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Sample records for lamina-specific axon targeting

  1. Versican in the developing brain: lamina-specific expression in interneuronal subsets and role in presynaptic maturation.

    PubMed

    Yamagata, Masahito; Sanes, Joshua R

    2005-09-14

    Chondroitin sulfate proteoglycans (CSPGs) of the extracellular matrix help stabilize synaptic connections in the postnatal brain and impede regeneration after injury. Here, we show that a CSPG of the lectican family, versican, also promotes presynaptic maturation in the developing brain. In the embryonic chick optic tectum, versican is expressed selectively by subsets of interneurons confined to the retinorecipient laminae, in which retinal axons arborize and form synapses. It is a major receptor for the Vicia villosa B4 lectin (VVA), shown previously to inhibit invasion of the retinorecipient lamina by retinal axons (Inoue and Sanes, 1997). In vitro, versican promotes enlargement of presynaptic varicosities in retinal axons. Depletion of versican in ovo, by RNA interference, results in retinal arbors with smaller than normal varicosities. We propose that versican provides a lamina-specific cue for presynaptic maturation and discuss the related but distinct effects of versican depletion and VVA blockade. PMID:16162928

  2. Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina.

    PubMed

    Yamagata, Masahito; Weiner, Joshua A; Sanes, Joshua R

    2002-09-01

    A major determinant of specific connectivity in the central nervous system is that synapses made by distinct afferent populations are restricted to particular laminae in their target area. We identify Sidekick (Sdk)-1 and -2, homologous transmembrane immunoglobulin superfamily molecules that mediate homophilic adhesion in vitro and direct laminar targeting of neurites in vivo. sdk-1 and -2 are expressed by nonoverlapping subsets of retinal neurons; each sdk is expressed by presynaptic (amacrine and bipolar) and postsynaptic (ganglion) cells that project to common inner plexiform (synaptic) sublaminae. Sdk proteins are concentrated at synaptic sites, and Sdk-positive synapses are restricted to the 2 (of > or =10) sublaminae to which sdk-expressing cells project. Ectopic expression of Sdk in Sdk-negative cells redirects their processes to a Sdk-positive sublamina. These results implicate Sdks as determinants of lamina-specific synaptic connectivity. PMID:12230981

  3. Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions.

    PubMed

    Sweeney, Lora B; Couto, Africa; Chou, Ya-Hui; Berdnik, Daniela; Dickson, Barry J; Luo, Liqun; Komiyama, Takaki

    2007-01-18

    Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. Here, we show that in the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. We propose that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons. PMID:17224402

  4. Missed connections: photoreceptor axon seeks target neuron for synaptogenesis.

    PubMed

    Astigarraga, Sergio; Hofmeyer, Kerstin; Treisman, Jessica E

    2010-08-01

    Extending axons must choose the appropriate synaptic target cells in order to assemble functional neural circuitry. The axons of the Drosophila color-sensitive photoreceptors R7 and R8 project as a single fascicle from each ommatidium, but their terminals are segregated into distinct layers within their target region. Recent studies have begun to reveal the molecular mechanisms that establish this projection pattern. Both homophilic adhesion molecules and specific ligand-receptor interactions make important contributions to stabilizing R7 and R8 terminals in the appropriate target layers. These cell recognition molecules are regulated by the same transcription factors that control R7 and R8 cell fates. Autocrine and repulsive signaling mechanisms prevent photoreceptor terminals from encroaching on their neighbors, preserving the spatial resolution of visual information. PMID:20434326

  5. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

  6. Role of GPR55 during Axon Growth and Target Innervation.

    PubMed

    Cherif, Hosni; Argaw, Anteneh; Cécyre, Bruno; Bouchard, Alex; Gagnon, Jonathan; Javadi, Pasha; Desgent, Sébastien; Mackie, Ken; Bouchard, Jean-François

    2015-01-01

    Guidance molecules regulate the navigation of retinal ganglion cell (RGC) projections toward targets in the visual thalamus. In this study, we demonstrate that the G-protein-coupled receptor 55 (GPR55) is expressed in the retina during development, and regulates growth cone (GC) morphology and axon growth. In vitro, neurons obtained from gpr55 knock-out (gpr55(-/-) ) mouse embryos have smaller GCs, less GC filopodia, and have a decreased outgrowth compared with gpr55(+/+) neurons. When gpr55(+/+) neurons were treated with GPR55 agonists, lysophosphatidylinositol (LPI) and O-1602, we observed a chemo-attractive effect and an increase in GC size and filopodia number. In contrast, cannabidiol (CBD) decreased the GC size and filopodia number inducing chemo-repulsion. In absence of the receptor (gpr55(-/-) ), no pharmacologic effects of the GPR55 ligands were observed. In vivo, compared to their wild-type (WT) littermates, gpr55(-/-) mice revealed a decreased branching in the dorsal terminal nucleus (DTN) and a lower level of eye-specific segregation of retinal projections in the superior colliculus (SC) and in the dorsal lateral geniculate nucleus (dLGN). Moreover, a single intraocular injection of LPI increased branching in the DTN, whereas treatment with CBD, an antagonist of GPR55, decreased it. These results indicate that GPR55 modulates the growth rate and the targets innervation of retinal projections and highlight, for the first time, an important role of GPR55 in axon refinement during development. PMID:26730399

  7. Role of GPR55 during Axon Growth and Target Innervation

    PubMed Central

    Cherif, Hosni; Argaw, Anteneh; Cécyre, Bruno; Bouchard, Alex; Gagnon, Jonathan; Javadi, Pasha; Desgent, Sébastien; Mackie, Ken

    2015-01-01

    Abstract Guidance molecules regulate the navigation of retinal ganglion cell (RGC) projections toward targets in the visual thalamus. In this study, we demonstrate that the G-protein-coupled receptor 55 (GPR55) is expressed in the retina during development, and regulates growth cone (GC) morphology and axon growth. In vitro, neurons obtained from gpr55 knock-out (gpr55-/-) mouse embryos have smaller GCs, less GC filopodia, and have a decreased outgrowth compared with gpr55+/+ neurons. When gpr55+/+ neurons were treated with GPR55 agonists, lysophosphatidylinositol (LPI) and O-1602, we observed a chemo-attractive effect and an increase in GC size and filopodia number. In contrast, cannabidiol (CBD) decreased the GC size and filopodia number inducing chemo-repulsion. In absence of the receptor (gpr55-/-), no pharmacologic effects of the GPR55 ligands were observed. In vivo, compared to their wild-type (WT) littermates, gpr55-/- mice revealed a decreased branching in the dorsal terminal nucleus (DTN) and a lower level of eye-specific segregation of retinal projections in the superior colliculus (SC) and in the dorsal lateral geniculate nucleus (dLGN). Moreover, a single intraocular injection of LPI increased branching in the DTN, whereas treatment with CBD, an antagonist of GPR55, decreased it. These results indicate that GPR55 modulates the growth rate and the targets innervation of retinal projections and highlight, for the first time, an important role of GPR55 in axon refinement during development. PMID:26730399

  8. Target selection: invasion, mapping and cell choice.

    PubMed

    Holt, C E; Harris, W A

    1998-02-01

    Recent research has shown that changes in the concentration of particular molecules lead axons to invade their target, and that concentration changes in other molecules at the borders of the target prevent axons from leaving the target area. After invasion, topographic and lamina-specific cues guide axons to the correct location within the target field. At the level of a single cell or part of a cell, the evidence raises the possibility that axon targeting might be a combinatorial affair whereby specific axons compare the relative concentrations of several molecules on the surface of postsynaptic cells in order to choose a particular target. Both proteins and carbohydrates of various classes play major roles in these processes. PMID:9568397

  9. Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals

    PubMed Central

    Iyer, Shrivats M.; Deisseroth, Karl; Delp, Scott L.

    2013-01-01

    Optogenetic control of the peripheral nervous system (PNS) would enable novel studies of motor control, somatosensory transduction, and pain processing. Such control requires the development of methods to deliver opsins and light to targeted sub-populations of neurons within peripheral nerves. We report here methods to deliver opsins and light to targeted peripheral neurons and robust optogenetic modulation of motor neuron activity in freely moving, non-transgenic mammals. We show that intramuscular injection of adeno-associated virus serotype 6 enables expression of channelrhodopsin (ChR2) in motor neurons innervating the injected muscle. Illumination of nerves containing mixed populations of axons from these targeted neurons and from neurons innervating other muscles produces ChR2-mediated optogenetic activation restricted to the injected muscle. We demonstrate that an implanted optical nerve cuff is well-tolerated, delivers light to the sciatic nerve, and optically stimulates muscle in freely moving rats. These methods can be broadly applied to study PNS disorders and lay the groundwork for future therapeutic application of optogenetics. PMID:23991144

  10. Neural activity promotes long-distance, target-specific regeneration of adult retinal axons.

    PubMed

    Lim, Jung-Hwan A; Stafford, Benjamin K; Nguyen, Phong L; Lien, Brian V; Wang, Chen; Zukor, Katherine; He, Zhigang; Huberman, Andrew D

    2016-08-01

    Axons in the mammalian CNS fail to regenerate after injury. Here we show that if the activity of mouse retinal ganglion cells (RGCs) is increased by visual stimulation or using chemogenetics, their axons regenerate. We also show that if enhancement of neural activity is combined with elevation of the cell-growth-promoting pathway involving mammalian target of rapamycin (mTOR), RGC axons regenerate long distances and re-innervate the brain. Analysis of genetically labeled RGCs revealed that this regrowth can be target specific: RGC axons navigated back to their correct visual targets and avoided targets incorrect for their function. Moreover, these regenerated connections were successful in partially rescuing a subset of visual behaviors. Our findings indicate that combining neural activity with activation of mTOR can serve as powerful tool for enhancing axon regeneration, and they highlight the remarkable capacity of CNS neurons to re-establish accurate circuit connections in adulthood. PMID:27399843

  11. Parameter exploration of staircase-shape extracellular stimulation for targeted stimulation of myelinated axon.

    PubMed

    Ueno, Ayako; Karashima, Akihiro; Nakao, Mitsuyuki; Katayama, Norihiro

    2011-01-01

    Spatio-temporal dynamics of a mathematical model of myelinated axon in response to staircase-shape extracellular electrical stimulation, which was developed for selective nerve stimulation, is investigated by the computer simulation. It is shown that the response is classified into four types: subthreshold response, cathodic excitation, anodal block and anodal break excitation. Based on the simulation results, simple diagrams representing the response characteristics of the axon are constructed as functions of stimulation parameters and distance between the axon and electrode. The diagram would be useful for determining simulation parameters for dynamic targeted stimulation of myelinated axon. PMID:22254459

  12. Axon Targeting of Olfactory Receptor Neurons is Patterned by Coupled Hedgehog Signaling at Two Distinct Steps

    PubMed Central

    Chou, Ya-Hui; Zheng, Xiaoyan; Beachy, Philip A.; Luo, Liqun

    2010-01-01

    SUMMARY We present evidence for a novel, coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second-step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events. PMID:20850015

  13. Quantitative morphological comparison of axon-targeting strategies for gene therapies directed to the nigro-striatal projection.

    PubMed

    Padmanabhan, S; Kareva, T; Kholodilov, N; Burke, R E

    2014-02-01

    Cellular targeting of mRNAs and proteins to axons is essential for axon growth during development and is likely to be important for adult maintenance as well. Given the importance and potency of these axon-targeting motifs to the biology of axons, it seems possible that they can be used in the design of transgenes that are intended to enhance axon growth or maintenance, so as to improve potency and minimize off-target effects. To investigate this possibility, it is first essential to assess known motifs for their efficacy. We have therefore evaluated four axon-targeting motifs, using adeno-associated viral vector-mediated gene delivery in the nigro-striatal dopaminergic system, a projection that is predominantly affected in Parkinson's disease. We have tested two mRNA axonal zipcodes, the 3' untranslated region (UTR) of β-actin and 3' UTR of tau, and two axonal-targeting protein motifs, the palmitoylation signal sequence in GAP-43 and the last 15 amino acids in the amyloid precursor protein, to direct the expression of the fluorescent protein Tomato in axons. These sequences, fused to Tomato, were able to target its expression to dopaminergic axons. Based on quantification of Tomato-positive axons, and the density of striatal innervation, we conclude that the C-terminal of the amyloid precursor protein is the most effective axon-targeting motif. PMID:24305419

  14. Collagen XIXa1 is crucial for motor axon navigation at intermediate targets

    PubMed Central

    Hilario, Jona D.; Wang, Chunping; Beattie, Christine E.

    2010-01-01

    During development, motor axons navigate from the spinal cord to their muscle targets in the periphery using stereotyped pathways. These pathways are broken down into shorter segments by intermediate targets where axon growth cones are believed to coordinate guidance cues. In zebrafish stumpy mutants, embryonic development proceeds normally; however, as trunk motor axons stall at their intermediate targets, suggesting that Stumpy is needed specifically for motor axon growth cones to proceed past intermediate targets. Fine mapping and positional cloning revealed that stumpy was the zebrafish homolog of the atypical FACIT collagen collagenXIXa1 (colXIX). colXIX expression was observed in a temporal and spatial pattern, consistent with a role in motor axon guidance at intermediate targets. Knocking down zebrafish ColXIX phenocopied the stumpy phenotype and this morpholino phenotype could be rescued by adding back either mouse or zebrafish colXIX RNA. The stumpy phenotype was also partially rescued in mutants by first knocking down zebrafish ColXIX and adding back colXIX RNA, suggesting that the mutation is acting as a dominant negative. Together, these results demonstrate a novel function for a FACIT collagen in guiding vertebrate motor axons through intermediate targets. PMID:21098567

  15. A microfluidic device to investigate axon targeting by limited numbers of purified cortical projection neuron subtypes

    PubMed Central

    Tharin, Suzanne; Kothapalli, Chandrasekhar R.; Ozdinler, Pembe Hande; Pasquina, Lincoln; Chung, Seok; Varner, Johanna; DeValence, Sarra; Kamm, Roger; Macklis, Jeffrey D.

    2012-01-01

    While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth “choice” experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers. PMID:23034677

  16. Function and Mechanism of Axonal Targeting of Voltage-sensitive Potassium Channels

    PubMed Central

    Gu, Chen; Barry, Joshua

    2011-01-01

    Precise localization of various ion channels into proper subcellular compartments is crucial for neuronal excitability and synaptic transmission. Axonal K+ channels that are activated by depolarization of the membrane potential participate in the repolarizing phase of the action potential, and hence regulate action potential firing patterns, which encode output signals. Moreover, some of these channels can directly control neurotransmitter release at axonal terminals by constraining local membrane excitability and limiting Ca2+ influx. K+ channels differ not only in biophysical and pharmacological properties, but in expression and subcellular distribution as well. Importantly, proper targeting of channel proteins is a prerequisite for electrical and chemical functions of axons. In this review, we first highlight recent studies that demonstrate different roles of axonal K+ channels in the local regulation of axonal excitability. Next, we focus on research progress in identifying axonal targeting motifs and machinery of several different types of K+ channels present in axons. Regulation of K+ channel targeting and activity may underlie a novel form of neuronal plasticity. This research field can contribute to generating novel therapeutic strategies through manipulating neuronal excitability in treating neurological diseases, such as multiple sclerosis, neuropathic pain, and Alzheimer’s disease. PMID:21530607

  17. Axonal degeneration in multiple sclerosis: defining therapeutic targets by identifying the causes of pathology.

    PubMed

    Lee, Jae Young; Biemond, Melissa; Petratos, Steven

    2015-12-01

    Current therapeutics in multiple sclerosis (MS) target the putative inflammation and immune attack on CNS myelin. Despite their effectiveness in blunting the relapse rate in MS patients, such therapeutics do not prevent MS disease progression. Importantly, specific clinical dilemma arises through inability to predict MS progression and thereby therapeutically target axonal injury during MS, limiting permanent disability. The current review identifies immune and neurobiological principles that govern the sequelae of axonal degeneration during MS disease progression. Defining the specific disease arbiters, inflammatory and autoimmune, oligodendrocyte dystrophy and degenerative myelin, we discuss a basis for a molecular mechanism in axons that may be targeted therapeutically, in spatial and temporal manner to limit axonal degeneration and thereby halt progression of MS. PMID:26619755

  18. Poly(ADP-ribose) polymerase 1 is a novel target to promote axonal regeneration

    PubMed Central

    Brochier, Camille; Jones, James I.; Willis, Dianna E.; Langley, Brett

    2015-01-01

    Therapeutic options for the restoration of neurological functions after acute axonal injury are severely limited. In addition to limiting neuronal loss, effective treatments face the challenge of restoring axonal growth within an injury environment where inhibitory molecules from damaged myelin and activated astrocytes act as molecular and physical barriers. Overcoming these barriers to permit axon growth is critical for the development of any repair strategy in the central nervous system. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a previously unidentified and critical mediator of multiple growth-inhibitory signals. We show that exposure of neurons to growth-limiting molecules—such as myelin-derived Nogo and myelin-associated glycoprotein—or reactive astrocyte-produced chondroitin sulfate proteoglycans activates PARP1, resulting in the accumulation of poly(ADP-ribose) in the cell body and axon and limited axonal growth. Accordingly, we find that pharmacological inhibition or genetic loss of PARP1 markedly facilitates axon regeneration over nonpermissive substrates. Together, our findings provide critical insights into the molecular mechanisms of axon growth inhibition and identify PARP1 as an effective target to promote axon regeneration. PMID:26598704

  19. Interstitial branch formation within the red nucleus by deep cerebellar nuclei-derived commissural axons during target recognition.

    PubMed

    Hara, Satoshi; Kaneyama, Takeshi; Inamata, Yasuyuki; Onodera, Ryota; Shirasaki, Ryuichi

    2016-04-01

    Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing. PMID:26356789

  20. Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABAB Receptors

    PubMed Central

    Valdés, Viviana; Valenzuela, José Ignacio; Salas, Daniela A.; Jaureguiberry-Bravo, Matías; Otero, Carolina; Thiede, Christina; Schmidt, Christoph F.; Couve, Andrés

    2012-01-01

    In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABABRs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABABRs are obligatory heteromers constituted by two subunits, GABABR1 and GABABR2. GABABR1a and GABABR1b are the most abundant subunit variants. GABABR1b is located in the somatodendritic domain whereas GABABR1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABABR1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABABRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABABR1a operate in a pre-Golgi compartment. In the axon GABABR1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABABR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABABRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors. PMID:22952914

  1. Deletions within its subcellular targeting domain enhance the axon protective capacity of Nmnat2 in vivo

    PubMed Central

    Milde, Stefan; Fox, A. Nicole; Freeman, Marc R.; Coleman, Michael P.

    2013-01-01

    The NAD-synthesising enzyme Nmnat2 is a critical survival factor for axons in vitro and in vivo. We recently reported that loss of axonal transport vesicle association through mutations in its isoform-specific targeting and interaction domain (ISTID) reduces Nmnat2 ubiquitination, prolongs its half-life and boosts its axon protective capacity in primary culture neurons. Here, we report evidence for a role of ISTID sequences in tuning Nmnat2 localisation, stability and protective capacity in vivo. Deletion of central ISTID sequences abolishes vesicle association and increases protein stability of fluorescently tagged, transgenic Nmnat2 in mouse peripheral axons in vivo. Overexpression of fluorescently tagged Nmnat2 significantly delays Wallerian degeneration in these mice. Furthermore, while mammalian Nmnat2 is unable to protect transected Drosophila olfactory receptor neuron axons in vivo, mutant Nmnat2s lacking ISTID regions substantially delay Wallerian degeneration. Together, our results establish Nmnat2 localisation and turnover as a valuable target for modulating axon degeneration in vivo. PMID:23995269

  2. Response of olfactory axons to loss of synaptic targets in the adult mouse

    PubMed Central

    Ardiles, Yona; de la Puente, Rafael; Toledo, Rafael; Isgor, Ceylan; Guthrie, Kathleen

    2007-01-01

    Glomerular convergence has been proposed to rely on interactions between like olfactory axons, however topographic targeting is influenced by guidance molecules encountered in the olfactory bulb. Disruption of these cues during development misdirects sensory axons, however little is known about the role of bulb-derived signals in later life, as new axons arise during turnover of the olfactory sensory neuron (OSN) population. To evaluate the contribution of bulb neurons in maintaining topographic projections in adults, we ablated them with N-methyl-D-aspartate (NMDA) in P2-IRES-tauLacZ mice and examined how sensory axons responded to loss of their postsynaptic partners. NMDA lesion eliminated bulb neurons without damage to sensory axons or olfactory ensheathing glia. P2 axons contained within glomeruli at the time of lesion maintained convergence at these locations; there was no evidence of compensatory growth into the remnant tissue. Delayed apoptosis of OSNs in the target-deprived epithelium led to declines in P2 neuron number as well as the gradual atrophy, and in some cases complete loss, of P2 glomeruli in lesioned bulbs by three weeks. Increased cell proliferation in the epithelium partially restored the OSN population, and by eight weeks, new P2 axons distributed within diverse locations in the bulb remnant and within the anterior olfactory nucleus. Prior studies have suggested that initial development of olfactory topography does not rely on synapse formation with target neurons, however the present data demonstrate that continued maintenance of the sensory map requires the presence of sufficient numbers and/or types of available bulbar synaptic targets. PMID:17674970

  3. Precise lamination of retinal axons generates multiple parallel input pathways in the tectum

    PubMed Central

    Robles, Estuardo; Filosa, Alessandro; Baier, Herwig

    2013-01-01

    The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We have utilized Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum. PMID:23486973

  4. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    PubMed

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. PMID:25652545

  5. Neurofascin as a novel target for autoantibody-mediated axonal injury

    PubMed Central

    Mathey, Emily K.; Derfuss, Tobias; Storch, Maria K.; Williams, Kieran R.; Hales, Kimberly; Woolley, David R.; Al-Hayani, Abdulmonem; Davies, Stephen N.; Rasband, Matthew N.; Olsson, Tomas; Moldenhauer, Anja; Velhin, Sviataslau; Hohlfeld, Reinhard; Meinl, Edgar; Linington, Christopher

    2007-01-01

    Axonal injury is considered the major cause of disability in patients with multiple sclerosis (MS), but the underlying effector mechanisms are poorly understood. Starting with a proteomics-based approach, we identified neurofascin-specific autoantibodies in patients with MS. These autoantibodies recognize the native form of the extracellular domains of both neurofascin 186 (NF186), a neuronal protein concentrated in myelinated fibers at nodes of Ranvier, and NF155, the oligodendrocyte-specific isoform of neurofascin. Our in vitro studies with hippocampal slice cultures indicate that neurofascin antibodies inhibit axonal conduction in a complement-dependent manner. To evaluate whether circulating antineurofascin antibodies mediate a pathogenic effect in vivo, we cotransferred these antibodies with myelin oligodendrocyte glycoprotein–specific encephalitogenic T cells to mimic the inflammatory pathology of MS and breach the blood–brain barrier. In this animal model, antibodies to neurofascin selectively targeted nodes of Ranvier, resulting in deposition of complement, axonal injury, and disease exacerbation. Collectively, these results identify a novel mechanism of immune-mediated axonal injury that can contribute to axonal pathology in MS. PMID:17846150

  6. Pre-target sorting of retino-collicular axons in the mouse

    PubMed Central

    Plas, Daniel T.; Lopez, Joshua E.; Crair, Michael C.

    2008-01-01

    The map of the retina onto the optic tectum is a highly conserved feature of the vertebrate visual system and the mechanism by which this mapping is accomplished during development is a long-standing problem of neurobiology. The early suggestion by Roger Sperry that the map is formed through interactions between retinal ganglion cell axons and target cells within the tectum has gained significant experimental support and wide spread acceptance. Nonetheless, reports in a variety of species indicate that some aspects of retinotopic order exist within the optic tract, leading to the suggestion that this ‘pre-ordering’ of retinal axons may play a role in the formation of the mature tectal map. A satisfactory account of pre-target order must provide the mechanism by which such axon order develops. Insofar as this mechanism must ultimately be genetically determined, the mouse suggests itself as the natural species in which to pursue these studies. Quantitative and repeatable methods are required to asses the contribution of candidate genes in mouse models. For these reasons, we have undertaken a quantitative study of the degree of retinotopic order within the optic tract and nerve of wild type mice both before and after the development of the retinotectal map. Our methods are based on tract-tracing using lipophilic dyes and our results indicate that there is a reestablishment of dorsoventral but not nasotemporal retinal order when the axons pass through the chiasm, and that this order is maintained throughout the subsequent tract. Furthermore, this dorsoventral retinotopic order is well-established by the day after birth, long before the final target zone is discernible within the tectum. We conclude that pretarget sorting of axons according to origin along the dorsoventral axis of the retina is both spatially and chronologically appropriate to contribute to the formation of the retinotectal map, and suggest that these methods be used to search for the molecular

  7. Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase

    SciTech Connect

    Read, David J.; Li Yong; Chao, Moses V.; Cavanagh, John B.; Glynn, Paul

    2010-05-15

    Single doses of organophosphorus compounds (OP) which covalently inhibit neuropathy target esterase (NTE) can induce lower-limb paralysis and distal damage in long nerve axons. Clinical signs of neuropathy are evident 3 weeks post-OP dose in humans, cats and chickens. By contrast, clinical neuropathy in mice following acute dosing with OPs or any other toxic compound has never been reported. Moreover, dosing mice with ethyloctylphosphonofluoridate (EOPF) - an extremely potent NTE inhibitor - causes a different (subacute) neurotoxicity with brain oedema. These observations have raised the possibility that mice are intrinsically resistant to neuropathies induced by acute toxic insult, but may incur brain oedema, rather than distal axonal damage, when NTE is inactivated. Here we provide the first report that hind-limb dysfunction and extensive axonal damage can occur in mice 3 weeks after acute dosing with a toxic compound, bromophenylacetylurea. Three weeks after acutely dosing mice with neuropathic OPs no clinical signs were observed, but distal lesions were present in the longest spinal sensory axons. Similar lesions were evident in undosed nestin-cre:NTEfl/fl mice in which NTE had been genetically-deleted from neural tissue. The extent of OP-induced axonal damage in mice was related to the duration of NTE inactivation and, as reported in chickens, was promoted by post-dosing with phenylmethanesulfonylfluoride. However, phenyldipentylphosphinate, another promoting compound in chickens, itself induced in mice lesions different from the neuropathic OP type. Finally, EOPF induced subacute neurotoxicity with brain oedema in both wild-type and nestin-cre:NTEfl/fl mice indicating that the molecular target for this effect is not neural NTE.

  8. The SUMO Protease Verloren Regulates Dendrite and Axon Targeting in Olfactory Projection Neurons

    PubMed Central

    Berdnik, Daniela; Favaloro, Vincenzo; Luo, Liqun

    2012-01-01

    Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and de-conjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO de-conjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival. PMID:22699913

  9. The SUMO protease Verloren regulates dendrite and axon targeting in olfactory projection neurons.

    PubMed

    Berdnik, Daniela; Favaloro, Vincenzo; Luo, Liqun

    2012-06-13

    Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and deconjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO deconjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival. PMID:22699913

  10. Ephrin-B3 coordinates timed axon targeting and amygdala spinogenesis for innate fear behaviour

    PubMed Central

    Zhu, Xiao-Na; Liu, Xian-Dong; Sun, Suya; Zhuang, Hanyi; Yang, Jing-Yu; Henkemeyer, Mark; Xu, Nan-Jie

    2016-01-01

    Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period. PMID:27008987

  11. Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve

    PubMed Central

    Phay, Monichan; Kim, Hak Hee; Yoo, Soonmoon

    2015-01-01

    Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neuro-degeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. PMID:26331719

  12. Contactin-4 mediates axon-target specificity and functional development of the accessory optic system.

    PubMed

    Osterhout, Jessica A; Stafford, Benjamin K; Nguyen, Phong L; Yoshihara, Yoshihiro; Huberman, Andrew D

    2015-05-20

    The mammalian eye-to-brain pathway includes more than 20 parallel circuits, each consisting of precise long-range connections between specific sets of retinal ganglion cells (RGCs) and target structures in the brain. The mechanisms that drive assembly of these parallel connections and the functional implications of their specificity remain unresolved. Here we show that in the absence of contactin 4 (CNTN4) or one of its binding partners, amyloid precursor protein (APP), a subset of direction-selective RGCs fail to target the nucleus of the optic tract (NOT)--the accessory optic system (AOS) target controlling horizontal image stabilization. Conversely, ectopic expression of CNTN4 biases RGCs to arborize in the NOT, and that process also requires APP. Our data reveal critical and novel roles for CNTN4/APP in promoting target-specific axon arborization, and they highlight the importance of this process for functional development of a behaviorally relevant parallel visual pathway. PMID:25959733

  13. Active mechanistic target of rapamycin plays an ancillary rather than essential role in zebrafish CNS axon regeneration

    PubMed Central

    Diekmann, Heike; Kalbhen, Pascal; Fischer, Dietmar

    2015-01-01

    The developmental decrease of the intrinsic regenerative ability of the mammalian central nervous system (CNS) is associated with reduced activity of mechanistic target of rapamycin (mTOR) in mature neurons such as retinal ganglion cells (RGCs). While mTOR activity is further decreased upon axonal injury, maintenance of its pre-injury level, for instance by genetic deletion of the phosphatase and tensin homolog (PTEN), markedly promotes axon regeneration in mammals. The current study now addressed the question whether active mTOR might generally play a central role in axon regeneration by analyzing its requirement in regeneration-competent zebrafish. Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals. Hardly any activity was detected in naïve RGCs, whereas it was markedly increased upon axotomy in vivo as well as in dissociated cell cultures. After a short burst, mTOR activity was quickly attenuated, which is contrary to the requirements for axon regeneration in mammals. Surprisingly, mTOR activity was not essential for axonal growth per se, but correlated with cytokine- and PTEN inhibitor-induced neurite extension in vitro. Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury. Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species. PMID:26217179

  14. Attractant and repellent cues cooperate in guiding a subset of olfactory sensory axons to a well-defined protoglomerular target.

    PubMed

    Taku, Alemji A; Marcaccio, Christina L; Ye, Wenda; Krause, Gregory J; Raper, Jonathan A

    2016-01-01

    Olfactory sensory axons target well-defined intermediate targets in the zebrafish olfactory bulb called protoglomeruli well before they form odorant receptor-specific glomeruli. A subset of olfactory sensory neurons are labeled by expression of the or111-7:IRES:GAL4 transgene whose axons terminate in the central zone (CZ) protoglomerulus. Previous work has shown that some of these axons misproject to the more dorsal and anterior dorsal zone (DZ) protoglomerulus in the absence of Netrin 1/Dcc signaling. In search of additional cues that guide these axons to the CZ, we found that Semaphorin 3D (Sema3D) is expressed in the anterior bulb and acts as a repellent that pushes them towards the CZ. Further analysis indicates that Sema3D signaling is mediated through Nrp1a, while Nrp2b also promotes CZ targeting but in a Sema3D-independent manner. nrp1a, nrp2b and dcc transcripts are detected in or111-7 transgene-expressing neurons early in development and both Nrp1a and Dcc act cell-autonomously in sensory neurons to promote accurate targeting to the CZ. dcc and nrp1a double mutants have significantly more DZ misprojections than either single mutant, suggesting that the two signaling systems act independently and in parallel to direct a specific subset of sensory axons to their initial protoglomerular target. PMID:26732841

  15. Liprin-alpha has LAR-independent functions in R7 photoreceptor axon targeting.

    PubMed

    Hofmeyer, Kerstin; Maurel-Zaffran, Corinne; Sink, Helen; Treisman, Jessica E

    2006-08-01

    In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. Here we identify a null mutation in the Liprin-alpha gene based on a similar R7 projection defect. Liprin-alpha physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-alpha nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-alpha proteins have been reported to control the localization of LAR, we find that LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-alpha. In addition, Liprin-alpha overexpression or loss of function can affect R7 targeting in the complete absence of LAR. We conclude that Liprin-alpha does not simply act by regulating LAR localization but also has LAR-independent functions. PMID:16864797

  16. Therapeutic Targeting of the Axonal and Microvascular Change Associated with Repetitive Mild Traumatic Brain Injury

    PubMed Central

    Miyauchi, Takashi; Wei, Enoch P.

    2013-01-01

    Abstract Recent interest in mild traumatic brain injury (mTBI) has increased the recognition that repetitive mTBI occurring within the sports and military settings can exacerbate the adverse consequences of the initial injury. While multiple studies have recently reported the pathological, metabolic, and functional changes associated with repetitive mTBI, no consideration has been given to the development of therapeutic approaches to attenuate these abnormalities. In this study, we used the model of repetitive impact acceleration insult previously reported by our laboratory to cause no initial structural and functional changes, yet evoke dramatic change following second insult of the same intensity. Using this model, we employed established neuroprotective agents including FK506 and hypothermia that were administered 1 h after the second insult. Following either therapeutic intervention, changes of cerebral vascular reactivity to acetylcholine were assessed through a cranial window. Following the completion of the vascular studies, the animals were prepared to access the numbers of amyloid precursor protein (APP) positive axons, a marker of axonal damage. Following repetitive injury, cerebral vascular reactivity was dramatically preserved by either therapeutic intervention or the combination thereof compared to control group in which no intervention was employed. Similarly, APP density was significantly lower in the therapeutic intervention group compared in controls. Although the individual use of FK506 or hypothermia exerted significant protection, no additive benefit was found when both therapies were combined. In sum, the current study demonstrates that the exacerbated pathophysiological changes associated with repetitive mTBI can be therapeutically targeted. PMID:23796228

  17. Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration*

    PubMed Central

    Cho, Yongcheol; Di Liberto, Valentina; Carlin, Dan; Abe, Namiko; Li, Kathy H.; Burlingame, Alma L.; Guan, Shenheng; Michaelevski, Izhak; Cavalli, Valeria

    2014-01-01

    Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration. PMID:24737317

  18. Toll Receptors Instruct Axon and Dendrite Targeting and Participate in Synaptic Partner Matching in a Drosophila Olfactory Circuit

    PubMed Central

    Ward, Alex; Hong, Weizhe; Favaloro, Vincenzo; Luo, Liqun

    2015-01-01

    SUMMARY Our understanding of the mechanisms that establish wiring specificity of complex neural circuits is far from complete. During Drosophila olfactory circuit assembly, axons of 50 olfactory receptor neuron (ORN) classes and dendrites of 50 projection neuron (PN) classes precisely target to 50 discrete glomeruli, forming parallel information-processing pathways. Here we show that Toll-6 and Toll-7, members of the Toll receptor family best known for functions in innate immunity and embryonic patterning, cell-autonomously instruct the targeting of specific classes of PN dendrites and ORN axons, respectively. The canonical ligands and downstream partners of Toll receptors in embryonic patterning and innate immunity are not required for the function of Toll-6/Toll-7 in wiring specificity, nor are their cytoplasmic domains. Interestingly, both Toll-6 and Toll-7 participate in synaptic partner matching between ORN axons and PN dendrites. Our investigations reveal that olfactory circuit assembly involves dynamic and long-range interactions between PN dendrites and ORN axons. PMID:25741726

  19. Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit.

