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BioRxiv : the Preprint Server For... Nov 2023To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic...
To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet, the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell (SAC) dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate for the first time that RGCs, the first neuron-type born within the retina, play an active role in functional specialization of the IPL. Retinal ganglion cell-dependent regulation of OFF and ON starburst amacrine cell dendritic scaffold segregation prevents blurring of OFF versus ON functional domains in the murine inner plexiform layer.
PubMed: 38014224
DOI: 10.1101/2023.11.18.567662 -
BioRxiv : the Preprint Server For... Nov 2023In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during...
In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using newly-generated transgenic tools and single cell labeling, we identify a transient developmental period when starburst somata receive extensive contacts from neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing, and suggest that this could be a general mechanism for mosaic patterning across many cell types and species.
PubMed: 38014021
DOI: 10.1101/2023.11.17.567616 -
Cell Reports Nov 2023Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we...
Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.
Topics: Mice; Animals; Amacrine Cells; Synapses; Retina; Retinal Bipolar Cells; Synaptic Transmission
PubMed: 37976158
DOI: 10.1016/j.celrep.2023.113440 -
BioRxiv : the Preprint Server For... Nov 2023Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applications ,...
Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for neurodegenerative diseases. Despite successful applications , implementation has been hampered by low efficiency. In this study, we present a highly efficient strategy for reprogramming retinal glial cells into neurons by simultaneously inhibiting key negative regulators. By suppressing Notch signaling through the removal of its central mediator we induced mature Müller glial cells to reprogram into bipolar and amacrine neurons in uninjured adult mouse retinas, and observed that this effect was further enhanced by retinal injury. We found that specific loss of function of and receptors in Müller glia mimicked the effect of deletion on Müller glia-derived neurogenesis. Integrated analysis of multiome (scRNA- and scATAC-seq) and CUT&Tag data revealed that Rbpj directly activates Notch effector genes and genes specific to mature Müller glia while also indirectly represses the expression of neurogenic bHLH factors. Furthermore, we found that combined loss of function of and resulted in a robust conversion of nearly all Müller glia to neurons. Finally, we demonstrated that inducing Müller glial proliferation by AAV (adeno-associated virus)-mediated overexpression of dominant- active Yap supports efficient levels of Müller glia-derived neurogenesis in both - and - deficient Müller glia. These findings demonstrate that, much like in zebrafish, Notch signaling actively represses neurogenic competence in mammalian Müller glia, and suggest that inhibition of Notch signaling and in combination with overexpression of activated Yap could serve as an effective component of regenerative therapies for degenerative retinal diseases.
PubMed: 37961663
DOI: 10.1101/2023.10.29.560483 -
BioRxiv : the Preprint Server For... Nov 2023Vision is initiated by the reception of light by photoreceptors and subsequent processing via parallel retinal circuits. Proper circuit organization depends on the...
Vision is initiated by the reception of light by photoreceptors and subsequent processing via parallel retinal circuits. Proper circuit organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. Indeed, similar deficits were observed in mice lacking the bipolar cell glutamate receptors GRIK1 (OFF-bipolar cells) and GRM6 (ON-bipolar cells). Mechanistically, FAT3 binds to the synaptic protein PTPσ and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. How FAT3 impacts ON-cone bipolar cell function at high temporal frequency remains to be uncovered. These findings expand the repertoire of FAT3's functions and reveal the importance of both ON- and OFF-bipolar cells for high frequency light response.
PubMed: 37961274
DOI: 10.1101/2023.11.02.565326 -
Stem Cells Translational Medicine Jan 2024Cone cell death is a characteristic shared by various retinal degenerative disorders, such as cone-rod dystrophy, Stargardt disease, achromatopsia, and retinitis...
Cone cell death is a characteristic shared by various retinal degenerative disorders, such as cone-rod dystrophy, Stargardt disease, achromatopsia, and retinitis pigmentosa. This leads to conditions like color blindness and permanently impaired visual acuity. Stem cell therapy focused on photoreceptor replacement holds promise for addressing these conditions. However, identifying surface markers that aid in enriching retinal progenitor cells (RPCs) capable of differentiating into cones remains a complex task. In this study, we employed single-cell RNA sequencing to scrutinize the transcriptome of developing retinas in C57BL/6J mice. This revealed the distinctive expression of somatostatin receptor 2 (Sstr2), a surface protein, in late-stage RPCs exhibiting the potential for photoreceptor differentiation. In vivo lineage tracing experiments verified that Sstr2+ cells within the late embryonic retina gave rise to cones, amacrine and horizontal cells during the developmental process. Furthermore, Sstr2+ cells that were isolated from the late embryonic mouse retina displayed RPC markers and exhibited the capability to differentiate into cones in vitro. Upon subretinal transplantation into both wild-type and retinal degeneration 10 (rd10) mice, Sstr2+ cells survived and expressed cone-specific markers. This study underscores the ability of Sstr2 to enrich late-stage RPCs primed for cone differentiation to a large extent. It proposes the utility of Sstr2 as a biomarker for RPCs capable of generating cones for transplantation purposes.
