Insulin signalling promotes dendrite and synapse regeneration and restores circuit function after axonal injury.

Dendrite pathology and synapse disassembly are critical features of chronic neurodegenerative diseases. In spite of this, the capacity of injured neurons to regenerate dendrites has been largely ignored. Here, we show that, upon axonal injury, retinal ganglion cells undergo rapid dendritic retraction and massive synapse loss that preceded neuronal death. Human recombinant insulin, administered as eye drops or systemically after dendritic arbour shrinkage and prior to cell loss, promoted robust regeneration of dendrites and successful reconnection with presynaptic targets. Insulin-mediated regeneration of excitatory postsynaptic sites on retinal ganglion cell dendritic processes increased neuronal survival and rescued light-triggered retinal responses. Further, we show that axotomy-induced dendrite retraction triggered substantial loss of the mammalian target of rapamycin (mTOR) activity exclusively in retinal ganglion cells, and that insulin fully reversed this response. Targeted loss-of-function experiments revealed that insulin-dependent activation of mTOR complex 1 (mTORC1) is required for new dendritic branching to restore arbour complexity, while complex 2 (mTORC2) drives dendritic process extension thus re-establishing field area. Our findings demonstrate that neurons in the mammalian central nervous system have the intrinsic capacity to regenerate dendrites and synapses after injury, and provide a strong rationale for the use of insulin and/or its analogues as pro-regenerative therapeutics for intractable neurodegenerative diseases including glaucoma.

Cell type-specific changes in retinal ganglion cell function induced by rod death and cone reorganization in rats.

We have determined the impact of rod death and cone reorganization on the spatiotemporal receptive fields (RFs) and spontaneous activity of distinct retinal ganglion cell (RGC) types. We compared RGC function between healthy and retinitis pigmentosa (RP) model rats (S334ter-3) at a time when nearly all rods were lost but cones remained. This allowed us to determine the impact of rod death on cone-mediated visual signaling, a relevant time point because the diagnosis of RP frequently occurs when patients are nightblind but daytime vision persists. Following rod death, functionally distinct RGC types persisted; this indicates that parallel processing of visual input remained largely intact. However, some properties of cone-mediated responses were altered ubiquitously across RGC types, such as prolonged temporal integration and reduced spatial RF area. Other properties changed in a cell type-specific manner, such as temporal RF shape (dynamics), spontaneous activity, and direction selectivity. These observations identify the extent of functional remodeling in the retina following rod death but before cone loss. They also indicate new potential challenges to restoring normal vision by replacing lost rod photoreceptors.NEW & NOTEWORTHY This study provides novel and therapeutically relevant insights to retinal function following rod death but before cone death. To determine changes in retinal output, we used a large-scale multielectrode array to simultaneously record from hundreds of retinal ganglion cells (RGCs). These recordings of large-scale neural activity revealed that following the death of all rods, functionally distinct RGCs remain. However, the receptive field properties and spontaneous activity of these RGCs are altered in a cell type-specific manner.

TIMP-1 affects the spatial distribution of dendritic processes of second-order neurons in a rat model of Retinitis Pigmentosa.

Retinitis Pigmentosa (RP) is an inherited disorder that may lead to blindness. In the rhodopsin S334ter-line-3 rat model of RP, the death of rods induces spatial rearrangement of cones into regular ring mosaics. Using this model, we discovered that the ring mosaics are restored to a homogeneous distribution upon application of tissue inhibitor of metalloproteinase-1 (TIMP-1). In this study, we further investigated the cone migration and spatial distribution of second-order neurons and their connections to cones in the presence or absence of TIMP-1 using immunohistochemistry to identify retinal neurons and their connections with cones. M-opsin cell bodies and their outer segments were evaluated to determine whether TIMP-1 delays the degeneration of outer segments of cones. We observed that during cone rearrangement into ring mosaics in RP retina, dendritic processes of second-order neurons undergo remodeling to maintain their synaptic connections with the cones in the rings. TIMP-1 treatment induced the cones to rearrange and dendritic processes of second-order neurons to return to a more homogeneous spatial distribution. In addition, TIMP-1 treatment protected the outer segments of cones at later stages of retinal degeneration. Our findings clearly demonstrate that despite their dramatic spatial rearrangement, cones and second-order neuron processes maintain their synaptic connections before and after TIMP-1 treatment.

