The retina's neurovascular unit: Müller glial sheaths and neuronal contacts.

The neurovascular unit (NVU), comprising vascular, glial and neural elements, supports the energetic demands of neural computation, but this aspect of the retina's trilaminar vessel network is poorly understood. Only the innermost vessel layer - the superficial vascular plexus (SVP) - is ensheathed by astrocytes, like brain capillaries, whereas glial ensheathment in other layers derives from radial Müller glia. Using serial electron microscopy reconstructions from mouse and primate retina, we find that Müller processes cover capillaries in a tessellating pattern, mirroring the tiled astrocytic endfeet wrapping brain capillaries. However, gaps in the Müller sheath, found mainly in the intermediate vascular plexus (IVP), permit different neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with a reduced or absent vascular basement lamina. Focal application of adenosine triphosphate (ATP) to the vitreal surface evoked Ca2+ signals in Müller sheaths in all three vascular layers. Pharmacological experiments confirmed that Müller sheaths express purinergic receptors that, when activated, trigger intracellular Ca2+ signals that are amplified by IP3-controlled intracellular Ca2+ stores. When rod photoreceptors die in a mouse model of retinitis pigmentosa (rd10), Müller sheaths dissociate from the deep vascular plexus (DVP) but are largely unchanged within the IVP or SVP. Thus, Müller glia interact with retinal vessels in a laminar, compartmentalized manner: glial sheathes are virtually complete in the SVP but fenestrated in the IVP, permitting direct neural-to-vascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.

Predictably manipulating photoreceptor light responses to reveal their role in downstream visual responses.

Computation in neural circuits relies on judicious use of nonlinear circuit components. In many cases, multiple nonlinear components work collectively to control circuit outputs. Separating the contributions of these different components is difficult, and this hampers our understanding of the mechanistic basis of many important computations. Here, we introduce a tool that permits the design of light stimuli that predictably alter rod and cone phototransduction currents - including stimuli that compensate for nonlinear properties such as light adaptation. This tool, based on well-established models for the rod and cone phototransduction cascade, permits the separation of nonlinearities in phototransduction from those in downstream circuits. This will allow, for example, direct tests of how adaptation in rod and cone phototransduction affects downstream visual signals and perception.

Regional variation in the organization and connectivity of the first synapse in the primate night vision pathway.

Sensitivity of primate daylight vision varies across the visual field. This is attributed to regional variations in cone photoreceptor density and synaptic connectivity of the underlying circuitry. In contrast, we have limited understanding of how synapse organization of the primate night vision pathway changes across space. Using serial electron microscopy, we reconstructed the first synapse of the night vision pathway between rod photoreceptors and second-order neurons, at multiple locations from the central part of the primate retina, fovea, to the periphery. We find that most facets of the rod synapse connectivity vary across retinal regions. However, rod synaptic divergence and convergence patterns do not change in the same manner across locations. Moreover, patterns of rod synapse organization are tightly correlated with photoreceptor density. Such regional heterogeneities revise the connectivity diagram of the primate rod synapse which will shape synapse function and sensitivity of the night vision pathway across visual space.

Sensory deprivation arrests cellular and synaptic development of the night-vision circuitry in the retina.

Experience regulates synapse formation and function across sensory circuits. How inhibitory synapses in the mammalian retina are sculpted by visual cues remains unclear. By use of a sensory deprivation paradigm, we find that visual cues regulate maturation of two GABA synapse types (GABAA and GABAC receptor synapses), localized across the axon terminals of rod bipolar cells (RBCs)-second-order retinal neurons integral to the night-vision circuit. Lack of visual cues causes GABAA synapses at RBC terminals to retain an immature receptor configuration with slower response profiles and prevents receptor recruitment at GABAC synapses. Additionally, the organizing protein for both these GABA synapses, LRRTM4, is not clustered at dark-reared RBC synapses. Ultrastructurally, the total number of ribbon-output/inhibitory-input synapses across RBC terminals remains unaltered by sensory deprivation, although ribbon synapse output sites are misarranged when the circuit develops without visual cues. Intrinsic electrophysiological properties of RBCs and expression of chloride transporters across RBC terminals are additionally altered by sensory deprivation. Introduction to normal 12-h light-dark housing conditions facilitates maturation of dark-reared RBC GABA synapses and restoration of intrinsic RBC properties, unveiling a new element of light-dependent retinal cellular and synaptic plasticity.

