between SACs and other retinal ganglion cell types. These novel connections were confirmed using physiological recordings. VSV transsynaptic tracing enables cell type-specific dissection of neural circuitry and can reveal synaptic relationships among neurons that are otherwise obscured due to the complexity and density of neuropil.

NMDA and AMPA receptors contribute similarly to temporal processing in mammalian retinal ganglion cells.

Postsynaptic AMPA- and NMDA-type glutamate receptors (AMPARs, NMDARs) are commonly expressed at the same synapses. AMPARs are thought to mediate the majority of fast excitatory neurotransmission whereas NMDARs, with their relatively slower kinetics and higher Ca(2+) permeability, are thought to mediate synaptic plasticity, especially in neural circuits devoted to learning and memory. In sensory neurons, however, the roles of AMPARs and NMDARs are less well understood. Here, we tested in the in vitro guinea pig retina whether AMPARs and NMDARs differentially support temporal contrast encoding by two ganglion cell types. In both OFF Alpha and Delta ganglion cells, contrast stimulation evoked an NMDAR-mediated response with a characteristic J-shaped I-V relationship. In OFF Delta cells, AMPAR- and NMDAR-mediated responses could be modulated at low frequencies but were suppressed during 10 Hz stimulation, when responses were instead shaped by synaptic inhibition. With inhibition blocked, both AMPAR- and NMDAR-mediated responses could be modulated at 10 Hz, indicating that NMDAR kinetics do not limit temporal encoding. In OFF Alpha cells, NMDAR-mediated responses followed stimuli at frequencies up to ∼18 Hz. In both cell types, NMDAR-mediated responses to contrast modulation at 9-18 Hz showed delays of <10 ms relative to AMPAR-mediated responses. Thus, NMDARs combine with AMPARs to encode rapidly modulated glutamate release, and NMDAR kinetics do not limit temporal coding by OFF Alpha and Delta ganglion cells substantially. Furthermore, glutamatergic transmission is differentially regulated across bipolar cell pathways: in some, release is suppressed at high temporal frequencies by presynaptic inhibition.

Form and function of the M4 cell, an intrinsically photosensitive retinal ganglion cell type contributing to geniculocortical vision.

The photopigment melanopsin confers photosensitivity upon a minority of retinal output neurons. These intrinsically photosensitive retinal ganglion cells (ipRGCs) are more diverse than once believed, comprising five morphologically distinct types, M1 through M5. Here, in mouse retina, we provide the first in-depth characterization of M4 cells, including their structure, function, and central projections. M4 cells apparently correspond to ON α cells of earlier reports, and are easily distinguished from other ipRGCs by their very large somata. Their dendritic arbors are more radiate and highly branched than those of M1, M2, or M3 cells. The melanopsin-based intrinsic photocurrents of M4 cells are smaller than those of M1 and M2 cells, presumably because melanopsin is more weakly expressed; we can detect it immunohistochemically only with strong amplification. Like M2 cells, M4 cells exhibit robust, sustained, synaptically driven ON responses and dendritic stratification in the ON sublamina of the inner plexiform layer. However, their stratification patterns are subtly different, with M4 dendrites positioned just distal to those of M2 cells and just proximal to the ON cholinergic band. M4 receptive fields are large, with an ON center, antagonistic OFF surround and nonlinear spatial summation. Their synaptically driven photoresponses lack direction selectivity and show higher ultraviolet sensitivity in the ventral retina than in the dorsal retina, echoing the topographic gradient in S- and M-cone opsin expression. M4 cells are readily labeled by retrograde transport from the dorsal lateral geniculate nucleus and thus likely contribute to the pattern vision that persists in mice lacking functional rods and cones.

Molecular identification of wide-field amacrine cells in mouse retina that encode stimulus orientation.