A presynaptic source drives differing levels of surround suppression in two mouse retinal ganglion cell types.

In early sensory systems, cell-type diversity generally increases from the periphery into the brain, resulting in a greater heterogeneity of responses to the same stimuli. Surround suppression is a canonical visual computation that begins within the retina and is found at varying levels across retinal ganglion cell types. Our results show that heterogeneity in the level of surround suppression occurs subcellularly at bipolar cell synapses. Using single-cell electrophysiology and serial block-face scanning electron microscopy, we show that two retinal ganglion cell types exhibit very different levels of surround suppression even though they receive input from the same bipolar cell types. This divergence of the bipolar cell signal occurs through synapse-specific regulation by amacrine cells at the scale of tens of microns. These findings indicate that each synapse of a single bipolar cell can carry a unique visual signal, expanding the number of possible functional channels at the earliest stages of visual processing.

A scalable and modular computational pipeline for axonal connectomics: automated tracing and assembly of axons across serial sections.

Progress in histological methods and in microscope technology has enabled dense staining and imaging of axons over large brain volumes, but tracing axons over such volumes requires new computational tools for 3D reconstruction of data acquired from serial sections. We have developed a computational pipeline for automated tracing and volume assembly of densely stained axons imaged over serial sections, which leverages machine learning-based segmentation to enable stitching and alignment with the axon traces themselves. We validated this segmentation-driven approach to volume assembly and alignment of individual axons over centimeter-scale serial sections and show the application of the output traces for analysis of local orientation and for proofreading over aligned volumes. The pipeline is scalable, and combined with recent advances in experimental approaches, should enable new studies of mesoscale connectivity and function over the whole human brain.

Distinctive synaptic structural motifs link excitatory retinal interneurons to diverse postsynaptic partner types.

Neurons make converging and diverging synaptic connections with distinct partner types. Whether synapses involving separate partners demonstrate similar or distinct structural motifs is not yet well understood. We thus used serial electron microscopy in mouse retina to map output synapses of cone bipolar cells (CBCs) and compare their structural arrangements across bipolar types and postsynaptic partners. Three presynaptic configurations emerge-single-ribbon, ribbonless, and multiribbon synapses. Each CBC type exploits these arrangements in a unique combination, a feature also found among rabbit ON CBCs. Though most synapses are dyads, monads and triads are also seen. Altogether, mouse CBCs exhibit at least six motifs, and each CBC type uses these in a stereotypic pattern. Moreover, synapses between CBCs and particular partner types appear biased toward certain motifs. Our observations reveal synaptic strategies that diversify the output within and across CBC types, potentially shaping the distinct functions of retinal microcircuits.

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.

Development of ON and OFF cholinergic amacrine cells in the human fetal retina.

Choline acetyltransferase (ChAT) expressing retinal amacrine cells are present across vertebrates. These interneurons play important roles in the development of retinal projections to the brain and in motion detection, specifically in generating direction-selective responses to moving stimuli. ChAT amacrine cells typically comprise two spatially segregated populations that form circuits in the 'ON' or 'OFF' synaptic layers of the inner retina. This stereotypic arrangement is also found across the adult human retina, with the notable exception that ChAT expression is evident in the ON but not OFF layer of the fovea, a region specialized for high-acuity vision. We thus investigated whether the human fovea exhibits a developmental path for ON and OFF ChAT cells that is retinal location-specific. Our analysis shows that at each retinal location, human ON and OFF ChAT cells differentiate, form their separate synaptic layers, and establish non-random mosaics at about the same time. However, unlike in the adult fovea, ChAT immunostaining is initially robust in both ON and OFF populations, up until at least mid-gestation. ChAT expression in the OFF layer in the fovea is therefore significantly reduced after mid-gestation. OFF ChAT cells in the human fovea and in the retinal periphery thus follow distinct maturational paths.

Dynamic assembly of ribbon synapses and circuit maintenance in a vertebrate sensory system.

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.

Assembly and maintenance of GABAergic and Glycinergic circuits in the mammalian nervous system.