New insights into retinal circuits through EM connectomics: what we have learnt and what remains to be learned.

The retinal neural circuit is intricately wired for efficient processing of visual signals. This is well-supported by the specialized connections between retinal neurons at both the functional and ultrastructural levels. Through 3D electron microscopic (EM) reconstructions of retinal neurons and circuits we have learnt much about the specificities of connections within the retinal layers including new insights into how retinal neurons establish connections and perform sophisticated visual computations. This mini-review will summarize the retinal circuitry and provide details about the novel insights EM connectomics has brought into our understanding of the retinal circuitry. We will also discuss unresolved questions about the retinal circuitry that can be addressed by EM connectomics in the future.

Cone photoreceptors in human stem cell-derived retinal organoids demonstrate intrinsic light responses that mimic those of primate fovea.

High-definition vision in humans and nonhuman primates is initiated by cone photoreceptors located within a specialized region of the retina called the fovea. Foveal cone death is the ultimate cause of central blindness in numerous retinal dystrophies, including macular degenerative diseases. 3D retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) hold tremendous promise to model and treat such diseases. To achieve this goal, RO cones should elicit robust and intrinsic light-evoked electrical responses (i.e., phototransduction) akin to adult foveal cones, which has not yet been demonstrated. Here, we show strong, graded, repetitive, and wavelength-specific light-evoked responses from RO cones. The photoresponses and membrane physiology of a significant fraction of these lab-generated cones are comparable with those of intact ex vivo primate fovea. These results greatly increase confidence in ROs as potential sources of functional human cones for cell replacement therapies, drug testing, and in vitro models of retinal dystrophies.

Transient expression of a GABA receptor subunit during early development is critical for inhibitory synapse maturation and function.

Developing neural circuits, including GABAergic circuits, switch receptor types. But the role of early GABA receptor expression for establishment of functional inhibitory circuits remains unclear. Tracking the development of GABAergic synapses across axon terminals of retinal bipolar cells (BCs), we uncovered a crucial role of early GABAA receptor expression for the formation and function of presynaptic inhibitory synapses. Specifically, early α3-subunit-containing GABAA (GABAAα3) receptors are a key developmental organizer. Before eye opening, GABAAα3 gives way to GABAAα1 at individual BC presynaptic inhibitory synapses. The developmental downregulation of GABAAα3 is independent of GABAAα1 expression. Importantly, lack of early GABAAα3 impairs clustering of GABAAα1 and formation of functional GABAA synapses across mature BC terminals. This impacts the sensitivity of visual responses transmitted through the circuit. Lack of early GABAAα3 also perturbs aggregation of LRRTM4, the organizing protein at GABAergic synapses of rod BC terminals, and their arrangement of output ribbon synapses.

GABAA presynaptic inhibition regulates the gain and kinetics of retinal output neurons.

Output signals of neural circuits, including the retina, are shaped by a combination of excitatory and inhibitory signals. Inhibitory signals can act presynaptically on axon terminals to control neurotransmitter release and regulate circuit function. However, it has been difficult to study the role of presynaptic inhibition in most neural circuits due to lack of cell type-specific and receptor type-specific perturbations. In this study, we used a transgenic approach to selectively eliminate GABAA inhibitory receptors from select types of second-order neurons - bipolar cells - in mouse retina and examined how this affects the light response properties of the well-characterized ON alpha ganglion cell retinal circuit. Selective loss of GABAA receptor-mediated presynaptic inhibition causes an enhanced sensitivity and slower kinetics of light-evoked responses from ON alpha ganglion cells thus highlighting the role of presynaptic inhibition in gain control and temporal filtering of sensory signals in a key neural circuit in the mammalian retina.

