Retinal circuit assembly relies on the precise timing and positioning of key molecules between neuronal partners to mediate proper synapse formation. In the outer retina, horizontal cells (HCs) are important interneurons that make the first contacts with photoreceptors and begin to segregate visual information into two distinct pathways by selectively forming synapses to the different types of photoreceptors. Dendrites of HCs synapse exclusively to cone photoreceptors, whereas the axon terminal synapses to rod photoreceptors. Failure to properly form these early connections disrupts the downstream connectivity of other postsynaptic neurons and leads to abnormal visual function. Although these early events are critical for proper synapse development, little is known about how this process is coordinated in the outer retina. In the present study, we performed single-cell RNA sequencing and uncovered members of the cytoskeletal scaffolding family of ankyrins to be differentially expressed in HCs during development. Specifically, we found ankyrin-B to be highly expressed in HCs at early time points and ankyrin-G to be expressed at later stages. Genetic deletion of both ankyrin-B and ankyrin-G disrupts outer retinal synaptic integrity and leads to impaired in vivo retinal responses. In summary, our findings uncovered a new requirement for ankyrin-B and ankyrin-G in maintaining synaptic connectivity in the outer retina essential for normal visual function.

Computations in sensory circuits rely on specialized synapses. In photoreceptors and retinal second-order bipolar neurons, light modulates continuous neurotransmitter release from a ribbon, a large planar structure at the synaptic release site. How ribbons regulate retinal circuit assembly and shape spatiotemporal coding properties of neural circuits remains unclear. Using a combinatorial structure–function analysis in a ribbon loss-of-function mouse model, we reveal that ribbons are required for proper organization and partner connectivity at photoreceptor synapses and for accruing of synaptic proteins at inner retinal synapses. Lack of synaptic ribbons leads to an enlargement of the dendritic field size of the retinal second-order neurons, bipolar cells, which alters spatial encoding for a key retinal circuit, ONα ganglion cell. We further demonstrate that such structural alterations of synapses in the absence of ribbons impact both tonic and light-evoked neurotransmitter release as well as recovery of synapse function after depression. These perturbations in synaptic structure and function in the absence of ribbons lead to alterations in salient visual computations such as light adaptation, receptive field organization, and feature detection across time and space in the ONα ganglion cell circuit. We further reveal that a lack of ribbons impairs the encoding of naturalistic changes in visual contrast and luminance for this retinal circuit. Our findings thus reveal synaptic ribbons to be core determinants of the mammalian retina, acting not only as neurotransmitter release machines but also as organizers of circuit assembly and regulators of visual encoding.

How afferent input shapes synaptic connections is fundamental to our understanding of cues that govern assembly of sensory circuits. In the retina, photoreceptors provide afferent visual information to second-order bipolar cells (BCs) that in turn transfer signals to output neurons. BCs have distinct inhibitory synapses at dendrites and axons but the role of afferent input for regulating the composition and function of these synapses remains unknown. We used a photoreceptor degeneration murine transgenic with labeled BCs and combined immunohistochemical assessment of synaptic proteins across timepoints with single-cell electrophysiology and transcriptomics to address how photoreceptor input regulates BC synapses. We find that inhibitory synapses across BCs have distinct dependencies on afferent input, with axonal synapses reacting first to deafferentation even though the dendritic synapses are at the site of deafferentation. Synapses were altered in a BC-type specific manner and deafferentation differentially impacted expression of synaptic proteins vs. RNA transcripts for synaptic genes revealing disrupted synaptic trafficking pathways. Loss of afferent input also prompted production of nonfunctional receptor proteins and led to withdrawal of BC output synapses. Our findings thus reveal susceptible and resilient retinal synapse types upon deafferentation and uncover how afferent input differentially regulates synapses across second-order neurons.

Rapid and high local calcium (Ca 2+ ) signals are essential for triggering neurotransmitter release from presynaptic terminals. In specialized bipolar ribbon synapses of the retina, these local Ca 2+ signals control multiple processes, including the priming, docking, and translocation of vesicles on the ribbon before exocytosis, endocytosis, and the replenishment of release-ready vesicles to the fusion sites for sustained neurotransmission. However, our knowledge about Ca 2+ signals along the axis of the ribbon active zone is limited. Here, we used fast confocal quantitative dual-color ratiometric line-scan imaging of a fluorescently labeled ribbon binding peptide and Ca 2+ indicators to monitor the spatial and temporal aspects of Ca 2+ transients of individual ribbon active zones in zebrafish retinal rod bipolar cells (RBCs). We observed that a Ca 2+ transient elicited a much greater fluorescence amplitude when the Ca 2+ indicator was conjugated to a ribeye-binding peptide than when using a soluble Ca 2+ indicator, and the estimated Ca 2+ levels at the ribbon active zone exceeded 26 μM in response to a 10-millisecond stimulus, as measured by a ribbon-bound low-affinity Ca 2+ indicator. Our quantitative modeling of Ca 2+ diffusion and buffering is consistent with this estimate and provides a detailed view of the spatiotemporal [Ca 2+ ] dynamics near the ribbon. Importantly, our data demonstrates that the local Ca 2+ levels may vary between ribbons of different RBCs and within the same cells. The variation in local Ca 2+ signals is found to correlate with ribbon size and active zone extent. Our serial electron microscopy results provide new information about the heterogeneity in ribbon size, shape, and area of the ribbon in contact with the plasma membrane.