Inhibition in the central nervous systems (CNS) is mediated by two neurotransmitters: gamma-aminobutyric acid (GABA) and glycine. Inhibitory synapses are generally GABAergic or glycinergic, although there are synapses that co-release both neurotransmitter types. Compared to excitatory circuits, much less is known about the cellular and molecular mechanisms that regulate synaptic partner selection and wiring patterns of inhibitory circuits. Recent work, however, has begun to fill this gap in knowledge, providing deeper insight into whether GABAergic and glycinergic circuit assembly and maintenance rely on common or distinct mechanisms. Here we summarize and contrast the developmental mechanisms that regulate the selection of synaptic partners, and that promote the formation, refinement, maturation and maintenance of GABAergic and glycinergic synapses and their respective wiring patterns. We highlight how some parts of the CNS demonstrate developmental changes in the type of inhibitory transmitter or receptor composition at their inhibitory synapses. We also consider how perturbation of the development or maintenance of one type of inhibitory connection affects other inhibitory synapse types in the same circuit. Mechanistic insight into the development and maintenance of GABAergic and glycinergic inputs, and inputs that co-release both these neurotransmitters could help formulate comprehensive therapeutic strategies for treating disorders of synaptic inhibition.

Synaptic Convergence Patterns onto Retinal Ganglion Cells Are Preserved despite Topographic Variation in Pre- and Postsynaptic Territories.

Sensory processing can be tuned by a neuron's integration area, the types of inputs, and the proportion and number of connections with those inputs. Integration areas often vary topographically to sample space differentially across regions. Here, we highlight two visual circuits in which topographic changes in the postsynaptic retinal ganglion cell (RGC) dendritic territories and their presynaptic bipolar cell (BC) axonal territories are either matched or unmatched. Despite this difference, in both circuits, the proportion of inputs from each BC type, i.e., synaptic convergence between specific BCs and RGCs, remained constant across varying dendritic territory sizes. Furthermore, synapse density between BCs and RGCs was invariant across topography. Our results demonstrate a wiring design, likely engaging homotypic axonal tiling of BCs, that ensures consistency in synaptic convergence between specific BC types onto their target RGCs while enabling independent regulation of pre- and postsynaptic territory sizes and synapse number between cell pairs.

Protective effect of clusterin on rod photoreceptor in rat model of retinitis pigmentosa.

Retinitis Pigmentosa (RP) begins with the death of rod photoreceptors and is slowly followed by a gradual loss of cones and a rearrangement of the remaining retinal neurons. Clusterin is a chaperone protein that protects cells and is involved in various pathophysiological stresses, including retinal degeneration. Using a well-established transgenic rat model of RP (rhodopsin S334ter), we investigated the effects of clusterin on rod photoreceptor survival. To investigate the role of clusterin in S334ter-line3 retinas, Voronoi analysis and immunohistochemistry were used to evaluate the geometry of rod distribution. Additionally, immunoblot analysis, Bax activation, STAT3 and Akt phosphorylation were used to evaluate the pathway involved in rod cell protection. In this study, clusterin (10µg/ml) intravitreal treatment produced robust preservation of rod photoreceptors in S334ter-line3 retina. The mean number of rods in 1mm2 was significantly greater in clusterin injected RP retinas (postnatal (P) 30, P45, P60, & P75) than in age-matched saline injected RP retinas (P<0.01). Clusterin activated Akt, STAT3 and significantly reduced Bax activity; in addition to inducing phosphorylated STAT3 in Müller cells, which suggests it may indirectly acts on photoreceptors. Thus, clusterin treatment may interferes with mechanisms leading to rod death by suppressing cell death through activation of Akt and STAT3, followed by Bax suppression. Novel insights into the pathway of how clusterin promotes the rod cell survival suggest this treatment may be a potential therapeutic strategy to slow progression of vision loss in human RP.

Reshaping the Cone-Mosaic in a Rat Model of Retinitis Pigmentosa: Modulatory Role of ZO-1 Expression in DL-Alpha-Aminoadipic Acid Reshaping.