Organization and emergence of a mixed GABA-glycine retinal circuit that provides inhibition to mouse ON-sustained alpha retinal ganglion cells.

In the retina, amacrine interneurons inhibit retinal ganglion cell (RGC) dendrites to shape retinal output. Amacrine cells typically use either GABA or glycine to exert synaptic inhibition. Here, we combined transgenic tools with immunohistochemistry, electrophysiology, and 3D electron microscopy to determine the composition and organization of inhibitory synapses across the dendritic arbor of a well-characterized RGC type in the mouse retina: the ON-sustained alpha RGC. We find mixed GABA-glycine receptor synapses across this RGC type, unveiling the existence of "mixed" inhibitory synapses in the retinal circuit. Presynaptic amacrine boutons with dual release sites are apposed to ON-sustained alpha RGC postsynapses. We further reveal the sequence of postsynaptic assembly for these mixed synapses: GABA receptors precede glycine receptors, and a lack of early GABA receptor expression impedes the recruitment of glycine receptors. Together our findings uncover the organization and developmental profile of an additional motif of inhibition in the mammalian retina.

LRRTM4: A Novel Regulator of Presynaptic Inhibition and Ribbon Synapse Arrangements of Retinal Bipolar Cells.

LRRTM4 is a transsynaptic adhesion protein regulating glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, we find that LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs). Knockout of LRRTM4 reduces RBC axonal GABAA and GABAC receptor clustering and disrupts presynaptic inhibition onto RBC terminals. LRRTM4 removal also perturbs the stereotyped output synapse arrangement at RBC terminals. Synaptic ribbons are normally apposed to two distinct postsynaptic "dyad" partners, but in the absence of LRRTM4, "monad" and "triad" arrangements are also formed. RBCs from retinas deficient in GABA release also demonstrate dyad mis-arrangements but maintain LRRTM4 expression, suggesting that defects in dyad organization in the LRRTM4 knockout could originate from reduced GABA receptor function. LRRTM4 is thus a key synapse organizing molecule at RBC terminals, where it regulates function of GABAergic synapses and assembly of RBC synaptic dyads.

S-cone photoreceptors in the primate retina are functionally distinct from L and M cones.

Daylight vision starts with signals in three classes of cone photoreceptors sensitive to short (S), middle (M), and long (L) wavelengths. Psychophysical studies show that perceptual sensitivity to rapidly varying inputs differs for signals originating in S cones versus L and M cones; notably, S-cone signals appear perceptually delayed relative to L- and M-cone signals. These differences could originate in the cones themselves or in the post-cone circuitry. To determine if the cones could contribute to these and related perceptual phenomena, we compared the light responses of primate S, M, and L cones. We found that S cones generate slower light responses than L and M cones, show much smaller changes in response kinetics as background-light levels increase, and are noisier than L and M cones. It will be important to incorporate these differences into descriptions of how cone signaling shapes human visual perception.

Cellular and Circuit Mechanisms Shaping the Perceptual Properties of the Primate Fovea.

The fovea is a specialized region of the retina that dominates the visual perception of primates by providing high chromatic and spatial acuity. While the foveal and peripheral retina share a similar core circuit architecture, they exhibit profound functional differences whose mechanisms are unknown. Using intracellular recordings and structure-function analyses, we examined the cellular and synaptic underpinnings of the primate fovea. Compared to peripheral vision, the fovea displays decreased sensitivity to rapid variations in light inputs; this difference is reflected in the responses of ganglion cells, the output cells of the retina. Surprisingly, and unlike in the periphery, synaptic inhibition minimally shaped the responses of foveal midget ganglion cells. This difference in inhibition cannot however, explain the differences in the temporal sensitivity of foveal and peripheral midget ganglion cells. Instead, foveal cone photoreceptors themselves exhibited slower light responses than peripheral cones, unexpectedly linking cone signals to perceptual sensitivity.

