The sodium-bicarbonate cotransporter Slc4a5 mediates feedback at the first synapse of vision.

Feedback at the photoreceptor synapse is the first neuronal circuit computation in vision, which influences downstream activity patterns within the visual system. Yet, the identity of the feedback signal and the mechanism of synaptic transmission are still not well understood. Here, we combined perturbations of cell-type-specific genes of mouse horizontal cells with two-photon imaging of the result of light-induced feedback in cones and showed that the electrogenic bicarbonate transporter Slc4a5, but not the electroneutral bicarbonate transporter Slc4a3, both expressed specifically in horizontal cells, is necessary for horizontal cell-to-cone feedback. Pharmacological blockage of bicarbonate transporters and buffering pH also abolished the feedback but blocking sodium-proton exchangers and GABA receptors did not. Our work suggests an unconventional mechanism of feedback at the first visual synapse: changes in horizontal cell voltage modulate bicarbonate transport to the cell, via Slc4a5, which leads to the modulation of feedback to cones.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

Rare variant analyses across multiethnic cohorts identify novel genes for refractive error.

Refractive error, measured here as mean spherical equivalent (SER), is a complex eye condition caused by both genetic and environmental factors. Individuals with strong positive or negative values of SER require spectacles or other approaches for vision correction. Common genetic risk factors have been identified by genome-wide association studies (GWAS), but a great part of the refractive error heritability is still missing. Some of this heritability may be explained by rare variants (minor allele frequency [MAF] ≤ 0.01.). We performed multiple gene-based association tests of mean Spherical Equivalent with rare variants in exome array data from the Consortium for Refractive Error and Myopia (CREAM). The dataset consisted of over 27,000 total subjects from five cohorts of Indo-European and Eastern Asian ethnicity. We identified 129 unique genes associated with refractive error, many of which were replicated in multiple cohorts. Our best novel candidates included the retina expressed PDCD6IP, the circadian rhythm gene PER3, and P4HTM, which affects eye morphology. Future work will include functional studies and validation. Identification of genes contributing to refractive error and future understanding of their function may lead to better treatment and prevention of refractive errors, which themselves are important risk factors for various blinding conditions.

Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq.

We present FLASH-seq (FS), a full-length single-cell RNA sequencing (scRNA-seq) method with increased sensitivity and reduced hands-on time compared to Smart-seq3. The entire FS protocol can be performed in ~4.5 hours, is simple to automate and can be easily miniaturized to decrease resource consumption. The FS protocol can also use unique molecular identifiers (UMIs) for molecule counting while displaying reduced strand-invasion artifacts. FS will be especially useful for characterizing gene expression at high resolution across multiple samples.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Restoring light sensitivity using tunable near-infrared sensors.

Enabling near-infrared light sensitivity in a blind human retina may supplement or restore visual function in patients with regional retinal degeneration. We induced near-infrared light sensitivity using gold nanorods bound to temperature-sensitive engineered transient receptor potential (TRP) channels. We expressed mammalian or snake TRP channels in light-insensitive retinal cones in a mouse model of retinal degeneration. Near-infrared stimulation increased activity in cones, ganglion cell layer neurons, and cortical neurons, and enabled mice to perform a learned light-driven behavior. We tuned responses to different wavelengths, by using nanorods of different lengths, and to different radiant powers, by using engineered channels with different temperature thresholds. We targeted TRP channels to human retinas, which allowed the postmortem activation of different cell types by near-infrared light.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

Distinct subcomponents of mouse retinal ganglion cell receptive fields are differentially altered by light adaptation.

The remarkable dynamic range of vision is facilitated by adaptation of retinal sensitivity to ambient lighting conditions. An important mechanism of sensitivity adaptation is control of the spatial and temporal window over which light is integrated. The retina accomplishes this by switching between parallel synaptic pathways with differing kinetics and degrees of synaptic convergence. However, the relative shifts in spatial and temporal integration are not well understood - particularly in the context of the antagonistic spatial surround. Here, we resolve these issues by characterizing the adaptation-induced changes to spatiotemporal integration in the linear receptive field center and surround of mouse retinal ganglion cells. While most ganglion cells lose their antagonistic spatial surround under scotopic conditions, a strong surround is maintained in a subset. We then applied a novel technique that allowed us to analyze the receptive field as a triphasic temporal filter in the center and a biphasic filter in the surround. The temporal tuning of the surround was relatively maintained across adaptation conditions compared to the center, which greatly increased its temporal integration. Though all phases of the center's triphasic temporal response slowed, some shifted significantly less. Additionally, adaptation differentially shifted ON and OFF pathway temporal tuning, reducing their asymmetry under scotopic conditions. Finally, spatial integration was significantly increased by dark adaptation in some cells while it decreased it in others. These findings provide novel insight into how adaptation adjusts visual information processing by altering fundamental properties of ganglion cell receptive fields, such as center-surround antagonism and space-time integration.

