scholarly journals Microglial trogocytosis and the complement system regulate axonal pruning in vivo

2020 ◽  
Author(s):  
Tony K.Y. Lim ◽  
Edward S. Ruthazer
Keyword(s):  
Behavioral Response ◽  
Neural Circuit ◽  
Ex Vivo ◽  
Complement C3 ◽  
Retinal Ganglion ◽  
Inhibitory Molecule ◽  
Membrane Bound ◽  
Circuit Development ◽  
The Complement System

AbstractPartial phagocytosis—called trogocytosis—of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here we directly observe axon trogocytosis by microglia in vivo in the developing Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. We identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally-expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.

scholarly journals Microglial trogocytosis and the complement system regulate axonal pruning in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tony KY Lim ◽  
Edward S Ruthazer
Keyword(s):  
Behavioral Response ◽  
Neural Circuit ◽  
Ex Vivo ◽  
Complement C3 ◽  
Retinal Ganglion ◽  
Inhibitory Molecule ◽  
Membrane Bound ◽  
Circuit Development ◽  
The Complement System

Partial phagocytosis—called trogocytosis—of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here, we directly observe axon trogocytosis by microglia in vivo in the developing Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. Using bioinformatics, we identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.

scholarly journals A method for single-neuron chronic recording from the retina in awake mice

Science ◽  
2018 ◽  
Vol 360 (6396) ◽  
pp. 1447-1451 ◽  
Author(s):  
Guosong Hong ◽  
Tian-Ming Fu ◽  
Mu Qiao ◽  
Robert D. Viveros ◽  
Xiao Yang ◽  
...  
Keyword(s):  
Retinal Ganglion Cells ◽  
Visual Information ◽  
Single Neuron ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Neural Circuitry ◽  
Retinal Ganglion ◽  
Chronic Recording ◽  
The Brain

The retina, which processes visual information and sends it to the brain, is an excellent model for studying neural circuitry. It has been probed extensively ex vivo but has been refractory to chronic in vivo electrophysiology. We report a nonsurgical method to achieve chronically stable in vivo recordings from single retinal ganglion cells (RGCs) in awake mice. We developed a noncoaxial intravitreal injection scheme in which injected mesh electronics unrolls inside the eye and conformally coats the highly curved retina without compromising normal eye functions. The method allows 16-channel recordings from multiple types of RGCs with stable responses to visual stimuli for at least 2 weeks, and reveals circadian rhythms in RGC responses over multiple day/night cycles.

scholarly journals Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity

2019 ◽  
Vol 20 (12) ◽  
pp. 3012 ◽  
Author(s):  
Beatriz Vidal-Villegas ◽  
Johnny Di Pierdomenico ◽  
Juan A Miralles de Imperial-Ollero ◽  
Arturo Ortín-Martínez ◽  
Francisco M Nadal-Nicolás ◽  
...  
Keyword(s):  
Intravitreal Injection ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Retinal Ganglion ◽  
Long Term Effects ◽  
Pou Domain ◽  
Short And Long Term ◽  
Sd Oct

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

scholarly journals A projection specific logic to sampling visual inputs in mouse superior colliculus

2018 ◽  
Author(s):  
Katja Reinhard ◽  
Chen Li ◽  
Quan Do ◽  
Emily Burke ◽  
Steven Heynderickx ◽  
...  
Keyword(s):  
Superior Colliculus ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Sensory Information ◽  
Cell Types ◽  
Brain Regions ◽  
Visual Response ◽  
Retinal Ganglion ◽  
Parabigeminal Nucleus

AbstractUsing sensory information to trigger different behaviours relies on circuits that pass-through brain regions. However, the rules by which parallel inputs are routed to different downstream targets is poorly understood. The superior colliculus mediates a set of innate behaviours, receiving input from ~30 retinal ganglion cell types and projecting to behaviourally important targets including the pulvinar and parabigeminal nucleus. Combining transsynaptic circuit tracing with in-vivo and ex-vivo electrophysiological recordings we observed a projection specific logic where each collicular output pathway sampled a distinct set of retinal inputs. Neurons projecting to the pulvinar or parabigeminal nucleus uniquely sampled 4 and 7 cell types, respectively. Four others innervated both pathways. The visual response properties of retinal ganglion cells correlated well with those of their disynaptic targets. These findings suggest that projection specific sampling of retinal inputs forms a mechanistic basis for the selective triggering of visually guided behaviours by the superior colliculus.

scholarly journals Cancer immunotherapy using cells modified with cytokine genes.

2003 ◽  
Vol 50 (3) ◽  
pp. 613-624 ◽  
Author(s):  
Dariusz W Kowalczyk ◽  
Piotr J Wysocki ◽  
Andrzej Mackiewicz
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Antitumor Agents ◽  
Ex Vivo ◽  
Cell Types ◽  
Cancer Gene Therapy ◽  
Cytokine Gene ◽  
Cytokine Genes ◽  
Membrane Bound

The ability of various cytokines to hamper tumor growth or to induce anti-tumor immune response has resulted in their study as antitumor agents in gene therapy approaches. In this review we will concentrate on the costimulation of antitumor immune responses using modification of various cell types by cytokine genes. Several strategies have emerged such as (i). modification of tumor cells with cytokine genes ex vivo (whole tumor cell vaccines), (ii). ex vivo modification of other cell types for cytokine gene delivery, (iii). delivery of cytokine genes into tumor microenvironment in vivo, (iv). modification of dendritic cells with cytokine genes ex vivo. Originally single cytokine genes were used. Subsequently, multiple cytokine genes were applied simultaneously, or in combination with other factors such as chemokines, membrane bound co-stimulatory molecules, or tumor associated antigens. In this review we discuss these strategies and their use in cancer treatment as well as the promises and limitations of cytokine based cancer gene therapy. Clinical trials, including our own experience, employing the above strategies are discussed.