Using Fluorescent Markers to Estimate Synaptic Connectivity In Situ.

Labeling fixed brain tissue with fluorescent synaptic and cellular markers can help assess circuit connectivity. Despite the diffraction-limited resolution of light microscopy there are several approaches to identify synaptic contacts onto a cell-of-interest. Understanding which image quantification methods can be applied to estimate cellular and synaptic connectivity at the light microscope level is beneficial to answer a range of questions, from mapping appositions between cellular structures or synaptic proteins to assessing synaptic contact density onto a cell-of-interest. This chapter provides the reader with details of the image analysis methods that can be applied to quantify in situ connectivity patterns at the level of cellular contacts and synaptic appositions.

Lack of CaBP1/Caldendrin or CaBP2 Leads to Altered Ganglion Cell Responses.

Calcium-binding proteins (CaBPs) form a subfamily of calmodulin-like proteins that were cloned from the retina. CaBP4 and CaBP5 have been shown to be important for normal visual function. Although CaBP1/caldendrin and CaBP2 have been shown to modulate various targets in vitro, it is not known whether they contribute to the transmission of light responses through the retina. Therefore, we generated mice that lack CaBP2 or CaBP1/caldendrin (Cabp2-/- and Cabp1-/- ) to test whether these CaBPs are essential for normal retinal function. By immunohistochemistry, the overall morphology of Cabp1-/- and Cabp2-/- retinas and the number of synaptic ribbons appear normal; transmission electron microscopy shows normal tethered ribbon synapses and synaptic vesicles as in wild-type retinas. However, whole-cell patch clamp recordings showed that light responses of retinal ganglion cells of Cabp2-/- and Cabp1-/- mice differ in amplitude and kinetics from those of wild-type mice. We conclude that CaBP1/caldendrin and CaBP2 are not required for normal gross retinal and synapse morphology but are necessary for the proper transmission of light responses through the retina; like other CaBPs, CaBP1/caldendrin and CaBP2 likely act by modulating presynaptic Ca2+-dependent signaling mechanisms.

Neurotransmission plays contrasting roles in the maturation of inhibitory synapses on axons and dendrites of retinal bipolar cells.

Neuronal output is modulated by inhibition onto both dendrites and axons. It is unknown whether inhibitory synapses at these two cellular compartments of an individual neuron are regulated coordinately or separately during in vivo development. Because neurotransmission influences synapse maturation and circuit development, we determined how loss of inhibition affects the expression of diverse types of inhibitory receptors on the axon and dendrites of mouse retinal bipolar cells. We found that axonal GABA but not glycine receptor expression depends on neurotransmission. Importantly, axonal and dendritic GABAA receptors comprise distinct subunit compositions that are regulated differentially by GABA release: Axonal GABAA receptors are down-regulated but dendritic receptors are up-regulated in the absence of inhibition. The homeostatic increase in GABAA receptors on bipolar cell dendrites is pathway-specific: Cone but not rod bipolar cell dendrites maintain an up-regulation of receptors in the transmission deficient mutants. Furthermore, the bipolar cell GABAA receptor alterations are a consequence of impaired vesicular GABA release from amacrine but not horizontal interneurons. Thus, inhibitory neurotransmission regulates in vivo postsynaptic maturation of inhibitory synapses with contrasting modes of action specific to synapse type and location.

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses.

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.

A readily retrievable pool of synaptic vesicles.

Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive zone in hippocampal boutons, which represent putative sites of endocytosis.

A common origin of synaptic vesicles undergoing evoked and spontaneous fusion.

There is a longstanding controversy on the identity of synaptic vesicles undergoing spontaneous versus evoked release. A recent study, introducing a new genetic probe, suggested that spontaneous release is driven by a resting pool of synaptic vesicles refractory to stimulation. We found that cross-depletion of spontaneously or actively recycling synaptic vesicle pools occurred on stimulation in rat hippocampal neurons and identified the recycling pool as a major source of spontaneous release.