Space-time codependence of retinal ganglion cells can be explained by novel and separable components of their receptive fields.

Reverse correlation methods such as spike-triggered averaging consistently identify the spatial center in the linear receptive fields (RFs) of retinal ganglion cells (GCs). However, the spatial antagonistic surround observed in classical experiments has proven more elusive. Tests for the antagonistic surround have heretofore relied on models that make questionable simplifying assumptions such as space-time separability and radial homogeneity/symmetry. We circumvented these, along with other common assumptions, and observed a linear antagonistic surround in 754 of 805 mouse GCs. By characterizing the RF's space-time structure, we found the overall linear RF's inseparability could be accounted for both by tuning differences between the center and surround and differences within the surround. Finally, we applied this approach to characterize spatial asymmetry in the RF surround. These results shed new light on the spatiotemporal organization of GC linear RFs and highlight a major contributor to its inseparability.

Effects of Chronic and Acute Intraocular Pressure Elevation on Scotopic and Photopic Contrast Sensitivity in Mice.

PURPOSE: To compare the impact of intraocular pressure (IOP) elevation on scotopic and photopic contrast sensitivity in mice. METHODS: We chronically elevated the IOP of wild-type mice via injection of polystyrene beads or acutely via injection of highly cohesive sodium hyaluronate. Some eyes with chronically elevated IOP were treated with either topical brimonidine tartrate 0.1% or brinzolamide 1%. Scotopic and photopic contrast sensitivity was assessed at peak spatiotemporal frequencies at multiple time points, with an established optokinetic technique. Retinal ganglion cell (RGC) counts were determined with an antibody to class III beta-tubulin. Correlations among IOP level, RGC count, and scotopic or photopic contrast sensitivity were performed. RESULTS: Six weeks of IOP elevation caused a generalized reduction of photopic contrast sensitivity and a preferential reduction of scotopic contrast sensitivity at peak spatiotemporal frequencies. The administration of brinzolamide but not brimonidine caused a significant reduction in cumulative IOP, whereas brimonidine, but not brinzolamide, prevented RGC loss. Both brimonidine and brinzolamide prevented contrast sensitivity loss, but brimonidine did so at earlier time points and across a wider range of lighting conditions. Following either chronic or acute IOP elevation, scotopic contrast sensitivity was impacted most prominently by IOP level and not by RGC count, while photopic contrast sensitivity was impacted by a combination of factors. CONCLUSIONS: It is possible that scotopic-specific retinal circuitry is altered preferentially by IOP elevation, and that changes in scotopic contrast sensitivity will assist with glaucoma detection. Brimonidine appears to prevent RGC loss via an IOP-independent mechanism.

Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes.

Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα(-/-) mice, but indistinguishable from controls in Cx36(-/-) and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα(-/-) mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36(-/-) mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night vision's ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways.

The ON Crossover Circuitry Shapes Spatiotemporal Profile in the Center and Surround of Mouse OFF Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are often grouped based on their functional properties. Many of these functional properties, such as receptive field (RF) size, are driven by specific retinal circuits. In this report, we determined the role of the ON bipolar cell (BC) mediated crossover circuitry in shaping the center and surround of OFF RGCs. We recorded from a large population of mouse RGCs using a multielectrode array (MEA) while pharmacologically removing the ON BC-mediated crossover circuit. OFF sustained and transient responses to whole field stimuli are lost under scotopic conditions, but maintained under photopic conditions. Though photopic light responses were grossly maintained, we found that photopic light response properties were altered. Using linear RF mapping, we found a significant reduction in the antagonistic surround and a decrease in size of the RF center. Using a novel approach to separate the distinct temporal filters present in the RF center, we see that the crossover pathway contributes specifically to the sluggish antagonistic filter in the center. These results provide new insight into the role of crossover pathways in driving RGCs and also demonstrate that the distinct inputs driving the RF center can be isolated and assayed by RGC activity.