    PubMed

    Ward, Alex; Hong, Weizhe; Favaloro, Vincenzo; Luo, Liqun

    2015-03-01

    Our understanding of the mechanisms that establish wiring specificity of complex neural circuits is far from complete. During Drosophila olfactory circuit assembly, axons of 50 olfactory receptor neuron (ORN) classes and dendrites of 50 projection neuron (PN) classes precisely target to 50 discrete glomeruli, forming parallel information-processing pathways. Here we show that Toll-6 and Toll-7, members of the Toll receptor family best known for functions in innate immunity and embryonic patterning, cell autonomously instruct the targeting of specific classes of PN dendrites and ORN axons, respectively. The canonical ligands and downstream partners of Toll receptors in embryonic patterning and innate immunity are not required for the function of Toll-6/Toll-7 in wiring specificity, nor are their cytoplasmic domains. Interestingly, both Toll-6 and Toll-7 participate in synaptic partner matching between ORN axons and PN dendrites. Our investigations reveal that olfactory circuit assembly involves dynamic and long-range interactions between PN dendrites and ORN axons. PMID:25741726

  20. Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

    PubMed Central

    Lyman, Mathew G.; Curanovic, Dusica; Enquist, Lynn W.

    2008-01-01

    The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system. PMID:18483549

  1. Do growth-stimulated retinal ganglion cell axons find their central targets after optic nerve injury? New insights by three-dimensional imaging of the visual pathway.

    PubMed

    Diekmann, Heike; Leibinger, Marco; Fischer, Dietmar

    2013-10-01

    Retinal ganglion cells (RGCs) do not normally regenerate injured axons. However, several strategies to transform RGCs into a potent regenerative state have been developed in recent years. Intravitreal CNTF application combined with conditional PTEN and SOCS3 deletion or zymosan-induced inflammatory stimulation together with cAMP analogue injection and PTEN-deletion in RGCs induce long-distance regeneration into the optic nerve of adult mice. A recent paper by the Benowitz group (de Lima et al.) claimed that the latter treatment enables full-length regeneration, with axons correctly navigating to their central target zones and partial recovery of visual behaviors. To gain a more detailed view of the extent and the trajectories of regenerating axons, Luo et al. applied a tissue clearing method and fluorescent microscopy to allow the tracing of naïve and regenerating RGC axons in whole ON and all the way to their brain targets. Using this approach, the authors found comparable axon regeneration in the optic nerve after both above-mentioned experimental treatments. Regeneration was accompanied by prevalent aberrant axon growth in the optic nerve and significant axonal misguidance at the optic chiasm. Less than 120 axons per animal reached the optic chiasm and only few entered the correct optic tract. Importantly, no axons reached visual targets in the olivary pretectal nucleus, the lateral geniculate nucleus or the superior colliculus, thereby contradicting and challenging previous claims by the Benowitz group. The data provided by Luo et al. rather suggest that potent stimulation of axonal growth per se is insufficient to achieve functional recovery and underscore the need to investigate regeneration-relevant axon guidance mechanisms in the mature visual system. PMID:23816572

  2. Sim1 and Sim2 are required for the correct targeting of mammillary body axons.

    PubMed

    Marion, Jean-François; Yang, Chun; Caqueret, Aurore; Boucher, Francine; Michaud, Jacques L

    2005-12-01

    The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo. PMID:16291793

  3. Anosmin-1a is required for fasciculation and terminal targeting of olfactory sensory neuron axons in the zebrafish olfactory system.

    PubMed

    Yanicostas, Constantin; Herbomel, Eric; Dipietromaria, Aurélie; Soussi-Yanicostas, Nadia

    2009-11-27

    The KAL-1 gene underlies the X-linked form of Kallmann syndrome (KS), a neurological disorder that impairs the development of the olfactory and GnRH systems. KAL-1 encodes anosmin-1, a cell matrix protein that shows cell adhesion, neurite outgrowth, and axon-guidance and -branching activities. We used zebrafish embryos as model to better understand the role of this protein during olfactory system (OS) development. First, we detected the protein in olfactory sensory neurons from 22 h post-fertilization (hpf) onward, i.e. prior their pioneer axons reached presumptive olfactory bulbs (OBs). We found that anosmin-1a depletion impaired the fasciculation of olfactory axons and their terminal targeting within OBs. Last, we showed that kal1a inactivation induced a severe decrease in the number of GABAergic and dopaminergic OB neurons. Though the phenotypes induced following anosmin-1a depletion in zebrafish embryos did not match precisely the defects observed in KS patients, our results provide the first demonstration of a direct requirement for anosmin-1 in OS development in vertebrates and stress the role of OB innervation on OB neuron differentiation. PMID:19464344

  4. Orthodenticle Is Required for the Expression of Principal Recognition Molecules That Control Axon Targeting in the Drosophila Retina.

    PubMed

    Mencarelli, Chiara; Pichaud, Franck

    2015-06-01

    Parallel processing of neuronal inputs relies on assembling neural circuits into distinct synaptic-columns and layers. This is orchestrated by matching recognition molecules between afferent growth cones and target areas. Controlling the expression of these molecules during development is crucial but not well understood. The developing Drosophila visual system is a powerful genetic model for addressing this question. In this model system, the achromatic R1-6 photoreceptors project their axons in the lamina while the R7 and R8 photoreceptors, which are involved in colour detection, project their axons to two distinct synaptic-layers in the medulla. Here we show that the conserved homeodomain transcription factor Orthodenticle (Otd), which in the eye is a main regulator of rhodopsin expression, is also required for R1-6 photoreceptor synaptic-column specific innervation of the lamina. Our data indicate that otd function in these photoreceptors is largely mediated by the recognition molecules flamingo (fmi) and golden goal (gogo). In addition, we find that otd regulates synaptic-layer targeting of R8. We demonstrate that during this process, otd and the R8-specific transcription factor senseless/Gfi1 (sens) function as independent transcriptional inputs that are required for the expression of fmi, gogo and the adhesion molecule capricious (caps), which govern R8 synaptic-layer targeting. Our work therefore demonstrates that otd is a main component of the gene regulatory network that regulates synaptic-column and layer targeting in the fly visual system. PMID:26114289

  5. Functional and structural characterization of axonal opioid receptors as targets for analgesia

    PubMed Central

    Mambretti, Egle M; Kistner, Katrin; Mayer, Stefanie; Massotte, Dominique; Kieffer, Brigitte L; Hoffmann, Carsten; Reeh, Peter W; Brack, Alexander; Asan, Esther

    2016-01-01

    Background Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. Results Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl

  6. The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

    PubMed Central

    Conte, Ivan; Merella, Stefania; Garcia-Manteiga, Jose Manuel; Migliore, Chiara; Lazarevic, Dejan; Carrella, Sabrina; Marco-Ferreres, Raquel; Avellino, Raffaella; Davidson, Nathan Paul; Emmett, Warren; Sanges, Remo; Bockett, Nicholas; Van Heel, David; Meroni, Germana; Bovolenta, Paola; Stupka, Elia; Banfi, Sandro

    2014-01-01

    Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. PMID:24895435

  7. MiR-133b ameliorates axon degeneration induced by MPP(+) via targeting RhoA.

    PubMed

    Niu, M; Xu, R; Wang, J; Hou, B; Xie, A

    2016-06-14

    Increasing evidence suggests that microRNAs (miRs) play a significant role in the pathogenesis of Parkinson's disease (PD). MiR-133b, which is significantly decreased in the PD midbrain, has recently been shown to promote neurite outgrowth and enhance neural functional recovery. However, the role of miR-133b in PD has not been clearly established. Here, using a well-established PD model culture based on the neurotoxin 1-methyl-4-phenyl-pyridinium (MPP(+)), we demonstrated that miR-133b could promote axon outgrowth in dopaminergic neurons (DNs) and ameliorated MPP(+)-induced axon degeneration. Additional experiments suggested that the mechanisms of this miR-133b-mediated effect might rely on RhoA inhibition. We demonstrated that RhoA, an inhibitor of axonal growth, was increased in DNs under MPP(+) treatment, and this increase could be attenuated by miR-133b overexpression. Moreover, we demonstrated that the induced expression of miR-133b could inhibit α-synuclein, which is critically involved in the pathological process of PD. Furthermore, we found that overexpression of miR-133b abrogated the MPP(+)-induced decrease in the Bcl-2/Bax ratio and upregulated phosphorylated Akt (p-Akt), which is a pro-survival kinase. Together these findings reveal novel roles for miR-133b in the pathogenesis of PD and provide new therapeutic avenues for the treatment of the disease. PMID:27012608

  8. Viral vector-based improvement of optic nerve regeneration: characterization of individual axons' growth patterns and synaptogenesis in a visual target.

    PubMed

    Yungher, B J; Luo, X; Salgueiro, Y; Blackmore, M G; Park, K K

    2015-10-01

    Lack of axon growth ability in the central nervous system poses a major barrier to achieving functional connectivity after injury. Thus, a non-transgenic regenerative approach to reinnervating targets has important implications in clinical and research settings. Previous studies using knockout (KO) mice have demonstrated long-distance axon regeneration. Using an optic nerve injury model, here we evaluate the efficacy of viral, RNA interference (RNAi) and pharmacological approaches that target the phosphatase and tensin homolog (PTEN) and signal transducer and activator of transcription-3 pathways to improve long-distance axon regeneration in wild-type mice. Our data show that adeno-associated virus (AAV) expressing short hairpin RNA (shRNA) against PTEN (shPTEN) enhances retinal ganglion cell axon regeneration after crush injury. However, compared with the previous data in PTEN KO mice, AAV-shRNA results in a lesser degree of regeneration, likely due to incomplete gene silencing inherent to RNAi. In comparison, an extensive enhancement in regeneration is seen when AAV-shPTEN is coupled to AAV encoding ciliary neurotrophic factor (CNTF) and to a cyclic adenosine monophosphate (cAMP) analog, allowing axons to travel long distances and reach their target. We apply whole-tissue imaging that facilitates three-dimensional visualization of single regenerating axons and document heterogeneous terminal patterns in the targets. This shows that some axonal populations generate extensive arbors and make synapses with the target neurons. Collectively, we show a combinatorial viral RNAi and pharmacological strategy that improves long-distance regeneration in wild-type animals and provide single fiber projection data that indicates a degree of preservation of target recognition. PMID:26005861

  9. Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis

    PubMed Central

    Pinatel, Delphine; Hivert, Bruno; Boucraut, José; Saint-Martin, Margaux; Rogemond, Véronique; Zoupi, Lida; Karagogeos, Domna; Honnorat, Jérôme; Faivre-Sarrailh, Catherine

    2015-01-01

    Contactin-associated protein-like 2 (Caspr2), also known as CNTNAP2, is a cell adhesion molecule that clusters voltage-gated potassium channels (Kv1.1/1.2) at the juxtaparanodes of myelinated axons and may regulate axonal excitability. As a component of the Kv1 complex, Caspr2 has been identified as a target in neuromyotonia and Morvan syndrome, but also in some cases of autoimmune limbic encephalitis (LE). How anti-Caspr2 autoimmunity is linked with the central neurological symptoms is still elusive. In the present study, using anti-Caspr2 antibodies from seven patients affected by pure LE, we determined that IgGs in the cerebrospinal fluid of four out seven patients were selectively directed against the N-terminal Discoïdin and LamininG1 modules of Caspr2. Using live immunolabeling of cultured hippocampal neurons, we determined that serum IgGs in all patients strongly targeted inhibitory interneurons. Caspr2 was highly detected on GAD65-positive axons that are surrounding the cell bodies and at the VGAT-positive inhibitory presynaptic contacts. Functional assays indicated that LE autoantibodies may induce alteration of Gephyrin clusters at inhibitory synaptic contacts. Next, we generated a Caspr2-Fc chimera to reveal Caspr2 receptors on hippocampal neurons localized at the somato-dendritic compartment and post-synapse. Caspr2-Fc binding was strongly increased on TAG-1-transfected neurons and conversely, Caspr2-Fc did not bind hippocampal neurons from TAG-1-deficient mice. Our data indicate that Caspr2 may participate as a cell recognition molecule in the dynamics of inhibitory networks. This study provides new insight into the potential pathogenic effect of anti-Caspr2 autoantibodies in central hyperexcitability that may be related with perturbation of inhibitory interneuron activity. PMID:26217189

  10. Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration.

    PubMed

    Sellers, Drew L; Bergen, Jamie M; Johnson, Russell N; Back, Heidi; Ravits, John M; Horner, Philip J; Pun, Suzie H

    2016-03-01

    A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics. PMID:26888285

  11. Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration

    PubMed Central

    Sellers, Drew L.; Bergen, Jamie M.; Johnson, Russell N.; Back, Heidi; Ravits, John M.; Horner, Philip J.; Pun, Suzie H.

    2016-01-01

    A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics. PMID:26888285

  12. Phenotyping the Function of TRPV1-Expressing Sensory Neurons by Targeted Axonal Silencing

    PubMed Central

    Brenneis, Christian; Kistner, Katrin; Puopolo, Michelino; Segal, David; Roberson, David; Sisignano, Marco; Labocha, Sandra; Ferreirós, Nerea; Strominger, Amanda; Cobos, Enrique J.; Ghasemlou, Nader; Geisslinger, Gerd; Reeh, Peter W.; Bean, Bruce P.; Woolf, Clifford J.

    2013-01-01

    Specific somatosensations may be processed by different subsets of primary afferents. C-fibers expressing heat-sensitive TRPV1 channels are proposed, for example, to be heat but not mechanical pain detectors. To phenotype in rats the sensory function of TRPV1+ afferents, we rapidly and selectively silenced only their activity, by introducing the membrane-impermeant sodium channel blocker QX-314 into these axons via the TRPV1 channel pore. Using tandem mass spectrometry we show that upon activation with capsaicin, QX-314 selectively accumulates in the cytosol only of TRPV1-expressing cells, and not in control cells. Exposure to QX-314 and capsaicin induces in small DRG neurons a robust sodium current block within 30 s. In sciatic nerves, application of extracellular QX-314 with capsaicin persistently reduces C-fiber but not A-fiber compound action potentials and this effect does not occur in TRPV1−/− mice. Behavioral phenotyping after selectively silencing TRPV1+ sciatic nerve axons by perineural injections of QX-314 and capsaicin reveals deficits in heat and mechanical pressure but not pinprick or light touch perception. The response to intraplantar capsaicin is substantially reduced, as expected. During inflammation, silencing TRPV1+ axons abolishes heat, mechanical, and cold hyperalgesia but tactile and cold allodynia remain following peripheral nerve injury. These results indicate that TRPV1-expressing sensory neurons process particular thermal and mechanical somatosensations, and that the sensory channels activated by mechanical and cold stimuli to produce pain in naive/inflamed rats differ from those in animals after peripheral nerve injury. PMID:23283344

  13. Axonal Targeting of the Serotonin Transporter in Cultured Rat Dorsal Raphe Neurons Is Specified by SEC24C-Dependent Export from the Endoplasmic Reticulum

    PubMed Central

    Sucic, Sonja; Koban, Florian; Schüchner, Stefan; Ogris, Egon; Sitte, Harald H.; Freissmuth, Michael

    2015-01-01

    Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT–607RI608–AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C–D796V/D797N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein–SERT from axons to the somatodendritic region. (4) SERT–K610Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes. PMID:24790205

  14. Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

    PubMed

    Montgomery, Therese R; Steinkellner, Thomas; Sucic, Sonja; Koban, Florian; Schüchner, Stefan; Ogris, Egon; Sitte, Harald H; Freissmuth, Michael

    2014-04-30

    Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes. PMID:24790205

  15. Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

    PubMed Central

    Mohr, E; Morris, J F; Richter, D

    1995-01-01

    It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7753814

  16. The receptor protein tyrosine phosphatase LAR promotes R7 photoreceptor axon targeting by a phosphatase-independent signaling mechanism

    PubMed Central

    Hofmeyer, Kerstin; Treisman, Jessica E.

    2009-01-01

    Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors. PMID:19889974

  17. Time dependent integration of matrix metalloproteinases and their targeted substrates directs axonal sprouting and synaptogenesis following central nervous system injury.

    PubMed

    Phillips, Linda L; Chan, Julie L; Doperalski, Adele E; Reeves, Thomas M

    2014-02-15

    Over the past two decades, many investigators have reported how extracellular matrix molecules act to regulate neuroplasticity. The majority of these studies involve proteins which are targets of matrix metalloproteinases. Importantly, these enzyme/substrate interactions can regulate degenerative and regenerative phases of synaptic plasticity, directing axonal and dendritic reorganization after brain insult. The present review first summarizes literature support for the prominent role of matrix metalloproteinases during neuroregeneration, followed by a discussion of data contrasting adaptive and maladaptive neuroplasticity that reveals time-dependent metalloproteinase/substrate regulation of postinjury synaptic recovery. The potential for these enzymes to serve as therapeutic targets for enhanced neuroplasticity after brain injury is illustrated with experiments demonstrating that metalloproteinase inhibitors can alter adaptive and maladaptive outcome. Finally, the complexity of metalloproteinase role in reactive synaptogenesis is revealed in new studies showing how these enzymes interact with immune molecules to mediate cellular response in the local regenerative environment, and are regulated by novel binding partners in the brain extracellular matrix. Together, these different examples show the complexity with which metalloproteinases are integrated into the process of neuroregeneration, and point to a promising new angle for future studies exploring how to facilitate brain plasticity. PMID:25206824

  18. Axon-axon interactions in neuronal circuit assembly: lessons from olfactory map formation.

    PubMed

    Imai, Takeshi; Sakano, Hitoshi

    2011-11-01

    During the development of the nervous system, neurons often connect axons and dendrites over long distances, which are navigated by chemical cues. During the past few decades, studies on axon guidance have focused on chemical cues provided by the axonal target or intermediate target. However, recent studies have shed light on the roles and mechanisms underlying axon-axon interactions during neuronal circuit assembly. The roles of axon-axon interactions are best exemplified in recent studies on olfactory map formation in vertebrates. Pioneer-follower interaction is essential for the axonal pathfinding process. Pre-target axon sorting establishes the anterior-posterior map order. The temporal order of axonal projection is converted to dorsal-ventral topography with the aid of secreted molecules provided by early-arriving axons. An activity-dependent process to form a discrete map also depends on axon sorting. Thus, an emerging principle of olfactory map formation is the 'self-organisation' of axons rather than the 'lock and key' matching between axons and targets. In this review, we discuss how axon-axon interactions contribute to neuronal circuit assembly. PMID:22103421

  19. Semaphorin 3F Is a Bifunctional Guidance Cue for Dopaminergic Axons and Controls Their Fasciculation, Channeling, Rostral Growth, and Intracortical Targeting

    PubMed Central

    Kolk, Sharon M.; Gunput, Rou-Afza F.; Tran, Tracy S.; van den Heuvel, Dianne M. A.; Prasad, Asheeta A.; Hellemons, Anita J. C. G. M.; Adolfs, Youri; Ginty, David D.; Kolodkin, Alex L.; Burbach, J. Peter H.; Smidt, Marten P.; Pasterkamp, R. Jeroen

    2010-01-01

    Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F−/−, npn-2−/−, and npn-2−/−;TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease. PMID:19812329

  20. Increased Chondroitin Sulfate Proteoglycan Expression in Denervated Brainstem Targets Following Spinal Cord Injury Creates a Barrier to Axonal Regeneration Overcome by Chondroitinase ABC and Neurotrophin-3

    PubMed Central

    Massey, James M.; Amps, Jeremy; Viapiano, Mariano S.; Matthews, Russell. T.; Wagoner, Michelle R.; Whitaker, Christopher M.; Alilain, Warren; Yonkof, Alicia L.; Khalyfa, Abdelnaby; Cooper, Nigel G. F.; Silver, Jerry; Onifer, Stephen M.

    2008-01-01

    Increased chondroitin sulfate proteoglycan (CSPG) expression in the vicinity of a spinal cord injury (SCI) is a primary participant in axonal regeneration failure. However, the presence of similar increases of CSPG expression in denervated synaptic targets well away from the primary lesion and the subsequent impact on regenerating axons attempting to approach deafferented neurons has not been studied. Constitutively expressed CSPGs within the extracellular matrix and perineuronal nets of the adult rat dorsal column nuclei (DCN) were characterized using real-time PCR, western blot analysis and immunohistochemistry. We show for the first time that by 2 days and through 3 weeks following SCI, the levels of NG2, neurocan and brevican associated with reactive glia throughout the DCN were dramatically increased throughout the DCN despite being well beyond areas of trauma-induced blood brain barrier breakdown. Importantly, regenerating axons from adult sensory neurons microtransplanted 2 weeks following SCI between the injury site and the DCN were able to regenerate rapidly within white matter (as shown previously by Davies et al., 1999) but were unable to enter the denervated DCN. Application of chondroitinase ABC or neurotrophin 3-expressing lentivirus in the DCN partially overcame this inhibition. When the treatments were combined, entrance by regenerating axons into the DCN was significantly augmented. These results demonstrate both an additional challenge and potential treatment strategy for successful functional pathway reconstruction after SCI. PMID:17540369

  1. Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation

    PubMed Central

    Lewandowska, Marta K.; Radivojević, Miloš; Jäckel, David; Müller, Jan; Hierlemann, Andreas R.

    2016-01-01

    Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10–20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations. PMID:27013945

  2. Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation.

    PubMed

    Lewandowska, Marta K; Radivojević, Miloš; Jäckel, David; Müller, Jan; Hierlemann, Andreas R

    2016-01-01

    Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10-20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations. PMID:27013945

  3. Cellular strategies of axonal pathfinding.

    PubMed

    Raper, Jonathan; Mason, Carol

    2010-09-01

    Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment. PMID:20591992

  4. Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

    PubMed Central

    Akin, Elizabeth J.; Solé, Laura; Dib-Hajj, Sulayman D.; Waxman, Stephen G.; Tamkun, Michael M.

    2015-01-01

    During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS. PMID:25874799

  5. Electrophysiology of Axonal Constrictions

    NASA Astrophysics Data System (ADS)

    Johnson, Christopher; Jung, Peter; Brown, Anthony

    2013-03-01

    Axons of myelinated neurons are constricted at the nodes of Ranvier, where they are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions are generated by local regulation of the kinetics of neurofilaments the most important cytoskeletal elements of the axon. In this paper we discuss how this shape affects the electrophysiological function of the neuron. Specifically, although the nodes are short (about 1 μm) in comparison to the distance between nodes (hundreds of μm) they have a substantial influence on the conduction velocity of neurons. We show through computational modeling that nodal constrictions (all other features such as numbers of ion channels left constant) reduce the required fiber diameter for a given target conduction velocity by up to 50% in comparison to an unconstricted axon. We further show that the predicted optimal fiber morphologies closely match reported fiber morphologies. Supported by The National Science Foundation (IOS 1146789)

  6. Rules Ventral Prefrontal Cortical Axons Use to Reach Their Targets: Implications for Diffusion Tensor Imaging Tractography and Deep Brain Stimulation for Psychiatric Illness

    PubMed Central

    Lehman, Julia F.; Greenberg, Benjamin D.; McIntyre, Cameron C.; Rasmussen, Steve A.; Haber, Suzanne N.

    2011-01-01

    The ventral prefrontal cortex (vPFC) is involved in reinforcement-based learning and is associated with depression, obsessive-compulsive disorder, and addiction. Neuroimaging is increasingly used to develop models of vPFC connections, to examine white matter (WM) integrity, and to target surgical interventions, including deep brain stimulation. We used primate (Macaca nemestrina/Macaca fascicularis) tracing studies and 3D reconstructions of WM tracts to delineate the rules vPFC projections follow to reach their targets. vPFC efferent axons travel through the uncinate fasciculus, connecting different vPFC regions and linking different functional regions. The uncinate fasciculus also is a conduit for vPFC fibers to reach other cortical bundles. Fibers in the internal capsule are organized according to destination. Thalamic fibers from each vPFC region travel dorsal to their brainstem fibers. The results show regional differences in the trajectories of fibers from different vPFC areas. Overall, the medial/lateral vPFC position dictates the route that fibers take to enter major WM tracts, as well as the position within specific tracts: axons from medial vPFC regions travel ventral to those from more lateral areas. This arrangement, coupled with dorsal/ventral organization of thalamic/brainstem fibers through the internal capsule, results in a complex mingling of thalamic and brainstem axons from different vPFC areas. Together, these data provide the foundation for dividing vPFC WM bundles into functional components and for predicting what is likely to be carried at different points through each bundle. These results also help determine the specific connections that are likely to be captured at different neurosurgical targets. PMID:21753016

  7. Rules ventral prefrontal cortical axons use to reach their targets: implications for diffusion tensor imaging tractography and deep brain stimulation for psychiatric illness.

    PubMed

    Lehman, Julia F; Greenberg, Benjamin D; McIntyre, Cameron C; Rasmussen, Steve A; Haber, Suzanne N

    2011-07-13

    The ventral prefrontal cortex (vPFC) is involved in reinforcement-based learning and is associated with depression, obsessive-compulsive disorder, and addiction. Neuroimaging is increasingly used to develop models of vPFC connections, to examine white matter (WM) integrity, and to target surgical interventions, including deep brain stimulation. We used primate (Macaca nemestrina/Macaca fascicularis) tracing studies and 3D reconstructions of WM tracts to delineate the rules vPFC projections follow to reach their targets. vPFC efferent axons travel through the uncinate fasciculus, connecting different vPFC regions and linking different functional regions. The uncinate fasciculus also is a conduit for vPFC fibers to reach other cortical bundles. Fibers in the internal capsule are organized according to destination. Thalamic fibers from each vPFC region travel dorsal to their brainstem fibers. The results show regional differences in the trajectories of fibers from different vPFC areas. Overall, the medial/lateral vPFC position dictates the route that fibers take to enter major WM tracts, as well as the position within specific tracts: axons from medial vPFC regions travel ventral to those from more lateral areas. This arrangement, coupled with dorsal/ventral organization of thalamic/brainstem fibers through the internal capsule, results in a complex mingling of thalamic and brainstem axons from different vPFC areas. Together, these data provide the foundation for dividing vPFC WM bundles into functional components and for predicting what is likely to be carried at different points through each bundle. These results also help determine the specific connections that are likely to be captured at different neurosurgical targets. PMID:21753016

  8. Light and electron microscopic analysis of enkephalin-like immunoreactivity in the basolateral amygdala, including evidence for convergence of enkephalin-containing axon terminals and norepinephrine transporter-containing axon terminals onto common targets.

    PubMed

    Zhang, Jingyi; McDonald, Alexander J

    2016-04-01

    Modulatory interactions of opioids and norepinephrine (NE) in the anterior subdivision of the basolateral nucleus of the amygdala (BLa) are critical for the consolidation of memories of emotionally arousing experiences. Although there have been several studies of the noradrenergic system in the amygdalar basolateral nuclear complex (BLC), little is known about the chemical neuroanatomy of opioid systems in this region. To address this knowledge gap the present study first examined the distribution of met-enkephalin-like immunoreactivity (ENK-ir) in the BLC at the light microscopic level, and then utilized dual-labeling immunocytochemistry combined with electron microscopy to investigate the extent of convergence of NE and ENK terminals onto common structures in the BLa. Antibodies to ENK and the norepinephrine transporter (NET) were used in these studies. Light microscopic examination revealed that a subpopulation of small nonpyramidal neurons expressed ENK-ir in all nuclei of the BLC. In addition, the somata of some pyramidal cells exhibited light to moderate ENK-ir. ENK+ axon terminals were also observed. Ultrastructural analysis confined to the BLa revealed that most ENK+ axon terminals formed asymmetrical synapses that mainly contacted spines and shafts of thin dendrites. ENK+ terminals forming symmetrical synapses mainly contacted dendritic shafts. Approximately 20% of NET+ terminals contacted a structure that was also contacted by an ENK+ terminal and 6% of NET+ terminals contacted an ENK+ terminal. These findings suggest that ENK and NE terminals in the BLa may interact by targeting common dendrites and by direct interactions between the two types of terminals. PMID:26835559

  9. Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

    PubMed

    Wang, Jiajing; Hmadcha, Abdelkrim; Zakarian, Vaagn; Song, Fei; Loeb, Jeffrey A

    2015-09-01

    The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions. PMID:25913151

  10. Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons

    PubMed Central

    Neumann, Brent; Nguyen, Ken C. Q.; Hall, David H.; Ben-Yakar, Adela; Hilliard, Massimo A.

    2011-01-01

    Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In C. elegans following laser-induced axotomy, the axon still attached to the cell body is able to regrow and reconnect with its separated distal fragment. Here we show that reconnection of separated axon fragments during regeneration of C. elegans mechanosensory neurons occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re-establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. We also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re-growing axon recognizing its own separated distal fragment. Thus, efficient axonal regeneration can occur by selective reconnection and fusion of separated axonal fragments beyond an injury site, with restoration of the damaged neuronal tract. PMID:21416556

  11. Giant Axonal Neuropathy

    MedlinePlus

    ... Diversity Find People About NINDS NINDS Giant Axonal Neuropathy Information Page Table of Contents (click to jump ... done? Clinical Trials Organizations What is Giant Axonal Neuropathy? Giant axonal neuropathy (GAN) is a rare inherited ...

  12. Degeneration and regeneration of ganglion cell axons.

    PubMed

    Weise, J; Ankerhold, R; Bähr, M

    2000-01-15

    The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed. PMID:10649506

  13. Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development

    PubMed Central

    Ma, Jingqun; Brennan, Kaelan J.; D’Aloia, Mitch R.; Pascuzzi, Pete E.; Weake, Vikki M.

    2016-01-01

    The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia. PMID:27261002

  14. Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

    PubMed

    Ma, Jingqun; Brennan, Kaelan J; D'Aloia, Mitch R; Pascuzzi, Pete E; Weake, Vikki M

    2016-01-01

    The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia. PMID:27261002

  15. NRP1 and NRP2 cooperate to regulate gangliogenesis, axon guidance and target innervation in the sympathetic nervous system.

    PubMed

    Maden, Charlotte H; Gomes, John; Schwarz, Quenten; Davidson, Kathryn; Tinker, Andrew; Ruhrberg, Christiana

    2012-09-15

    The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A. PMID:22790009

  16. Beyond Columnar Organization: Cell Type- and Target Layer-Specific Principles of Horizontal Axon Projection Patterns in Rat Vibrissal Cortex

    PubMed Central

    Narayanan, Rajeevan T.; Egger, Robert; Johnson, Andrew S.; Mansvelder, Huibert D.; Sakmann, Bert; de Kock, Christiaan P.J.; Oberlaender, Marcel

    2015-01-01

    Vertical thalamocortical afferents give rise to the elementary functional units of sensory cortex, cortical columns. Principles that underlie communication between columns remain however unknown. Here we unravel these by reconstructing in vivo-labeled neurons from all excitatory cell types in the vibrissal part of rat primary somatosensory cortex (vS1). Integrating the morphologies into an exact 3D model of vS1 revealed that the majority of intracortical (IC) axons project far beyond the borders of the principal column. We defined the corresponding innervation volume as the IC-unit. Deconstructing this structural cortical unit into its cell type-specific components, we found asymmetric projections that innervate columns of either the same whisker row or arc, and which subdivide vS1 into 2 orthogonal [supra-]granular and infragranular strata. We show that such organization could be most effective for encoding multi whisker inputs. Communication between columns is thus organized by multiple highly specific horizontal projection patterns, rendering IC-units as the primary structural entities for processing complex sensory stimuli. PMID:25838038

  17. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function

    PubMed Central

    Kuhn, Peer-Hendrik; Colombo, Alessio Vittorio; Schusser, Benjamin; Dreymueller, Daniela; Wetzel, Sebastian; Schepers, Ute; Herber, Julia; Ludwig, Andreas; Kremmer, Elisabeth; Montag, Dirk; Müller, Ulrike; Schweizer, Michaela; Saftig, Paul; Bräse, Stefan; Lichtenthaler, Stefan F

    2016-01-01

    Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain. DOI: http://dx.doi.org/10.7554/eLife.12748.001 PMID:26802628

  18. Beyond Columnar Organization: Cell Type- and Target Layer-Specific Principles of Horizontal Axon Projection Patterns in Rat Vibrissal Cortex.

    PubMed

    Narayanan, Rajeevan T; Egger, Robert; Johnson, Andrew S; Mansvelder, Huibert D; Sakmann, Bert; de Kock, Christiaan P J; Oberlaender, Marcel

    2015-11-01

    Vertical thalamocortical afferents give rise to the elementary functional units of sensory cortex, cortical columns. Principles that underlie communication between columns remain however unknown. Here we unravel these by reconstructing in vivo-labeled neurons from all excitatory cell types in the vibrissal part of rat primary somatosensory cortex (vS1). Integrating the morphologies into an exact 3D model of vS1 revealed that the majority of intracortical (IC) axons project far beyond the borders of the principal column. We defined the corresponding innervation volume as the IC-unit. Deconstructing this structural cortical unit into its cell type-specific components, we found asymmetric projections that innervate columns of either the same whisker row or arc, and which subdivide vS1 into 2 orthogonal [supra-]granular and infragranular strata. We show that such organization could be most effective for encoding multi whisker inputs. Communication between columns is thus organized by multiple highly specific horizontal projection patterns, rendering IC-units as the primary structural entities for processing complex sensory stimuli. PMID:25838038

  19. IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories.

    PubMed

    Preitner, Nicolas; Quan, Jie; Li, Xinmin; Nielsen, Finn C; Flanagan, John G

    2016-08-01

    RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing mouse brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate development. PMID:27385015

  20. Targeted next-generation sequencing reveals further genetic heterogeneity in axonal Charcot–Marie–Tooth neuropathy and a mutation in HSPB1

    PubMed Central

    Ylikallio, Emil; Johari, Mridul; Konovalova, Svetlana; Moilanen, Jukka S; Kiuru-Enari, Sari; Auranen, Mari; Pajunen, Leila; Tyynismaa, Henna

    2014-01-01

    Charcot–Marie–Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype–phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches. PMID:23963299

  1. Calcium Release from Intra-Axonal Endoplasmic Reticulum Leads to Axon Degeneration through Mitochondrial Dysfunction

    PubMed Central

    Villegas, Rosario; Martinez, Nicolas W.; Lillo, Jorge; Pihan, Phillipe; Hernandez, Diego; Twiss, Jeffery L.