In S334ter-line-3 rat model of Retinitis Pigmentosa (RP), rod cell death induces the rearrangement of cones into mosaics of rings while the fibrotic processes of Müller cells remodel to fill the center of the rings. In contrast, previous work established that DL-alpha-aminoadipic-acid (AAA), a compound that transiently blocks Müller cell metabolism, abolishes these highly structured cone rings. Simultaneously, adherens-junction associated protein, Zonula occludens-1 (ZO-1) expression forms in a network between the photoreceptor segments and Müller cells processes. Thus, we hypothesized that AAA treatment alters the cone mosaic rings by disrupting the distal sealing formed by these fibrotic processes, either directly or indirectly, by down regulating the expression of ZO-1. Therefore, we examined these processes and ZO-1 expression at the outer retina after intravitreal injection of AAA and observed that AAA treatment transiently disrupts the distal glial sealing in RP retina, plus induces cones in rings to become more homogeneous. Moreover, ZO-1 expression is actively suppressed after 3 days of AAA treatment, which coincided with cone ring disruption. Similar modifications of glial sealing and cone distribution were observed after injection of siRNA to inhibit ZO-1 expression. These findings support our hypothesis and provide additional information about the critical role played by ZO-1 in glial sealing and shaping the ring mosaic in RP retina. These studies represent important advancements in the understanding of retinal degeneration's etiology and pathophysiology.

Inhibition of Matrix Metalloproteinase 9 Enhances Rod Survival in the S334ter-line3 Retinitis Pigmentosa Model.

Retinitis Pigmentosa (RP) is one of the most common forms of inherited visual loss with the initial degeneration of rod photoreceptors, followed by a progressive cone photoreceptor deterioration. Coinciding with this visual loss, the extracellular matrix (ECM) is reorganized, which alters matrix metalloproteinase (MMP) activity levels. A potential pathological role of MMPs, MMP-9 in particular, involves an excitotoxicity-mediated physiological response. In the current study, we examine the MMP-9 and MMP-2 expression levels in the rhodopsin S334ter-line3 RP rat model and investigate the impact of treatment with SB-3CT, a specific MMP-9 and MMP-2 inhibitor, on rod cell survival was tested. Retinal MMP-9 and MMP-2 expression levels were quantified by immunoblot analysis from S334ter-line3 rats compared to controls. Gelatinolytic activities of MMP-9 and MMP-2 by zymography were examined. The geometry of rod death was further evaluated using Voronoi analysis. Our results revealed that MMP-9 was elevated while MMP-2 was relatively unchanged when S334ter-line 3 retinas were compared to controls. With SB-3CT treatment, we observed gelatinolytic activity of both MMPs was decreased and diminished clustering associated with rod death, in addition to a robust preservation of rod photoreceptors. These results demonstrate that up-regulation of MMP-9 in retinas of S334ter-line3 are associated with rod death. The application of SB-3CT dramatically interferes with mechanisms leading to apoptosis in an MMP-9-dependent manner. Future studies will determine the feasibility of using SB-3CT as a potential therapeutic strategy to slow progression of vision loss in genetic inherited forms of human RP.

The effect of TIMP-1 on the cone mosaic in the retina of the rat model of retinitis pigmentosa.

PURPOSE: The array of photoreceptors found in normal retinas provides uniform and regular sampling of the visual space. In contrast, cones in retinas of the S334ter-line-3 rat model for RP migrate to form a mosaic of rings, leaving large holes with few or no photoreceptors. Similar mosaics appear in human patients with other forms of retinal dystrophy. In the current study, we aimed to investigate the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1) on the mosaic of cones in S334ter-line-3 rat retinas. We focused on TIMP-1 because it is one of the regulators of the extracellular matrix important for cellular migration. METHODS: Immunohistochemistry was performed to reveal M-opsin cone cells (M-cone) and the results were quantified to test statistically whether or not TIMP-1 restores the mosaics to normal. In particular, the tests focused on the Voronoi and nearest-neighbor distance analyses. RESULTS: Our tests indicated that TIMP-1 led to significant disruption of the M-opsin cone rings in S334ter-line-3 rat retinas and resulted in almost complete homogeneous mosaics. In addition, TIMP-1 induced the M-cone spatial distribution to become closer to random with decreased regularity in S334ter-line-3 rat retinas. CONCLUSIONS: These findings confirm that TIMP-1 induced M-cone mosaics in S334ter-line-3 to gain homogeneity without reaching the degree of regularity seen in normal retinal mosaics. Even if TIMP-1 fails to promote regularity, the effects of this drug on homogeneity appear to be so dramatic that TIMP-1 may be a potential therapeutic agent. TIMP-1 improves sampling of the visual field simply by causing homogeneity.