ABSTRACT
Neurons of the mammalian central nervous system fail to regenerate. Substantial progress has been made toward identifying the cellular and molecular mechanisms that underlie regenerative failure and how altering those pathways can promote cell survival and/or axon regeneration. Here, we summarize those findings while comparing the regenerative process in the central versus the peripheral nervous system. We also highlight studies that advance our understanding of the mechanisms underlying neural degeneration in response to injury, as many of these mechanisms represent primary targets for restoring functional neural circuits.
Subject(s)
Axons/metabolism , Central Nervous System/metabolism , Nerve Regeneration/physiology , Neurons/metabolism , Signal Transduction/physiology , Animals , Humans , Peripheral Nervous System/metabolismABSTRACT
The assembly of functional neural circuits requires the combined action of progressive and regressive events. Regressive events encompass a variety of inhibitory developmental processes, including axon and dendrite pruning, which facilitate the removal of exuberant neuronal connections. Most axon pruning involves the removal of axons that had already made synaptic connections; thus, axon pruning is tightly associated with synapse elimination. In many instances, these developmental processes are regulated by the interplay between neurons and glial cells that act instructively during neural remodeling. Owing to the importance of axon and dendritic pruning, these remodeling events require precise spatial and temporal control, and this is achieved by a range of distinct molecular mechanisms. Disruption of these mechanisms results in abnormal pruning, which has been linked to brain dysfunction. Therefore, understanding the mechanisms of axon and dendritic pruning will be instrumental in advancing our knowledge of neural disease and mental disorders.
Subject(s)
Axons/physiology , Neuronal Plasticity/physiology , Neurons/physiology , Animals , Humans , Neuroglia/physiology , Signal Transduction/physiology , Synapses/physiologyABSTRACT
Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3ß serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3ß/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3ß can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code.
Subject(s)
Carrier Proteins , Tubulin , Animals , Tubulin/metabolism , Carrier Proteins/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Neurons/metabolism , Microtubules/metabolism , Axons/metabolism , Cells, Cultured , MammalsABSTRACT
Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) ß2-Chimaerin (ß2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds ß2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. ß2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/ß2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.
Subject(s)
Axons/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Neoplasm Proteins/metabolism , Neuropeptides/metabolism , Signal Transduction , rac GTP-Binding Proteins/metabolism , Animals , Dentate Gyrus/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Synapses , rac1 GTP-Binding ProteinABSTRACT
Visual system function depends upon the elaboration of precise connections between retinal ganglion cell (RGC) axons and their central targets in the brain. Though some progress has been made in defining the molecules that regulate RGC connectivity required for the assembly and function of image-forming circuitry, surprisingly little is known about factors required for intrinsically photosensitive RGCs (ipRGCs) to target a principal component of the non-image-forming circuitry: the suprachiasmatic nucleus (SCN). Furthermore, the molecules required for forming circuits critical for circadian behaviors within the SCN are not known. We observe here that the adhesion molecule teneurin-3 (Tenm3) is highly expressed in vasoactive intestinal peptide (VIP) neurons located in the core region of the SCN. Since Tenm3 is required for other aspects of mammalian visual system development, we investigate roles for Tenm3 in regulating ipRGC-SCN connectivity and function. Our results show that Tenm3 negatively regulates association between VIP and arginine vasopressin (AVP) neurons within the SCN and is essential for M1 ipRGC axon innervation to the SCN. Specifically, in Tenm3-/- mice, we find a reduction in ventro-medial innervation to the SCN. Despite this reduction, Tenm3-/- mice have higher sensitivity to light and faster re-entrainment to phase advances, probably due to the increased association between VIP and AVP neurons. These data show that Tenm3 plays key roles in elaborating non-image-forming visual system circuitry and that it influences murine responses to phase-advancing light stimuli.
Subject(s)
Axons , Retinal Ganglion Cells , Animals , Mice , Axons/metabolism , Circadian Rhythm/physiology , Mammals/metabolism , Retinal Ganglion Cells/physiology , Suprachiasmatic Nucleus/metabolism , Vasoactive Intestinal Peptide/metabolismABSTRACT
The retina is a tremendously complex image processor, containing numerous cell types that form microcircuits encoding different aspects of the visual scene. Each microcircuit exhibits a distinct pattern of synaptic connectivity. The developmental mechanisms responsible for this patterning are just beginning to be revealed. Furthermore, signals processed by different retinal circuits are relayed to specific, often distinct, brain regions. Thus, much work has focused on understanding the mechanisms that wire retinal axonal projections to their appropriate central targets. Here, we highlight recently discovered cellular and molecular mechanisms that together shape stereotypic wiring patterns along the visual pathway, from within the retina to the brain. Although some mechanisms are common across circuits, others play unconventional and circuit-specific roles. Indeed, the highly organized connectivity of the visual system has greatly facilitated the discovery of novel mechanisms that establish precise synaptic connections within the nervous system.