    2014-01-01

    Axonal degeneration represents an early pathological event in neurodegeneration, constituting an important target for neuroprotection. Regardless of the initial injury, which could be toxic, mechanical, metabolic, or genetic, degeneration of axons shares a common mechanism involving mitochondrial dysfunction and production of reactive oxygen species. Critical steps in this degenerative process are still unknown. Here we show that calcium release from the axonal endoplasmic reticulum (ER) through ryanodine and IP3 channels activates the mitochondrial permeability transition pore and contributes to axonal degeneration triggered by both mechanical and toxic insults in ex vivo and in vitro mouse and rat model systems. These data reveal a critical and early ER-dependent step during axonal degeneration, providing novel targets for axonal protection in neurodegenerative conditions. PMID:24849352

  2. Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

    PubMed Central

    Milde, Stefan; Gilley, Jonathan; Coleman, Michael P.

    2013-01-01

    Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, WldS. The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles. PMID:23610559

  3. Peripheral nerve axons contain machinery for co-translational secretion of axonally-generated proteins.

    PubMed

    Merianda, Tanuja; Twiss, Jeffery

    2013-08-01

    The axonal compartment of developing neurons and mature peripheral nervous system (PNS) neurons has the capacity to locally synthesize proteins. Axonally-synthesized proteins have been shown to facilitate axonal pathfinding and maintenance in developing central nervous system (CNS) and PNS neurons, and to facilitate the regeneration of mature PNS neurons. RNA-profiling studies of the axons of cultured neurons have shown a surprisingly complex population of mRNAs that encode proteins for a myriad of functions. Although classic-appearing rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (ER) and Golgi apparatus have not been documented in axons by ultrastructural studies, axonal RNA profiling studies show several membrane and secreted protein-encoding mRNAs whose translation products would need access to a localized secretory mechanism. We previously showed that the axons of cultured neurons contain functional equivalents of RER and Golgi apparatus. Here, we show that markers for the signal-recognition particle, RER, ER, and Golgi apparatus are present in PNS axons in vivo. Co-localization of these proteins mirrors that seen for cultured axons where locally-translated proteins are localized to the axoplasmic membrane. Moreover, nerve injury increases the levels and/or aggregation of these proteins, suggesting that the regenerating axon has an increased capacity for membrane targeting of locally synthesized proteins. PMID:23839054

  4. Sensory activity affects sensory axon development in C. elegans.

    PubMed

    Peckol, E L; Zallen, J A; Yarrow, J C; Bargmann, C I

    1999-05-01

    The simple nervous system of the nematode C. elegans consists of 302 neurons with highly reproducible morphologies, suggesting a hard-wired program of axon guidance. Surprisingly, we show here that sensory activity shapes sensory axon morphology in C. elegans. A class of mutants with deformed sensory cilia at their dendrite endings have extra axon branches, suggesting that sensory deprivation disrupts axon outgrowth. Mutations that alter calcium channels or membrane potential cause similar defects. Cell-specific perturbations of sensory activity can cause cell-autonomous changes in axon morphology. Although the sensory axons initially reach their targets in the embryo, the mutations that alter sensory activity cause extra axon growth late in development. Thus, perturbations of activity affect the maintenance of sensory axon morphology after an initial pattern of innervation is established. This system provides a genetically tractable model for identifying molecular mechanisms linking neuronal activity to nervous system structure. PMID:10101123

  5. Regulating Axonal Responses to Injury: The Intersection between Signaling Pathways Involved in Axon Myelination and The Inhibition of Axon Regeneration

    PubMed Central

    Rao, Sudheendra N. R.; Pearse, Damien D.

    2016-01-01

    Following spinal cord injury (SCI), a multitude of intrinsic and extrinsic factors adversely affect the gene programs that govern the expression of regeneration-associated genes (RAGs) and the production of a diversity of extracellular matrix molecules (ECM). Insufficient RAG expression in the injured neuron and the presence of inhibitory ECM at the lesion, leads to structural alterations in the axon that perturb the growth machinery, or form an extraneous barrier to axonal regeneration, respectively. Here, the role of myelin, both intact and debris, in antagonizing axon regeneration has been the focus of numerous investigations. These studies have employed antagonizing antibodies and knockout animals to examine how the growth cone of the re-growing axon responds to the presence of myelin and myelin-associated inhibitors (MAIs) within the lesion environment and caudal spinal cord. However, less attention has been placed on how the myelination of the axon after SCI, whether by endogenous glia or exogenously implanted glia, may alter axon regeneration. Here, we examine the intersection between intracellular signaling pathways in neurons and glia that are involved in axon myelination and axon growth, to provide greater insight into how interrogating this complex network of molecular interactions may lead to new therapeutics targeting SCI. PMID:27375427

  6. Regulation of Axonal Transport by Protein Kinases.

    PubMed

    Gibbs, Katherine L; Greensmith, Linda; Schiavo, Giampietro

    2015-10-01

    The intracellular transport of organelles, proteins, lipids, and RNA along the axon is essential for neuronal function and survival. This process, called axonal transport, is mediated by two classes of ATP-dependent motors, kinesins, and cytoplasmic dynein, which carry their cargoes along microtubule tracks. Protein kinases regulate axonal transport through direct phosphorylation of motors, adapter proteins, and cargoes, and indirectly through modification of the microtubule network. The misregulation of axonal transport by protein kinases has been implicated in the pathogenesis of several nervous system disorders. Here, we review the role of protein kinases acting directly on axonal transport and discuss how their deregulation affects neuronal function, paving the way for the exploitation of these enzymes as novel drug targets. PMID:26410600

  7. Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity.

    PubMed

    Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M; Shen, Kang

    2016-05-01

    The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools. PMID:27151641

  8. Axonal interferon responses and alphaherpesvirus neuroinvasion

    NASA Astrophysics Data System (ADS)

    Song, Ren

    Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at a peripheral epithelial surface and continues into the peripheral nervous system (PNS) that innervates this tissue. Inflammatory responses are induced at the infected peripheral site prior to viral invasion of the PNS. PNS neurons are highly polarized cells with long axonal processes that connect to distant targets. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which include type I interferon (e.g. IFNbeta) and type II interferon (i.e. IFNgamma). IFNbeta can be produced by all types of cells, while IFNgamma is secreted by some specific types of immune cells. And both types of IFN induce antiviral responses in surrounding cells that express the IFN receptors. The fundamental question is how do PNS neurons respond to the inflammatory milieu experienced only by their axons. Axons must act as potential front-line barriers to prevent PNS infection and damage. Using compartmented cultures that physically separate neuron axons from cell bodies, I found that pretreating isolated axons with IFNbeta or IFNgamma significantly diminished the number of HSV-1 and PRV particles moving from axons to the cell bodies in an IFN receptor-dependent manner. Furthermore, I found the responses in axons are activated differentially by the two types of IFNs. The response to IFNbeta is a rapid, axon-only response, while the response to IFNgamma involves long distance signaling to the PNS cell body. For example, exposing axons to IFNbeta induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFNgamma induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated IFNgamma-, but not IFNbeta-mediated antiviral effects. Proteomic analysis of IFNbeta- or IFNgamma-treated axons identified several differentially regulated proteins. Therefore

  9. Axon fasciculation in the developing olfactory nerve

    PubMed Central

    2010-01-01

    Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and extend toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses only 1 of approximately 1,200 odor receptors (ORs). OSNs expressing the same OR are distributed in restricted zones of the OE. However, within a zone, the OSNs expressing a specific OR are not contiguous - distribution appears stochastic. Upon reaching the OB the OSN axons expressing the same OR reproducibly coalesce into two to three glomeruli. While ORs appear necessary for appropriate convergence of axons, a variety of adhesion associated molecules and activity-dependent mechanisms are also implicated. Recent data suggest pre-target OSN axon sorting may influence glomerular convergence. Here, using regional and OR-specific markers, we addressed the spatio-temporal properties associated with the onset of homotypic fasciculation in embryonic mice and assessed the degree to which subpopulations of axons remain segregated as they extend toward the nascent OB. We show that immediately upon crossing the basal lamina, axons uniformly turn sharply, usually at an approximately 90° angle toward the OB. Molecularly defined subpopulations of axons show evidence of spatial segregation within the nascent nerve by embryonic day 12, within 48 hours of the first OSN axons crossing the basal lamina, but at least 72 hours before synapse formation in the developing OB. Homotypic fasciculation of OSN axons expressing the same OR appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, the final convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB. PMID:20723208

  10. Imaging axon pathfinding in zebrafish in vivo.

    PubMed

    Leung, Louis; Holt, Christine E

    2012-09-01

    Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques, one can gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The major advantage of using zebrafish lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the method for stabilizing and preparing live zebrafish embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. It has been used for retinotectal axon pathfinding, but can be adapted to visualize other axon tracts of interest. PMID:22949713

  11. Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease

    PubMed Central

    Selvaraj, Bhuvaneish Thangaraj; Frank, Nicolas; Bender, Florian L.P.; Asan, Esther

    2012-01-01

    Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration. PMID:23109669

  12. Genetic labeling of both the axons of transduced, glutamatergic neurons in rat postrhinal cortex and their postsynaptic neurons in other neocortical areas by Herpes Simplex Virus vectors that coexpress an axon-targeted ß-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter

    PubMed Central

    Zhang, Guo-rong; Cao, Haiyan; Li, Xu; Zhao, Hua; Geller, Alfred I.

    2010-01-01

    Neuronal circuits comprise the foundation for neuronal physiology and synaptic plasticity, and thus for consequent behaviors and learning, but our knowledge of neocortical circuits is incomplete. Mapping neocortical circuits is a challenging problem because these circuits contain large numbers of neurons, a high density of synapses, and numerous classes and subclasses of neurons that form many different types of synapses. Expression of specific genetic tracers in small numbers of specific subclasses of neocortical neurons has potential to map neocortical circuits. Suitable genetic tracers have been established in neurons in subcortical areas, but application to neocortical circuits has been limited. Enabling this approach, Herpes Simplex Virus (HSV-1) plasmid (amplicon) vectors can transduce small numbers of neurons in a specific neocortical area. Further, expression of a particular genetic tracer can be restricted to specific subclasses of neurons; in particular, the vesicular glutamate transporter-1 (VGLUT1) promoter supports expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex. Here, we show that expression of an axon-targeted ß-galactosidase (ß-gal) from such vectors supports mapping specific commissural and associative projections of the transduced neurons in POR cortex. Further, coexpression of wheat germ agglutinin (WGA) and an axon-targeted ß-gal supports mapping both specific projections of the transduced neurons and identifying specific postsynaptic neurons for the transduced neurons. The neocortical circuit mapping capabilities developed here may support mapping specific neocortical circuits that have critical roles in cognitive learning. PMID:20849834

  13. microRNAs in axon guidance

    PubMed Central

    Iyer, Archana N.; Bellon, Anaïs; Baudet, Marie-Laure

    2014-01-01

    Brain wiring is a highly intricate process in which trillions of neuronal connections are established. Its initial phase is particularly crucial in establishing the general framework of neuronal circuits. During this early step, differentiating neurons extend axons, which reach their target by navigating through a complex environment with extreme precision. Research in the past 20 years has unraveled a vast and complex array of chemotropic cues that guide the leading tip of axons, the growth cone, throughout its journey. Tight regulation of these cues, and of their receptors and signaling pathways, is necessary for the high degree of accuracy required during circuit formation. However, little is known about the nature of regulatory molecules or mechanisms fine-tuning axonal cue response. Here we review recent, and somewhat fragmented, research on the possibility that microRNAs (miRNAs) could be key fine-tuning regulatory molecules in axon guidance. miRNAs appear to shape long-range axon guidance, fasciculation and targeting. We also present several lines of evidence suggesting that miRNAs could have a compartmentalized and differential action at the cell soma, and within axons and growth cones. PMID:24672429

  14. Branch management: mechanisms of axon branching in the developing vertebrate CNS

    PubMed Central

    Kalil, Katherine; Dent, Erik W.

    2014-01-01

    The remarkable ability of a single axon to extend multiple branches and form terminal arbors allows vertebrate neurons to integrate information from divergent regions of the nervous system. Axons select appropriate pathways during development, but it is the branches that extend interstitially from the axon shaft and arborize at specific targets that are responsible for virtually all of the synaptic connectivity in the vertebrate CNS. How do axons form branches at specific target regions? Recent studies have identified molecular cues that activate intracellular signalling pathways in axons and mediate dynamic reorganization of the cytoskeleton to promote the formation of axon branches. PMID:24356070

  15. Astrocytes Block Axonal Regeneration in Mammals by Activating the Physiological Stop Pathway

    NASA Astrophysics Data System (ADS)

    Liuzzi, Francis J.; Lasek, Raymond J.

    1987-08-01

    Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells.

  16. Imaging axon pathfinding in Xenopus in vivo.

    PubMed

    Leung, Louis; Holt, Christine E

    2012-09-01

    Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques allows one to gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The main advantage of using Xenopus lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the methods for stabilizing and preparing live Xenopus embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. This approach can been used to investigate how the knockdown of certain gene functions can affect the speed of navigation through the well-studied Xenopus retinotectal pathway. It can be adapted to visualize other axon tracts of interest. PMID:22949712

  17. Regulation of conduction time along axons.

    PubMed

    Seidl, A H

    2014-09-12

    speed of signal propagation, i.e. the speed at which an action potential travels. Conduction time refers to the time it takes for a specific signal to travel from its origin to its target, i.e. neuronal cell body to axonal terminal. PMID:23820043

  18. The back and forth of axonal injury and repair after stroke

    PubMed Central

    Hinman, Jason D.

    2015-01-01

    Purpose of review The axon plays a central role in both the injury and repair phases after stroke. This review highlights emerging principles in the study of axonal injury in stroke and the role of the axon in neural repair after stroke. Recent findings Ischemic stroke produces a rapid and significant loss of axons in the acute phase. This early loss of axons results from a primary ischemic injury that triggers a wave of calcium signaling, activating proteolytic mechanisms and downstream signaling cascades. A second progressive phase of axonal injury occurs during the subacute period and damages axons that survive the initial ischemic insult but go on to experience a delayed axonal degeneration driven in part by changes in axoglial contact and axonal energy metabolism. Recovery from stroke is dependent on axonal sprouting and reconnection that occurs during a third degenerative/regenerative phase. Despite this central role played by the axon, comparatively little is understood about the molecular pathways that contribute to early and subacute axonal degeneration after stroke. Recent advances in axonal neurobiology and signaling suggest new targets that hold promise as potential molecular therapeutics including axonal calcium signaling, axoglial energy metabolism and cell adhesion as well as retrograde axonal mitogen-activated protein kinase pathways. These novel pathways must be modeled appropriately as the type and severity of axonal injury vary by stroke subtype. Summary Stroke-induced injury to axons occurs in three distinct phases each with a unique molecular underpinning. A wealth of new data about the molecular organization and molecular signaling within axons is available but not yet robustly applied to the study of axonal injury after stroke. Identifying the spatiotemporal patterning of molecular pathways within the axon that contribute to injury and repair may offer new therapeutic strategies for the treatment of stroke. PMID:25364952

  19. Traffic lights for axon growth: proteoglycans and their neuronal receptors

    PubMed Central

    Shen, Yingjie

    2014-01-01

    Axon growth is a central event in the development and post-injury plasticity of the nervous system. Growing axons encounter a wide variety of environmental instructions. Much like traffic lights in controlling the migrating axons, chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs) often lead to “stop” and “go” growth responses in the axons, respectively. Recently, the LAR family and NgR family molecules were identified as neuronal receptors for CSPGs and HSPGs. These discoveries provided molecular tools for further study of mechanisms underlying axon growth regulation. More importantly, the identification of these proteoglycan receptors offered potential therapeutic targets for promoting post-injury axon regeneration. PMID:25206823

  20. Axons take a dive

    PubMed Central

    Tong, Cheuk Ka; Cebrián-Silla, Arantxa; Paredes, Mercedes F; Huang, Eric J; García-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2015-01-01

    In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular–subventricular zone (V–SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells. PMID:26413556

  1. Specificity of peripheral nerve regeneration: interactions at the axon level.

    PubMed

    Allodi, Ilary; Udina, Esther; Navarro, Xavier

    2012-07-01

    Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs. PMID:22609046

  2. White-matter astrocytes, axonal energy metabolism, and axonal degeneration in multiple sclerosis

    PubMed Central

    Cambron, Melissa; D'Haeseleer, Miguel; Laureys, Guy; Clinckers, Ralph; Debruyne, Jan; De Keyser, Jacques

    2012-01-01

    In patients with multiple sclerosis (MS), a diffuse axonal degeneration occurring throughout the white matter of the central nervous system causes progressive neurologic disability. The underlying mechanism is unclear. This review describes a number of pathways by which dysfunctional astrocytes in MS might lead to axonal degeneration. White-matter astrocytes in MS show a reduced metabolism of adenosine triphosphate-generating phosphocreatine, which may impair the astrocytic sodium potassium pump and lead to a reduced sodium-dependent glutamate uptake. Astrocytes in MS white matter appear to be deficient in β2 adrenergic receptors, which are involved in stimulating glycogenolysis and suppressing inducible nitric oxide synthase (NOS2). Glutamate toxicity, reduced astrocytic glycogenolysis leading to reduced lactate and glutamine production, and enhanced nitric oxide (NO) levels may all impair axonal mitochondrial metabolism, leading to axonal degeneration. In addition, glutamate-mediated oligodendrocyte damage and impaired myelination caused by a decreased production of N-acetylaspartate by axonal mitochondria might also contribute to axonal loss. White-matter astrocytes may be considered as a potential target for neuroprotective MS therapies. PMID:22214904

  3. Retrograde and Wallerian Axonal Degeneration Occur Synchronously after Retinal Ganglion Cell Axotomy

    PubMed Central

    Kanamori, Akiyasu; Catrinescu, Maria-Magdalena; Belisle, Jonathan M.; Costantino, Santiago; Levin, Leonard A.

    2013-01-01

    Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration. PMID:22642911

  4. Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

    SciTech Connect

    Menelaou, Evdokia; Paul, Latoya T.; Perera, Surangi N.; Svoboda, Kurt R.

    2015-04-01

    Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15–30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. - Highlights: • Embryonic nicotine exposure can specifically affect secondary motoneuron axons in a dose-dependent manner.

  5. Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

    PubMed

    Louveau, Antoine; Nerrière-Daguin, Véronique; Vanhove, Bernard; Naveilhan, Philippe; Neunlist, Michel; Nicot, Arnaud; Boudin, Hélène

    2015-12-01

    Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia. PMID:26212105

  6. Clinical Progression in Parkinson's Disease and the Neurobiology of Axons

    PubMed Central

    Cheng, Hsiao-Chun; Ulane, Christina M.; Burke, Robert E.

    2010-01-01

    In spite of tremendous growth in recent years in our knowledge of the molecular basis of Parkinson's disease and the molecular pathways of cell injury and death, we remain without therapies that forestall disease progression. While there are many possible explanations for this lack of success, one is that experimental therapeutics to date have not adequately focused on an important component of the disease process, that of axon degeneration. It remains unknown what neuronal compartment, either the soma or the axon, is involved at disease onset, although some have proposed that it is the axons and their terminals that take the initial brunt of injury. Nevertheless, this concept has not been formally incorporated into many of the current theories of disease pathogenesis, and it has not achieved a wide consensus. More importantly, in view of growing evidence that the molecular mechanisms of axon degeneration are separate and distinct from the canonical pathways of programmed cell death that mediate soma destruction, the possibility of early involvement of axons in PD has not been adequately emphasized as a rationale to explore the neurobiology of axons for novel therapeutic targets. We propose that it is ongoing degeneration of axons, not cell bodies, that is the primary determinant of clinically apparent progression of disease, and that future experimental therapeutics intended to forestall disease progression will benefit from a new focus on the distinct mechanisms of axon degeneration. PMID:20517933

  7. Myelin-Associated Inhibitors in Axonal Growth After CNS Injury

    PubMed Central

    Geoffroy, Cédric G.; Zheng, Binhai

    2014-01-01

    There are multiple barriers to axonal growth after CNS injury. Myelin-associated inhibitors represent one group of barriers extrinsic to the injured neurons. Nogo, MAG and OMgp are three prototypical myelin inhibitors that signal through multiple neuronal receptors to exert growth inhibition. Targeting myelin inhibition alone modulates the compensatory sprouting of uninjured axons but the effect on the regeneration of injured axons is limited. Meanwhile, modulating sprouting, a naturally occurring repair mechanism, may be a more attainable therapeutic goal for promoting functional repair after CNS injury in the near term. PMID:24608164

  8. Local axonal protection by WldS as revealed by conditional regulation of protein stability.

    PubMed

    Wang, Jack T; Medress, Zachary A; Vargas, Mauricio E; Barres, Ben A

    2015-08-18

    The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment. PMID:26209654

  9. Local axonal protection by WldS as revealed by conditional regulation of protein stability

    PubMed Central

    Wang, Jack T.; Medress, Zachary A.; Vargas, Mauricio E.; Barres, Ben A.

    2015-01-01

    The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4–5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD+, the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment. PMID:26209654

  10. Minimizing the caliber of myelinated axons by means of nodal constrictions.

    PubMed

    Johnson, Christopher; Holmes, William R; Brown, Anthony; Jung, Peter

    2015-09-01

    In myelinated axons, most of the voltage-gated ion channels are concentrated at the nodes of Ranvier, which are short gaps in the myelin sheath. This arrangement leads to saltatory conduction and a larger conduction velocity than in nonmyelinated axons. Intriguingly, axons in the peripheral nervous system that exceed about 2 μm in diameter exhibit a characteristic narrowing of the axon at nodes that results in a local reduction of the axonal cross-sectional area. The extent of constriction increases with increasing internodal axonal caliber, reaching a threefold reduction in diameter for the largest axons. In this paper, we use computational modeling to investigate the effect of nodal constrictions on axonal conduction velocity. For a fixed number of ion channels, we find that there is an optimal extent of nodal constriction which minimizes the internodal axon caliber that is required to achieve a given target conduction velocity, and we show that this is sensitive to the precise geometry of the axon and myelin sheath in the flanking paranodal regions. Thus axonal constrictions at nodes of Ranvier appear to be a biological adaptation to minimize axonal volume, thereby maximizing the spatial and metabolic efficiency of these processes, which can be a significant evolutionary constraint. We show that the optimal nodal morphologies are relatively insensitive to changes in the number of nodal sodium channels. PMID:26224772

  11. Sensory axon regeneration: rebuilding functional connections in the spinal cord

    PubMed Central

    Smith, George M.; Falone, Anthony E.; Frank, Eric

    2011-01-01

    Functional regeneration within the adult spinal cord remains a formidable task. A major barrier to regeneration of sensory axons into the spinal cord is the dorsal root entry zone. This region displays many of the inhibitory features characteristic of other central nervous system injuries. Several experimental treatments, including inactivation of inhibitory molecules (such as Nogo and chondroitin sulfate proteoglycans) or administration of neurotrophic factors (such as nerve growth factor, neurotrophin3, glial derived neurotrophic factor and artemin), have been found to promote anatomical and functional regeneration across this barrier. There have been relatively few experiments, however, to determine if regenerating axons project back to their appropriate target areas within the spinal cord. This review focuses on recent advances in sensory axon regeneration, including studies assessing the ability of sensory axons to reconnect with their original synaptic targets. PMID:22137336

  12. A model of axonal transport drug delivery

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey

    2012-04-01

    In this paper a model of targeted drug delivery by means of active (motor-driven) axonal transport is developed. The model is motivated by recent experimental research by Filler et al. (A.G. Filler, G.T. Whiteside, M. Bacon, M. Frederickson, F.A. Howe, M.D. Rabinowitz, A.J. Sokoloff, T.W. Deacon, C. Abell, R. Munglani, J.R. Griffiths, B.A. Bell, A.M.L. Lever, Tri-partite complex for axonal transport drug delivery achieves pharmacological effect, Bmc Neuroscience 11 (2010) 8) that reported synthesis and pharmacological efficiency tests of a tri-partite complex designed for axonal transport drug delivery. The developed model accounts for two populations of pharmaceutical agent complexes (PACs): PACs that are transported retrogradely by dynein motors and PACs that are accumulated in the axon at the Nodes of Ranvier. The transitions between these two populations of PACs are described by first-order reactions. An analytical solution of the coupled system of transient equations describing conservations of these two populations of PACs is obtained by using Laplace transform. Numerical results for various combinations of parameter values are presented and their physical significance is discussed.

  13. Axonal PPARγ promotes neuronal regeneration after injury.

    PubMed

    Lezana, Juan Pablo; Dagan, Shachar Y; Robinson, Ari; Goldstein, Ronald S; Fainzilber, Mike; Bronfman, Francisca C; Bronfman, Miguel

    2016-06-01

    PPARγ is a ligand-activated nuclear receptor best known for its involvement in adipogenesis and glucose homeostasis. PPARγ activity has also been associated with neuroprotection in different neurological disorders, but the mechanisms involved in PPARγ effects in the nervous system are still unknown. Here we describe a new functional role for PPARγ in neuronal responses to injury. We found both PPAR transcripts and protein within sensory axons and observed an increase in PPARγ protein levels after sciatic nerve crush. This was correlated with increased retrograde transport of PPARγ after injury, increased association of PPARγ with the molecular motor dynein, and increased nuclear accumulation of PPARγ in cell bodies of sensory neurons. Furthermore, PPARγ antagonists attenuated the response of sensory neurons to sciatic nerve injury, and inhibited axonal growth of both sensory and cortical neurons in culture. Thus, axonal PPARγ is involved in neuronal injury responses required for axonal regeneration. Since PPARγ is a major molecular target of the thiazolidinedione (TZD) class of drugs used in the treatment of type II diabetes, several pharmaceutical agents with acceptable safety profiles in humans are available. Our findings provide motivation and rationale for the evaluation of such agents for efficacy in central and peripheral nerve injuries. PMID:26446277

  14. Microfluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Black, Bryan; Ordonez, Simon; Mondal, Argha; Jain, Ankur; Mohanty, Samarendra

    2014-10-01

    The precision of axonal pathfinding and the accurate formation of functional neural circuitry are crucial for an organism during development as well as during adult central and peripheral nerve regeneration. While chemical cues are believed to be primarily responsible for axonal pathfinding, we hypothesize that forces due to localized fluid flow may directly affect neuronal guidance during early organ development. Here, we report direct evidence of fluid flow influencing axonal migration, producing turning angles of up to 90°. Microfluidic flow simulations indicate that an axon may experience significant bending force due to cross-flow, which may contribute to the observed axonal turning. This method of flow-based guidance was successfully used to fasciculate one advancing axon onto another, showcasing the potential of this technique to be used for the formation of in vitro neuronal circuits.

  15. Intrinsic Control of Axon Regeneration.

    PubMed

    He, Zhigang; Jin, Yishi

    2016-05-01

    A determinant of axon regeneration is the intrinsic growth ability of injured neurons, which dictates a battery of injury responses in axons and cell bodies. While some of these regulatory mechanisms are evolutionarily conserved, others are unique to the mammalian central nervous system (CNS) where spontaneous regeneration usually does not occur. Here we examine our current understanding of these mechanisms at cellular and molecular terms and discuss their potential implications for promoting axon regeneration and functional recovery after nerve injury. PMID:27151637

  16. Regulation of axon regeneration by the RNA repair and splicing pathway.

    PubMed

    Song, Yuanquan; Sretavan, David; Salegio, Ernesto A; Berg, Jim; Huang, Xi; Cheng, Tong; Xiong, Xin; Meltzer, Shan; Han, Chun; Nguyen, Trong-Tuong; Bresnahan, Jacqueline C; Beattie, Michael S; Jan, Lily Yeh; Jan, Yuh Nung

    2015-06-01

    Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. PMID:25961792

  17. Axonal injury and regeneration in the adult brain of Drosophila

    PubMed Central

    Ayaz, Derya; Leyssen, Maarten; Koch, Marta; Yan, Jiekun; Srahna, Mohammed; Sheeba, Vasu; Fogle, Keri J.; Holmes, Todd C.; Hassan, Bassem A.

    2009-01-01

    Drosophila melanogaster is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the Central Nervous System (CNS). We developed a versatile and physiologically robust preparation for the long-term culture of the whole Drosophila brain. We use this method to develop a novel Drosophila model for CNS axonal injury and regeneration. We first show that, similar to mammalian CNS axons, injured adult wild type fly CNS axons fail to regenerate, whereas adult-specific enhancement of Protein Kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms following injury. We next exploit this model to explore pathways that induce robust regeneration and find that adult-specific activation of JNK signalling is sufficient for de novo CNS axonal regeneration after injury, including the growth of new axons past the lesion site and into the normal target area. PMID:18524906

  18. Membrane turnover and receptor trafficking in regenerating axons.

    PubMed

    Hausott, Barbara; Klimaschewski, Lars

    2016-02-01

    Peripheral axonal regeneration requires surface-expanding membrane addition. The continuous incorporation of new membranes into the axolemma allows the pushing force of elongating microtubules to drive axonal growth cones forwards. Hence, a constant supply of membranes and cytoskeletal building blocks is required, often for many weeks. In human peripheral nerves, axonal tips may be more than 1 m away from the neuronal cell body. Therefore, in the initial phase of regeneration, membranes are derived from pre-existing vesicles or synthesised locally. Only later stages of axonal regeneration are supported by membranes and proteins synthesised in neuronal cell bodies, considering that the fastest anterograde transport mechanisms deliver cargo at 20 cm/day. Whereas endocytosis and exocytosis of membrane vesicles are balanced in intact axons, membrane incorporation exceeds membrane retrieval during regeneration to compensate for the loss of membranes distal to the lesion site. Physiological membrane turnover rates will not be established before the completion of target reinnervation. In this review, the current knowledge on membrane traffic in axonal outgrowth is summarised, with a focus on endosomal vesicles as the providers of membranes and carriers of growth factor receptors required for initiating signalling pathways to promote the elongation and branching of regenerating axons in lesioned peripheral nerves. PMID:26222895

  19. Astrocyte scar formation aids central nervous system axon regeneration.

    PubMed

    Anderson, Mark A; Burda, Joshua E; Ren, Yilong; Ao, Yan; O'Shea, Timothy M; Kawaguchi, Riki; Coppola, Giovanni; Khakh, Baljit S; Deming, Timothy J; Sofroniew, Michael V

    2016-04-14

    Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration. PMID:27027288

  20. White matter involvement after TBI: Clues to axon and myelin repair capacity.

    PubMed

    Armstrong, Regina C; Mierzwa, Amanda J; Marion, Christina M; Sullivan, Genevieve M

    2016-01-01

    Impact-acceleration forces to the head cause traumatic brain injury (TBI) with damage in white matter tracts comprised of long axons traversing the brain. White matter injury after TBI involves both traumatic axonal injury (TAI) and myelin pathology that evolves throughout the post-injury time course. The axon response to initial mechanical forces and secondary insults follows the process of Wallerian degeneration, which initiates as a potentially reversible phase of intra-axonal damage and proceeds to an irreversible phase of axon fragmentation. Distal to sites of axon disconnection, myelin sheaths remain for prolonged periods, which may activate neuroinflammation and inhibit axon regeneration. In addition to TAI, TBI can cause demyelination of intact axons. These evolving features of axon and myelin pathology also represent opportunities for repair. In experimental TBI, demyelinated axons exhibit remyelination, which can serve to both protect axons and facilitate recovery of function. Myelin remodeling may also contribute to neuroplasticity. Efficient clearance of myelin debris is a potential target to attenuate the progression of chronic pathology. During the early phase of Wallerian degeneration, interventions that prevent the transition from reversible damage to axon disconnection warrant the highest priority, based on the poor regenerative capacity of axons in the CNS. Clinical evaluation of TBI will need to address the challenge of accurately detecting the extent and stage of axon damage. Distinguishing the complex white matter changes associated with axons and myelin is necessary for interpreting advanced neuroimaging approaches and for identifying a broader range of therapeutic opportunities to improve outcome after TBI. PMID:25697845

  1. Fiber tractography of the axonal pathways linking the basal ganglia and cerebellum in Parkinson disease: implications for targeting in deep brain stimulation

    PubMed Central

    Sweet, Jennifer A.; Walter, Benjamin L.; Gunalan, Kabilar; Chaturvedi, Ashutosh; Mcintyre, Cameron C.; Miller, Jonathan P.

    2015-01-01

    Object Stimulation of white matter pathways near targeted structures may contribute to therapeutic effects of deep brain stimulation (DBS) for patients with Parkinson disease (PD). Two tracts linking the basal ganglia and cerebellum have been described in primates: the subthalamopontocerebellar tract (SPCT) and the dentatothalamic tract (DTT). The authors used fiber tractography to evaluate white matter tracts that connect the cerebellum to the region of the basal ganglia in patients with PD who were candidates for DBS. Methods Fourteen patients with advanced PD underwent 3-T MRI, including 30-directional diffusion-weighted imaging sequences. Diffusion tensor tractography was performed using 2 regions of interest: ipsilateral subthalamic and red nuclei, and contralateral cerebellar hemisphere. Nine patients underwent subthalamic DBS, and the course of each tract was observed relative to the location of the most effective stimulation contact and the volume of tissue activated. Results In all patients 2 distinct tracts were identified that corresponded closely to the described anatomical features of the SPCT and DTT, respectively. The mean overall distance from the active contact to the DTT was 2.18 ± 0.35 mm, and the mean proportional distance relative to the volume of tissue activated was 1.35 ± 0.48. There was a nonsignificant trend toward better postoperative tremor control in patients with electrodes closer to the DTT. Conclusions The SPCT and the DTT may be related to the expression of symptoms in PD, and this may have implications for DBS targeting. The use of tractography to identify the DTT might assist with DBS targeting in the future. PMID:24484226

  2. Synaptic Democracy and Vesicular Transport in Axons

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Levien, Ethan

    2015-04-01

    Synaptic democracy concerns the general problem of how regions of an axon or dendrite far from the cell body (soma) of a neuron can play an effective role in neuronal function. For example, stimulated synapses far from the soma are unlikely to influence the firing of a neuron unless some sort of active dendritic processing occurs. Analogously, the motor-driven transport of newly synthesized proteins from the soma to presynaptic targets along the axon tends to favor the delivery of resources to proximal synapses. Both of these phenomena reflect fundamental limitations of transport processes based on a localized source. In this Letter, we show that a more democratic distribution of proteins along an axon can be achieved by making the transport process less efficient. This involves two components: bidirectional or "stop-and-go" motor transport (which can be modeled in terms of advection-diffusion), and reversible interactions between motor-cargo complexes and synaptic targets. Both of these features have recently been observed experimentally. Our model suggests that, just as in human societies, there needs to be a balance between "efficiency" and "equality".