scholarly journals A projection specific logic to sampling visual inputs in mouse superior colliculus

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Katja Reinhard ◽  
Chen Li ◽  
Quan Do ◽  
Emily G Burke ◽  
Steven Heynderickx ◽  
...  
Keyword(s):  
Superior Colliculus ◽  
Ex Vivo ◽  
Sensory Information ◽  
Cell Types ◽  
Brain Regions ◽  
Biased Sampling ◽  
Visual Response ◽  
Retinal Ganglion ◽  
Parabigeminal Nucleus

Using sensory information to trigger different behaviors relies on circuits that pass through brain regions. The rules by which parallel inputs are routed to downstream targets are poorly understood. The superior colliculus mediates a set of innate behaviors, receiving input from >30 retinal ganglion cell types and projecting to behaviorally important targets including the pulvinar and parabigeminal nucleus. Combining transsynaptic circuit tracing with in vivo and ex vivo electrophysiological recordings, we observed a projection-specific logic where each collicular output pathway sampled a distinct set of retinal inputs. Neurons projecting to the pulvinar or the parabigeminal nucleus showed strongly biased sampling from four cell types each, while six others innervated both pathways. The visual response properties of retinal ganglion cells correlated well with those of their disynaptic targets. These findings open the possibility that projection-specific sampling of retinal inputs forms a basis for the selective triggering of behaviors by the superior colliculus.

scholarly journals Diabetes Is Associated with Increased Autoreactivity of Mannan-Binding Lectin

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Esben Axelgaard ◽  
Jakob Appel Østergaard ◽  
Steffen Thiel ◽  
Troels Krarup Hansen
Keyword(s):  
Diabetic Nephropathy ◽  
Complement System ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Lectin Pathway ◽  
Urine Albumin ◽  
Mannan Binding Lectin ◽  
The Complement System ◽  
Binding Lectin

Mannan-binding lectin (MBL) has been reported to be involved in the pathophysiology of diabetic nephropathy. MBL is a pattern-recognition molecule of the innate immune system that initiates the lectin pathway of the complement system upon recognition of evolutionary conserved pathogen-associated molecular patterns or to altered self-tissue. Our group have previously shown direct effects of MBL on diabetes-induced kidney damage, and we hypothesized that MBL may cause autoactivation of the complement system via binding to neoepitopes induced by hyperglycemia. In the present study, we induced diabetes in MBL knockout mice and in wild type C57BL/6J mice by low-dose streptozotocin injection and measured blood glucose and urine albumin-to-creatinine ratio to monitor development of diabetes. After 24 weeks, fluorescently labelled recombinant MBL was injected intravenously in diabetic MBL knockout mice after which the distribution was investigated using in vivo fluorescence imaging. Mice were subjected to in vivo and ex vivo imaging 24 hours after injection. MBL was found to accumulate in the kidneys of diabetic mice as compared to healthy control mice (p<0.0001). These findings support the hypothesis of a significant role of MBL and the complement system in the pathophysiology of diabetic nephropathy.

scholarly journals A potential role for somatostatin signaling in regulating retinal neurogenesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kurt Weir ◽  
Dong Won Kim ◽  
Seth Blackshaw
Keyword(s):  
Ganglion Cells ◽  
Ex Vivo ◽  
Retinal Ganglion ◽  
Pharmacological Agents ◽  
Embryonic Mouse ◽  
Relative Fraction ◽  
Dependent Inhibition ◽  
Retinal Explants ◽  
Retinal Neurogenesis

AbstractNeuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.

scholarly journals TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 305-317 ◽  
Author(s):  
Carlos Lizama ◽  
Diego Rojas-Benítez ◽  
Marcelo Antonelli ◽  
Andreas Ludwig ◽  
Ximena Bustamante-Marín ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Cell Apoptosis ◽  
Ex Vivo ◽  
Extracellular Domain ◽  
Germ Cell Apoptosis ◽  
Protein Levels ◽  
Kit Receptor ◽  
Membrane Bound

The pathways leading to male germ cell apoptosisin vivoare poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myristate 13-acetate (PMA) induced germ cell apoptosis, which was prevented when an inhibitor of TACE/ADAM17 was present in the assay.Ex-vivorat testis culture showed that PMA induced the cleavage of the KIT extracellular domain. Isolation of apoptotic germ cells showed that even though protein levels of TACE/ADAM17 were higher in apoptotic germ cells than in nonapoptotic cells, the contrary was observed for ADAM10. These results suggest that TACE/ADAM17 is one of the elements triggering physiological germ cell apoptosis during the first wave of spermatogenesis.

scholarly journals Regulation of retinal neurogenesis by somatostatin signaling

2020 ◽  
Author(s):  
Kurt Weir ◽  
Dong Won Kim ◽  
Seth Blackshaw
Keyword(s):  
Ganglion Cells ◽  
Ex Vivo ◽  
Retinal Ganglion ◽  
Pharmacological Agents ◽  
Embryonic Mouse ◽  
Relative Fraction ◽  
Dependent Inhibition ◽  
Retinal Explants ◽  
Retinal Neurogenesis

AbstractNeuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a dose-dependent inhibition of photoreceptor generation while increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants and abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, although in vivo Sstr2 is dispensable for normal retinal development.

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