Visual information processing is sculpted by a diverse group of inhibitory interneurons in the retina called amacrine cells. Yet, for most of the >60 amacrine cell types, molecular identities and specialized functional attributes remain elusive. Here, we developed an intersectional genetic strategy to target a group of wide-field amacrine cells (WACs) in mouse retina that co-express the transcription factor Bhlhe22 and the Kappa Opioid Receptor (KOR; B/K WACs). B/K WACs feature straight, unbranched dendrites spanning over 0.5 mm (∼15° visual angle) and produce non-spiking responses to either light increments or decrements. Two-photon dendritic population imaging reveals Ca 2+ signals tuned to the physical orientations of B/K WAC dendrites, signifying a robust structure-function alignment. B/K WACs establish divergent connections with multiple retinal neurons, including unexpected connections with non-orientation-tuned ganglion cells and bipolar cells. Our work sets the stage for future comprehensive investigations of the most enigmatic group of retinal neurons: WACs.

Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells.

The retina uses two photoreceptor types to encode the wide range of light intensities in the natural environment. Rods mediate vision in dim light, whereas cones mediate vision in bright light. Mouse photoreceptors include only 3% cones, and the majority of these coexpress two opsins (short- and middle-wavelength sensitive, S and M), with peak sensitivity to either ultraviolet (360 nm) or green light (508 nm). The M/S-opsin ratio varies across the retina but has not been characterized functionally, preventing quantitative study of cone-mediated vision. Furthermore, physiological and behavioral measurements suggested that mouse retina supports relatively slow temporal processing (peak sensitivity, ∼ 2-5 Hz) compared to primates; however, past studies used visible wavelengths that are inefficient at stimulating mouse S-opsin. Here, we measured the M/S-opsin expression ratio across the mouse retina, as reflected by ganglion cell responses in vitro, and probed cone-mediated ganglion cell temporal properties using ultraviolet light stimulation and linear systems analysis. From recordings in mice lacking rod function (Gnat1(-/-), Rho(-/-)), we estimate ∼ 70% M-opsin expression in far dorsal retina, dropping to <5% M-opsin expression throughout ventral retina. In mice lacking cone function (Gnat2(cpfl3)), light-adapted rod-mediated responses peaked at ∼ 5-7 Hz. In wild-type mice, cone-mediated responses peaked at ∼ 10 Hz, with substantial responsiveness up to ∼ 30 Hz. Therefore, despite the small percentage of cones, cone-mediated responses in mouse ganglion cells are fast and robust, similar to those in primates. These measurements enable quantitative analysis of cone-mediated responses at all levels of the visual system.

A synaptic mechanism for retinal adaptation to luminance and contrast.

The gain of signaling in primary sensory circuits is matched to the stimulus intensity by the process of adaptation. Retinal neural circuits adapt to visual scene statistics, including the mean (background adaptation) and the temporal variance (contrast adaptation) of the light stimulus. The intrinsic properties of retinal bipolar cells and synapses contribute to background and contrast adaptation, but it is unclear whether both forms of adaptation depend on the same cellular mechanisms. Studies of bipolar cell synapses identified synaptic mechanisms of gain control, but the relevance of these mechanisms to visual processing is uncertain because of the historical focus on fast, phasic transmission rather than the tonic transmission evoked by ambient light. Here, we studied use-dependent regulation of bipolar cell synaptic transmission evoked by small, ongoing modulations of membrane potential (V(M)) in the physiological range. We made paired whole-cell recordings from rod bipolar (RB) and AII amacrine cells in a mouse retinal slice preparation. Quasi-white noise voltage commands modulated RB V(M) and evoked EPSCs in the AII. We mimicked changes in background luminance or contrast, respectively, by depolarizing the V(M) or increasing its variance. A linear systems analysis of synaptic transmission showed that increasing either the mean or the variance of the presynaptic V(M) reduced gain. Further electrophysiological and computational analyses demonstrated that adaptation to mean potential resulted from both Ca channel inactivation and vesicle depletion, whereas adaptation to variance resulted from vesicle depletion alone. Thus, background and contrast adaptation apparently depend in part on a common synaptic mechanism.