Neurons rely on molecular interactions typically mediated by transmembrane adhesion proteins to locate appropriate partners and establish connections. A recent study finds that a member of the cadherin family of cell adhesion proteins organizes color-preferring connections in the part of the retinal neural circuit designated for encoding light decrements.

Mitochondria are critical for synaptic function. At the synapse, mitochondria produce ATP and buffer calcium, both of which are required for synapse function. Defects in mitochondrial maintenance are linked to neurodegenerative disease, yet we know little about what regulates the need for mitophagy at the synapse. We assessed the impact of neuron type, activity, and mitochondrial damage on mitophagy rate in axons of larval zebrafish. Using electron and confocal microscopy, we show that mitophagy occurs in the axon terminal of postsynaptic sensory neurons and presynaptic motor neurons at similar rates. Increasing neuronal activity or mitochondria damage does not impact the amount of mitophagy in axons. Only by combining neuronal activity and mitochondrial damage does the rate of mitophagy increase in the axon and this increase requires Pink1. Together, our data support a model in which increased mitophagic demand in axons is rare and uniquely sensitive to Pink1 disruption. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Neurological Disorders and Stroke, https://ror.org/01s5ya894, R01NS124692 University of Wisconsin-Madison Department of Integrative Biology Weinreb Predoctoral Fellowship Wisconsin Alumni Research Foundation, https://ror.org/00hwxbz16 UW 2020: WARF Discovery Initiative Award

Fixed tissue analyses of synaptic density and connectivity rely on analyses of image stacks containing fluorescence signals. This chapter details image analysis routines that can be implemented to determine connectivity between labeled cell types and procedures for quantification of synaptic density across a tissue volume or specifically within a labeled cell of interest. The chapter details open-source software and practices that can be implemented for these analyses as well as considerations for validation of detected signals, estimation of background noise in images, and determination of non-specific detections.

Rapid and high local calcium (Ca2+) signals are essential for triggering neurotransmitter release from presynaptic terminals. In specialized bipolar ribbon synapses of the retina, these local Ca2+ signals control multiple processes, including the priming, docking, and translocation of vesicles on the ribbon before exocytosis, endocytosis, and the replenishment of release-ready vesicles to the fusion sites for sustained neurotransmission. However, our knowledge about Ca2+ signals along the axis of the ribbon active zone is limited. Here, we used fast confocal quantitative dual-color ratiometric line-scan imaging of a fluorescently labeled ribbon binding peptide and Ca2+ indicators to monitor the spatial and temporal aspects of Ca2+ transients of individual ribbon active zones in zebrafish retinal rod bipolar cells (RBCs). We observed that a Ca2+ transient elicited a much greater fluorescence amplitude when the Ca2+ indicator was conjugated to a ribeye-binding peptide than when using a soluble Ca2+ indicator, and the estimated Ca2+ levels at the ribbon active zone exceeded 26 mu M in response to a 10 millisecond stimulus, as measured by a ribbon-bound low-affinity Ca2+ indicator. Our quantitative modeling of Ca2+ diffusion and buffering is consistent with this estimate and provides a detailed view of the spatiotemporal [Ca2+] dynamics near the ribbon. Importantly, our data demonstrates that the local Ca2+ levels may vary between ribbons of different RBCs and within the same cells. The variation in local Ca2+ signals is found to correlate with ribbon size and active zone extent. Our serial electron microscopy results provide new information about the heterogeneity in ribbon size, shape, and area of the ribbon in contact with the plasma membrane.