Subject(s)
Brain/physiology , Neurons/physiology , Retina/physiology , Visual Pathways/physiology , Animals , Brain/metabolism , Humans , Neurons/metabolism , Photic Stimulation , Retina/metabolism , Visual Pathways/metabolismABSTRACT
Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage both male and female mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of Layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and overexpression (OE) manipulations in an in vivo candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating Layer II/III cortical projection neuron (CPN) collateral axon branching in vitro LOF experiments show that Dbn1 is necessary to suppress the elongation of Layer II/III CPN collateral axon branches within Layer IV, where axon branching by Layer II/III CPNs is normally absent. Conversely, Dbn1 OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1S142 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon Dbn1 OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation.SIGNIFICANCE STATEMENT Laminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory Layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons in vivo, we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in vivo and in vitro may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in autism spectrum disorder and epilepsy.
Subject(s)
Autism Spectrum Disorder , Neuropeptides , Animals , Female , Male , Mice , Autism Spectrum Disorder/metabolism , Axons/physiology , Cytoskeletal Proteins/metabolism , Neurons/metabolism , Neuropeptides/metabolismSubject(s)
Axons/physiology , Animals , Central Nervous System/embryology , Humans , Neurons/metabolism , Signal TransductionSubject(s)
Axons/physiology , Animals , Humans , Nerve Tissue Proteins/metabolism , Neurogenesis , Neurons/metabolismABSTRACT
The vertebrate retina contains an array of neural circuits that detect distinct features in visual space. Direction-selective (DS) circuits are an evolutionarily conserved retinal circuit motif - from zebrafish to rodents to primates - specialized for motion detection. During retinal development, neuronal subtypes that wire DS circuits form exquisitely precise connections with each other to shape the output of retinal ganglion cells tuned for specific speeds and directions of motion. In this review, we follow the chronology of DS circuit development in the vertebrate retina, including the cellular, molecular, and activity-dependent mechanisms that regulate the formation of DS circuits, from cell birth and migration to synapse formation and refinement. We highlight recent findings that identify genetic programs critical for specifying neuronal subtypes within DS circuits and molecular interactions essential for responses along the cardinal axes of motion. Finally, we discuss the roles of DS circuits in visual behavior and in certain human visual disease conditions. As one of the best-characterized circuits in the vertebrate retina, DS circuits represent an ideal model system for studying the development of neural connectivity at the level of individual genes, cells, and behavior.
Subject(s)
Retina/embryology , Retina/physiology , Vertebrates/physiology , Visual Pathways , Animals , Humans , Neurogenesis , Neurons/physiology , Nystagmus, Pathologic/genetics , Retina/cytology , Retinal Ganglion Cells/physiology , Synapses , Vertebrates/embryologyABSTRACT
The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. Here, we uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.