  3. Signaling from axon guidance receptors.

    PubMed

    Bashaw, Greg J; Klein, Rüdiger

    2010-05-01

    Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues--netrins, semaphorins, ephrins, and slits--have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance. PMID:20452961

  4. Presynaptic Type III Neuregulin 1 Is Required for Sustained Enhancement of Hippocampal Transmission by Nicotine and for Axonal Targeting of α7 Nicotinic Acetylcholine Receptors

    PubMed Central

    Zhong, Chongbo; Du, Chuang; Hancock, Melissa; Mertz, Marjolijn; Talmage, David A.; Role, Lorna W.

    2009-01-01

    Both the neuregulin 1 (Nrg1) and α7 nicotinic acetylcholine receptor (α7*nAChRs) genes have been linked to schizophrenia and associated sensory–motor gating deficits. The prominence of nicotine addiction in schizophrenic patients is reflected in the normalization of gating deficits by nicotine self-administration. To assess the role of presynaptic type III Nrg1 at hippocampal–accumbens synapses, an important relay in sensory–motor gating, we developed a specialized preparation of chimeric circuits in vitro. Synaptic relays from Nrg1tm1Lwr heterozygote ventral hippocampal slices to wild-type (WT) nucleus accumbens neurons (1) lack a sustained, α7*nAChRs-mediated phase of synaptic potentiation seen in comparable WT/WT circuits and (2) are deficient in targeting α7*nAChRs to presynaptic sites. Thus, selective alteration of the level of presynaptic type III Nrg1 dramatically affects the modulation of glutamatergic transmission at ventral hippocampal to nucleus accumbens synapses. PMID:18784291

  5. The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons.

    PubMed

    Caton, A; Hacker, A; Naeem, A; Livet, J; Maina, F; Bladt, F; Klein, R; Birchmeier, C; Guthrie, S

    2000-04-01

    During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies. PMID:10725250

  6. Axonal model for temperature stimulation.

    PubMed

    Fribance, Sarah; Wang, Jicheng; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2016-10-01

    Recent studies indicate that a rapid increase in local temperature plays an important role in nerve stimulation by laser. To analyze the temperature effect, our study modified the classical HH axonal model by incorporating a membrane capacitance-temperature relationship. The modified model successfully simulated the generation and propagation of action potentials induced by a rapid increase in local temperature when the Curie temperature of membrane capacitance is below 40 °C, while the classical model failed to simulate the axonal excitation by temperature stimulation. The new model predicts that a rapid increase in local temperature produces a rapid increase in membrane capacitance, which causes an inward membrane current across the membrane capacitor strong enough to depolarize the membrane and generate an action potential. If the Curie temperature of membrane capacitance is 31 °C, a temperature increase of 6.6-11.2 °C within 0.1-2.6 ms is required for axonal excitation and the required increase is smaller for a faster increase. The model also predicts that: (1) the temperature increase could be smaller if the global axon temperature is higher; (2) axons of small diameter require a smaller temperature increase than axons of large diameter. Our study indicates that the axonal membrane capacitance-temperature relationship plays a critical role in inducing the transient membrane depolarization by a rapidly increasing temperature, while the effects of temperature on ion channel kinetics cannot induce depolarization. The axonal model developed in this study will be very useful for analyzing the axonal response to local heating induced by pulsed infrared laser. PMID:27342462

  7. Giant axonal neuropathy: MRS findings.

    PubMed

    Alkan, Alpay; Kutlu, Ramazan; Sigirci, Ahmet; Baysal, Tamer; Altinok, Tayfun; Yakinci, Cengiz

    2003-10-01

    Giant axonal neuropathy (GAN) is a rare genetic disease of childhood involving the central and peripheral nervous systems. Axonal loss with several giant axons filled with neurofilaments is the main histopathological feature of peripheral nerve biopsies in this disease. Routine neuroimaging studies reveal diffuse hyperintensities in cerebral and cerebellar white matter. In this case report, the authors present the brain magnetic resonance spectroscopic features (normal N-acetylaspartate/creatine and increased choline/creatine and myoinositol/creatine ratios), which might indicate the absence of neuroaxonal loss and the presence of significant demyelination and glial proliferation in white matter, of an 11-year-old boy diagnosed with GAN. PMID:14569833

  8. Retinopetal Axons in Mammals: Emphasis on Histamine and Serotonin

    PubMed Central

    Gastinger, Matthew J.; Tian, Ning; Horvath, Tamas; Marshak, David W.

    2012-01-01

    Since 1892, anatomical studies have demonstrated that the retinas of mammals, including humans, receive input from the brain via axons emerging from the optic nerve. There are only a small number of these retinopetal axons, but their branches in the inner retina are very extensive. More recently, the neurons in the brain stem that give rise to these axons have been localized, and their neurotransmitters have been identified. One set of retinopetal axons arises from perikarya in the posterior hypothalamus and uses histamine, and the other arises from perikarya in the dorsal raphe and uses serotonin. These serotonergic and histaminergic neurons are not specialized to supply the retina; rather, they are a subset of the neurons that project via collaterals to many other targets in the central nervous system, as well. They are components of the ascending arousal system, firing most rapidly when the animal is awake and active. The contributions of these retinopetal axons to vision may be predicted from the known effects of serotonin and histamine on retinal neurons. There is also evidence suggesting that retinopetal axons play a role in the etiology of retinal diseases. PMID:16877274

  9. Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation

    PubMed Central

    Petralia, Ronald S.; Ott, Carolyn; Wang, Ya-Xian; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2015-01-01

    The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. PMID:26658865

  10. Abeta, tau and ApoE4 in Alzheimer's disease: the axonal connection.

    PubMed

    Adalbert, Robert; Gilley, Jonathan; Coleman, Michael P

    2007-04-01

    Mutations in amyloid precursor protein (APP), tau and apolipoprotein E4 (ApoE4) lead to Alzheimer's disease (AD) or related pathologies. Pathogenesis and interactions between these pathways have been studied in mouse models. Here, we highlight the fact that axons are important sites of cellular pathology in each pathway and propose that pathway convergence at the molecular level might occur in axons. Recent developments suggest that axonal transport of APP influences beta-amyloid deposition and that tau regulates axonal transport. ApoE4 influences both axonal tau phosphorylation and amyloid-induced neurite pathology. Thus, a better understanding of axonal events in AD might help connect the pathogenic mechanisms of beta-amyloid, ApoE4 and tau, indicating the most important steps for therapeutic targeting. PMID:17344096

  11. Fusimotor axons in the kitten.

    PubMed

    Gregory, J E; Proske, U

    1986-11-01

    In kittens 1- to 23-days old growth of axons in the soleus nerve has been studied using the structural parameters nerve length, internodal length, and axonal diameter. In addition, single functional fusimotor axons were isolated in lumbosacral ventral roots, and the responses of muscle spindles in soleus were studied during fusimotor stimulation. While nerve length over the soleus nerve to lumbar spinal root increased from 41 to 76 mm during the 22 days, mean internodal length increased from 250 to 410 microns. Mean axon diameter increased from 2.1 to 4.1 microns. In the youngest animals values for both internodal length and axon diameter were distributed uniformly about the mean. From day 11 onward the distributions became bimodal, including a growing number of new axons in the small-myelinated range. Filaments of ventral root were isolated that on repetitive stimulation had a specific excitatory effect on the discharge of muscle spindles. The responses could be attributed to axons that were not associated with measurable tension and were therefore likely to be fusimotor fibers. Measurements of the conduction velocity of skeletomotor and fusimotor axons showed that conduction speed increased progressively with age for both groups, but the rate of increase was more than three times faster in the most rapidly conducting skeletomotor axons compared with the fusimotor axons. The distribution of conduction velocities for fusimotor fibers showed two peaks, one in the range typical for conduction in unmyelinated fibers, 0.5-1.0 m/s, the second at 3-4 m/s. The small number of values in the range of 1-2 m/s was attributed to the process of myelination. It is suggested that conduction speed increases discontinuously over this part of the range as impulse conduction changes from continuous propagation to saltatory transmission. Eighteen fusimotor axons could be classified as having either a static or a dynamic action on spindle discharge. Repetitive stimulation of fusimotor

  12. Dynamic Changes in Local Protein Synthetic Machinery in Regenerating Central Nervous System Axons after Spinal Cord Injury

    PubMed Central

    Sachdeva, Rahul; Farrell, Kaitlin; McMullen, Mary-Katharine; Twiss, Jeffery L.; Houle, John D.

    2016-01-01

    Intra-axonal localization of mRNAs and protein synthesis machinery (PSM) endows neurons with the capacity to generate proteins locally, allowing precise spatiotemporal regulation of the axonal response to extracellular stimuli. A number of studies suggest that this local translation is a promising target to enhance the regenerative capacity of damaged axons. Using a model of central nervous system (CNS) axons regenerating into intraspinal peripheral nerve grafts (PNGs) we established that adult regenerating CNS axons contain several different mRNAs and protein synthetic machinery (PSM) components in vivo. After lower thoracic level spinal cord transection, ascending sensory axons regenerate into intraspinal PNGs but axon growth is stalled when they reach the distal end of the PNG (3 versus 7 weeks after grafting, resp.). By immunofluorescence with optical sectioning of axons by confocal microscopy, the total and phosphorylated forms of PSMs are significantly lower in stalled compared with actively regenerating axons. Reinjury of these stalled axons increased axonal localization of the PSM proteins, indicative of possible priming for a subcellular response to axotomy. These results suggest that axons downregulate protein synthetic capacity as they cease growing, yet they retain the ability to upregulate PSM after a second injury. PMID:27375904

  13. Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding.

    PubMed

    Moret, Frédéric; Renaudot, Christelle; Bozon, Muriel; Castellani, Valérie

    2007-12-01

    Class III semaphorins (SemaIIIs) are intercellular cues secreted by surrounding tissues to guide migrating cells and axons in the developing organism. This chemotropic activity is crucial for the formation of nerves and vasculature. Intriguingly, SemaIIIs are also synthesized by neurons during axon pathfinding, but their function as intrinsic cues remains unknown. We have explored the role of Sema3A expression in motoneurons during spinal nerve development. Loss- and gain-of-function in the neural tube of the chick embryo were undertaken to target Sema3A expression in motoneurons while preserving Sema3A sources localized in peripheral tissues, known to provide important repulsive information for delineating the routes of motor axons towards their ventral or dorsal targets. Strikingly, Sema3A overexpression induced defasciculation and exuberant growth of motor axon projections into these normally non-permissive territories. Moreover, knockdown studies showed that motoneuronal Sema3A is required for correct spinal nerve compaction and dorsal motor axon extension. Further analysis of Sema3A gain- and loss-of-function in ex vivo models revealed that Sema3A in motoneurons sets the level of sensitivity of their growth cones to exogenous Sema3A exposure. This regulation is associated with post-transcriptional and local control of the availability of the Sema3A receptor neuropilin 1 at the growth cone surface. Thus, by modulating the strength of Sema3A-mediated environmental repulsive constraints, Sema3A in motoneurons enables axons to extend more or less far away from these repulsive sources. Such interplay between intrinsic and extrinsic Sema3A may represent a fundamental mechanism in the accurate specification of axon pathways. PMID:18039974

  14. Equivalent Activities of Repulsive Axon Guidance Receptors

    PubMed Central

    Long, Hong; Yoshikawa, Shingo

    2016-01-01

    Receptors on the growth cone at the leading edge of elongating axons play critical guidance roles by recognizing cues via their extracellular domains and transducing signals via their intracellular domains, resulting in changes in direction of growth. An important concept to have emerged in the axon guidance field is the importance of repulsion as a major guidance mechanism. Given the number and variety of different repulsive receptors, it is generally thought that there are likely to be qualitative differences in the signals they transduce. However, the nature of these possible differences is unknown. By creating chimeras using the extracellular and intracellular domains of three different Drosophila repulsive receptors, Unc5, Roundabout (Robo), and Derailed (Drl) and expressing them in defined cells within the embryonic nervous system, we examined the responses elicited by their intracellular domains systematically. Surprisingly, we found no qualitative differences in growth cone response or axon growth, suggesting that, despite their highly diverged sequences, each intracellular domain elicits repulsion via a common pathway. In terms of the signaling pathway(s) used by the repulsive receptors, mutations in the guanine nucleotide exchange factor Trio strongly enhance the repulsive activity of all three intracellular domains, suggesting that repulsion by Unc5, Robo, and Drl, and perhaps repulsion in general, involves Trio activity. SIGNIFICANCE STATEMENT A prevailing concept that has emerged in the axon guidance field is the importance of repulsion as a guidance mechanism for steering axons to their appropriate targets. Given the number and variety of different repulsive receptors, it is generally thought that there are differences in the signals that they transduce. However, this has never been tested directly. We have used the advanced genetics of Drosophila to compare directly the outputs of different repulsive receptors. Surprisingly, we found no qualitative

  15. The axonal cytoskeleton: from organization to function

    PubMed Central

    Kevenaar, Josta T.; Hoogenraad, Casper C.

    2015-01-01

    The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the structural basis for several specialized axonal structures, such as the axon initial segment (AIS) and presynaptic boutons. Emerging evidence suggest that the unique cytoskeleton organization in the axon is essential for its structure and integrity. In addition, the increasing number of neurodevelopmental and neurodegenerative diseases linked to defect in actin- and microtubule-dependent processes emphasizes the importance of a properly regulated cytoskeleton for normal axonal functioning. Here, we provide an overview of the current understanding of actin and microtubule organization within the axon and discuss models for the functional role of the cytoskeleton at specialized axonal structures. PMID:26321907

  16. Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points

    PubMed Central

    Pittman, Andrew J.; Law, Mei-Yee; Chien, Chi-Bin

    2008-01-01

    Summary Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before—pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared to axon-autonomous receptor function has not been assessed. Here we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have coequal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts. PMID:18653554

  17. Rapid in vivo forward genetic approach for identifying axon death genes in Drosophila

    PubMed Central

    Neukomm, Lukas J.; Burdett, Thomas C.; Gonzalez, Michael A.; Züchner, Stephan; Freeman, Marc R.

    2014-01-01

    Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFP-labeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss. PMID:24958874

  18. MicroRNA-210 promotes sensory axon regeneration of adult mice in vivo and in vitro.

    PubMed

    Hu, Yi-Wen; Jiang, Jing-Jing; Yan-Gao; Wang, Rui-Ying; Tu, Guan-Jun

    2016-05-27

    Axon regeneration as a critical step in nerve repairing and remodeling after peripheral nerve injury relies on regulation of gene expression. MicroRNAs are emerging to be important epigenetic regulators of gene expression to control axon regeneration. Here we used a novel in vivo electroporation approach to transfect microRNA-210 (miR-210) or siRNAs to adult mice dorsal root ganglion (DRG) neurons, measured the axon length 3days after sciatic nerve crush or dissociated DRG cultures in vitro to detect the effect of miR-210 in sensory axon regeneration. Importantly, we found that miR-210 overexpression could promote sensory axon regeneration and inhibit apoptsosis by ephrin-A3 (EFNA3). In addition, inhibition of endogenous miR-210 in DRG neurons impaired axon regeneration in vitro and in vivo, the regulatory effect of miR-210 was mediated by increased expression of EFNA3 because downregulation of EFNA3 fully rescued axon regeneration. We thus demonstrate that miR-210 is a new physiological regulator of sensory axon regeneration, and EFNA3 may be the functional target of miR-210. We conclude that miR-210 may play an important role in sensory axon regeneration. PMID:27102143

  19. Motoneuron axon pathfinding errors in zebrafish: differential effects related to concentration and timing of nicotine exposure.

    PubMed

    Menelaou, Evdokia; Paul, Latoya T; Perera, Surangi N; Svoboda, Kurt R

    2015-04-01

    Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15-30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. PMID:25668718

  20. Multiple Transcription Factor Families Regulate Axon Growth and Regeneration

    PubMed Central

    Moore, Darcie L.; Goldberg, Jeffrey L.

    2011-01-01

    Understanding axon regenerative failure remains a major goal in neuroscience, and reversing this failure remains a major goal for clinical neurology. While an inhibitory CNS environment clearly plays a role, focus on molecular pathways within neurons has begun to yield fruitful insights. Initial steps forward investigated the receptors and signaling pathways immediately downstream of environmental cues, but recent work has also shed light on transcriptional control mechanisms that regulate intrinsic axon growth ability, presumably through whole cassettes of gene target regulation. Here we will discuss transcription factors that regulate neurite growth in vitro and in vivo, including p53, SnoN, E47, CREB, STAT3, NFAT, c-Jun, ATF3, Sox11, NFκB, and Kruppel-like factors (KLFs). Revealing the similarities and differences among the functions of these transcription factors may further our understanding of the mechanisms of transcriptional regulation in axon growth and regeneration. PMID:21674813

  1. Inhibition of kinesin-5 improves regeneration of injured axons by a novel microtubule-based mechanism.

    PubMed

    Baas, Peter W; Matamoros, Andrew J

    2015-06-01

    Microtubules have been identified as a powerful target for augmenting regeneration of injured adult axons in the central nervous system. Drugs that stabilize microtubules have shown some promise, but there are concerns that abnormally stabilizing microtubules may have only limited benefits for regeneration, while at the same time may be detrimental to the normal work that microtubules perform for the axon. Kinesin-5 (also called kif11 or Eg5), a molecular motor protein best known for its crucial role in mitosis, acts as a brake on microtubule movements by other motor proteins in the axon. Drugs that inhibit kinesin-5, originally developed to treat cancer, result in greater mobility of microtubules in the axon and an overall shift in the forces on the microtubule array. As a result, the axon grows faster, retracts less, and more readily enters environments that are inhibitory to axonal regeneration. Thus, drugs that inhibit kinesin-5 offer a novel microtubule-based means to boost axonal regeneration without the concerns that accompany abnormal stabilization of the microtubule array. Even so, inhibiting kinesin-5 is not without its own caveats, such as potential problems with navigation of the regenerating axon to its target, as well as morphological effects on dendrites that could affect learning and memory if the drugs reach the brain. PMID:26199587

  2. Fas-associated factor 1 as a regulator of olfactory axon guidance.

    PubMed

    Cheng, Kai; Bai, Li; Belluscio, Leonardo

    2011-08-17

    Axon guidance is a crucial part of neural circuit formation. While precise axonal targeting forms the basis of accurate information delivery, the mechanisms that regulate this process are still unclear. Apoptotic signaling molecules have been identified in the axon terminal, but their specific role in axon guidance is not well understood. Here we use the mouse olfactory system as an in vivo model to demonstrate that by modulating Fas-associated factor 1 (FAF1), an apoptosis regulatory molecule, we can rewire axonal projections. Interestingly, FAF1 is highly expressed in the developing mouse olfactory system, but its expression is downregulated postnatally. Using a tetracycline-inducible promoter Tet-Off system, we generated transgenic mice in which FAF1 is specifically expressed in immature olfactory sensory neurons (OSNs) and show that overexpression of FAF1 not only misroutes OSN axons to deep layers of the olfactory bulb but also leads to widespread disruption of the glomerular layer. In addition, we also demonstrate that the specific convergence of P2 receptor OSN axons is completely distorted in the FAF1 mice. Strikingly, all of the mutant phenotypes can be recovered by shutting down FAF1 expression through the administration of doxycycline. Together, our study provides clear in vivo evidence that an apoptotic molecule can indeed regulate axon targeting and that OSNs can restore their organization even after broad disruption. PMID:21849551

  3. Imaging Axonal Transport of Mitochondria

    PubMed Central

    Wang, Xinnan; Schwarz, Thomas L.

    2010-01-01

    Neuronal mitochondria need to be transported and distributed in axons and dendrites in order to ensure an adequate energy supply and provide sufficient Ca2+ buffering in each portion of these highly extended cells. Errors in mitochondrial transport are implicated in neurodegenerative diseases. Here we present useful tools to analyze axonal transport of mitochondria both in vitro in cultured rat neurons and in vivo in Drosophila larval neurons. These methods enable investigators to take advantage of both systems to study the properties of mitochondrial motility under normal or pathological conditions. PMID:19426876

  4. Asthma as an axon reflex.

    PubMed

    Barnes, P J

    1986-02-01

    In asthma, damage to airway epithelium, possibly caused by eosinophil products, exposes C-fibre afferent nerve endings. Stimulation of these endings by inflammatory mediators such as bradykinin may result in an axon (local) reflex with antidromic conduction down afferent nerve collaterals and release of sensory neuropeptides such as substance P, neurokinin A, and calcitonin gene-related peptide. These peptides are potent inducers of airway smooth muscle contraction, bronchial oedema, extravasation of plasma, mucus hypersecretion, and possibly inflammatory cell infiltration and secretion. Thus, axon reflexes could account for at least some of the pathophysiology of asthma and this concept might lead to new strategies for treatment. PMID:2418322

  5. Optofluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Ordonez, Simon; Black, Bryan; Mohanty, Samarendra K.

    2013-03-01

    Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc) methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite particles were placed separately in one chamber and single particle was transported through microfluidic channel to another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.

  6. BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori.

    PubMed

    Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

    2016-02-15

    Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm. PMID:26625973

  7. Passive microfluidic chamber for long-term imaging of axon guidance in response to soluble gradients.

    PubMed

    Taylor, A M; Menon, S; Gupton, S L

    2015-07-01

    Understanding how axons are guided to target locations within the brain is of fundamental importance for neuroscience, and is a widely studied area of research. Biologists have an unmet need for reliable and easily accessible methods that generate stable, soluble molecular gradients for the investigation of axon guidance. Here we developed a microfluidic device with contiguous media-filled compartments that uses gravity-driven flow to generate a stable and highly reproducible gradient within a viewing compartment only accessible to axons. This device uses high-resistance microgrooves to both direct the growth of axons into an isolated region and to generate a stable gradient within the fluidically isolated axon viewing compartment for over 22 h. Establishing a stable gradient relies on a simple and quick pipetting procedure with no external pump or tubing. Since the axons extend into the axonal compartment through aligned microgrooves, the analysis of turning is simplified. Further, the multiple microgrooves in parallel alignment serve to increase sample sizes, improving statistical analyses. We used this method to examine growth cone turning in response to the secreted axon guidance cue netrin-1. We report the novel finding that growth cones of embryonic mouse cortical axons exhibited attractive turning in the lower concentrations of netrin-1, but were repulsed when exposed to higher concentrations. We also performed immunocytochemistry in growth cones exposed to a netrin-1 gradient within the axon viewing compartment and show that netrin receptors associated with both attraction and repulsion, DCC and UNC5H, localized to these growth cones. Together, we developed an accessible gradient chamber for higher throughput axon guidance studies and demonstrated its capabilities. PMID:26000554

  8. N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development.

    PubMed

    Brusés, Juan L

    2011-06-15

    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. PMID:21452216

  9. N-cadherin regulates primary motor axons growth and branching during zebrafish embryonic development

    PubMed Central

    Brusés, Juan L

    2013-01-01

    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type specific pathway selection. Analysis of N-cadherin mutants (cdh2hi3644Tg) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ~40% of the somitic hemisegments, and an ~150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point which abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to abnormally stall at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin depleted embryos the majority of primary motor axons innervated their appropriate myotomal territories indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. PMID:21452216

  10. The gene ten-1 contributes to axon regeneration accuracy following femtosecond laser axotomy in C. elegans

    NASA Astrophysics Data System (ADS)

    Stevens, Dylan T.; Mathew, Manoj; Goksör, Mattias; Pilon, Marc

    2012-10-01

    The precise cutting of axons in C. elegans using short laser pulses permits the investigation of parameters that may influence axonal regeneration. This study began by building and optimizing a femtosecond laser axotomy setup that we first used to monitor the effect of cutting axons near or far from the cell body of the PLM mechanosensory neurons in C. elegans. To assess regeneration, we developed a scoring system where the angle between the regenerating trajectory and its direct line to the target is measured; we called this measurement the "angle of regeneration". The results indicate that axons cut near the cell body regenerate better than those cut far from the cell body but nearer their target. The role of teneurins, which are transmembrane proteins with a large extracellular domain that are thought to regulate the remodelling of the extracellular matrix, has not yet been explored as a potential contributor to axon regeneration. We cut PLM axons in wild-type or ten-1 mutant worms, and measured the angle of regeneration 48 hours later, and the frequency of reconnection to the target. Our results show that functional ten-1 contributes to successful axon regeneration.

  11. Importance of timing of olfactory receptor-axon outgrowth for glomerulus development in Manduca sexta.

    PubMed

    Rössler, W; Tolbert, L P; Hildebrand, J G

    2000-09-18

    In the moth Manduca sexta, development of glomeruli in the antennal (olfactory) lobes (ALs) follows a precise timetable and involves interactions of olfactory receptor cell (ORC) axons with AL glial cells and neurons. To study the importance of timing for these intercellular interactions, we experimentally desynchronized the development of the ALs and the ORCs by altering the temperature of the developing antenna and brain for defined periods of time during development. Selective cooling of the antenna relative to the body resulted in a delay of ORC-axon outgrowth, and slightly warming the antenna while cooling the body caused precocious ingrowth of axons into the AL. Whereas cooling of the antenna for 24 hours caused only a delay in the formation of glomeruli, cooling for 48 hours led to significant disruption of glomerular development. Glial cells did not form normal glomerular borders, and glomeruli were shaped abnormally. Axons of pheromone-specific ORCs projected to their correct target, but terminal branches within the macroglomerular complex (MGC) were not clearly segregated. The results suggest that proper formation of glial glomerular borders requires interaction of ORC axons and glial cells within a sensitive period, whereas targeting of ORC axons appears to be effective over extended periods in development. Precocious ingrowth of ORC axons after warming the antenna and cooling the body for 48 hours resulted in enlarged protoglomeruli. Glial borders formed normally, but a subpopulation of MGC-specific ORC axons grew past the MGC. The decreased accuracy of targeting in these cases suggests that targeting mechanisms are not fully developed before the time when ORC axons normally would enter the brain. PMID:10954842

  12. CLUSTERED K+ CHANNEL COMPLEXES IN AXONS

    PubMed Central

    Rasband, Matthew N.

    2010-01-01

    Voltage-gated K+ (Kv) channels regulate diverse neuronal properties including action potential threshold, amplitude, and duration, frequency of firing, neurotransmitter release, and resting membrane potential. In axons, Kv channels are clustered at a variety of functionally important sites including axon initial segments, juxtaparanodes of myelinated axons, nodes of Ranvier, and cerebellar basket cell terminals. These channels are part of larger protein complexes that include cell adhesion molecules and scaffolding proteins. These interacting proteins play important roles in recruiting K+ channels to distinct axonal domains. Here, I review the composition, functions, and mechanism of localization of these K+ channel complexes in axons. PMID:20816921

  13. Netrin1-DCC-Mediated Attraction Guides Post-Crossing Commissural Axons in the Hindbrain

    PubMed Central

    Shoja-Taheri, Farnaz; DeMarco, Arielle

    2015-01-01

    Commissural axons grow along precise trajectories that are guided by several cues secreted from the ventral midline. After initial attraction to the floor plate using Netrin1 activation of its main attractive receptor, DCC (deleted in colorectal cancer), axons cross the ventral midline, and many turn to grow longitudinally on the contralateral side. After crossing the midline, axons are thought to lose their responsiveness to Netrin1 and become sensitive to midline Slit-Robo repulsion. We aimed to address the in vivo significance of Netrin1 in guiding post-crossing axon trajectories in mouse embryos. Surprisingly, in contrast to the spinal cord, Netrin1 and DCC mutants had abundant commissural axons crossing in the hindbrain. In Netrin1 and DCC mutants, many post-crossing axons made normal turns to grow longitudinally, but projected abnormally at angles away from the midline. In addition, exposure of cultured hindbrain explants to ectopic Netrin1 caused attractive deflection of post-crossing axons. Thus, Netrin1-DCC signaling is not required to attract pre-crossing axons toward the hindbrain floor plate, but is active in post-crossing guidance. Also in contrast with spinal cord, analysis of hindbrain post-crossing axons in Robo1/2 mutant embryos showed that Slit-Robo repulsive signaling was not required for post-crossing trajectories. Our findings show that Netrin1-DCC attractive signaling, but not Slit-Robo repulsive signaling, remains active in hindbrain post-crossing commissural axons to guide longitudinal trajectories, suggesting surprising regional diversity in commissural axon guidance mechanisms. SIGNIFICANCE STATEMENT The left and right sides of the brainstem and spinal cord are connected primarily by axon fibers that grow across the ventral midline, and then away on the other side to their targets. Based on spinal cord, axons are initially attracted by diffusible attractive protein signals to approach and cross the midline, and then are thought to switch

  14. Soluble adenylyl cyclase is not required for axon guidance to netrin-1.

    PubMed

    Moore, Simon W; Lai Wing Sun, Karen; Xie, Fang; Barker, Philip A; Conti, Marco; Kennedy, Timothy E

    2008-04-01

    During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1. PMID:18400890

  15. N-docosahexaenoylethanolamine regulates Hedgehog signaling and promotes growth of cortical axons

    PubMed Central

    Kharebava, Giorgi; Rashid, Mohammad A.; Lee, Ji-Won; Sarkar, Sarmila; Kevala, Karl; Kim, Hee-Yong

    2015-01-01

    ABSTRACT Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3) that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine), an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1) transcription and sonic hedgehog (Shh) target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO) agonist (SAG) or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway. PMID:26545965

  16. NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System

    PubMed Central

    Leung, Louis C.; Harris, William A.; Holt, Christine E.; Piper, Michael

    2015-01-01

    Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway. PMID:26489017

  17. Human intraretinal myelination: Axon diameters and axon/myelin thickness ratios

    PubMed Central

    FitzGibbon, Thomas; Nestorovski, Zoran

    2013-01-01

    Purpose: Human intraretinal myelination of ganglion cell axons occurs in about 1% of the population. We examined myelin thickness and axon diameter in human retinal specimens containing myelinated retinal ganglion cell axons. Materials and Methods: Two eyes containing myelinated patches were prepared for electron microscopy. Two areas were examined in one retina and five in the second retina. Measurements were compared to normal retinal and optic nerve samples and the rabbit retina, which normally contains myelinated axons. Measurements were made using a graphics tablet. Results: Mean axon diameter of myelinated axons at all locations were significantly larger than unmyelinated axons (P ≤ 0.01). Myelinated axons within the patches were significantly larger than axons within the optic nerve (P < 0.01). The relationship between axon diameter/fiber diameter (the G-ratio) seen in the retinal sites differed from that in the nerve. G-ratios were higher and myelin thickness was positively correlated to axon diameter (P < 0.01) in the retina but negatively correlated to axon diameter in the nerve (P < 0.001). Conclusion: Intraretinally myelinated axons are larger than non-myelinated axons from the same population and suggests that glial cells can induce diameter changes in retinal axons that are not normally myelinated. This effect is more dramatic on intraretinal axons compared with the normal transition zone as axons enter the optic nerve and these changes are abnormal. Whether intraretinal myelin alters axonal conduction velocity or blocks axonal conduction remains to be clarified and these issues may have different clinical outcomes. PMID:24212308

  18. Axon degeneration: context defines distinct pathways.

    PubMed

    Geden, Matthew J; Deshmukh, Mohanish

    2016-08-01

    Axon degeneration is an essential part of development, plasticity, and injury response and has been primarily studied in mammalian models in three contexts: 1) Axotomy-induced Wallerian degeneration, 2) Apoptosis-induced axon degeneration (axon apoptosis), and 3) Axon pruning. These three contexts dictate engagement of distinct pathways for axon degeneration. Recent advances have identified the importance of SARM1, NMNATs, NAD+ depletion, and MAPK signaling in axotomy-induced Wallerian degeneration. Interestingly, apoptosis-induced axon degeneration and axon pruning have many shared mechanisms both in signaling (e.g. DLK, JNKs, GSK3α/β) and execution (e.g. Puma, Bax, caspase-9, caspase-3). However, the specific mechanisms by which caspases are activated during apoptosis versus pruning appear distinct, with apoptosis requiring Apaf-1 but not caspase-6 while pruning requires caspase-6 but not Apaf-1. PMID:27197022

  19. Pioneer Axon Navigation Is Controlled by AEX-3, a Guanine Nucleotide Exchange Factor for RAB-3 in Caenorhabditis elegans.

    PubMed

    Bhat, Jaffar M; Hutter, Harald

    2016-07-01

    Precise and accurate axon tract formation is an essential aspect of brain development. This is achieved by the migration of early outgrowing axons (pioneers) allowing later outgrowing axons (followers) to extend toward their targets in the embryo. In Caenorhabditis elegans the AVG neuron pioneers the right axon tract of the ventral nerve cord, the major longitudinal axon tract. AVG is essential for the guidance of follower axons and hence organization of the ventral nerve cord. In an enhancer screen for AVG axon guidance defects in a nid-1/Nidogen mutant background, we isolated an allele of aex-3 aex-3 mutant animals show highly penetrant AVG axon navigation defects. These defects are dependent on a mutation in nid-1/Nidogen, a basement membrane component. Our data suggest that AEX-3 activates RAB-3 in the context of AVG axon navigation. aex-3 genetically acts together with known players of vesicular exocytosis: unc-64/Syntaxin, unc-31/CAPS, and ida-1/IA-2. Furthermore our genetic interaction data suggest that AEX-3 and the UNC-6/Netrin receptor UNC-5 act in the same pathway, suggesting AEX-3 might regulate the trafficking and/or insertion of UNC-5 at the growth cone to mediate the proper guidance of the AVG axon. PMID:27116976

  20. Hts, the Drosophila homologue of Adducin, physically interacts with the transmembrane receptor Golden goal to guide photoreceptor axons.