The mammalian visual system consists of two distinct pathways: rod- and cone-driven vision. The rod pathway is responsible for dim light vision whereas the cone pathway mediates daylight vision and color perception. The distinct processing of visual information begins at the first synapse of rod and cone photoreceptors. The unique composition and organization of the rod and cone synapse is what allows information to be parsed into the different visual pathways. Although this is a critical process for vision, little is known about the key molecules responsible for establishing and maintaining the distinct synaptic architecture of the rod and cone synapse. In the present study, we uncovered a new role for Ankyrins in maintaining the synaptic integrity of the rod and cone synapse. Loss of Ankyrin-B and Ankyrin-G results in connectivity defects between photoreceptors and their synaptic partners. Ultrastructure analysis of the rod and cone synapse revealed impaired synaptic innervation, abnormal terminal morphology, and disruption of synaptic connections. Consistent with these findings, functional studies revealed impaired in vivo retinal responses in animals with loss of Ankyrin-B and Ankyrin-G. Taken together, our data supports a new role for Ankyrins in maintaining synaptic integrity and organization of photoreceptor synapses in the mouse outer retina. SIGNFICANCE STATEMENT:The first synapse in the outer retina begins to process visual information into two distinct pathways. This is largely attributed to the different composition and organization of the rod and cone synapse. Although the structural integrity of the rod and cone synapse is critical for normal vision, little is known about the key molecules responsible for maintaining the unique structure of the different photoreceptor synapses. In this study, we demonstrate a new function for the cytoskeletal scaffolding proteins, Ankryin-B and Ankyrin-G in the mouse outer retina. We found Ankyrin-B and Ankyrin-G are both required for proper retinal connectivity, where loss of these molecules leads to synaptic defects and impaired retinal responses.

Sensory circuits can exhibit remarkable resilience to disruption, often maintaining function through recruitment of compensatory mechanisms. In the mammalian retina, the balance between ON and OFF pathways that encode distinct luminance profiles is essential for processing visual information. How selective disruption of one input stream can trigger adaptive and/or compensatory measures in retinal output neurons is not fully understood. To determine how retinal output circuits can adapt to different degrees of input suppression, we genetically suppressed the ON pathway input in two models with partial (50%) and complete (100%) ON pathway blockade. We used single-cell electrophysiology to record intrinsic properties, synaptic inputs, and spike outputs of alpha retinal ganglion cell (RGC) types that serve as primary output channels. Complementary immunohistochemistry assessed structural changes in excitatory and inhibitory synaptic protein expression within individual RGCs. We found that 50% ON pathway suppression triggers adaptive scaling of excitatory synaptic proteins in ON and OFF RGCs that are aimed at preserving visual function. In contrast, complete suppression leads to maladaptive intrinsic alterations and cyclical instability in specific OFF-RGC types, impairing visual processing. We also observed luminance-level-dependent alterations in the OFF pathway output and contrast-encoding abilities after ON pathway suppression. Our findings reveal that the extent of input suppression determines whether compensatory mechanisms are beneficial or detrimental, offering new insights into retinal plasticity mechanisms. Uncovering these mechanisms expands our knowledge of sensory neuroplasticity, revealing potential therapeutic strategies for ameliorating dysfunction in disease conditions of ON pathway suppression, such as congenital stationary night blindness.

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 associated with astrocytes, like brain capillaries, whereas radial Müller glia interact with vessels in the other layers. Using serial electron microscopic reconstructions from mouse and primate retina, we find that Müller processes cover capillaries in a tessellating pattern, mirroring the wrapping of brain capillaries by tiled astrocytic endfeet. Gaps in the Müller sheath, found mainly in the intermediate vascular plexus (IVP), permit diverse neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with reduced or absent vascular basement lamina. Focal application of 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 inositol triphosphate (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 sheaths are virtually complete in the SVP but fenestrated in the IVP, permitting direct neurovascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.

Congenital stationary night blindness type 2 (CSNB2) is caused by mutations affecting the Cav1.4 Ca2+ channel. Although Cav1.4 is required for synaptic release by both rods and cones, visual phenotypes of CSNB2 are generally mild and often involve selective dysfunction of rods. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (KI) mouse expressing a non-conducting Cav1.4 mutant, which both lack Ca2+ currents and synaptic responses in rods. Surprisingly, aberrant Cav3 currents were detected in cones of both KI and KO mice.

Synapse formation within the retinal circuit ensures that distinct neuronal types can communicate efficiently to process visual signals. Synapses thus form the core of the visual computations performed by the retinal circuit. Retinal synapses are diverse but can be broadly categorized into multipartner ribbon synapses and 1:1 conventional synapses. In this article, we review our current understanding of the cellular and molecular mechanisms that regulate the functional establishment of mammalian retinal synapses, including the role of adhesion proteins, synaptic proteins, extracellular matrix and cytoskeletal-associated proteins, and activity-dependent cues. We outline future directions and areas of research that will expand our knowledge of these mechanisms. Understanding the regulators moderating synapse formation and function not only reveals the integrated developmental processes that establish retinal circuits, but also divulges the identity of mechanisms that could be engaged during disease and degeneration.

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.

In congenital stationary night blindness, type 2 (CSNB2)—a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel—visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrels, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e. cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.