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The postsynaptic adhesion proteins Neuroligins (NLs) are essential for proper synapse function, and their alterations are associated with a variety of neurodevelopmental disorders. It is increasingly clear that each NL isoform occupies specific subsets of synapses and is able to regulate the function of discrete networks. Studies of NL2 and NL4 in the retina in particular have contributed towards uncovering their role in inhibitory synapse function. In this study we show that NL3 is also predominantly expressed at inhibitory postsynapses in the retinal inner plexiform layer (IPL), where it colocalizes with both GABAA- and glycinergic receptor clusters in a 3:2 ratio. In the NL3 deletion-mutant (knockout or KO) mouse, we uncovered a dramatic reduction of the number of GABAAα2-subunit containing GABAA receptor clusters at the IPL. Retinal activity was thereafter assessed in KO and wild-type (WT) littermates by multi-electrode-array recordings of the output cells of retina, the retinal ganglion cells (RGCs). RGCs in the NL3 KO showed reduced spontaneous activity and an altered response to white noise stimulation. Moreover, upon application of light flashes, the proportion of cells firing at light offset (OFF RGCs) was significantly lower in the NL3 KO compared to WT littermates, whereas the relative number of cells firing at light onset (ON RGCs) increased. Interestingly, although GABAAα2-bearing receptors have been related to direction-selective circuits of the retina, features of direction selective-retinal ganglion cells recorded remained unperturbed in the NL3 KO. Together our data underscore the importance of NL3 for the integrity of specific GABAAergic retinal circuits and identifies NL3 as an important regulator of retinal activity. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the role of Neuroligin 2 (NL2) in synaptic transmission and network function using the mouse retina as a model circuit. We show that NL2 is preferentially located at GABAergic rather than glycinergic or glutamatergic postsynapses. The absence of NL2 from the retina resulted in a severe reduction of GABAA receptor clustering, and in subtle alterations of the retinal circuitry. Light processing was impaired accordingly, and retinal ganglion cells, the output neurons of the retina, showed increased basal activity and altered coding of visual information. Together, our data indicate that NL2 is essential for the functional integrity of GABAergic signaling and as a consequence, for information processing in the retina. [ABSTRACT FROM AUTHOR]

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.

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 correlated to ribbon size, which in turn correlates with active zone extent, as serial electron microscopy provides new information about the heterogeneity in ribbon size, shape, and area of the ribbon in contact with the plasma membrane.

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.

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 squirrel, 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., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

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 Ca 2+ signals in Müller sheaths in all three vascular layers. Pharmacological experiments confirmed that Müller sheaths express purinergic receptors that, when activated, trigger intracellular Ca 2+ signals that are amplified by IP 3 -controlled intracellular Ca 2+ 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.

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View ® ) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.