    PubMed

    Ohler, Stephan; Hakeda-Suzuki, Satoko; Suzuki, Takashi

    2011-01-01

    Neurons steer their axons towards their proper targets during development. Molecularly, a number of guidance receptors have been identified. The transmembrane protein Golden goal (Gogo) was reported previously to guide photoreceptor (R) axons in the Drosophila visual system. Here, we show that Hts, the Drosophila homologue of Adducin, physically interacts with Gogo's cytoplasmic domain via its head-neck domain. hts null mutants show similar defects in R axon guidance as do gogo mutants. Rescue experiments suggest that the C-terminal tail but not the MARCKS homology domain of Hts is required. Overexpression of either gogo or hts causes abnormally thick swellings of R8 axons in the medulla, but if both are co-overexpressed, R8 axons appear normal and the amount of excessive Hts is reduced. Our results fit with a model where Gogo both positively and negatively regulates Hts that affects the Actin-Spectrin cytoskeleton in growth cone filopodia, thereby guiding R axons. PMID:21128303

  1. Promoting axon regeneration in the adult CNS by modulation of the melanopsin/GPCR signaling

    PubMed Central

    Li, Songshan; Yang, Chao; Zhang, Li; Gao, Xin; Wang, Xuejie; Liu, Wen; Wang, Yuqi; Jiang, Songshan; Wong, Yung Hou; Zhang, Yifeng; Liu, Kai

    2016-01-01

    Cell-type–specific G protein-coupled receptor (GPCR) signaling regulates distinct neuronal responses to various stimuli and is essential for axon guidance and targeting during development. However, its function in axonal regeneration in the mature CNS remains elusive. We found that subtypes of intrinsically photosensitive retinal ganglion cells (ipRGCs) in mice maintained high mammalian target of rapamycin (mTOR) levels after axotomy and that the light-sensitive GPCR melanopsin mediated this sustained expression. Melanopsin overexpression in the RGCs stimulated axonal regeneration after optic nerve crush by up-regulating mTOR complex 1 (mTORC1). The extent of the regeneration was comparable to that observed after phosphatase and tensin homolog (Pten) knockdown. Both the axon regeneration and mTOR activity that were enhanced by melanopsin required light stimulation and Gq/11 signaling. Specifically, activating Gq in RGCs elevated mTOR activation and promoted axonal regeneration. Melanopsin overexpression in RGCs enhanced the amplitude and duration of their light response, and silencing them with Kir2.1 significantly suppressed the increased mTOR signaling and axon regeneration that were induced by melanopsin. Thus, our results provide a strategy to promote axon regeneration after CNS injury by modulating neuronal activity through GPCR signaling. PMID:26831088

  2. Polarized domains of myelinated axons.

    PubMed

    Salzer, James L

    2003-10-01

    The entire length of myelinated axons is organized into a series of polarized domains that center around nodes of Ranvier. These domains, which are crucial for normal saltatory conduction, consist of distinct multiprotein complexes of cell adhesion molecules, ion channels, and scaffolding molecules; they also differ in their diameter, organelle content, and rates of axonal transport. Juxtacrine signals from myelinating glia direct their sequential assembly. The composition, mechanisms of assembly, and function of these molecular domains will be reviewed. I also discuss similarities of this domain organization to that of polarized epithelia and present emerging evidence that disorders of domain organization and function contribute to the axonopathies of myelin and other neurologic disorders. PMID:14556710

  3. Pretarget sorting of retinocollicular axons in the mouse.

    PubMed

    Plas, Daniel T; Lopez, Joshua E; Crair, Michael C

    2005-10-31

    The map of the retina onto the optic tectum is a highly conserved feature of the vertebrate visual system; the mechanism by which this mapping is accomplished during development is a long-standing problem of neurobiology. The early suggestion by Roger Sperry that the map is formed through interactions between retinal ganglion cell axons and target cells within the tectum has gained significant experimental support and widespread acceptance. Nonetheless, reports in a variety of species indicate that some aspects of retinotopic order exist within the optic tract, leading to the suggestion that this "preordering" of retinal axons may play a role in the formation of the mature tectal map. A satisfactory account of pretarget order must provide the mechanism by which such axon order develops. Insofar as this mechanism must ultimately be determined genetically, the mouse suggests itself as the natural species in which to pursue these studies. Quantitative and repeatable methods are required to assess the contribution of candidate genes in mouse models. For these reasons, we have undertaken a quantitative study of the degree of retinotopic order within the optic tract and nerve of wild-type mice both before and after the development of the retinotectal map. Our methods are based on tract tracing using lipophilic dyes, and our results indicate that there is a reestablishment of dorsoventral but not nasotemporal retinal order when the axons pass through the chiasm and that this order is maintained throughout the subsequent tract. Furthermore, this dorsoventral retinotopic order is well established by the day after birth, long before the final target zone is discernible within the tectum. We conclude that pretarget sorting of axons according to origin along the dorsoventral axis of the retina is both spatially and chronologically appropriate to contribute to the formation of the retinotectal map, and we suggest that these methods be used to search for the molecular basis of

  4. MicroRNA 146a locally mediates distal axonal growth of dorsal root ganglia neurons under high glucose and sildenafil conditions.

    PubMed

    Jia, Longfei; Wang, Lei; Chopp, Michael; Zhang, Yi; Szalad, Alexandra; Zhang, Zheng Gang

    2016-08-01

    Axonal loss contributes to induction of diabetic peripheral neuropathy. Sildenafil, a phosphodiesterase type 5 inhibitor, ameliorates neurological dysfunction in diabetic peripheral neuropathy. However, the direct effect of high glucose and sildenafil on axonal growth has not been extensively investigated. Using rat primary dorsal root ganglia (DRG) neurons cultured in a microfluidic chamber, we investigated the effect of axonal application of high glucose and sildenafil on distal axonal growth. We found that axonal, but not cell body, application of high glucose locally inhibited distal axonal growth. However, axonal application of sildenafil overcame high glucose-reduced axonal growth. Quantitative real-time RT-PCR (qRT-PCR) and Western blot analysis of distal axonal samples revealed that high glucose reduced axonal miR-146a levels and substantially increased miR-146a target genes, IRAK1 and TRAF6 in the axon. In contrast, sildenafil significantly reversed high glucose-reduced miR-146a levels and high glucose-increased IRAK1 and TRAF6. Gain- and loss-of function of miR-146a in DRG neurons revealed that miR-146a mediated the local effect of high glucose on the distal axonal growth. These in vitro data provide new insights into molecular mechanisms of diabetic peripheral neuropathy. PMID:27167084

  5. On the Universality of Axon P Systems.

    PubMed

    Zhang, Xingyi; Pan, Linqiang; Paun, Andrei

    2015-11-01

    Axon P systems are computing models with a linear structure in the sense that all nodes (i.e., computing units) are arranged one by one along the axon. Such models have a good biological motivation: an axon in a nervous system is a complex information processor of impulse signals. Because the structure of axon P systems is linear, the computational power of such systems has been proved to be greatly restricted; in particular, axon P systems are not universal as language generators. It remains open whether axon P systems are universal as number generators. In this paper, we prove that axon P systems are universal as both number generators and function computing devices, and investigate the number of nodes needed to construct a universal axon P system. It is proved that four nodes (respectively, nine nodes) are enough for axon P systems to achieve universality as number generators (respectively, function computing devices). These results illustrate that the simple linear structure is enough for axon P systems to achieve a desired computational power. PMID:25680218

  6. The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

    PubMed Central

    Tienari, P J; De Strooper, B; Ikonen, E; Simons, M; Weidemann, A; Czech, C; Hartmann, T; Ida, N; Multhaup, G; Masters, C L; Van Leuven, F; Beyreuther, K; Dotti, C G

    1996-01-01

    We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP. Images PMID:8895567

  7. Human Genetic Disorders of Axon Guidance

    PubMed Central

    Engle, Elizabeth C.

    2010-01-01

    This article reviews symptoms and signs of aberrant axon connectivity in humans, and summarizes major human genetic disorders that result, or have been proposed to result, from defective axon guidance. These include corpus callosum agenesis, L1 syndrome, Joubert syndrome and related disorders, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia. Genes mutated in these disorders can encode axon growth cone ligands and receptors, downstream signaling molecules, and axon transport motors, as well as proteins without currently recognized roles in axon guidance. Advances in neuroimaging and genetic techniques have the potential to rapidly expand this field, and it is feasible that axon guidance disorders will soon be recognized as a new and significant category of human neurodevelopmental disorders. PMID:20300212

  8. Borderless regulates glial extension and axon ensheathment.

    PubMed

    Cameron, Scott; Chen, Yixu; Rao, Yong

    2016-06-15

    Ensheathment of axons by glial processes is essential for normal brain function. While considerable progress has been made to define molecular and cellular mechanisms underlying the maintenance of axon ensheathment, less is known about molecular details of early events for the wrapping of axons by glial processes in the developing nervous system. In this study, we investigate the role of the transmembrane protein Borderless (Bdl) in the developing Drosophila visual system. Bdl belongs to the immunoglobulin (Ig) superfamily, and its in vivo function is unknown. We show that Bdl is expressed in wrapping glia (WG) in the developing eye disc. Cell-type-specific transgene rescue and knockdown indicate that Bdl is specifically required in WG for the extension of glial processes along photoreceptor axons in the optic lobe, and axon ensheathment. Our results identify Bdl as a novel glia-specific cell-surface recognition molecule in regulating glial extension and axon ensheathment. PMID:27131624

  9. The influence of activity on axon pathfinding in the optic tectum.

    PubMed

    Kita, Elizabeth M; Scott, Ethan K; Goodhill, Geoffrey J

    2015-06-01

    The relative importance of neural activity versus activity-independent cues in shaping the initial wiring of the brain is still largely an open question. While activity is clearly critical for circuit rearrangements after initial connections have been made, whether it also plays a role in initial axon pathfinding remains to be determined. Here, we investigated this question using the guidance of zebrafish retinal ganglion cell axons to their targets in the tectum as a model. Recent results have implicated biased branching as a key feature of pathfinding in the zebrafish tectum. Using tetrodotoxin to silence neural activity globally, we found a decrease in the area covered by axon branches during pathfinding. After reaching the target, there were dynamic differences in axon length, area and the number of branches between conditions. However, other aspects of pathfinding were unaffected by silencing, including the ratio of branches directed toward the target, length, and number of branches, as well as turning angle, velocity, and number of growth cones per axon. These results challenge the hypothesis that neural connections develop in sequential stages of molecularly guided pathfinding and activity-based refinement. Despite a maintenance of overall guidance, axon pathfinding dynamics can nevertheless be altered by activity loss. PMID:25556913

  10. Regulation of axon regeneration by the RNA repair/splicing pathway

    PubMed Central

    Song, Yuanquan; Sretavan, David; Salegio, Ernesto A; Berg, Jim; Huang, Xi; Cheng, Tong; Xiong, Xin; Meltzer, Shan; Han, Chun; Nguyen, Trong-Tuong; Bresnahan, Jacqueline C.; Beattie, Michael S.; Jan, Lily Yeh; Jan, Yuh Nung

    2015-01-01

    Mechanisms governing a neuron’s regenerative ability are important but not well understood. We identified Rtca, RNA 3′-terminal phosphate cyclase, as an inhibitor for axon regeneration. Removal of dRtca cell-autonomously enhanced axon regrowth in the Drosophila central nervous system, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair/splicing pathway important for stress induced mRNA splicing, including that of Xbp1, a cellular stress sensor. dRtca and dArchease had opposing effects on Xbp1 splicing, and deficiency of dArchease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. PMID:25961792

  11. Ontophyletics of the nervous system: development of the corpus callosum and evolution of axon tracts.

    PubMed Central

    Katz, M J; Lasek, R J; Silver, J

    1983-01-01

    The evolution of nervous systems has included significant changes in the axon tracts of the central nervous system. These evolutionary changes required changes in axonal growth in embryos. During development, many axons reach their targets by following guidance cues that are organized as pathways in the embryonic substrate, and the overall pattern of the major axon tracts in the adult can be traced back to the fundamental pattern of such substrate pathways. Embryological and comparative anatomical studies suggest that most axon tracts, such as the anterior commissure, have evolved by the modified use of preexisting substrate pathways. On the other hand, recent developmental studies suggest that a few entirely new substrate pathways have arisen during evolution; these apparently provided opportunities for the formation of completely new axon tracts. The corpus callosum, which is found only in placental mammals, may be such a truly new axon tract. We propose that the evolution of the corpus callosum is founded on the emergence of a new preaxonal substrate pathway, the "glial sling," which bridges the two halves of the embryonic forebrain only in placental mammals. Images PMID:6577462

  12. The BMP roof plate chemorepellent regulates the rate of commissural axonal growth

    PubMed Central

    Phan, Keith D.; Hazen, Virginia M.; Frendo, Michele; Jia, Zhengping; Butler, Samantha J.

    2010-01-01

    Commissural spinal axons extend away from the roof plate (RP) in response to a chemorepellent mediated by the Bone Morphogenetic Proteins (BMPs). Previous studies have focused on the ability of commissural axons to translate a spatial gradient of BMPs into directional information in vitro. However, a notable feature of this system in vivo is that the gradient of BMPs is thought to act from behind the commissural cell bodies, making it possible for the BMPs to have a continued effect on commissural axons as they grow away from the RP. Here, we demonstrate that BMPs activate the cofilin regulator Limk1 to control the rate of commissural axon extension in the dorsal spinal cord. By modulating Limk1 activity in both rodent and chicken commissural neurons, the rate of axon growth can either be stalled or accelerated. Altering the activation state of Limk1 also influences subsequent guidance decisions: accelerated axons make rostrocaudal projection errors while navigating their intermediate target, the floor plate. These results suggest that guidance cues can specify information about the rate of growth, to ensure that axons reach subsequent signals either at particular times or speeds during development. PMID:21084599

  13. Serial Section Registration of Axonal Confocal Microscopy Datasets for Long-Range Neural Circuit Reconstruction

    SciTech Connect

    Hogrebe, Luke; Paiva, Antonio R.; Jurrus, Elizabeth R.; Christensen, Cameron; Bridge, Michael; Dai, Li; Pfeiffer, Rebecca; Hof, Patrick; Roysam, Badrinath; Korenberg, Julie; Tasdizen, Tolga

    2012-06-15

    In the context of long-range digital neural circuit reconstruction, this paper investigates an approach for registering axons across histological serial sections. Tracing distinctly labeled axons over large distances allows neuroscientists to study very explicit relationships between the brain's complex interconnects and, for example, diseases or aberrant development. Large scale histological analysis requires, however, that the tissue be cut into sections. In immunohistochemical studies thin sections are easily distorted due to the cutting, preparation, and slide mounting processes. In this work we target the registration of thin serial sections containing axons. Sections are first traced to extract axon centerlines, and these traces are used to define registration landmarks where they intersect section boundaries. The trace data also provides distinguishing information regarding an axon's size and orientation within a section. We propose the use of these features when pairing axons across sections in addition to utilizing the spatial relationships amongst the landmarks. The global rotation and translation of an unregistered section are accounted for using a random sample consensus (RANSAC) based technique. An iterative nonrigid refinement process using B-spline warping is then used to reconnect axons and produce the sought after connectivity information.

  14. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis.

    PubMed

    Zaimi, Aldo; Duval, Tanguy; Gasecka, Alicja; Côté, Daniel; Stikov, Nikola; Cohen-Adad, Julien

    2016-01-01

    Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg. PMID:27594833

  15. Demyelination increases axonal stationary mitochondrial size and the speed of axonal mitochondrial transport

    PubMed Central

    Kiryu-Seo, Sumiko; Ohno, Nobuhiko; Kidd, Grahame J.; Komuro, Hitoshi; Trapp, Bruce D.

    2010-01-01

    Axonal degeneration contributes to permanent neurological disability in inherited and acquired diseases of myelin. Mitochondrial dysfunction has been proposed as a major contributor to this axonal degeneration. It remains to be determined, however, if myelination, demyelination or remyelination alter the size and distribution of axonal mitochondrial stationary sites or the rates of axonal mitochondrial transport. Using live myelinated rat dorsal root ganglion (DRG) cultures, we investigated whether myelination and lysolecithin-induced demyelination affect axonal mitochondria. Myelination increased the size of axonal stationary mitochondrial sites by 2.3 fold. Following demyelination, the size of axonal stationary mitochondrial sites was increased by an additional 2.2 fold and the transport velocity of motile mitochondria was increased by 47%. These measures returned to the levels of myelinated axons following remyelination. Demyelination induced activating transcription factor (ATF) 3 in DRG neurons. Knockdown of neuronal ATF3 by shRNA abolished the demyelination-induced increase in axonal mitochondrial transport and increased nitrotyrosine immunoreactivity in axonal mitochondria, suggesting that neuronal ATF3 expression and increased mitochondrial transport protect demyelinated axons from oxidative damage. In response to insufficient ATP production, demyelinated axons increase the size of stationary mitochondrial sites and thereby balance ATP production with the increased energy needs of nerve conduction. PMID:20463228

  16. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis

    PubMed Central

    Zaimi, Aldo; Duval, Tanguy; Gasecka, Alicja; Côté, Daniel; Stikov, Nikola; Cohen-Adad, Julien

    2016-01-01

    Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg. PMID:27594833

  17. Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing

    PubMed Central

    Hernandez-Enriquez, Berenice; Wu, Zhuhao; Martinez, Edward; Olsen, Olav; Kaprielian, Zaven; Maness, Patricia F.; Yoshida, Yutaka; Tessier-Lavigne, Marc; Tran, Tracy S.

    2015-01-01

    Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons. PMID:26680304

  18. Development of a glia-rich axon-sorting zone in the olfactory pathway of the moth Manduca sexta.

    PubMed

    Rössler, W; Oland, L A; Higgins, M R; Hildebrand, J G; Tolbert, L P

    1999-11-15

    Olfactory receptor cells (ORCs) of a particular odor tuning are dispersed in the olfactory epithelium, but their axons converge on distinct glomeruli in primary olfactory centers. As a consequence, axon associations must change to bring axons of ORCs with the same odor specificity together. Studies in Manduca sexta have indicated that just before they enter the antennal lobe (AL), ORC axons undergo extreme reorganization, finally entering the AL in fascicles destined for subsets of glomeruli. This axon-sorting zone is heavily populated by glial cells, and ORC axon growth cones often are in close physical contact with the glia. In moths rendered glia deficient, ORC axons fail to fasciculate in this region. Using propidium iodide to label nuclei and 5-bromo-2'-deoxyuridine to monitor proliferation, we found that the glia in the sorting zone arise from the AL, appearing shortly after the first ORC axons arrive. Experimental removal of some or all of the sensory innervation revealed that proliferation of sorting-zone glia is triggered by ORC axons. A second set of glia arises in the antenna and migrates along the antennal nerve toward the brain, populating the nerve after the establishment of the sorting zone. Development of this type of glial cell is independent of contact of the ORC axons with their central targets. We conclude that the sorting zone arises from CNS glia in response to ingrowth of ORC axons, and a critical number of glia must be present in the sorting zone for axons to correctly establish new neighbor-neighbor associations. PMID:10559396

  19. Three-dimensional evaluation of retinal ganglion cell axon regeneration and pathfinding in whole mouse tissue after injury.

    PubMed

    Luo, Xueting; Salgueiro, Yadira; Beckerman, Samuel R; Lemmon, Vance P; Tsoulfas, Pantelis; Park, Kevin K

    2013-09-01

    Injured retinal ganglion cell (RGC) axons do not regenerate spontaneously, causing loss of vision in glaucoma and after trauma. Recent studies have identified several strategies that induce long distance regeneration in the optic nerve. Thus, a pressing question now is whether regenerating RGC axons can find their appropriate targets. Traditional methods of assessing RGC axon regeneration use histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. To unequivocally evaluate regenerating RGC axons, here we apply tissue clearance and light sheet fluorescence microscopy (LSFM) to image whole optic nerve and brain without physical sectioning. In mice with PTEN/SOCS3 deletion, a condition known to promote robust regeneration, axon growth followed tortuous paths through the optic nerve, with many axons reversing course and extending towards the eye. Such aberrant growth was prevalent in the proximal region of the optic nerve where strong astroglial activation is present. In the optic chiasms of PTEN/SOCS3 deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons project to the opposite optic nerve or to the ipsilateral optic tract. Following bilateral optic nerve crush, similar divergent trajectory is seen at the optic chiasm compared to unilateral crush. Centrally, axonal projection is limited predominantly to the hypothalamus. Together, we demonstrate the applicability of LSFM for comprehensive assessment of optic nerve regeneration, providing in-depth analysis of the axonal trajectory and pathfinding. Our study indicates significant axon misguidance in the optic nerve and brain, and underscores the need for investigation of axon guidance mechanisms during optic nerve regeneration in adults. PMID:23510761

  20. Competition with Primary Sensory Afferents Drives Remodeling of Corticospinal Axons in Mature Spinal Motor Circuits

    PubMed Central

    Jiang, Yu-Qiu; Zaaimi, Boubker

    2016-01-01

    Injury to the mature motor system drives significant spontaneous axonal sprouting instead of axon regeneration. Knowing the circuit-level determinants of axonal sprouting is important for repairing motor circuits after injury to achieve functional rehabilitation. Competitive interactions are known to shape corticospinal tract axon outgrowth and withdrawal during development. Whether and how competition contributes to reorganization of mature spinal motor circuits is unclear. To study this question, we examined plastic changes in corticospinal axons in response to two complementary proprioceptive afferent manipulations: (1) enhancing proprioceptive afferents activity by electrical stimulation; or (2) diminishing their input by dorsal rootlet rhizotomy. Experiments were conducted in adult rats. Electrical stimulation produced proprioceptive afferent sprouting that was accompanied by significant corticospinal axon withdrawal and a decrease in corticospinal connections on cholinergic interneurons in the medial intermediate zone and C boutons on motoneurons. In contrast, dorsal rootlet rhizotomy led to a significant increase in corticospinal connections, including those on cholinergic interneurons; C bouton density increased correspondingly. Motor cortex-evoked muscle potentials showed parallel changes to those of corticospinal axons, suggesting that reciprocal corticospinal axon changes are functional. Using the two complementary models, we showed that competitive interactions between proprioceptive and corticospinal axons are an important determinant in the organization of mature corticospinal axons and spinal motor circuits. The activity- and synaptic space-dependent properties of the competition enables prediction of the remodeling of spared corticospinal connection and spinal motor circuits after injury and informs the target-specific control of corticospinal connections to promote functional recovery. SIGNIFICANCE STATEMENT Neuroplasticity is limited in maturity

  1. Neuronal activity biases axon selection for myelination in vivo

    PubMed Central

    Hines, Jacob H.; Ravanelli, Andrew M.; Schwindt, Rani; Scott, Ethan K.; Appel, Bruce

    2015-01-01

    An essential feature of vertebrate neural development is ensheathment of axons with myelin, an insulating membrane formed by oligodendrocytes. Not all axons are myelinated, but mechanisms directing myelination of specific axons are unknown. Using zebrafish we show that activity-dependent secretion stabilizes myelin sheath formation on select axons. When VAMP2-dependent exocytosis is silenced in single axons, oligodendrocytes preferentially ensheath neighboring axons. Nascent sheaths formed on silenced axons are shorter in length, but when activity of neighboring axons is also suppressed, inhibition of sheath growth is relieved. Using in vivo time-lapse microscopy, we show that only 25% of oligodendrocyte processes that initiate axon wrapping are stabilized during normal development, and that initiation does not require activity. Instead, oligodendrocyte processes wrapping silenced axons are retracted more frequently. We propose that axon selection for myelination results from excessive and indiscriminate initiation of wrapping followed by refinement that is biased by activity-dependent secretion from axons. PMID:25849987

  2. The axonal transport of mitochondria

    PubMed Central

    Saxton, William M.; Hollenbeck, Peter J.

    2012-01-01

    Vigorous transport of cytoplasmic components along axons over substantial distances is crucial for the maintenance of neuron structure and function. The transport of mitochondria, which serves to distribute mitochondrial functions in a dynamic and non-uniform fashion, has attracted special interest in recent years following the discovery of functional connections among microtubules, motor proteins and mitochondria, and their influences on neurodegenerative diseases. Although the motor proteins that drive mitochondrial movement are now well characterized, the mechanisms by which anterograde and retrograde movement are coordinated with one another and with stationary axonal mitochondria are not yet understood. In this Commentary, we review why mitochondria move and how they move, focusing particularly on recent studies of transport regulation, which implicate control of motor activity by specific cell-signaling pathways, regulation of motor access to transport tracks and static microtubule–mitochondrion linkers. A detailed mechanism for modulating anterograde mitochondrial transport has been identified that involves Miro, a mitochondrial Ca2+-binding GTPase, which with associated proteins, can bind and control kinesin-1. Elements of the Miro complex also have important roles in mitochondrial fission–fusion dynamics, highlighting questions about the interdependence of biogenesis, transport, dynamics, maintenance and degradation. PMID:22619228

  3. Gray Matter Axonal Connectivity Maps

    PubMed Central

    Bonilha, Leonardo; Gleichgerrcht, Ezequiel; Nesland, Travis; Rorden, Chris; Fridriksson, Julius

    2015-01-01

    Structural brain connectivity is generally assessed through methods that rely on pre-defined regions of interest (e.g., Brodmann’s areas), thus preventing analyses that are largely free from a priori anatomical assumptions. Here, we introduce a novel and practical technique to evaluate a voxel-based measure of axonal projections connecting gray matter tissue [gray matter axonal connectivity map (GMAC)]. GMACs are compatible with voxel-based statistical approaches, and can be used to assess whole brain, scale-free, gray matter connectivity. In this study, we demonstrate how whole-brain GMACs can be generated from conventional structural connectome methodology, describing each step in detail, as well as providing tools to allow for the calculation of GMAC. To illustrate the utility of GMAC, we demonstrate the relationship between age and gray matter connectivity, using voxel-based analyses of GMAC. We discuss the potential role of GMAC in further analyses of cortical connectivity in healthy and clinical populations. PMID:25798111

  4. Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons.

    PubMed

    Pijak, D S; Hall, G F; Tenicki, P J; Boulos, A S; Lurie, D I; Selzer, M E

    1996-05-13

    It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 microns. Antibodies specific for highly phosphorylated NFs labeled only large axons (> 10 microns), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5-10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of

  5. Rapid signalling in distinct dopaminergic axons during locomotion and reward.

    PubMed

    Howe, M W; Dombeck, D A

    2016-07-28

    Dopaminergic projection axons from the midbrain to the striatum are crucial for motor control, as their degeneration in Parkinson disease results in profound movement deficits. Paradoxically, most recording methods report rapid phasic dopamine signalling (~100-ms bursts) in response to unpredicted rewards, with little evidence for movement-related signalling. The leading model posits that phasic signalling in striatum-targeting dopamine neurons drives reward-based learning, whereas slow variations in firing (tens of seconds to minutes) in these same neurons bias animals towards or away from movement. However, current methods have provided little evidence to support or refute this model. Here, using new optical recording methods, we report the discovery of rapid phasic signalling in striatum-targeting dopaminergic axons that is associated with, and capable of triggering, locomotion in mice. Axons expressing these signals were largely distinct from those that responded to unexpected rewards. These results suggest that dopaminergic neuromodulation can differentially impact motor control and reward learning with sub-second precision, and indicate that both precise signal timing and neuronal subtype are important parameters to consider in the treatment of dopamine-related disorders. PMID:27398617

  6. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

    Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  7. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  8. The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability.

    PubMed

    Cosker, Katharina E; Fenstermacher, Sara J; Pazyra-Murphy, Maria F; Elliott, Hunter L; Segal, Rosalind A

    2016-05-01

    To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (splicing factor, poly-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts in nuclei, cytoplasm and axons to regulate functionally related mRNAs essential for axon survival. Notably, SFPQ is required for coassembly of LaminB2 (Lmnb2) and Bclw (Bcl2l2) mRNAs in RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability. PMID:27019013

  9. The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

    PubMed

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I; Collins, Catherine A

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  10. Microtechnologies for studying the role of mechanics in axon growth and guidance

    PubMed Central

    Kilinc, Devrim; Blasiak, Agata; Lee, Gil U.

    2015-01-01

    The guidance of axons to their proper targets is not only a crucial event in neurodevelopment, but also a potential therapeutic target for neural repair. Axon guidance is mediated by various chemo- and haptotactic cues, as well as the mechanical interactions between the cytoskeleton and the extracellular matrix (ECM). Axonal growth cones, dynamic ends of growing axons, convert external stimuli to biochemical signals, which, in turn, are translated into behavior, e.g., turning or retraction, via cytoskeleton–matrix linkages. Despite the inherent mechanical nature of the problem, the role of mechanics in axon guidance is poorly understood. Recent years has witnessed the application of a range of microtechnologies in neurobiology, from microfluidic circuits to single molecule force spectroscopy. In this mini-review, we describe microtechnologies geared towards dissecting the mechanical aspects of axon guidance, divided into three categories: controlling the growth cone microenvironment, stimulating growth cones with externally applied forces, and measuring forces exerted by the growth cones. A particular emphasis is given to those studies that combine multiple techniques, as dictated by the complexity of the problem. PMID:26283918

  11. Commissural axons of the mouse cochlear nucleus.

    PubMed

    Brown, M Christian; Drottar, Marie; Benson, Thane E; Darrow, Keith

    2013-05-01

    The axons of commissural neurons that project from one cochlear nucleus to the other were studied after labeling with anterograde tracer. Injections were made into the dorsal subdivision of the cochlear nucleus in order to restrict labeling only to the group of commissural neurons that gave off collaterals to, or were located in, this subdivision. The number of labeled commissural axons in each injection was correlated with the number of labeled radiate multipolar neurons, suggesting radiate neurons as the predominant origin of the axons. The radiate commissural axons are thick and myelinated, and they exit the dorsal acoustic stria of the injected cochlear nucleus to cross the brainstem in the dorsal half, near the crossing position of the olivocochlear bundle. They enter the opposite cochlear nucleus via the dorsal and ventral acoustic stria and at its medial border. Reconstructions of single axons demonstrate that terminations are mostly in the core and typically within a single subdivision of the cochlear nucleus. Extents of termination range from narrow to broad along both the dorsoventral (i.e., tonotopic) and the rostrocaudal dimensions. In the electron microscope, labeled swellings form synapses that are symmetric (in that there is little postsynaptic density), a characteristic of inhibitory synapses. Our labeled axons do not appear to include excitatory commissural axons that end in edge regions of the nucleus. Radiate commissural axons could mediate the broadband inhibition observed in responses to contralateral sound, and they may balance input from the two ears with a quick time course. PMID:23124982

  12. Cable energy function of cortical axons

    PubMed Central

    Ju, Huiwen; Hines, Michael L.; Yu, Yuguo

    2016-01-01

    Accurate estimation of action potential (AP)-related metabolic cost is essential for understanding energetic constraints on brain connections and signaling processes. Most previous energy estimates of the AP were obtained using the Na+-counting method, which seriously limits accurate assessment of metabolic cost of ionic currents that underlie AP conduction along the axon. Here, we first derive a full cable energy function for cortical axons based on classic Hodgkin-Huxley (HH) neuronal equations and then apply the cable energy function to precisely estimate the energy consumption of AP conduction along axons with different geometric shapes. Our analytical approach predicts an inhomogeneous distribution of metabolic cost along an axon with either uniformly or nonuniformly distributed ion channels. The results show that the Na+-counting method severely underestimates energy cost in the cable model by 20–70%. AP propagation along axons that differ in length may require over 15% more energy per unit of axon area than that required by a point model. However, actual energy cost can vary greatly depending on axonal branching complexity, ion channel density distributions, and AP conduction states. We also infer that the metabolic rate (i.e. energy consumption rate) of cortical axonal branches as a function of spatial volume exhibits a 3/4 power law relationship. PMID:27439954

  13. Cable energy function of cortical axons.

    PubMed

    Ju, Huiwen; Hines, Michael L; Yu, Yuguo

    2016-01-01

    Accurate estimation of action potential (AP)-related metabolic cost is essential for understanding energetic constraints on brain connections and signaling processes. Most previous energy estimates of the AP were obtained using the Na(+)-counting method, which seriously limits accurate assessment of metabolic cost of ionic currents that underlie AP conduction along the axon. Here, we first derive a full cable energy function for cortical axons based on classic Hodgkin-Huxley (HH) neuronal equations and then apply the cable energy function to precisely estimate the energy consumption of AP conduction along axons with different geometric shapes. Our analytical approach predicts an inhomogeneous distribution of metabolic cost along an axon with either uniformly or nonuniformly distributed ion channels. The results show that the Na(+)-counting method severely underestimates energy cost in the cable model by 20-70%. AP propagation along axons that differ in length may require over 15% more energy per unit of axon area than that required by a point model. However, actual energy cost can vary greatly depending on axonal branching complexity, ion channel density distributions, and AP conduction states. We also infer that the metabolic rate (i.e. energy consumption rate) of cortical axonal branches as a function of spatial volume exhibits a 3/4 power law relationship. PMID:27439954

  14. Early events in axon/dendrite polarization.

    PubMed

    Cheng, Pei-lin; Poo, Mu-ming

    2012-01-01

    Differentiation of axons and dendrites is a critical step in neuronal development. Here we review the evidence that axon/dendrite formation during neuronal polarization depends on the intrinsic cytoplasmic asymmetry inherited by the postmitotic neuron, the exposure of the neuron to extracellular chemical factors, and the action of anisotropic mechanical forces imposed by the environment. To better delineate the functions of early signals among a myriad of cellular components that were shown to influence axon/dendrite formation, we discuss their functions by distinguishing their roles as determinants, mediators, or modulators and consider selective degradation of these components as a potential mechanism for axon/dendrite polarization. Finally, we examine whether these early events of axon/dendrite formation involve local autocatalytic activation and long-range inhibition, as postulated by Alan Turing for the morphogenesis of patterned biological structure. PMID:22715881

  15. Dynamics of Mitochondrial Transport in Axons.

    PubMed

    Niescier, Robert F; Kwak, Sang Kyu; Joo, Se Hun; Chang, Karen T; Min, Kyung-Tai

    2016-01-01

    The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons. PMID:27242435

  16. Dynamics of Mitochondrial Transport in Axons

    PubMed Central

    Niescier, Robert F.; Kwak, Sang Kyu; Joo, Se Hun; Chang, Karen T.; Min, Kyung-Tai

    2016-01-01

    The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons. PMID:27242435

  17. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

    PubMed Central

    Salinas, Patricia C.

    2015-01-01

    Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling. PMID:26252776

  18. Mapping mean axon diameter and axonal volume fraction by MRI using temporal diffusion spectroscopy

    PubMed Central

    Xu, Junzhong; Li, Hua; Harkins, Kevin D.; Jiang, Xiaoyu; Xie, Jingping; Kang, Hakmook; Does, Mark D.; Gore, John C.