Topics: Animals; Mice; Mice, Inbred C57BL; Receptors, Somatostatin; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Stem Cells
PubMed: 37935630
DOI: 10.1093/stcltm/szad073 -
Cell Reports Nov 2023In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways...
In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways via different bipolar cells, enabling AIIs to operate at night and during the day. Previous work has examined luminance-dependent changes in AII gap junction connectivity, but less is known about how surrounding circuitry shapes AII receptive fields across light levels. Here, we report that moderate contrast stimuli elicit surround inhibition in AIIs under all but the dimmest visual conditions, due to actions of horizontal cells and at least two ACs that inhibit presynaptic bipolar cells. Under photopic (daylight) conditions, surround inhibition transforms AII response kinetics, which are inherited by downstream ganglion cells. Ablating neuronal nitric oxide synthase type-1 (nNOS-1) ACs removes AII surround inhibition under mesopic (dusk/dawn), but not photopic, conditions. Our findings demonstrate how multiple layers of neural circuitry interact to encode signals across a wide physiological range.
Topics: Amacrine Cells; Retina; Retinal Cone Photoreceptor Cells; Gap Junctions
PubMed: 37930888
DOI: 10.1016/j.celrep.2023.113390 -
ENeuro Nov 2023The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified...
The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified to promote different cell fates. In , is required for specifying photoreceptors. In mice, there are two closely related homologs, and While is known to promote the genesis of retinal ganglion cells, there is no study on the function of in retinal development. Here, we crossed mice to mice carrying a Cre-dependent TdTomato reporter to track potential -lineage neurons in retinas. We characterized a heterogeneous group of TdTomato retinal neurons that were detected at the postnatal stage, including glutamatergic amacrine cells, AII amacrine cells, and BC3b bipolar cells. Unexpectedly, we did not observe TdTomato retinal neurons in the mice with an knock-in allele and a Flp-dependent TdTomato reporter, suggesting is not expressed in the mouse retina. Consistent with these data, conditional removal of in the retina did not cause any observable phenotypes. Importantly, we did not detect expression in the retina at multiple ages using mice with knock-in allele. Therefore, we conclude that mice have ectopic Cre expression in the retina and that is not required for retinal development.
Topics: Mice; Animals; Amacrine Cells; Mice, Transgenic; Alleles; Retina; Basic Helix-Loop-Helix Transcription Factors
PubMed: 37923392
DOI: 10.1523/ENEURO.0307-23.2023 -
PloS One 2023The retina is an intricately organized neural tissue built on cone and rod pathways for color and night vision. Genetic mutations that disrupt the proper function of the...
The retina is an intricately organized neural tissue built on cone and rod pathways for color and night vision. Genetic mutations that disrupt the proper function of the rod circuit contribute to blinding diseases including retinitis pigmentosa and congenital stationary night blindness (CSNB). Down Syndrome cell adhesion molecule like 1 (Dscaml1) is expressed by rods, rod bipolar cells (RBCs), and sub-populations of amacrine cells, and has been linked to a middle age onset of CSNB in humans. However, how Dscaml1 contributes to this visual deficit remains unexplored. Here, we probed Dscaml1's role in the maintenance of the rod-to-RBC synapse using a loss of function mouse model. We used immunohistochemistry to investigate the anatomical formation and maintenance of the rod-to-RBC synapse in the young, adult, and aging retina. We generated 3D reconstructions, using serial electron micrographs, of rod spherules and RBCs to measure the number of invaginating neurites, RBC dendritic tip number, and RBC mitochondrial morphology. We find that while rod-to-RBC synapses form and are maintained, similar to wildtype, that there is an increase in the number of invaginating neurites in rod spherules, a reduction in RBC dendritic tips, and reduced mitochondrial volume and complexity in the Dscaml1 mutant retina compared to controls. We also observed precocious sprouting of RBC dendrites into the outer nuclear layer (ONL) of the Dscaml1 mutant retina compared to controls. These results contribute to our knowledge of Dscaml1's role in rod circuit development and maintenance and give additional insight into possible genetic therapy targets for blinding diseases and disorders like CSNB.
Topics: Humans; Mice; Animals; Retina; Synapses; Retinal Cone Photoreceptor Cells; Retinal Bipolar Cells; Aging
PubMed: 37910517
DOI: 10.1371/journal.pone.0290257 -
Proceedings of the National Academy of... Nov 2023Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to...
Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.
Topics: Animals; Humans; Mice; Diabetes Mellitus; Diabetic Retinopathy; Inflammation; Microglia; Retina; Signal Transduction
PubMed: 37903272
DOI: 10.1073/pnas.2308214120