Subject(s)
Cytoskeleton/metabolism , Drosophila Proteins/metabolism , Filamins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanylate Cyclase/metabolism , Membrane Proteins/metabolism , Semaphorins/metabolism , Signal Transduction/physiology , Amino Acid Motifs , Animals , Cytoskeleton/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Filamins/genetics , Guanine Nucleotide Exchange Factors/genetics , Guanylate Cyclase/genetics , Membrane Proteins/genetics , Protein Domains , Semaphorins/geneticsABSTRACT
The Drosophila transmembrane semaphorin-1a (Sema-1a) is a repulsive guidance cue that uses the Plexin A (PlexA) receptor during neural development. Sema-1a is required in axons to facilitate motor axon defasciculation at guidance choice points. We found that mutations in the trol gene strongly suppress Sema-1a-mediated repulsive axon guidance. trol encodes the phylogenetically conserved secreted heparan sulfate proteoglycan (HSPG) perlecan, a component of the extracellular matrix. Motor axon guidance defects in perlecan mutants resemble those observed in Sema-1a- and PlexA-null mutant embryos, and perlecan mutants genetically interact with PlexA and Sema-1a. Perlecan protein is found in both the CNS and the periphery, with higher expression levels in close proximity to motor axon trajectories and pathway choice points. Restoring perlecan to mutant motor neurons rescues perlecan axon guidance defects. Perlecan augments the reduction in phospho-focal adhesion kinase (phospho-FAK) levels that result from treating insect cells in vitro with Sema-1a, and genetic interactions among integrin, Sema-1a, and FAK in vivo support an antagonistic relationship between Sema-1a and integrin signaling. Therefore, perlecan is required for Sema-1a-PlexA-mediated repulsive guidance, revealing roles for extracellular matrix proteoglycans in modulating transmembrane guidance cue signaling during neural development.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Extracellular Matrix/metabolism , Heparan Sulfate Proteoglycans/metabolism , Motor Neurons/cytology , Semaphorins/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Heparan Sulfate Proteoglycans/genetics , Motor Neurons/metabolism , Mutation , Phosphorylation , Signal TransductionABSTRACT
The second messengers cAMP and cGMP modulate attraction and repulsion mediated by neuronal guidance cues. We find that the Drosophila receptor guanylyl cyclase Gyc76C genetically interacts with Semaphorin 1a (Sema-1a) and physically associates with the Sema-1a receptor plexin A (PlexA). PlexA regulates Gyc76C catalytic activity in vitro, and each distinct Gyc76C protein domain is crucial for regulating Gyc76C activity in vitro and motor axon guidance in vivo. The cytosolic protein dGIPC interacts with Gyc76C and facilitates Sema-1a-PlexA/Gyc76C-mediated motor axon guidance. These findings provide an in vivo link between semaphorin-mediated repulsive axon guidance and alteration of intracellular neuronal cGMP levels.
Subject(s)
Axons/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Guanylate Cyclase/metabolism , Motor Neurons/physiology , Nerve Tissue Proteins/metabolism , Neurogenesis , Receptors, Cell Surface/metabolism , Semaphorins/metabolism , Animals , Axons/metabolism , Carrier Proteins/metabolism , Catalysis , Cells, Cultured , Cyclic GMP/metabolism , Drosophila Proteins/biosynthesis , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Guanylate Cyclase/biosynthesis , Guanylate Cyclase/genetics , Motor Neurons/metabolism , Protein Binding , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Sequence Deletion/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.
Subject(s)
Cell Membrane/metabolism , Neurons/cytology , Neurons/metabolism , Retina/cytology , Retina/metabolism , Semaphorins/metabolism , Signal Transduction , Amacrine Cells/enzymology , Amacrine Cells/metabolism , Animals , Calbindins , Dopamine/metabolism , Gene Expression Profiling , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Nerve Tissue Proteins , Neurites/metabolism , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Retina/embryology , Retinal Ganglion Cells/metabolism , Rod Opsins/metabolism , S100 Calcium Binding Protein G/metabolism , Semaphorins/deficiency , Semaphorins/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.
Subject(s)
Animal Structures/physiology , Cytoskeletal Proteins/metabolism , Drosophila/physiology , Gene Expression Regulation, Developmental , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/physiology , Amino Acid Sequence , Animal Structures/metabolism , Animal Structures/ultrastructure , Animals , Binding Sites , Cell Membrane/metabolism , Cell Membrane/physiology , Cell-Matrix Junctions/metabolism , Cell-Matrix Junctions/physiology , Cytoskeletal Proteins/genetics , Drosophila/anatomy & histology , Drosophila/genetics , Drosophila/metabolism , Electrophysiological Phenomena , Genome, Insect , Hearing Disorders/genetics , Hearing Disorders/pathology , Hearing Disorders/veterinary , Integrins/metabolism , Larva/genetics , Larva/metabolism , Larva/physiology , Larva/ultrastructure , Mechanotransduction, Cellular , Microscopy, Electron, Transmission , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Muscles/cytology , Muscles/metabolism , Protein Interaction Mapping , Sequence Homology, Amino Acid , Signal Transduction , Talin/genetics , Talin/metabolism , Vibration , Vinculin/genetics , Vinculin/metabolism , src Homology DomainsABSTRACT
Most excitatory synapses in the mammalian brain are formed on dendritic spines, and spine density has a profound impact on synaptic transmission, integration, and plasticity. Membrane-associated guanylate kinase (MAGUK) proteins are intracellular scaffolding proteins with well established roles in synapse function. However, whether MAGUK proteins are required for the formation of dendritic spines in vivo is unclear. We isolated a novel disc large-5 (Dlg5) allele in mice, Dlg5(LP), which harbors a missense mutation in the DLG5 SH3 domain, greatly attenuating its ability to interact with the DLG5 GUK domain. We show here that DLG5 is a MAGUK protein that regulates spine formation, synaptogenesis, and synaptic transmission in cortical neurons. DLG5 regulates synaptogenesis by enhancing the cell surface localization of N-cadherin, revealing a key molecular mechanism for regulating the subcellular localization of this cell adhesion molecule during synaptogenesis.