    2014-01-01

    Mapping mean axon diameter and intra-axonal volume fraction may have significant clinical potential because nerve conduction velocity is directly dependent on axon diameter, and several neurodegenerative diseases affect axons of specific sizes and alter axon counts. Diffusion-weighted MRI methods based on the pulsed gradient spin echo (PGSE) sequence have been reported to be able to assess axon diameter and volume fraction non-invasively. However, due to the relatively long diffusion times used, e.g. > 20 ms, the sensitivity to small axons (diameter < 2 µm) is low, and the derived mean axon diameter has been reported to be overestimated. In the current study, oscillating gradient spin echo (OGSE) diffusion sequences with variable frequency gradients were used to assess rat spinal white matter tracts with relatively short effective diffusion times (1 – 5 ms). In contrast to previous PGSE-based methods, the extra-axonal diffusion cannot be modeled as hindered (Gaussian) diffusion when short diffusion times are used. Appropriate frequency-dependent rates are therefore incorporated into our analysis and validated by histology-based computer simulation of water diffusion. OGSE data were analyzed to derive mean axon diameters and intra-axonal volume fractions of rat spinal white matter tracts (mean axon diameter ~ 1.27 – 5.54 µm). The estimated values were in good agreement with histology, including the small axon diameters (< 2.5 µm). This study establishes a framework for quantification of nerve morphology using the OGSE method with high sensitivity to small axons. PMID:25225002

  19. Why do axons differ in caliber?

    PubMed Central

    Perge, János A.; Niven, Jeremy E.; Mugnaini, Enrico; Balasubramanian, Vijay; Sterling, Peter

    2012-01-01

    CNS axons differ in diameter (d) by nearly 100-fold (~ 0.1 to 10μm); therefore they differ in cross-sectional area (d2) and volume by nearly 10,000-fold. If, as found for optic nerve, mitochondrial volume-fraction is constant with axon diameter, energy capacity would rise with axon volume, also as d2. Given constraints on space and energy, we asked what functional requirements set an axon’s diameter? Surveying 16 fiber groups spanning nearly the full range of diameters in five species (guinea pig, rat, monkey, locust, octopus), we found that: (i) thin axons are most numerous; (ii) mean firing frequencies, estimated for 9 of the identified axon classes, are low for thin fibers and high for thick ones, ranging from ~1 to >100Hz; (iii) a tract’s distribution of fiber diameters, whether narrow or broad, and whether symmetric or skewed, reflects heterogeneity of information rates conveyed by its individual fibers; (iv) mitochondrial volume/axon length, rises ≥ d2. To explain the pressure towards thin diameters we note an established law of diminishing returns: an axon, to double its information rate, must more than double its firing rate. Since diameter is apparently linear with firing rate, doubling information rate would more than quadruple an axon’s volume and energy use. Thicker axons may be needed to encode features that cannot be efficiently decoded if their information is spread over several low-rate channels. Thus information rate may be the main variable that sets axon caliber - with axons constrained to deliver information at the lowest acceptable rate. PMID:22238098

  20. c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy

    PubMed Central

    Hantke, Janina; Carty, Lucy; Wagstaff, Laura J.; Turmaine, Mark; Wilton, Daniel K.; Quintes, Susanne; Koltzenburg, Martin; Baas, Frank; Mirsky, Rhona

    2014-01-01

    Charcot–Marie–Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot–Marie–Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot–Marie–Tooth disease type 1A. In line with our previous findings in humans with Charcot–Marie–Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun−/− mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot–Marie–Tooth disease type 1A: on the one hand they are the genetic source of

  1. Intraretinal projection of retinal ganglion cell axons as a model system for studying axon navigation

    PubMed Central

    Bao, Zheng-Zheng

    2008-01-01

    The initial step of retinal ganglion cell (RGC) axon pathfinding involves directed growth of RGC axons toward the center of the retina, the optic disc, a process termed “intraretinal guidance”. Due to the accessibility of the system, and with various embryological, molecular, and genetic approaches, significant progress has been made in recent years toward understanding the mechanisms involved in the precise guidance of the RGC axons. As axons are extending from RGCs located throughout the retina, a multitude of factors expressed along with the differentiation wave are important for the guidance of the RGC axons. To ensure that the RGC axons are oriented correctly, restricted to the optic fiber layer (OFL) of the retina, and exit the eye properly, different sets of positive and negative factors cooperate in the process. Fasciculation mediated by a number of cell adhesion molecules (CAMs) and modulation of axonal response to guidance factors provide additional mechanisms to ensure proper guidance of the RGC axons. The intraretinal axon guidance thus serves as an excellent model system for studying how different signals are regulated, modulated and integrated for guiding a large number of axons in three-dimensional space. PMID:17320832

  2. Emerging brain morphologies from axonal elongation

    PubMed Central

    Holland, Maria A.; Miller, Kyle E.; Kuhl, Ellen

    2015-01-01

    Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy - as an emergent property from directional axonal growth - intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function. PMID:25824370

  3. Cargo distributions differentiate pathological axonal transport impairments

    PubMed Central

    Mitchell, Cassie S.; Lee, Robert H.; Coulter, Wallace H.

    2012-01-01

    Axonal transport is an essential process in neurons, analogous to shipping goods, by which energetic and cellular building supplies are carried downstream (anterogradely) and wastes are carried upstream (retrogradely) by molecular motors, which act as cargo porters. Impairments in axonal transport have been linked to devastating and often lethal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntington’s, and Alzheimer’s. Axonal transport impairment types include a decrease in available motors for cargo transport (motor depletion), the presence of defective or non-functional motors (motor dilution), and the presence of increased or larger cargos (protein aggregation). An impediment to potential treatment identification has been the inability to determine what type(s) of axonal transport impairment candidates that could be present in a given disease. In this study, we utilize a computational model and common axonal transport experimental metrics to reveal the axonal transport impairment general characteristics or “signatures” that result from three general defect types of motor depletion, motor dilution, and protein aggregation. Our results not only provide a means to discern these general impairments types, they also reveal key dynamic and emergent features of axonal transport, which potentially underlie multiple impairment types. The identified characteristics, as well as the analytical method, can be used to help elucidate the axonal transport impairments observed in experimental and clinical data. For example, using the model-predicted defect signatures, we identify the defect candidates, which are most likely to be responsible for the axonal transport impairments in the G93A SOD1 mouse model of ALS. PMID:22285784

  4. Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured peripheral nervous system

    PubMed Central

    Farah, Mohamed H.; Pan, Bao Han; Hoffman, Paul N.; Ferraris, Dana; Tsukamoto, Takashi; Nguyen, Thien; Wong, Philip C.; Price, Donald L.; Slusher, Barbara S.; Griffin, John W.

    2012-01-01

    β- site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid β peptides that are present in plaques of Alzheimer's Disease. BACE1 has been an attractive target for drug development. In cultured embryonic neurons BACE1-cleaved N-terminal APP is further processed to generate a fragment that can trigger axonal degeneration, suggesting a vital role for BACE1 in axonal health. In addition, BACE1 cleaves neuregulin 1 type III, a protein critical for myelination of peripheral axons by Schwann cells during development. Here, we asked if axonal degeneration or axonal regeneration in adult nerves might be affected by inhibition or elimination of BACE1. We report that BACE1 knockout and wild-type nerves degenerated at a similar rate after axotomy and to a similar extent in the experimental neuropathies produced by administration of paclitaxel and acrylamide. These data indicate N-APP is not the sole culprit in axonal degeneration in adult nerves. Unexpectedly, however, we observed that BACE1 knockout mice had markedly enhanced clearance of axonal and myelin debris from degenerated fibers, accelerated axonal regeneration, and earlier reinnervation of neuromuscular junctions, compared to littermate controls. These observations were reproduced in part by pharmacological inhibition of BACE1. These data suggest BACE1 inhibition as a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage. PMID:21490216

  5. MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3β expression.

    PubMed

    Jiang, J-J; Liu, C-M; Zhang, B-Y; Wang, X-W; Zhang, M; Saijilafu; Zhang, S-R; Hall, P; Hu, Y-W; Zhou, F-Q

    2015-01-01

    MicroRNAs are emerging to be important epigenetic factors that control axon regeneration. Here, we report that microRNA-26a (miR-26a) is a physiological regulator of mammalian axon regeneration in vivo. We demonstrated that endogenous miR-26a acted to target specifically glycogen synthase kinase 3β (GSK3β) in adult mouse sensory neurons in vitro and in vivo. Inhibition of endogenous miR-26a in sensory neurons impaired axon regeneration in vitro and in vivo. Moreover, the regulatory effect of miR-26a was mediated by increased expression of GSK3β because downregulation or pharmacological inhibition of GSK3β fully rescued axon regeneration. Our results also suggested that the miR-26a-GSK3β pathway regulated axon regeneration at the neuronal soma by controlling gene expression. We provided biochemical and functional evidences that the regeneration-associated transcription factor Smad1 acted downstream of miR-26a and GSK3β to control sensory axon regeneration. Our study reveals a novel miR-26a-GSK3β-Smad1 signaling pathway in the regulation of mammalian axon regeneration. Moreover, we provide the first evidence that, in addition to inhibition of GSK3β kinase activity, maintaining a lower protein level of GSK3β in neurons by the microRNA is necessary for efficient axon regeneration. PMID:26313916

  6. Diapause Formation and Downregulation of Insulin-Like Signaling via DAF-16/FOXO Delays Axonal Degeneration and Neuronal Loss

    PubMed Central

    Calixto, Andrea; Jara, Juan S.; Court, Felipe A.

    2012-01-01

    Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO–dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment. PMID:23300463

  7. Variability and Reliabiltiy in Axon Growth Cone Navigation Decision Making

    NASA Astrophysics Data System (ADS)

    Garnelo, Marta; Ricoult, Sébastien G.; Juncker, David; Kennedy, Timothy E.; Faisal, Aldo A.

    2015-03-01

    The nervous system's wiring is a result of axon growth cones navigating through specific molecular environments during development. In order to reach their target, growth cones need to make decisions under uncertainty as they are faced with stochastic sensory information and probabilistic movements. The overall system therefore exhibits features of whole organisms (perception, decision making, action) in the subset of a single cell. We aim to characterise growth cone navigation in defined nano-dot guidance cue environments, by using the tools of computational neuroscience to conduct ``molecular psychophysics.'' We start with a generative model of growth cone behaviour and we 1. characterise sensory and internal sources of noise contributing to behavioural variables, by combining knowledge of the underlying stochastic dynamics in cue sensing and the growth of the cytoskeleton. This enables us to 2. produce bottom-up lower limit estimates of behavioural response reliability and visualise it as probability distributions over axon growth trajectories. Given this information we can match our in silico model's ``psychometric'' decision curves with empirical data. Finally we use a Monte-Carlo approach to predict response distributions of axon trajectories from our model.

  8. Genetic Dissection of the Function of Hindbrain Axonal Commissures

    PubMed Central

    Renier, Nicolas; Schonewille, Martijn; Giraudet, Fabrice; Badura, Aleksandra; Tessier-Lavigne, Marc; Avan, Paul; De Zeeuw, Chris I.; Chédotal, Alain

    2010-01-01

    In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre;Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes. PMID:20231872

  9. Dysregulated axonal RNA translation in amyotrophic lateral sclerosis.

    PubMed

    Yasuda, Kyota; Mili, Stavroula

    2016-09-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease that has been associated with a diverse array of genetic changes. Prominent among these are mutations in RNA-binding proteins (RBPs) or repeat expansions that give rise to toxic RNA species. RBPs are additionally central components of pathologic aggregates that constitute a disease hallmark, suggesting that dysregulation of RNA metabolism underlies disease progression. In the context of neuronal physiology, transport of RNAs and localized RNA translation in axons are fundamental to neuronal survival and function. Several lines of evidence suggest that axonal RNA translation is a central process perturbed by various pathogenic events associated with ALS. Dysregulated translation of specific RNA groups could underlie feedback effects that connect and reinforce disease manifestations. Among such candidates are RNAs encoding proteins involved in the regulation of microtubule dynamics. Further understanding of axonally dysregulated RNA targets and of the feedback mechanisms they induce could provide useful therapeutic insights. WIREs RNA 2016, 7:589-603. doi: 10.1002/wrna.1352 For further resources related to this article, please visit the WIREs website. PMID:27038103

  10. A model for fast axonal transport.

    PubMed

    Blum, J J; Reed, M C

    1985-01-01

    A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport. PMID:2416456

  11. Axonal and Schwann Cell BACE1 Is Equally Required for Remyelination of Peripheral Nerves

    PubMed Central

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce

    2015-01-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  12. Accelerating axonal growth promotes motor recovery after peripheral nerve injury in mice

    PubMed Central

    Ma, Chi Him Eddie; Omura, Takao; Cobos, Enrique J.; Latrémolière, Alban; Ghasemlou, Nader; Brenner, Gary J.; van Veen, Ed; Barrett, Lee; Sawada, Tomokazu; Gao, Fuying; Coppola, Giovanni; Gertler, Frank; Costigan, Michael; Geschwind, Dan; Woolf, Clifford J.

    2011-01-01

    Although peripheral nerves can regenerate after injury, proximal nerve injury in humans results in minimal restoration of motor function. One possible explanation for this is that injury-induced axonal growth is too slow. Heat shock protein 27 (Hsp27) is a regeneration-associated protein that accelerates axonal growth in vitro. Here, we have shown that it can also do this in mice after peripheral nerve injury. While rapid motor and sensory recovery occurred in mice after a sciatic nerve crush injury, there was little return of motor function after sciatic nerve transection, because of the delay in motor axons reaching their target. This was not due to a failure of axonal growth, because injured motor axons eventually fully re-extended into muscles and sensory function returned; rather, it resulted from a lack of motor end plate reinnervation. Tg mice expressing high levels of Hsp27 demonstrated enhanced restoration of motor function after nerve transection/resuture by enabling motor synapse reinnervation, but only within 5 weeks of injury. In humans with peripheral nerve injuries, shorter wait times to decompression surgery led to improved functional recovery, and, while a return of sensation occurred in all patients, motor recovery was limited. Thus, absence of motor recovery after nerve damage may result from a failure of synapse reformation after prolonged denervation rather than a failure of axonal growth. PMID:21965333

  13. Odorant receptors can mediate axonal identity and gene choice via cAMP-independent mechanisms

    PubMed Central

    Grosmaitre, Xavier; Feinstein, Paul

    2016-01-01

    Odorant receptors (ORs) control several aspects of cell fate in olfactory sensory neurons (OSNs), including singular gene choice and axonal identity. The mechanisms of OR-induced axon guidance have been suggested to principally rely on G-protein signalling. Here, we report that for a subset of OSNs, deleting G proteins or altering their levels of signalling does not affect axonal identity. Signalling-deficient ORs or surrogate receptors that are unable to couple to Gs/Golf still provide axons with distinct identities and the anterior–posterior targeting of axons does not correlate with the levels of cAMP produced by genetic modifications. In addition, we refine the models of negative feedback by showing that ectopic ORs can be robustly expressed without suppressing endogenous gene choice. In conclusion, our results uncover a new feature of ORs, showing that they can instruct axonal identity and regulate olfactory map formation independent of canonical G-protein signalling and cAMP production. PMID:27466441

  14. Prevention of Axonal Injury using Calpain Inhibitor in Chronic Progressive Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Hassen, Getaw Worku; Feliberti, Jason; Kesner, Leo; Stracher, Alfred; Mokhtarian, Foroozan

    2011-01-01

    Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS following relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Nav1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG) – induced disease model of MS]. Intraperitoneal application of CYLA (2mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis. PMID:18725211

  15. Axonal and Schwann cell BACE1 is equally required for remyelination of peripheral nerves.

    PubMed

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce; Yan, Riqiang

    2015-03-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  16. Odorant receptors can mediate axonal identity and gene choice via cAMP-independent mechanisms.

    PubMed

    Movahedi, Kiavash; Grosmaitre, Xavier; Feinstein, Paul

    2016-07-01

    Odorant receptors (ORs) control several aspects of cell fate in olfactory sensory neurons (OSNs), including singular gene choice and axonal identity. The mechanisms of OR-induced axon guidance have been suggested to principally rely on G-protein signalling. Here, we report that for a subset of OSNs, deleting G proteins or altering their levels of signalling does not affect axonal identity. Signalling-deficient ORs or surrogate receptors that are unable to couple to Gs/Golf still provide axons with distinct identities and the anterior-posterior targeting of axons does not correlate with the levels of cAMP produced by genetic modifications. In addition, we refine the models of negative feedback by showing that ectopic ORs can be robustly expressed without suppressing endogenous gene choice. In conclusion, our results uncover a new feature of ORs, showing that they can instruct axonal identity and regulate olfactory map formation independent of canonical G-protein signalling and cAMP production. PMID:27466441

  17. The neural adhesion molecule TAG-1 modulates responses of sensory axons to diffusible guidance signals.

    PubMed

    Law, Chris O; Kirby, Rebecca J; Aghamohammadzadeh, Soheil; Furley, Andrew J W

    2008-08-01

    When the axons of primary sensory neurons project into the embryonic mammalian spinal cord, they bifurcate and extend rostrocaudally before sending collaterals to specific laminae according to neuronal subclass. The specificity of this innervation has been suggested to be the result both of differential sensitivity to chemorepellants expressed in the ventral spinal cord and of the function of Ig-like neural cell adhesion molecules in the dorsal horn. The relationship between these mechanisms has not been addressed. Focussing on the pathfinding of TrkA+ NGF-dependent axons, we demonstrate for the first time that their axons project prematurely into the dorsal horn of both L1 and TAG-1 knockout mice. We show that axons lacking TAG-1, similar to those lacking L1, are insensitive to wild-type ventral spinal cord (VSC)-derived chemorepellants, indicating that adhesion molecule function is required in the axons, and that this loss of response is explained in part by loss of response to Sema3A. We present evidence that TAG-1 affects sensitivity to Sema3A by binding to L1 and modulating the endocytosis of the L1/neuropilin 1 Sema3A receptor complex. However, TAG-1 appears to affect sensitivity to other VSC-derived chemorepellants via an L1-independent mechanism. We suggest that this dependence of chemorepellant sensitivity on the functions of combinations of adhesion molecules is important to ensure that axons project via specific pathways before extending to their final targets. PMID:18550718

  18. Serial section registration of axonal confocal microscopy datasets for long-range neural circuit reconstruction

    PubMed Central

    Hogrebe, Luke; Paiva, Antonio R.C.; Jurrus, Elizabeth; Christensen, Cameron; Bridge, Michael; Dai, Li; Pfeiffer, Rebecca; Hof, Patrick R.; Roysam, Badrinath; Korenberg, Julie R.; Tasdizen, Tolga

    2014-01-01

    In the context of long-range digital neural circuit reconstruction, this paper investigates an approach for registering axons across histological serial sections. Tracing distinctly labeled axons over large distances allows neuroscientists to study very explicit relationships between the brain’s complex interconnects and, for example, diseases or aberrant development. Large scale histological analysis requires, however, that the tissue be cut into sections. In immunohistochemical studies thin sections are easily distorted due to the cutting, preparation, and slide mounting processes. In this work we target the registration of thin serial sections containing axons. Sections are first traced to extract axon centerlines, and these traces are used to define registration landmarks where they intersect section boundaries. The trace data also provides distinguishing information regarding an axon’s size and orientation within a section. We propose the use of these features when pairing axons across sections in addition to utilizing the spatial relationships among the landmarks. The global rotation and translation of an unregistered section are accounted for using a random sample consensus (RANSAC) based technique. An iterative nonrigid refinement process using B-spline warping is then used to reconnect axons and produce the sought after connectivity information. PMID:22465678

  19. Oxidized galectin-1 stimulates macrophages to promote axonal regeneration in peripheral nerves after axotomy.

    PubMed

    Horie, Hidenori; Kadoya, Toshihiko; Hikawa, Naoshi; Sango, Kazunori; Inoue, Hiroko; Takeshita, Kaori; Asawa, Reiko; Hiroi, Tomoko; Sato, Manami; Yoshioka, Tohru; Ishikawa, Yoshihiro

    2004-02-25

    Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration. PMID:14985427

  20. Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

    PubMed

    Ruschel, Jörg; Hellal, Farida; Flynn, Kevin C; Dupraz, Sebastian; Elliott, David A; Tedeschi, Andrea; Bates, Margaret; Sliwinski, Christopher; Brook, Gary; Dobrindt, Kristina; Peitz, Michael; Brüstle, Oliver; Norenberg, Michael D; Blesch, Armin; Weidner, Norbert; Bunge, Mary Bartlett; Bixby, John L; Bradke, Frank

    2015-04-17

    After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury. PMID:25765066

  1. Calcium/calmodulin-dependent protein kinase IIalpha in optic axons moves with slow axonal transport and undergoes posttranslational modification.

    PubMed

    Lund, L M; McQuarrie, I G

    2001-12-21

    In neurons, the mRNA for calcium/calmodulin-dependent protein kinase II alpha (CKIIalpha) is known to be targeted to dendrites-where the enzyme is synthesized and supports postsynaptic functions. We are interested in knowing how neuronal proteins enter axons from the nerve cell body, and the mechanism for protein transport to terminals. Because CKIIalpha immunofluorescence can be demonstrated in over 80% of retinal ganglion cells, we asked whether this regulatory protein is being transported into optic axons. Using Sprague-Dawley rats, [(35)S] methionine was injected into the vitreous humor of the eye. Four days later, the optic nerves, tracts, lateral geniculate ganglia, and superior colliculi were removed and processed for 2D-PAGE and Western blotting. Radiolabeled CKIIalpha appears to move with slow component b (SCb) of axonal transport, as is the case in rodent sciatic motor neurons. In addition, the radiolabeled CKIIalpha isoform that enters the optic nerve is found to be 4 kDa heavier (in SDS-PAGE molecular mass) than the isoform in the optic tract, superior colliculus, and lateral geniculate nucleus. This reduction is likely the result of dephosphorylation, which is a mechanism used to regulate the enzyme's activity. PMID:11741313

  2. Excitability tuning of axons in the central nervous system.

    PubMed

    Ohura, Shunsuke; Kamiya, Haruyuki

    2016-05-01

    The axon is a long neuronal process that originates from the soma and extends towards the presynaptic terminals. The pioneering studies on the squid giant axon or the spinal cord motoneuron established that the axon conducts action potentials faithfully to the presynaptic terminals with self-regenerative processes of membrane excitation. Recent studies challenged the notion that the fundamental understandings obtained from the study of squid giant axons are readily applicable to the axons in the mammalian central nervous system (CNS). These studies revealed that the functional and structural properties of the CNS axons are much more variable than previously thought. In this review article, we summarize the recent understandings of axon physiology in the mammalian CNS due to progress in the subcellular recording techniques which allow direct recordings from the axonal membranes, with emphasis on the hippocampal mossy fibers as a representative en passant axons typical for cortical axons. PMID:26493201

  3. Axon reflexes in human cold exposed fingers.

    PubMed

    Daanen, H A; Ducharme, M B

    2000-02-01

    Exposure of fingers to severe cold induces cold induced vasodilatation (CIVD). The mechanism of CIVD is still debated. The original theory states that an axon reflex causes CIVD. To test this hypothesis, axon reflexes were evoked by electrical stimulation of the middle fingers of hands immersed in water at either 5 degrees C or 35 degrees C. Axon reflexes were pronounced in the middle finger of the hand in warm water, but absent from the hand in cold water, even though the stimulation was rated as "rather painful" to "painful". These results showed that axon reflexes do not occur in a cold-exposed hand and thus are unlikely to explain the CIVD phenomenon. PMID:10638384

  4. CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins

    PubMed Central

    Chen, Lizhen; Liu, Zhijie; Zhou, Bing; Wei, Chaoliang; Zhou, Yu; Rosenfeld, Michael G; Fu, Xiang-Dong; Chisholm, Andrew D; Jin, Yishi

    2016-01-01

    Axon injury triggers dramatic changes in gene expression. While transcriptional regulation of injury-induced gene expression is widely studied, less is known about the roles of RNA binding proteins (RBPs) in post-transcriptional regulation during axon regeneration. In C. elegans the CELF (CUGBP and Etr-3 Like Factor) family RBP UNC-75 is required for axon regeneration. Using crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) we identify a set of genes involved in synaptic transmission as mRNA targets of UNC-75. In particular, we show that UNC-75 regulates alternative splicing of two mRNA isoforms of the SNARE Syntaxin/unc-64. In C. elegans mutants lacking unc-75 or its targets, regenerating axons form growth cones, yet are deficient in extension. Extending these findings to mammalian axon regeneration, we show that mouse Celf2 expression is upregulated after peripheral nerve injury and that Celf2 mutant mice are defective in axon regeneration. Further, mRNAs for several Syntaxins show CELF2 dependent regulation. Our data delineate a post-transcriptional regulatory pathway with a conserved role in regenerative axon extension. DOI: http://dx.doi.org/10.7554/eLife.16072.001 PMID:27253061

  5. Activity-dependent mismatch between axo-axonic synapses and the axon initial segment controls neuronal output

    PubMed Central

    Wefelmeyer, Winnie; Cattaert, Daniel; Burrone, Juan

    2015-01-01

    The axon initial segment (AIS) is a structure at the start of the axon with a high density of sodium and potassium channels that defines the site of action potential generation. It has recently been shown that this structure is plastic and can change its position along the axon, as well as its length, in a homeostatic manner. Chronic activity-deprivation paradigms in a chick auditory nucleus lead to a lengthening of the AIS and an increase in neuronal excitability. On the other hand, a long-term increase in activity in dissociated rat hippocampal neurons results in an outward movement of the AIS and a decrease in the cell’s excitability. Here, we investigated whether the AIS is capable of undergoing structural plasticity in rat hippocampal organotypic slices, which retain the diversity of neuronal cell types present at postnatal ages, including chandelier cells. These interneurons exclusively target the AIS of pyramidal neurons and form rows of presynaptic boutons along them. Stimulating individual CA1 pyramidal neurons that express channelrhodopsin-2 for 48 h leads to an outward shift of the AIS. Intriguingly, both the pre- and postsynaptic components of the axo-axonic synapses did not change position after AIS relocation. We used computational modeling to explore the functional consequences of this partial mismatch and found that it allows the GABAergic synapses to strongly oppose action potential generation, and thus downregulate pyramidal cell excitability. We propose that this spatial arrangement is the optimal configuration for a homeostatic response to long-term stimulation. PMID:26195803

  6. Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system.

    PubMed

    Deleuze, C; Runquist, M; Orcel, H; Rabié, A; Dayanithi, G; Alonso, G; Hussy, N

    2005-01-01

    Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments. PMID:16125853

  7. Delayed Feedback Model of Axonal Length Sensing

    PubMed Central

    Karamched, Bhargav R.; Bressloff, Paul C.

    2015-01-01

    A fundamental question in cell biology is how the sizes of cells and organelles are regulated at various stages of development. Size homeostasis is particularly challenging for neurons, whose axons can extend from hundreds of microns to meters (in humans). Recently, a molecular-motor-based mechanism for axonal length sensing has been proposed, in which axonal length is encoded by the frequency of an oscillating retrograde signal. In this article, we develop a mathematical model of this length-sensing mechanism in which advection-diffusion equations for bidirectional motor transport are coupled to a chemical signaling network. We show that chemical oscillations emerge due to delayed negative feedback via a Hopf bifurcation, resulting in a frequency that is a monotonically decreasing function of axonal length. Knockdown of either kinesin or dynein causes an increase in the oscillation frequency, suggesting that the length-sensing mechanism would produce longer axons, which is consistent with experimental findings. One major prediction of the model is that fluctuations in the transport of molecular motors lead to a reduction in the reliability of the frequency-encoding mechanism for long axons. PMID:25954897

  8. Mistargeting hippocampal axons by expression of a truncated Eph receptor

    PubMed Central

    Yue, Yong; Chen, Zhi-Yong; Gale, Nick W.; Blair-Flynn, Jan; Hu, Tian-Jing; Yue, Xin; Cooper, Margaret; Crockett, David P.; Yancopoulos, George D.; Tessarollo, Lino; Zhou, Renping

    2002-01-01

    Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections. PMID:12124402

  9. Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae

    PubMed Central

    2014-01-01

    Background Peripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells. Results Using pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored. Conclusion The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration. PMID:25326036

  10. Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

    PubMed Central

    2010-01-01

    Background Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. Results We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. Conclusion Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise

  11. Cocaine-induced expression changes of axon guidance molecules in the adult rat brain.

    PubMed

    Bahi, Amine; Dreyer, Jean-Luc

    2005-02-01

    Administration of drugs of abuse induces strong molecular adaptations and plasticity within the mesolimbic dopamine (DA) system, a pathway essential for reward-seeking behavior. Little is known about the specific targets involved in this neuroadaptation process, but there are indications that cocaine and other drugs of abuse share the ability to alter the morphology of neuronal dendrites and spines, the primary site of excitatory synapses in the brain. Axon guidance molecules, the very molecular cues that regulate the formation of axon-target connections during development, may mediate these alterations. To test this hypothesis, we investigated mRNA expression changes of 39 axon guidance molecules, including 17 Semaphorins, 12 Ephs, 8 Ephrins, and 2 neuropilins in the mesolimbic dopamine system of cocaine-treated animals under different paradigms by mean of DNA-Microarray and quantitative real-time PCR. In all cases, strong changes in gene expression are observed, yielding to up or downregulation of these axon guidance molecules. Our data suggest that cocaine treatment induces activation of a complex program of synaptic rearrangements, which may partly recapitulate the plastic changes occurring during development, and may underlie the important neuroplastic adaptations that occur in the reward- and memory-related brain centers following drug action. We conclude that in some brain regions, exposure to psychomotor-stimulant drugs produce expression changes in axon guidance molecules, which may contribute to cognitive deficits associated with drug abuse. PMID:15691709

  12. Paraventricular hypothalamic nucleus: axonal projections to the brainstem

    PubMed Central

    Geerling, Joel C.; Shin, Jung-Won; Chimenti, Peter C.; Loewy, Arthur D.

    2010-01-01

    The paraventricular hypothalamic nucleus (PVH) contains many neurons that innervate the brainstem, but information regarding their target sites remains incomplete. Here, we labeled neurons in the rat PVH with an anterograde axonal tracer, Phaseolus vulgaris leucoagglutinin (PHAL) and studied their descending projections in reference to specific neuronal subpopulations throughout the brainstem. While many of their target sites were identified previously, numerous new observations were made. Major findings include: (1) In the midbrain, the PVH projects lightly to the ventral tegmental area, Edinger-Westphal nucleus, ventrolateral periaqueductal gray matter, reticular formation, pedunculopontine tegmental nucleus, and dorsal raphe nucleus. (2) In the dorsal pons, the PVH projects heavily to the pre-locus coeruleus, yet very little to the catecholamine neurons in the locus coeruleus, and selectively targets the viscerosensory subregions of the parabrachial nucleus; (3) In the ventral medulla, the superior salivatory nucleus, retrotrapezoid nucleus, compact and external formations of the nucleus ambiguus, A1 and caudal C1 catecholamine neurons, and caudal pressor area receive dense axonal projections, generally exceeding the PVH projection to the rostral C1 region; (4) The medial nucleus of the solitary tract (including A2 noradrenergic and aldosterone-sensitive neurons) receives the most extensive projections of the PVH, substantially more than the dorsal vagal nucleus or area postrema. Our findings suggest that the PVH may modulate a range of homeostatic functions, including cerebral and ocular blood flow, corneal and nasal hydration, ingestive behavior, sodium intake, and glucose metabolism, as well as cardiovascular, gastrointestinal, and respiratory activities. PMID:20187136

  13. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration

    PubMed Central

    Tang-Schomer, Min D.; Patel, Ankur R.; Baas, Peter W.; Smith, Douglas H.

    2010-01-01

    Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the “normal” straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.—Tang-Schomer, M. D., Patel, A. R,, Baas, P. W., Smith, D. H. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration. PMID:20019243

  14. Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase

    PubMed Central

    Chen, Lizhen; Chuang, Marian; Koorman, Thijs; Boxem, Mike; Jin, Yishi; Chisholm, Andrew D

    2015-01-01

    Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In Caenorhabditis elegans the signaling protein Exchange Factor for ARF-6 (EFA-6) is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury. DOI: http://dx.doi.org/10.7554/eLife.08695.001 PMID:26339988

  15. Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons

    PubMed Central

    Balastik, Martin; Zhou, Xiao Zhen; Alberich-Jorda, Meritxell; Weissova, Romana; Žiak, Jakub; Pazyra-Murphy, Maria F.; Cosker, Katharina E; Machonova, Olga; Kozmikova, Iryna; Chen, Chun-Hau; Pastorino, Lucia; Asara, John M.; Cole, Adam; Sutherland, Calum; Segal, Rosalind A.; Lu, Kun Ping

    2015-01-01

    SUMMARY Axon guidance relies on precise translation of the gradients of the extracellular signals into local changes of cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here we show that during embryonic development in growing axons low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate, that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A level specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth, and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance. PMID:26489457

  16. Axon diameter and axonal transport: In vivo and in vitro effects of androgens

    PubMed Central

    Pesaresi, M; Soon-Shiong, R; French, L; Kaplan, DR; Miller, FD; Paus, T.

    2015-01-01

    Testosterone is a sex hormone involved in brain maturation via multiple molecular mechanisms. Previous human studies described age-related changes in the overall volume and morphological properties of white matter during male puberty. Based on this work, we have proposed that testosterone may induce an increase of radial growth and, possibly, modulate axonal transport. In order to determine whether this is the case we have used two different experimental approaches. With electron microscopy, we have evaluated sex differences in the structural properties of axons in the corpus callosum (splenium) of young rats, and tested consequences of castration carried out after weaning. Then we examined in vitro the effect of the non-aromatizable androgen Mibolerone on the structure and bidirectional transport of wheat-germ agglutinin vesicles in the axons of cultured sympathetic neurons. With electron microscopy, we found robust sex differences in axonal diameter (males>females) and g ratio (males>females). Removal of endogenous testosterone by castration was associated with lower axon diameter and lower g ratio in castrated (vs. intact) males. In vitro, Mibolerone influenced the axonal transport in a time- and dose-dependent manner, and increased the axon caliber as compared with vehicle-treated neurons. These findings are consistent with the role of testosterone in shaping the axon by regulating its radial growth, as predicted by the initial human studies. PMID:25956809

  17. Cytoplasmic structure in rapid-frozen axons

    PubMed Central

    1982-01-01

    Turtle optic nerves were rapid-frozen from the living state, fractured, etched, and rotary shadowed. Stereo views of fractured axons show that axoplasm consists of three types of longitudinally oriented domains. One type consists of neurofilament bundles in which individual filaments are interconnected by a cross-bridging network. Contiguous to neurofilament domains are domains containing microtubules suspended in a loose, granular matrix. A third domain is confined to a zone, 80-100 nm wide, next to the axonal membrane and consists of a dense filamentous network connecting the longitudinal elements of the axonal cytoskeleton to particles on the inner surface of the axolemma. Three classes of membrane-limited organelles are distinguished: axoplasmic reticulum, mitochondria, and discrete vesicular organelles. The vesicular organelles must include lysosomes, multivesicular bodies, and vesicles which are retrogradely transported in axons, though some vesicular organelles may be components of the axoplasmic reticulum. Organelles in each class have a characteristic relationship to the axonal cytoskeleton. The axoplasmic reticulum enters all three domains of axoplasm, but mitochondria and vesicular organelles are excluded from the neurofilament bundles, a distribution confirmed in thin sections of cryoembedded axons. Vesicular organelles differ from mitochondria in at least three ways with respect to their relationships to adjacent axoplasm: (a) one, or sometimes both, of their ends are associated with a gap in the surrounding granular axoplasm; (b) an appendage is typically associated with one of their ends; and (c) they are not attached or closely apposed to microtubules. Mitochondria, on the other hand, are only rarely associated with gaps in the axoplasm, do not have an appendage, and are virtually always attached to one or more microtubules by an irregular array of side-arms. We propose that the longitudinally oriented microtubule domains are channels within which

  18. Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons

    PubMed Central

    Ohno, Nobuhiko; Chiang, Hao; Mahad, Don J.; Kidd, Grahame J.; Liu, LiPing; Ransohoff, Richard M.; Sheng, Zu-Hang; Komuro, Hitoshi; Trapp, Bruce D.

    2014-01-01

    Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na+ channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS. PMID:24958879

  19. Axonal degeneration in multiple sclerosis: can we predict and prevent permanent disability?

    PubMed

    Lee, Jae Young; Taghian, Kasra; Petratos, Steven

    2014-01-01

    Axonal degeneration is a major determinant of permanent neurological impairment during multiple sclerosis (MS). Due to the variable course of clinical disease and the heterogeneity of MS lesions, the mechanisms governing axonal degeneration may differ between disease stages. While the etiology of MS remains elusive, there now exist potential prognostic biomarkers that can predict the conversion to clinically definite MS. Specialized imaging techniques identifying axonal injury and drop-out are becoming established in clinical practice as a predictive measure of MS progression, such as optical coherence tomography (OCT) or diffusion tensor imaging (DTI). However, these imaging techniques are still being debated as predictive biomarkers since controversy surrounds their lesion-specific association with expanded disability status scale (EDSS). A more promising diagnostic measure of axonal degeneration has been argued for the detection of reduced N-acetyl aspartate (NAA) and Creatine ratios via magnetic resonance spectroscopic (MRS) imaging, but again fail with its specificity for predicting actual axonal degeneration. Greater accuracy of predictive biomarkers is therefore warranted and may include CSF neurofilament light chain (NF-L) and neurofilament heavy chain (NF-H) levels, for progressive MS. Furthermore, defining the molecular mechanisms that occur during the neurodegenerative changes in the various subgroups of MS may in fact prove vital for the future development of efficacious neuroprotective therapies. The clinical translation of a combined Na+ and Ca2+ channel blocker may lead to the establishment of a bona fide neuroprotective agent for the treatment of progressive MS. However, more specific therapeutic targets to limit axonal damage in MS need investigation and may include such integral axonal proteins such as the collapsin response mediator protein-2 (CRMP-2), a molecule which upon post-translational modification may propagate axonal degeneration in MS

  20. USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth

    PubMed Central

    Yang, Seung Wook; Oh, Kyu Hee; Park, Esther; Chang, Hyun Min; Park, Jung Mi; Seong, Min Woo; Ka, Seung Hyeun; Song, Woo Keun; Park, Dong Eun; Baas, Peter W.

    2013-01-01

    Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation. PMID:23904609

  1. Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

    PubMed Central

    Marx, Astrid; Godinez, William J.; Tsimashchuk, Vasil; Bankhead, Peter; Rohr, Karl; Engel, Ulrike

    2013-01-01

    Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin–microtubule interactions. PMID:23515224

  2. Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

    PubMed

    Zhang, Peisu; Abdelmohsen, Kotb; Liu, Yong; Tominaga-Yamanaka, Kumiko; Yoon, Je-Hyun; Ioannis, Grammatikakis; Martindale, Jennifer L; Zhang, Yongqing; Becker, Kevin G; Yang, In Hong; Gorospe, Myriam; Mattson, Mark P

    2015-01-01

    Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release. PMID:26586091

  3. Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity

    PubMed Central

    Zhang, Peisu; Abdelmohsen, Kotb; Liu, Yong; Tominaga-Yamanaka, Kumiko; Yoon, Je-Hyun; Ioannis, Grammatikakis; Martindale, Jennifer L.; Zhang, Yongqing; Becker, Kevin G.; Yang, In Hong; Gorospe, Myriam; Mattson, Mark P.

    2015-01-01

    Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S–mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release. PMID:26586091

  4. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  5. Motor axon exit from the mammalian spinal cord is controlled by the homeodomain protein Nkx2.9 via Robo-Slit signaling

    PubMed Central

    Bravo-Ambrosio, Arlene; Mastick, Grant; Kaprielian, Zaven

    2012-01-01

    Mammalian motor circuits control voluntary movements by transmitting signals from the central nervous system (CNS) to muscle targets. To form these circuits, motor neurons (MNs) must extend their axons out of the CNS. Although exit from the CNS is an indispensable phase of motor axon pathfinding, the underlying molecular mechanisms remain obscure. Here, we present the first identification of a genetic pathway that regulates motor axon exit from the vertebrate spinal cord, utilizing spinal accessory motor neurons (SACMNs) as a model system. SACMNs are a homogeneous population of spinal MNs with axons that leave the CNS through a discrete lateral exit point (LEP) and can be visualized by the expression of the cell surface protein BEN. We show that the homeodomain transcription factor Nkx2.9 is selectively required for SACMN axon exit and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9; loss of Nkx2.9 leads to a reduction in Robo2 mRNA and protein within SACMNs and SACMN axons fail to exit the spinal cord in Robo2-deficient mice. Consistent with short-range interactions between Robo2 and Slit ligands regulating SACMN axon exit, Robo2-expressing SACMN axons normally navigate through LEP-associated Slits as they emerge from the spinal cord, and fail to exit in Slit-deficient mice. Our studies support the view that Nkx2.9 controls SACMN axon exit from the mammalian spinal cord by regulating Robo-Slit signaling. PMID:22399681

  6. CLEC-38, a transmembrane protein with C-type lectin-like domains, negatively regulates UNC-40-mediated axon outgrowth and promotes presynaptic development in Caenorhabditis elegans.

    PubMed

    Kulkarni, Gauri; Li, Haichang; Wadsworth, William G

    2008-04-23

    In the developing nervous system, axons respond to various guidance cues to find their targets. The effects guidance cues have on an axon may change as an axon undergoes morphological changes, such as branching, turning, and synapse formation. The means by which these changes are regulated are not well understood. In Caenorhabditis elegans, the UNC-40/DCC (deleted in colorectal cancer) receptor mediates responses to the UNC-6/netrin guidance cue. Here, we show that CLEC-38, a protein with predicted transmembrane and C-type lectin-like domains, regulates UNC-40-mediated axon outgrowth as well as the organization of presynaptic terminals. We observe that, in genetic backgrounds sensitized for axon guidance defects, loss of clec-38 function can suppress defects in an UNC-40-dependent manner. Within migrating axons, clec-38 acts cell autonomously. Furthermore, loss of clec-38 function alters UNC-40::GFP (green fluorescent protein) expression. We also observe that loss of clec-38 function disrupts presynaptic patterning in animals with normal axon guidance and that there are genetic interactions between clec-38 and rpm-1, which encodes a protein implicated in regulating presynaptic assembly and axon morphology. We suggest CLEC-38 plays a role in promoting synapse assembly and refining axon outgrowth activity. PMID:18434533

  7. Axonal isoforms of myosin-I.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2005-05-13

    We have examined spinal motor neurons in Sprague-Dawley rats to further characterize a mechanoenzyme, myosin-Igamma (myr4), which is found in high concentration during axon tract formation in neonates. We raised an antibody to myr4 and made riboprobes for in situ hybridization. Myr4 mRNA was abundant in spinal cord motor neurons (particularly during axon regrowth). Nerves undergoing Wallerian degeneration (from a crush 7 days earlier) showed anti-myr4 labeling of the axolemma and SER--after microtubules, neurofilaments, and F-actin had already been degraded--which is consistent with a described lipid-binding domain in the tail region of myosin-Is. Newly synthesized myr4 was carried in axons by the slow component (SC) of axonal transport at 1-8 mm/day, whereas, none was carried by the fast component (FC). We conclude that SC delivers myr4 to the cytoplasmic surfaces of stationary axonal membranes (SER and axolemma). This positioning would anchor the tail domain of myr4 and leave the catalytic head domain free to interact with F-actin. PMID:15809075

  8. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  9. How Schwann Cells Sort Axons: New Concepts.

    PubMed

    Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo

    2016-06-01

    Peripheral nerves contain large myelinated and small unmyelinated (Remak) fibers that perform different functions. The choice to myelinate or not is dictated to Schwann cells by the axon itself, based on the amount of neuregulin I-type III exposed on its membrane. Peripheral axons are more important in determining the final myelination fate than central axons, and the implications for this difference in Schwann cells and oligodendrocytes are discussed. Interestingly, this choice is reversible during pathology, accounting for the remarkable plasticity of Schwann cells, and contributing to the regenerative potential of the peripheral nervous system. Radial sorting is the process by which Schwann cells choose larger axons to myelinate during development. This crucial morphogenetic step is a prerequisite for myelination and for differentiation of Remak fibers, and is arrested in human diseases due to mutations in genes coding for extracellular matrix and linkage molecules. In this review we will summarize progresses made in the last years by a flurry of reverse genetic experiments in mice and fish. This work revealed novel molecules that control radial sorting, and contributed unexpected ideas to our understanding of the cellular and molecular mechanisms that control radial sorting of axons. PMID:25686621

  10. A novel closed-body model of spinal cord injury caused by high-pressure air blasts produces extensive axonal injury and motor impairments

    PubMed Central

    del Mar, Nobel; von Buttlar, Xinyu; Yu, Angela S.; Guley, Natalie H.; Reiner, Anton; Honig, Marcia G.

    2015-01-01

    Diffuse axonal injury is thought to be the basis of the functional impairments stemming from mild traumatic brain injury. To examine how axons are damaged by traumatic events, such as motor vehicle accidents, falls, sports activities, or explosive blasts, we have taken advantage of the spinal cord with its extensive white matter tracts. We developed a closed-body model of spinal cord injury in mice whereby high-pressure air blasts targeted to lower thoracic vertebral levels produce tensile, compressive, and shear forces within the parenchyma of the spinal cord and thereby cause extensive axonal injury. Markers of cytoskeletal integrity showed that spinal cord axons exhibited three distinct pathologies: microtubule breakage, neurofilament compaction, and calpain-mediated spectrin breakdown. The dorsally situated axons of the corticospinal tract primarily exhibited microtubule breakage, whereas all three pathologies were common in the lateral and ventral white matter. Individual axons typically demonstrated only one of the three pathologies during the first 24 h after blast injury, suggesting that the different perturbations are initiated independently of one another. For the first few days after blast, neurofilament compaction was frequently accompanied by autophagy, and subsequent to that, by the fragmentation of degenerating axons. TuJ1 immunolabeling and mice with YFP-reporter labeling each revealed more extensive microtubule breakage than did βAPP immunolabeling, raising doubts about the sensitivity of this standard approach for assessing axonal injury. Although motor deficits were mild and largely transient, some aspects of motor function gradually worsened over several weeks, suggesting that a low level of axonal degeneration continued past the initial wave. Our model can help provide further insight into how to intervene in the processes by which initial axonal damage culminates in axonal degeneration, to improve outcomes after traumatic injury. Importantly

  11. A novel closed-body model of spinal cord injury caused by high-pressure air blasts produces extensive axonal injury and motor impairments.

    PubMed

    del Mar, Nobel; von Buttlar, Xinyu; Yu, Angela S; Guley, Natalie H; Reiner, Anton; Honig, Marcia G

    2015-09-01

    Diffuse axonal injury is thought to be the basis of the functional impairments stemming from mild traumatic brain injury. To examine how axons are damaged by traumatic events, such as motor vehicle accidents, falls, sports activities, or explosive blasts, we have taken advantage of the spinal cord with its extensive white matter tracts. We developed a closed-body model of spinal cord injury in mice whereby high-pressure air blasts targeted to lower thoracic vertebral levels produce tensile, compressive, and shear forces within the parenchyma of the spinal cord and thereby cause extensive axonal injury. Markers of cytoskeletal integrity showed that spinal cord axons exhibited three distinct pathologies: microtubule breakage, neurofilament compaction, and calpain-mediated spectrin breakdown. The dorsally situated axons of the corticospinal tract primarily exhibited microtubule breakage, whereas all three pathologies were common in the lateral and ventral white matter. Individual axons typically demonstrated only one of the three pathologies during the first 24h after blast injury, suggesting that the different perturbations are initiated independently of one another. For the first few days after blast, neurofilament compaction was frequently accompanied by autophagy, and subsequent to that, by the fragmentation of degenerating axons. TuJ1 immunolabeling and mice with YFP-reporter labeling each revealed more extensive microtubule breakage than did βAPP immunolabeling, raising doubts about the sensitivity of this standard approach for assessing axonal injury. Although motor deficits were mild and largely transient, some aspects of motor function gradually worsened over several weeks, suggesting that a low level of axonal degeneration continued past the initial wave. Our model can help provide further insight into how to intervene in the processes by which initial axonal damage culminates in axonal degeneration, to improve outcomes after traumatic injury. Importantly

  12. Double-label confocal laser-scanning microscopy, image restoration, and real-time three-dimensional reconstruction to study axons in the central nervous system and their contacts with target neurons.

    PubMed

    Wouterlood, Floris G; van Haeften, Theo; Blijleven, Nico; Pérez-Templado, Pepa; Pérez-Templado, Helena

    2002-03-01

    The current double tracing-double confocal laser-scanning method was developed to reconstruct identified nerve fibers and their contacts with identified target neurons in the rat brain in three dimensions. It intends to fill the gap between conventional light microscopic and electron microscopic neuroanatomic tracing. The steps involved are as follows: (1) injection of two neuroanatomic tracers--Phaseolus vulgaris leucoagglutinin (PHA-L) to label fibers innervating a particular brain area and Neurobiotin to label prospective target neurons in that area; (2) immunofluorescence detection of the labeled fibers (fluorophore Cy5, infrared emission), together with fluorochromated avidin detection of the taken-up Neurobiotin (Cy2 or Alexa 488; green emission); (3) acquisition of Z-series of confocal images at high magnification with a laser-scanning microscope using the laser lines 488 nm and 647 nm; and (4) computer-processing and three-dimensional reconstruction of the labeled fibers and the presumed target dendrites. Rotation on the computer of the three-dimensional reconstructed fibers and dendrites along all three spatial axes enabled the authors to determine whether "true" or "false" contacts occur. In a true contact no space was present between the apposing structures, whereas a false contact consisted of two differently stained structures close to each other but separated by a narrow, optically empty space. One important phenomenon in the three-dimensional reconstruction of double-stained structures that needed correction was "twin image mismatch"--i.e., the observation that a three-dimensional reconstruction of a small test object (double-stained on purpose) produced two slightly shifted objects, each associated with its particular fluorochrome. To measure the actual twin image mismatch of the confocal instrument and to obtain accurate correction factors the authors took in each session in which they obtained image series of the real experiments, with both laser

  13. Transforming Growth Factor-β Promotes Axonal Regeneration After Chronic Nerve Injury.

    PubMed

    Sulaiman, Wale A R

    2016-04-01

    When spinal cord injury (SCI) occurs, injured cells must survive and regenerate to close gaps caused by the injury and to create functional motor units. After peripheral nerve injury, Wallerian degeneration in the distal nerve stump creates a neurotrophic and growth-supportive environment for injured neurons and axons via Schwann cells and secreted cytokines/neurotrophins. In both SCI and peripheral nerve injury, injured motor and sensory neurons must regenerate axons, eventually reaching and reinnervating target tissue (SDC Figure 1, http://links.lww.com/BRS/B116). This process is often unsuccessful after SCI, and the highly complex anatomy of branching axons and nerves in the peripheral nervous system leads to slow recovery of function, even with careful and appropriate techniques. PMID:27015069

  14. Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling

    PubMed Central

    Zschätzsch, Marlen; Oliva, Carlos; Langen, Marion; De Geest, Natalie; Özel, Mehmet Neset; Williamson, W Ryan; Lemon, William C; Soldano, Alessia; Munck, Sebastian; Hiesinger, P Robin; Sanchez-Soriano, Natalia; Hassan, Bassem A

    2014-01-01

    Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001 PMID:24755286

  15. A mathematical model explains saturating axon guidance responses to molecular gradients

    PubMed Central

    Nguyen, Huyen; Dayan, Peter; Pujic, Zac; Cooper-White, Justin; Goodhill, Geoffrey J

    2016-01-01

    Correct wiring is crucial for the proper functioning of the nervous system. Molecular gradients provide critical signals to guide growth cones, which are the motile tips of developing axons, to their targets. However, in vitro, growth cones trace highly stochastic trajectories, and exactly how molecular gradients bias their movement is unclear. Here, we introduce a mathematical model based on persistence, bias, and noise to describe this behaviour, constrained directly by measurements of the detailed statistics of growth cone movements in both attractive and repulsive gradients in a microfluidic device. This model provides a mathematical explanation for why average axon turning angles in gradients in vitro saturate very rapidly with time at relatively small values. This work introduces the most accurate predictive model of growth cone trajectories to date, and deepens our understanding of axon guidance events both in vitro and in vivo. DOI: http://dx.doi.org/10.7554/eLife.12248.001 PMID:26830461

  16. Microfluidic device for unidirectional axon growth

    NASA Astrophysics Data System (ADS)

    Malishev, E.; Pimashkin, A.; Gladkov, A.; Pigareva, Y.; Bukatin, A.; Kazantsev, V.; Mukhina, I.; Dubina, M.

    2015-11-01

    In order to better understand the communication and connectivity development of neuron networks, we designed microfluidic devices with several chambers for growing dissociated neuronal cultures from mice fetal hippocampus (E18). The chambers were connected with microchannels providing unidirectional axonal growth between “Source” and “Target” neural sub-networks. Experiments were performed in a hippocampal cultures plated in a poly-dimethylsiloxane (PDMS) microfluidic chip, aligned with a 60 microelectrode array (MEA). Axonal growth through microchannels was observed with brightfield, phase-contrast and fluorescence microscopy, and after 7 days in vitro electrical activity was recorded. Visual inspection and spike propagation analysis showed the predominant axonal growth in microchannels in a direction from “Source” to “Target”.

  17. Giant axonal neuropathy: visual and oculomotor deficits.

    PubMed

    Kirkham, T H; Guitton, D; Coupland, S G

    1980-08-01

    Giant axonal neuropathy, a generalised disorder or neurofilaments, presents as a chronic, progressive peripheral neuropathy in childhood. Evidence for central nervous system involvement is demonstrated in this study of four male patients with giant axonal neuropathy who had defective visual function and abnormal ocular motility. The visual system was studied by electroretinography, which showed normal retinal function, and by visual evoked potentials, which showed disease of both optic nerves and retrochiasmal visual pathways. The ocular motility disorder, studied by electrooculography, comprised defective pursuit, inability to maintain eccentric gaze with gaze paretic and rebound nystagmus, abnormal optokinetic responses and failure of suppression of the vestibulo-ocular reflex by fixation. These findings suggested involvement by giant axonal neuropathy of the cerebellar and brain stem pathways important in the control of ocular motility. PMID:7192592

  18. Axonal Fiber Terminations Concentrate on Gyri

    PubMed Central

    Nie, Jingxin; Guo, Lei; Li, Kaiming; Wang, Yonghua; Chen, Guojun; Li, Longchuan; Chen, Hanbo; Deng, Fan; Jiang, Xi; Zhang, Tuo; Huang, Ling; Faraco, Carlos; Zhang, Degang; Guo, Cong; Yap, Pew-Thian; Hu, Xintao; Li, Gang; Lv, Jinglei; Yuan, Yixuan; Zhu, Dajiang; Han, Junwei; Sabatinelli, Dean; Zhao, Qun; Miller, L. Stephen; Xu, Bingqian; Shen, Ping; Platt, Simon; Shen, Dinggang; Hu, Xiaoping

    2012-01-01

    Convoluted cortical folding and neuronal wiring are 2 prominent attributes of the mammalian brain. However, the macroscale intrinsic relationship between these 2 general cross-species attributes, as well as the underlying principles that sculpt the architecture of the cerebral cortex, remains unclear. Here, we show that the axonal fibers connected to gyri are significantly denser than those connected to sulci. In human, chimpanzee, and macaque brains, a dominant fraction of axonal fibers were found to be connected to the gyri. This finding has been replicated in a range of mammalian brains via diffusion tensor imaging and high–angular resolution diffusion imaging. These results may have shed some lights on fundamental mechanisms for development and organization of the cerebral cortex, suggesting that axonal pushing is a mechanism of cortical folding. PMID:22190432

  19. GSK3 and KIF5 regulate activity-dependent sorting of gephyrin between axons and dendrites.

    PubMed

    Rathgeber, Louisa; Gromova, Kira V; Schaefer, Irina; Breiden, Petra; Lohr, Christian; Kneussel, Matthias

    2015-01-01

    The kinesin KIF5 transports neuronal cargoes into axons and dendrites. Isolated KIF5 motor domains preferentially move into axons, however KIF5 binding to GRIP1 or gephyrin drives the motor into dendrites, to deliver AMPA receptors (AMPARs) or glycine receptors (GlyRs), respectively. At postsynaptic sites, gephyrin forms a multimeric scaffold to anchor GlyRs and GABAA receptors (GABAARs) in apposition to inhibitory presynaptic terminals. Here, we report the unexpected observation that increased intracellular calcium through chronic activation of AMPARs, steers a newly synthesized gephyrin fusion protein (tomato-gephyrin) to axons and interferes with its normal delivery into dendrites of cultured neurons. Axonal gephyrin clusters were not apposed to presynaptic terminals, but colocalized with GlyRs and neuroligin-2 (NLG2). Notably, functional blockade of glycogen synthase kinase-3 (GSK3) and KIF5 normalized gephyrin missorting into the axonal compartment. In contrast, mutagenesis of gephyrin S270, a GSK3 target, did not contribute to axo-dendritic sorting. Our data are consistent with previous observations, which report regulation of kinesin motility through GSK3 activity. They suggest that GSK3 regulates the sorting of GlyR/gephyrin and NLG2 complexes in a KIF5-dependent manner. PMID:25701174

  20. Roles of channels and receptors in the growth cone during PNS axonal regeneration.

    PubMed

    Shim, Sangwoo; Ming, Guo-li

    2010-05-01

    Neurons in the peripheral nervous system (PNS) are known to maintain a regenerative capacity and will normally regenerate their axons within a permissive growth environment. The success of regeneration in the PNS largely depends on maintenance of the supportive basal lamina membrane, efficient removal of axonal and myelin debris by macrophages and Schwann cells, expression of neurotrophic factors by Schwann cells, and up-regulation of the intrinsic growth program in PNS neurons. The PNS regenerative process is well characterized through initial Wallerian degeneration followed by axonal sprouting, formation of neuronal growth cones, active axonal growth to the target, and finally sensory and motor functional recovery. The initiation and maintenance of active growth cones during peripheral nerve regeneration recapitulate many aspects of early neural development and are achieved through the activation of complex signaling cascades, involving various receptors, channels, cytoplasmic signaling cascades, as well as transcriptional and translational programs. This review focuses on roles of cell surface ion channels and receptors in the growth cone during Wallerian degeneration and axon regeneration in the PNS. PMID:19833126

  1. Imaging Axonal Degeneration and Repair in Preclinical Animal Models of Multiple Sclerosis

    PubMed Central

    Yandamuri, Soumya S.; Lane, Thomas E.

    2016-01-01

    Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by chronic neuroinflammation, demyelination, and axonal damage. Infiltration of activated lymphocytes and myeloid cells are thought to be primarily responsible for white matter damage and axonopathy. Over time, this neurologic damage manifests clinically as debilitating motor and cognitive symptoms. Existing MS therapies focus on symptom relief and delay of disease progression through reduction of neuroinflammation. However, long-term strategies to remyelinate, protect, or regenerate axons have remained elusive, posing a challenge to treating progressive forms of MS. Preclinical mouse models and techniques, such as immunohistochemistry, flow cytometry, and genomic and proteomic analysis have provided advances in our understanding of discrete time-points of pathology following disease induction. More recently, in vivo and in situ two-photon (2P) microscopy has made it possible to visualize continuous real-time cellular behavior and structural changes occurring within the CNS during neuropathology. Research utilizing 2P imaging to study axonopathy in neuroinflammatory demyelinating disease has focused on five areas: (1) axonal morphologic changes, (2) organelle transport and health, (3) relationship to inflammation, (4) neuronal excitotoxicity, and (5) regenerative therapies. 2P imaging may also be used to identify novel therapeutic targets via identification and clarification of dynamic cellular and molecular mechanisms of axonal regeneration and remyelination. Here, we review tools that have made 2P accessible for imaging neuropathologies and advances in our understanding of axonal degeneration and repair in preclinical models of demyelinating diseases. PMID:27242796

  2. Imaging Axonal Degeneration and Repair in Preclinical Animal Models of Multiple Sclerosis.

    PubMed

    Yandamuri, Soumya S; Lane, Thomas E

    2016-01-01

    Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by chronic neuroinflammation, demyelination, and axonal damage. Infiltration of activated lymphocytes and myeloid cells are thought to be primarily responsible for white matter damage and axonopathy. Over time, this neurologic damage manifests clinically as debilitating motor and cognitive symptoms. Existing MS therapies focus on symptom relief and delay of disease progression through reduction of neuroinflammation. However, long-term strategies to remyelinate, protect, or regenerate axons have remained elusive, posing a challenge to treating progressive forms of MS. Preclinical mouse models and techniques, such as immunohistochemistry, flow cytometry, and genomic and proteomic analysis have provided advances in our understanding of discrete time-points of pathology following disease induction. More recently, in vivo and in situ two-photon (2P) microscopy has made it possible to visualize continuous real-time cellular behavior and structural changes occurring within the CNS during neuropathology. Research utilizing 2P imaging to study axonopathy in neuroinflammatory demyelinating disease has focused on five areas: (1) axonal morphologic changes, (2) organelle transport and health, (3) relationship to inflammation, (4) neuronal excitotoxicity, and (5) regenerative therapies. 2P imaging may also be used to identify novel therapeutic targets via identification and clarification of dynamic cellular and molecular mechanisms of axonal regeneration and remyelination. Here, we review tools that have made 2P accessible for imaging neuropathologies and advances in our understanding of axonal degeneration and repair in preclinical models of demyelinating diseases. PMID:27242796

  3. Axonal commissures in the central nervous system: how to cross the midline?

    PubMed

    Nawabi, Homaira; Castellani, Valérie

    2011-08-01

    Organisms with bilateral symmetry elaborate patterns of neuronal projections connecting both sides of the central nervous system at all levels of the neuraxis. During development, these so-called commissural projections navigate across the midline to innervate their contralateral targets. Commissural axon pathfinding has been extensively studied over the past years and turns out to be a highly complex process, implicating modulation of axon responsiveness to the various guidance cues that instruct axon trajectories towards, within and away from the midline. Understanding the molecular mechanisms allowing these switches of response to take place at the appropriate time and place is a major challenge for current research. Recent work characterized several instructive processes controlling the spatial and temporal fine-tuning of the guidance molecular machinery. These findings illustrate the molecular strategies by which commissural axons modulate their sensitivity to guidance cues during midline crossing and show that regulation at both transcriptional and post-transcriptional levels are crucial for commissural axon guidance. PMID:21538161

  4. X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

    PubMed Central

    Gross, Garrett G.; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker

    2013-01-01

    Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

  5. X11/Mint genes control polarized localization of axonal membrane proteins in vivo.

    PubMed

    Gross, Garrett G; Lone, G Mohiddin; Leung, Lok Kwan; Hartenstein, Volker; Guo, Ming

    2013-05-01

    Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

  6. Oligodendrocyte Lineage and Subventricular Zone Response to Traumatic Axonal Injury in the Corpus Callosum

    PubMed Central

    Sullivan, Genevieve M.; Mierzwa, Amanda J.; Kijpaisalratana, Naruchorn; Tang, *Haiying; Wang, Yong; Song, Sheng-Kwei; Selwyn, Reed

    2013-01-01

    Abstract Traumatic brain injury frequently causes traumatic axonal injury (TAI) in white matter tracts. Experimental TAI in the corpus callosum of adult mice was used to examine the effects on oligodendrocyte lineage cells and myelin in conjunction with neuroimaging. The injury targeted the corpus callosum over the subventricular zone, a source of neural stem/progenitor cells. Traumatic axonal injury was produced in the rostral body of the corpus callosum by impact onto the skull at the bregma. During the first week after injury, magnetic resonance diffusion tensor imaging showed that axial diffusivity decreased in the corpus callosum and that corresponding regions exhibited significant axon damage accompanied by hypertrophic microglia and reactive astrocytes. Oligodendrocyte progenitor proliferation increased in the subventricular zone and corpus callosum. Oligodendrocytes in the corpus callosum shifted toward upregulation of myelin gene transcription. Plp/CreERT:R26IAP reporter mice showed normal reporter labeling of myelin sheaths 0 to 2 days after injury but labeling was increased between 2 and 7 days after injury. Electron microscopy revealed axon degeneration, demyelination, and redundant myelin figures. These findings expand the cell types and responses to white matter injuries that inform diffusion tensor imaging evaluation and identify pivotal white matter changes after TAI that may affect axon vulnerability vs. recovery after brain injury. PMID:24226267

  7. Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ.

    PubMed

    Zarei, Kasra; Scheetz, Todd E; Christopher, Mark; Miller, Kathy; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G; Fingert, John H; Abràmoff, Michael David

    2016-01-01

    We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ's performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice. PMID:27226405

  8. Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

    NASA Astrophysics Data System (ADS)

    Zarei, Kasra; Scheetz, Todd E.; Christopher, Mark; Miller, Kathy; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G.; Fingert, John H.; Abràmoff, Michael David

    2016-05-01

    We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ’s performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice.

  9. Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

    PubMed Central

    Zarei, Kasra; Scheetz, Todd E.; Christopher, Mark; Miller, Kathy; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G.; Fingert, John H.; Abràmoff, Michael David

    2016-01-01

    We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ’s performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice. PMID:27226405

  10. High frequency stimulation can block axonal conduction.

    PubMed

    Jensen, Alicia L; Durand, Dominique M

    2009-11-01

    High frequency stimulation (HFS) is used to control abnormal neuronal activity associated with movement, seizure, and psychiatric disorders. Yet, the mechanisms of its therapeutic action are not known. Although experimental results have shown that HFS suppresses somatic activity, other data has suggested that HFS could generate excitation of axons. Moreover it is unclear what effect the stimulation has on tissue surrounding the stimulation electrode. Electrophysiological and computational modeling literature suggests that HFS can drive axons at the stimulus frequency. Therefore, we tested the hypothesis that unlike cell bodies, axons are driven by pulse train HFS. This hypothesis was tested in fibers of the hippocampus both in-vivo and in-vitro. Our results indicate that although electrical stimulation could activate and drive axons at low frequencies (0.5-25 Hz), as the stimulus frequency increased, electrical stimulation failed to continuously excite axonal activity. Fiber tracts were unable to follow extracellular pulse trains above 50 Hz in-vitro and above 125 Hz in-vivo. The number of cycles required for failure was frequency dependent but independent of stimulus amplitude. A novel in-vitro preparation was developed, in which, the alveus was isolated from the remainder of the hippocampus slice. The isolated fiber tract was unable to follow pulse trains above 75 Hz. Reversible conduction block occurred at much higher stimulus amplitudes, with pulse train HFS (>150 Hz) preventing propagation through the site of stimulation. This study shows that pulse train HFS affects axonal activity by: (1) disrupting HFS evoked excitation leading to partial conduction block of activity through the site of HFS; and (2) generating complete conduction block of secondary evoked activity, as HFS amplitude is increased. These results are relevant for the interpretation of the effects of HFS for the control of abnormal neural activity such as epilepsy and Parkinson's disease. PMID

  11. Retrograde Axonal Degeneration in Parkinson Disease

    PubMed Central

    Tagliaferro, Patricia; Burke, Robert E.

    2016-01-01

    In spite of tremendous research efforts we have not yet achieved two of our principal therapeutic goals in the treatment of Parkinson’s disease (PD), to prevent its onward progression and to provide restoration of systems that have already been damaged by the time of diagnosis. There are many possible reasons for our inability to make progress. One possibility is that our efforts thus far may not have been directed towards the appropriate cellular compartments. Up until now research has been largely focused on the loss of neurons in the disease. Thus, neuroprotection approaches have been largely aimed at blocking mechanisms that lead to destruction of the neuronal cell body. Attempts to provide neurorestoration have been almost entirely focused on replacement of neurons. We herein review the evidence that the axonal component of diseased neuronal systems merit more of our attention. Evidence from imaging studies, from postmortem neurochemical studies, and from genetic animal models suggests that the axons of the dopaminergic system are involved predominantly and early in PD. Since the mechanisms of axonal destruction are distinct from those of neuron cell body degeneration, a focus on axonal neurobiology will offer new opportunities for preventing their degeneration. At present these mechanisms remain largely obscure. However, defining them is likely to offer new opportunities for neuroprotection. In relation to neurorestoration, while it has been classically believed that neurons of the adult central nervous system are incapable of new axon growth, recent evidence shows that this is not true for the dopaminergic projection. In conclusion, the neurobiology of axons is likely to offer many new approaches to protective and restorative therapeutics. PMID:27003783

  12. Axonal transport of ribonucleoprotein particles (vaults).

    PubMed

    Li, J Y; Volknandt, W; Dahlstrom, A; Herrmann, C; Blasi, J; Das, B; Zimmermann, H

    1999-01-01

    RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon

  13. Axonal transport disruption in peripheral nerve disease

    PubMed Central

    Lloyd, Thomas E.