Subject(s)
Cadherins/metabolism , Dendritic Spines/physiology , Guanylate Kinases/physiology , Membrane Proteins/physiology , Neurogenesis/physiology , Synapses/physiology , Animals , Cells, Cultured , Cerebral Cortex/physiology , Cerebral Cortex/ultrastructure , Dendritic Spines/metabolism , Dendritic Spines/ultrastructure , Guanylate Kinases/genetics , Male , Membrane Proteins/genetics , Mice , Mutation, Missense , Primary Cell Culture , Synapses/ultrastructure , Synaptic Transmission/genetics , Synaptic Transmission/physiology , beta Catenin/metabolismABSTRACT
The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2(-/-) brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A-Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.
Subject(s)
Central Nervous System/growth & development , Central Nervous System/metabolism , Pyramidal Cells/cytology , Pyramidal Cells/growth & development , Semaphorins/metabolism , Synapses/physiology , Animals , Central Nervous System/cytology , Central Nervous System/drug effects , Central Nervous System/ultrastructure , Female , Gene Expression Regulation, Developmental , Male , Mice , Mice, Knockout , Neuropilin-1/metabolism , Neuropilin-2/metabolism , Pyramidal Cells/drug effects , Pyramidal Cells/ultrastructure , Recombinant Proteins/pharmacology , Semaphorins/genetics , Semaphorins/pharmacology , Signal Transduction , Synapses/drug effects , Synapses/ultrastructureABSTRACT
The study of behavioral responses to visual stimuli is a key component of understanding visual system function. One notable response is the optokinetic reflex (OKR), a highly conserved innate behavior necessary for image stabilization on the retina. The OKR provides a robust readout of image tracking ability and has been extensively studied to understand visual system circuitry and function in animals from different genetic backgrounds. The OKR consists of two phases: a slow tracking phase as the eye follows a stimulus to the edge of the visual plane and a compensatory fast phase saccade that resets the position of the eye in the orbit. Previous methods of tracking gain quantification, although reliable, are labor intensive and can be subjective or arbitrarily derived. To obtain more rapid and reproducible quantification of eye tracking ability, we have developed a novel semi-automated analysis program, PyOKR, that allows for quantification of two-dimensional eye tracking motion in response to any directional stimulus, in addition to being adaptable to any type of video-oculography equipment. This method provides automated filtering, selection of slow tracking phases, modeling of vertical and horizontal eye vectors, quantification of eye movement gains relative to stimulus speed, and organization of resultant data into a usable spreadsheet for statistical and graphical comparisons. This quantitative and streamlined analysis pipeline, readily accessible via PyPI import, provides a fast and direct measurement of OKR responses, thereby facilitating the study of visual behavioral responses.
Subject(s)
Eye-Tracking Technology , Animals , Nystagmus, Optokinetic/physiology , Eye Movements/physiologyABSTRACT
In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4â»/â» retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.
Subject(s)
Neurogenesis/physiology , Receptors, Cell Surface/physiology , Retinal Horizontal Cells/cytology , Retinal Horizontal Cells/ultrastructure , Retinal Photoreceptor Cell Outer Segment/physiology , Semaphorins/physiology , Synapses/physiology , Animals , Dendrites/ultrastructure , Female , Male , Mice , Mutation , Nerve Tissue Proteins , Photoreceptor Cells, Vertebrate/cytology , Photoreceptor Cells, Vertebrate/ultrastructure , Receptors, Cell Surface/genetics , Retinal Bipolar Cells/cytology , Retinal Bipolar Cells/ultrastructure , Retinal Photoreceptor Cell Outer Segment/ultrastructure , Semaphorins/genetics , Signal Transduction/physiology , Synapses/ultrastructureABSTRACT
Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through beta1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through alpha1beta1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and alpha1beta1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.