    2015-01-01

    Many neurodegenerative diseases and neuropathies have been proposed to be caused by a disruption of axonal transport. However, the mechanisms whereby impaired transport causes disease remain unclear. Proposed mechanisms include impairment in delivery of organelles such as mitochondria, defective retrograde neurotrophic signaling, and disruption of the synaptic vesicle cycle within the synaptic terminal. Simple model organisms such as the fruitfly, Drosophila melanogaster, allow live imaging of axonal transport to be combined with high-throughput genetic screens and are providing insights into the pathophysiology of peripheral nerve diseases. PMID:23279432

  14. Proper migration and axon outgrowth of zebrafish cranial motoneuron subpopulations require the cell adhesion molecule MDGA2A

    PubMed Central

    Ingold, Esther; vom Berg-Maurer, Colette M.; Burckhardt, Christoph J.; Lehnherr, André; Rieder, Philip; Keller, Philip J.; Stelzer, Ernst H.; Greber, Urs F.; Neuhauss, Stephan C. F.; Gesemann, Matthias

    2015-01-01

    ABSTRACT The formation of functional neuronal circuits relies on accurate migration and proper axonal outgrowth of neuronal precursors. On the route to their targets migrating cells and growing axons depend on both, directional information from neurotropic cues and adhesive interactions mediated via extracellular matrix molecules or neighbouring cells. The inactivation of guidance cues or the interference with cell adhesion can cause severe defects in neuronal migration and axon guidance. In this study we have analyzed the function of the MAM domain containing glycosylphosphatidylinositol anchor 2A (MDGA2A) protein in zebrafish cranial motoneuron development. MDGA2A is prominently expressed in distinct clusters of cranial motoneurons, especially in the ones of the trigeminal and facial nerves. Analyses of MDGA2A knockdown embryos by light sheet and confocal microscopy revealed impaired migration and aberrant axonal outgrowth of these neurons; suggesting that adhesive interactions mediated by MDGA2A are required for the proper arrangement and outgrowth of cranial motoneuron subtypes. PMID:25572423

  15. Molecular mechanisms of scar-sourced axon growth inhibitors

    PubMed Central

    Ohtake, Yosuke; Li, Shuxin

    2014-01-01

    Astrogliosis is a defense response of the CNS to minimize primary damage and to repair injured tissues, but it ultimately generates harmful effects by upregulating inhibitory molecules to suppress neuronal elongation and forming potent barriers to axon regeneration. Chondroitin sulfate proteoglycans (CSPGs) are highly expressed by reactive scars and are potent contributors to the non-permissive environment in mature CNS. Surmounting strong inhibition by CSPG-rich scar is an important therapeutic goal for achieving functional recovery after CNS injuries. Currently, enzymatic digestion of CSPGs with locally applied chondroitinase ABC is the main in vivo approach to overcome scar inhibition, but several disadvantages may prevent using this bacterial enzyme as a therapeutic option for patients. A better understanding of molecular mechanisms underlying CSPG function may facilitate development of new effective therapies to overcome scar-mediated inhibition. Previous studies support that CSPGs act by non-specifically hindering the binding of matrix molecules to their cell surface receptors through steric interactions, but two members of the leukocyte common antigen related (LAR) phosphatase subfamily, protein tyrosine phosphatase σ and LAR, are functional receptors that bind CSPGs with high affinity and mediate CSPG inhibition. CSPGs may also act by binding two receptors for myelin-associated growth inhibitors, Nogo receptors 1 and 3. Thus, CSPGs inhibit axon growth through multiple mechanisms, making them especially potent and difficult therapeutic targets. Identification of CSPG receptors is not only important for understanding the scar-mediated growth suppression, but also for developing novel and selective therapies to promote axon sprouting and/or regeneration after CNS injuries. PMID:25192646

  16. Hyperactivated Stat3 boosts axon regeneration in the CNS.

    PubMed

    Mehta, Saloni T; Luo, Xueting; Park, Kevin K; Bixby, John L; Lemmon, Vance P

    2016-06-01

    Axonal regeneration after spinal cord injury (SCI) is intrinsically and extrinsically inhibited by multiple factors. One major factor contributing to intrinsic regeneration failure is the inability of mature neurons in the central nervous system (CNS) to activate regeneration-associated transcription factors (TFs) post-injury. A prior study identified TFs overexpressed in neurons of the peripheral nervous system (PNS) compared to the CNS; some of these could be involved in the ability of PNS neurons to regenerate. Of these, signal transducer and activator of transcription 3 (STAT3), as well its downstream regeneration-associated targets, showed a significant upregulation in PNS neurons relative to CNS neurons, and a constitutively active variant of Stat3 (Stat3CA) promoted neurite growth when expressed in cerebellar neurons (Lerch et al., 2012; Smith et al., 2011). To further enhance STAT3's neurite outgrowth enhancing activity, Stat3CA was fused with a viral activation domain (VP16). VP16 hyperactivates TFs by recruiting transcriptional co-factors to the DNA binding domain (Hirai et al., 2010). Overexpression of this VP16-Stat3CA chimera in primary cortical neurons led to a significant increase of neurite outgrowth as well as Stat3 transcriptional activity in vitro. Furthermore, in vivo transduction of retinal ganglion cells (RGCs) with AAV constructs expressing VP16-Stat3CA resulted in regeneration of optic nerve axons after injury, to a greater degree than for those expressing Stat3CA alone. These findings confirm and extend the concept that overexpression of hyperactivated transcription factors identified as functioning in PNS regeneration can promote axon regeneration in the CNS. PMID:27060489

  17. SARM1 activation triggers axon degeneration locally via NAD+ destruction

    PubMed Central

    Gerdts, Josiah; Brace, E.J.; Sasaki, Yo; DiAntonio, Aaron

    2015-01-01

    Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of NAD+ after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-Interleukin Receptor (TIR) domain of SARM1 alone was sufficient to induce locally-mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD+, whereas SARM1-induced axon destruction could be counteracted by increased NAD+ synthesis. SARM1-induced depletion of NAD+ may explain the potent axon protection in Wallerian Degeneration slow (Wlds) mutant mice. PMID:25908823

  18. MSC p43 required for axonal development in motor neurons

    PubMed Central

    Zhu, Xiaodong; Liu, Yang; Yin, Yanqing; Shao, Aiyun; Zhang, Bo; Kim, Sunghoon; Zhou, Jiawei

    2009-01-01

    Neuron connectivity and correct neural function largely depend on axonal integrity. Neurofilaments (NFs) constitute the main cytoskeletal network maintaining the structural integrity of neurons and exhibit dynamic changes during axonal and dendritic growth. However, the mechanisms underlying axonal development and maintenance remain poorly understood. Here, we identify that multisynthetase complex p43 (MSC p43) is essential for NF assembly and axon maintenance. The MSC p43 protein was predominantly expressed in central neurons and interacted with NF light subunit in vivo. Mice lacking MSC p43 exhibited axon degeneration in motor neurons, defective neuromuscular junctions, muscular atrophy, and motor dysfunction. Furthermore, MSC p43 depletion in mice caused disorganization of the axonal NF network. Mechanistically, MSC p43 is required for maintaining normal phosphorylation levels of NFs. Thus, MSC p43 is indispensable in maintaining axonal integrity. Its dysfunction may underlie the NF disorganization and axon degeneration associated with motor neuron degenerative diseases. PMID:19717447

  19. Subcellular patterning: axonal domains with specialized structure and function

    PubMed Central

    Normand, Elizabeth A.; Rasband, Matthew N.

    2015-01-01

    Myelinated axons are patterned into discrete and often repeating domains responsible for the efficient and rapid transmission of electrical signals. These domains include nodes of Ranvier and axon initial segments. Disruption of axonal patterning leads to nervous system dysfunction. In this review we introduce the concept of subcellular patterning as applied to axons and discuss how these patterning events depend on both intrinsic, cytoskeletal mechanisms, and extrinsic, myelinating-glia dependent mechanisms. PMID:25710532

  20. Calcium/calmodulin-dependent protein kinase IIbeta isoform is expressed in motor neurons during axon outgrowth and is part of slow axonal transport.

    PubMed

    Lund, Linda M; McQuarrie, Irvine G

    2002-03-15

    Previously, we identified calcium/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) mRNA in spinal motor neurons with 372 bp inserted in what corresponds to the "association" domain of the protein. This was interesting because known additions and deletions to CaMKIIbeta mRNA are usually less than 100 bp in size and found in the "variable" region. Changes in the association domain of CaMKIIbeta could influence substrate specificity, activity or intracellular targeting. We show that three variations of this insert are found in CNS neurons or sciatic motor neurons of Sprague-Dawley rats. We used PCR and nucleic acid sequencing to identify inserts of 114, 243, or 372 bases. We also show that addition of the 372 bases is associated with outgrowth of the axon (the standard CaMKIIbeta downregulates when axon outgrowth occurs). Radiolabeling, immunoblots, and 2D PAGE identified this larger CaMKIIbeta as part of the group of soluble proteins moving at the slowest rate of axonal transport (SCa) in sciatic motor neurons (similar1 mm/day). This group is composed mainly of structural proteins (e.g., tubulin) used to assemble the cytoskeleton of regrowing axons. PMID:11891785

  1. Long-range projection neurons of the mouse ventral tegmental area: a single-cell axon tracing analysis

    PubMed Central

    Aransay, Ana; Rodríguez-López, Claudia; García-Amado, María; Clascá, Francisco; Prensa, Lucía

    2015-01-01

    Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four “subtypes” could be distinguished among the FPN cells based on their projection targets: (1) “Mesocorticolimbic” FPN projecting to both neocortex and basal forebrain; (2) “Mesocortical” FPN innervating the neocortex almost exclusively; (3) “Mesolimbic” FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) “Mesostriatal” FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and

  2. Long-range projection neurons of the mouse ventral tegmental area: a single-cell axon tracing analysis.

    PubMed

    Aransay, Ana; Rodríguez-López, Claudia; García-Amado, María; Clascá, Francisco; Prensa, Lucía

    2015-01-01

    Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four "subtypes" could be distinguished among the FPN cells based on their projection targets: (1) "Mesocorticolimbic" FPN projecting to both neocortex and basal forebrain; (2) "Mesocortical" FPN innervating the neocortex almost exclusively; (3) "Mesolimbic" FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) "Mesostriatal" FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structures

  3. Electrokinetic confinement of axonal growth for dynamically configurable neural networks.

    PubMed

    Honegger, Thibault; Scott, Mark A; Yanik, Mehmet F; Voldman, Joel

    2013-02-21

    Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 10(5) Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode 'gates' that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca(2+) imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks. PMID:23314575

  4. Electrokinetic confinement of axonal growth for dynamically configurable neural networks

    PubMed Central

    Honegger, Thibault; Scott, Mark A.; Yanik, Mehmet F.; Voldman, Joel

    2013-01-01

    Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 105 Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode `gates' that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca2+ imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks. PMID:23314575

  5. Ultra-rapid axon-axon ephaptic inhibition of cerebellar Purkinje cells by the pinceau.

    PubMed

    Blot, Antonin; Barbour, Boris

    2014-02-01

    Excitatory synaptic activity in the brain is shaped and balanced by inhibition. Because inhibition cannot propagate, it is often recruited with a synaptic delay by incoming excitation. Cerebellar Purkinje cells are driven by long-range excitatory parallel fiber inputs, which also recruit local inhibitory basket cells. The axon initial segment of each Purkinje cell is ensheathed by basket cell axons in a structure called the pinceau, which is largely devoid of chemical synapses. In mice, we found at the single-cell level that the pinceau mediates ephaptic inhibition of Purkinje cell firing at the site of spike initiation. The reduction of firing rate was synchronous with the presynaptic action potential, eliminating a synaptic delay and allowing granule cells to inhibit Purkinje cells without a preceding phase of excitation. Axon-axon ephaptic intercellular signaling can therefore mediate near-instantaneous feedforward and lateral inhibition. PMID:24413696

  6. Spatial temperature gradients guide axonal outgrowth.

    PubMed

    Black, Bryan; Vishwakarma, Vivek; Dhakal, Kamal; Bhattarai, Samik; Pradhan, Prabhakar; Jain, Ankur; Kim, Young-Tae; Mohanty, Samarendra

    2016-01-01

    Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects. PMID:27460512

  7. Spatial temperature gradients guide axonal outgrowth

    PubMed Central

    Black, Bryan; Vishwakarma, Vivek; Dhakal, Kamal; Bhattarai, Samik; Pradhan, Prabhakar; Jain, Ankur; Kim, Young-tae; Mohanty, Samarendra

    2016-01-01

    Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects. PMID:27460512

  8. Laser Nanosurgery of Cerebellar Axons In Vivo

    PubMed Central

    Allegra Mascaro, Anna L.; Sacconi, Leonardo; Pavone, Francesco Saverio

    2014-01-01

    Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9. This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury. PMID:25146130

  9. Early cellular signaling responses to axonal injury

    PubMed Central

    Lukas, Thomas J; Wang, Ai Ling; Yuan, Ming; Neufeld, Arthur H

    2009-01-01

    Background We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury. Results We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFα and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax). Conclusion We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration. PMID:19284657

  10. Spatial temperature gradients guide axonal outgrowth

    NASA Astrophysics Data System (ADS)

    Black, Bryan; Vishwakarma, Vivek; Dhakal, Kamal; Bhattarai, Samik; Pradhan, Prabhakar; Jain, Ankur; Kim, Young-Tae; Mohanty, Samarendra

    2016-07-01

    Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects.

  11. Mechanosensitivity in axon growth and guidance

    NASA Astrophysics Data System (ADS)

    Urbach, Jeff

    2013-03-01

    In the developing nervous system, axons respond to a diverse array of cues to generate the intricate connection network required for proper function. The growth cone, a highly motile structure at the tip of a growing axon, integrates information about the local environment and modulates outgrowth and guidance, but little is known about effects of external mechanical cues and internal mechanical forces on growth cone behavior. We have investigated axon outgrowth and force generation on soft elastic substrates for dorsal root ganglion (DRG) neurons (from the peripheral nervous system) and hippocampal neurons (from the central) to see how the mechanics of the microenvironment affect different populations. We find that force generation and stiffness-dependent outgrowth are strongly dependent on cell type. We also observe very different internal dynamics and substrate coupling in the two populations, suggesting that the difference in force generation is due to stronger adhesions and therefore stronger substrate engagement in the peripheral nervous system neurons. We will discuss the biological origins of these differences, and recent analyses of the dynamic aspects of growth cone force generation and the implications for the role of mechanosensitivity in axon guidance. In collaboration with D. Koch, W. Rosoff, and H. M. Geller. Supported by NINDS grant 1R01NS064250-01 (J.S.U.) and the NHLBI Intramural Research Program (H.M.G.).

  12. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  13. The axonal attractant Netrin-1 is an angiogenic factor.

    PubMed

    Park, Kye Won; Crouse, Dana; Lee, Mark; Karnik, Satyajit K; Sorensen, Lise K; Murphy, Kelly J; Kuo, Calvin J; Li, Dean Y

    2004-11-16

    Blood vessels and nerves often follow parallel trajectories, suggesting that distal targets use common cues that induce vascularization and innervation. Netrins are secreted by the floor plate and attract commissural axons toward the midline of the neural tube. Here, we show that Netrin-1 is also a potent vascular mitogen. Netrin-1 stimulates proliferation, induces migration, and promotes adhesion of endothelial cells and vascular smooth muscle cells with a specific activity comparable to vascular endothelial growth factor and platelet-derived growth factor. Our evidence indicates that the netrin receptor, Neogenin, mediates netrin signaling in vascular smooth muscle cells, but suggests that an unidentified receptor mediates the proangiogenic effects of Netrin-1 on endothelial cells. Netrin-1 also stimulates angiogenesis in vivo and augments the response to vascular endothelial growth factor. Thus, we demonstrate that Netrin-1 is a secreted neural guidance cue with the unique ability to attract both blood vessels and axons, and suggest that other cues may also function as vascular endothelial growth factors. PMID:15520390

  14. Structure and Activity of the Axon Guidance Protein MICAL

    SciTech Connect

    Nadella,M.; Bianchet, M.; Gabelli, S.; Barrila, J.; Amzel, L.

    2005-01-01

    During development, neurons are guided to their targets by short- and long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL{sub fd}) determined by x-ray diffraction to 2.0 Angstrom resolution. The structure shows that MICAL{sub fd} is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICALfd is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H{sub 2}O{sub 2} (Km, NAPDH = 222 {micro}M; k{sub cat} = 77 sec{sup -1}), a molecule with known signaling properties. We propose that the H{sub 2}O{sub 2} produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.

  15. Patch voltage clamp of squid axon membrane.

    PubMed

    Fishman, H M

    1975-12-01

    A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise. PMID:1214276

  16. AxonQuant: A Microfluidic Chamber Culture-Coupled Algorithm That Allows High-Throughput Quantification of Axonal Damage

    PubMed Central

    Li, Yang; Yang, Mengxue; Huang, Zhuo; Chen, Xiaoping; Maloney, Michael T.; Zhu, Li; Liu, Jianghong; Yang, Yanmin; Du, Sidan; Jiang, Xingyu; Wu, Jane Y.

    2014-01-01

    Published methods for imaging and quantitatively analyzing morphological changes in neuronal axons have serious limitations because of their small sample sizes, and their time-consuming and nonobjective nature. Here we present an improved microfluidic chamber design suitable for fast and high-throughput imaging of neuronal axons. We developed the Axon-Quant algorithm, which is suitable for automatic processing of axonal imaging data. This microfluidic chamber-coupled algorithm allows calculation of an ‘axonal continuity index’ that quantitatively measures axonal health status in a manner independent of neuronal or axonal density. This method allows quantitative analysis of axonal morphology in an automatic and nonbiased manner. Our method will facilitate large-scale high-throughput screening for genes or therapeutic compounds for neurodegenerative diseases involving axonal damage. When combined with imaging technologies utilizing different gene markers, this method will provide new insights into the mechanistic basis for axon degeneration. Our microfluidic chamber culture-coupled AxonQuant algorithm will be widely useful for studying axonal biology and neurodegenerative disorders. PMID:24603552

  17. Axon growth inhibition by RhoA/ROCK in the central nervous system

    PubMed Central

    Fujita, Yuki; Yamashita, Toshihide

    2014-01-01

    Rho kinase (ROCK) is a serine/threonine kinase and a downstream target of the small GTPase Rho. The RhoA/ROCK pathway is associated with various neuronal functions such as migration, dendrite development, and axonal extension. Evidence from animal studies reveals that RhoA/ROCK signaling is involved in various central nervous system (CNS) diseases, including optic nerve and spinal cord injuries, stroke, and neurodegenerative diseases. Given that RhoA/ROCK plays a critical role in the pathophysiology of CNS diseases, the development of therapeutic agents targeting this pathway is expected to contribute to the treatment of CNS diseases. The RhoA/ROCK pathway mediates the effects of myelin-associated axon growth inhibitors—Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and repulsive guidance molecule (RGM). Blocking RhoA/ROCK signaling can reverse the inhibitory effects of these molecules on axon outgrowth, and promotes axonal sprouting and functional recovery in animal models of CNS injury. To date, several RhoA/ROCK inhibitors have been under development or in clinical trials as therapeutic agents for neurological disorders. In this review, we focus on the RhoA/ROCK signaling pathway in neurological disorders. We also discuss the potential therapeutic approaches of RhoA/ROCK inhibitors for various neurological disorders. PMID:25374504

  18. The formation of axonal caliber and nodes of Ranvier

    NASA Astrophysics Data System (ADS)

    Li, Yinyun; Jung, Peter; Brown, Anthony

    2013-03-01

    A remarkable feature of myelinated neurons is that their axons are constricted at the nodes of Ranvier. These are the locations where axons are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions emerge during development and have been observed to reduce axonal cross sectional area by factors of more than 10. Combining fluorescent imaging methods with computational modeling, we describe how the nervous system regulates the local caliber of its axons through the regulation of the transport kinetics of its most important cytoskeletal elements, the neurofilaments, matching axon caliber and shape to its physiologic function. National Science Foundation IOS 1146789

  19. Changed distribution of sodium channels along demyelinated axons.

    PubMed

    England, J D; Gamboni, F; Levinson, S R; Finger, T E

    1990-09-01

    Voltage-gated sodium channels are largely localized to the nodes of Ranvier in myelinated axons, providing a physiological basis for saltatory conduction. What happens to these channels in demyelinated axons is not known with certainty. Experimentally demyelinated axons were examined by using a well-characterized, polyclonal antibody directed against sodium channels. Immunocytochemical and radioimmunoassay data were consistent with the distribution of an increased number of sodium channels along segments of previously internodal axon. These findings affirm the plasticity of sodium channels in demyelinated axolemma and may be relevant to understanding how axons recover conduction after demyelination. PMID:2168559

  20. Extension of the Caenorhabditis elegans Pharyngeal M1 Neuron Axon Is Regulated by Multiple Mechanisms

    PubMed Central

    Refai, Osama; Rohs, Patricia; Mains, Paul E.; Gaudet, Jeb

    2013-01-01

    The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism. PMID:24048649

  1. Regeneration of diabetic axons is enhanced by selective knockdown of the PTEN gene.

    PubMed

    Singh, Bhagat; Singh, Vandana; Krishnan, Anand; Koshy, Kurien; Martinez, Jose A; Cheng, Chu; Almquist, Chris; Zochodne, Douglas W

    2014-04-01

    Diabetes mellitus renders both widespread and localized irreversible damage to peripheral axons while imposing critical limitations on their ability to regenerate. A major failure of regenerative capacity thereby imposes a 'double hit' in diabetic patients who frequently develop focal neuropathies such as carpal tunnel syndrome in addition to generalized diffuse polyneuropathy. The mechanisms of diabetic neuron regenerative failure have been speculative and few approaches have offered therapeutic opportunities. In this work we identify an unexpected but major role for PTEN upregulation in diabetic peripheral neurons in attenuating axon regrowth. In chronic diabetic neuropathy models in mice, we identified significant PTEN upregulation in peripheral sensory neurons of messenger RNA and protein compared to littermate controls. In vitro, sensory neurons from these mice responded to PTEN knockdown with substantial rises in neurite outgrowth and branching. To test regenerative plasticity in a chronic diabetic model with established neuropathy, we superimposed an additional focal sciatic nerve crush injury and assessed morphological, electrophysiological and behavioural recovery. Knockdown of PTEN in dorsal root ganglia ipsilateral to the side of injury was achieved using a unique form of non-viral short interfering RNA delivery to the ipsilateral nerve injury site and paw. In comparison with scrambled sequence control short interfering RNA, PTEN short interfering RNA improved several facets of regeneration: recovery of compound muscle action potentials, reflecting numbers of reconnected motor axons to endplates, conduction velocities of both motor and sensory axons, reflecting their maturation during regrowth, numbers and calibre of regenerating myelinated axons distal to the injury site, reinnervation of the skin by unmyelinated epidermal axons and recovery of mechanical sensation. Collectively, these findings identify a novel therapeutic approach, potentially

  2. Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo☆

    PubMed Central

    Tripathi, Vineeta Bhasker; Baskaran, Pranetha; Shaw, Christopher E.; Guthrie, Sarah

    2014-01-01

    Intracellular inclusions of the TAR-DNA binding protein 43 (TDP-43) have been reported in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD-TDP). Rare mutations in TARDBP have been linked to both ALS and FTD-TDP suggesting that TDP-43 dysfunction is mechanistic in causing disease. TDP-43 is a predominantly nuclear protein with roles in regulating RNA transcription, splicing, stability and transport. In ALS, TDP-43 aberrantly accumulates in the cytoplasm of motor neurons where it forms aggregates. However it has until recently been unclear whether the toxic effects of TDP-43 involve recruitment to motor axons, and what effects this might have on axonal growth and integrity. Here we use chick embryonic motor neurons, in vivo and in vitro, to model the acute effects of TDP-43. We show that wild-type and two TDP-43 mutant proteins cause toxicity in chick embryonic motor neurons in vivo. Moreover, TDP-43 is increasingly mislocalised to axons over time in vivo, axon growth to peripheral targets is truncated, and expression of neurofilament-associated antigen is reduced relative to control motor neurons. In primary spinal motor neurons in vitro, a progressive translocation of TDP-43 to the cytoplasm occurs over time, similar to that observed in vivo. This coincides with the appearance of cytoplasmic aggregates, a reduction in the axonal length, and cellular toxicity, which was most striking for neurons expressing TDP-43 mutant forms. These observations suggest that the capacity of spinal motor neurons to produce and maintain an axon is compromised by dysregulation of TDP-43 and that the disruption of cytoskeletal integrity may play a role in the pathogenesis of ALS and FTD-TDP. PMID:24423647

  3. Netrin/DCC signaling guides olfactory sensory axons to their correct location in the olfactory bulb

    PubMed Central

    Lakhina, Vanisha; Marcaccio, Christina L.; Shao, Xin; Lush, Mark E.; Jain, Roshan A.; Fujimoto, Esther; Bonkowsky, Joshua L.; Granato, Michael; Raper, Jonathan A.

    2012-01-01

    Olfactory sensory neurons expressing particular olfactory receptors project to specific reproducible locations within the bulb. The axonal guidance cues that organize this precise projection pattern are only beginning to be identified. To aid in their identification and characterization, we generated a transgenic zebrafish line, OR111-7:IRES:Gal4, in which a small subset of olfactory sensory neurons is labeled. Most sensory neurons expressing the OR111-7 transgene project to a specific location within the bulb, the central zone protoglomerulus, while a smaller number project to the LG1 protoglomerulus. Inhibiting netrin/DCC signaling perturbs the ability of OR111-7 expressing axons to enter the olfactory bulb and alters their patterns of termination within the bulb. The netrin receptor DCC is expressed in olfactory sensory neurons around the time that they elaborate their axons, netrin1a is expressed near the medial-most margin of the olfactory bulb, and netrin1b is expressed within the ventral region of the bulb. Loss of netrin/DCC signaling components causes some OR111-7 expressing sensory axons to wander posteriorly after exiting the olfactory pit, away from netrin expressing areas in the bulb. OR111-7 expressing axons that enter the bulb target the central zone less precisely than normal, spreading away from netrin expressing regions. These pathfinding errors can be corrected by the re-expression of DCC within OR111-7 transgene expressing neurons in DCC morphant embryos. These findings implicate netrins as the only known attractants for olfactory sensory neurons, first drawing OR111-7 expressing axons into the bulb and then into the ventromedially positioned central zone protoglomerulus. PMID:22457493

  4. Dynamic Axonal Translation in Developing and Mature Visual Circuits.

    PubMed

    Shigeoka, Toshiaki; Jung, Hosung; Jung, Jane; Turner-Bridger, Benita; Ohk, Jiyeon; Lin, Julie Qiaojin; Amieux, Paul S; Holt, Christine E

    2016-06-30

    Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo. PMID:27321671

  5. Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion

    PubMed Central

    Song, Ren; Koyuncu, Orkide O.; Greco, Todd M.; Diner, Benjamin A.; Cristea, Ileana M.

    2016-01-01

    ABSTRACT Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion. PMID:26838720

  6. The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.

    PubMed

    Mi, Weiqian; Beirowski, Bogdan; Gillingwater, Thomas H; Adalbert, Robert; Wagner, Diana; Grumme, Daniela; Osaka, Hitoshi; Conforti, Laura; Arnhold, Stefan; Addicks, Klaus; Wada, Keiji; Ribchester, Richard R; Coleman, Michael P

    2005-02-01

    Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders. PMID:15644421

  7. The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo

    PubMed Central

    Gaynes, John A.; Otsuna, Hideo; Campbell, Douglas S.; Manfredi, John P.; Levine, Edward M.

    2015-01-01

    Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3’UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development. PMID:26325373

  8. Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

    PubMed Central

    Valakh, Vera; Frey, Erin; Babetto, Elisabetta; Walker, Lauren J; DiAntonio, Aaron

    2015-01-01

    Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage. PMID:25726747

  9. The Emerging Role of Forces in Axonal Elongation

    PubMed Central

    Suter, Daniel M.; Miller, Kyle E.

    2011-01-01

    An understanding of how axons elongate is needed to develop rational strategies to treat neurological diseases and nerve injury. Growth cone-mediated neuronal elongation is currently viewed as occurring through cytoskeletal dynamics involving the polymerization of actin and tubulin subunits at the tip of the axon. However, recent work suggests that axons and growth cones also generate forces (through cytoskeletal dynamics, kinesin, dynein, and myosin), forces induce axonal elongation, and axons lengthen by stretching. This review highlights results from various model systems (Drosophila, Aplysia, Xenopus, chicken, mouse, rat, and PC12 cells), supporting a role for forces, bulk microtubule movements, and intercalated mass addition in the process of axonal elongation. We think that a satisfying answer to the question, “How do axons grow?” will come by integrating the best aspects of biophysics, genetics, and cell biology. PMID:21527310

  10. Axon position within the corpus callosum determines contralateral cortical projection.

    PubMed

    Zhou, Jing; Wen, Yunqing; She, Liang; Sui, Ya-Nan; Liu, Lu; Richards, Linda J; Poo, Mu-Ming

    2013-07-16

    How developing axons in the corpus callosum (CC) achieve their homotopic projection to the contralateral cortex remains unclear. We found that axonal position within the CC plays a critical role in this projection. Labeling of nearby callosal axons in mice showed that callosal axons were segregated in an orderly fashion, with those from more medial cerebral cortex located more dorsally and subsequently projecting to more medial contralateral cortical regions. The normal axonal order within the CC was grossly disturbed when semaphorin3A/neuropilin-1 signaling was disrupted. However, the order in which axons were positioned within the CC still determined their contralateral projection, causing a severe disruption of the homotopic contralateral projection that persisted at postnatal day 30, when the normal developmental refinement of contralateral projections is completed in wild-type (WT) mice. Thus, the orderly positioning of axons within the CC is a primary determinant of how homotopic interhemispheric projections form in the contralateral cortex. PMID:23812756

  11. B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

    PubMed Central

    O’Donovan, Kevin J.; Ma, Kaijie; Guo, Hengchang; Wang, Chen; Sun, Fang; Han, Seung Baek; Kim, Hyukmin; Wong, Jamie K.; Charron, Jean; Zou, Hongyan; Son, Young-Jin; He, Zhigang

    2014-01-01

    Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems. PMID:24733831

  12. Electrogenic Tuning of the Axon Initial Segment

    PubMed Central

    Clark, Brian D.; Goldberg, Ethan M.; Rudy, Bernardo

    2010-01-01

    Action potentials (APs) provide the primary means of rapid information transfer in the nervous system. Where exactly these signals are initiated in neurons has been a basic question in neurobiology and the subject of extensive study. Converging lines of evidence indicate that APs are initiated in a discrete and highly specialized portion of the axon—the axon initial segment (AIS). The authors review key aspects of the organization and function of the AIS and focus on recent work that has provided important insights into its electrical signaling properties. In addition to its main role in AP initiation, the new findings suggest that the AIS is also a site of complex AP modulation by specific types of ion channels localized to this axonal domain. PMID:20007821

  13. Neuroserpin, an axonally secreted serine protease inhibitor.

    PubMed Central

    Osterwalder, T; Contartese, J; Stoeckli, E T; Kuhn, T B; Sonderegger, P

    1996-01-01

    We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult. Images PMID:8670795

  14. Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon.

    PubMed Central

    Goldberg, D J; Schwartz, J H; Sherbany, A A

    1978-01-01

    1. The axonal transport of pulses of [3H]serotonin was studied in an axon of the serotonergic giant cerebral neurone (GCN) of Aplysia californica. 2. [3H]serotonin was transported as a discrete peak which was followed by a relatively low, smooth trail. 3. The peak broadened as it moved along the axon, sometimes skewing in the proximal direction. 4. The velocity of the transport was highly dependent on temperature, but the rate of peak broadening was not. The velocity was 130 mm per day at 23 degrees C and 48 mm per day at 14 degrees C. The rate of broadening was 143 micrometer per mm transport at 23 degrees C and 156 micrometer per mm transport at 14 degrees C. 5. In another series of experiments, almost the entire length of the lip nerve, which contained the axon of GCN, was maintained at 1--3 degrees C to block transport. The GCN's cell body and the proximal few millimetres of the nerve were maintained at 23 degrees C. As a result, the amount of [3H]serotonin in the proximal segment of the nerve increased manyfold during periods of up to 4 hr. The concentrated pulse of [3H]serotonin resulting from this treatment was transported more slowly than normal after the cooling was terminated. Sometimes, a minor peak split from the major peak of radioactivity and was transported a normal velocity. 6. Incubation of the cerebral ganglion and nerves for 16 hr in the presence of anisomycin, an inhibitor of protein synthesis, reduced by nearly fourfold the amount of [3H]serotonin subsequently exported into the axon of the GCN. The transport velocity at this reduced concentration was less than half the normal value. If the concentration of [3H]serotonin in the axon was restored to normal in the presence of anisomycin, the velocity of transport was also returned to normal. 7. We conclude that the velocity of transport of serotonergic vesicles in the axon of the GCN is positively dependent on the local concentration of vesicles, except at very high concentrations, where the

  15. Neurotrophins and the control of axonal outgrowth.

    PubMed

    Tucker, K L

    2002-12-01

    The neurotrophins NGF, BDNF, NT-3, and NT-4/5 are well known for their neuron survival-promoting functions. This review focuses upon a less-investigated function of this gene family, namely that of controlling the growth of axons in vivo. Recent evidence is discussed that reveals a definitive requirement for neurotrophin signaling in the elongation of peripheral nerves during embryonic development. PMID:12434114

  16. Multifunctional Silk Nerve Guides for Axon Outgrowth

    NASA Astrophysics Data System (ADS)

    Tupaj, Marie C.

    Peripheral nerve regeneration is a critical issue as 2.8% of trauma patients present with this type of injury, estimating a total of 200,000 nerve repair procedures yearly in the United States. While the peripheral nervous system exhibits slow regeneration, at a rate of 0.5 mm -- 9 mm/day following trauma, this regenerative ability is only possible under certain conditions. Clinical repairs have changed slightly in the last 30 years and standard methods of treatment include suturing damaged nerve ends, allografting, and autografting, with the autograft the gold standard of these approaches. Unfortunately, the use of autografts requires a second surgery and there is a shortage of nerves available for grafting. Allografts are a second option however allografts have lower success rates and are accompanied by the need of immunosuppressant drugs. Recently there has been a focus on developing nerve guides as an "off the shelf" approach. Although some natural and synthetic guidance channels have been approved by the FDA, these nerve guides are unfunctionalized and repair only short gaps, less than 3 cm in length. The goal of this project was to identify strategies for functionalizing peripheral nerve conduits for the outgrowth of neuron axons in vitro . To accomplish this, two strategies (bioelectrical and biophysical) were indentified for increasing axon outgrowth and promoting axon guidance. Bioelectrical strategies exploited electrical stimulation for increasing neurite outgrowth. Biophysical strategies tested a range of surface topographies for axon guidance. Novel methods were developed for integrating electrical and biophysical strategies into silk films in 2D. Finally, a functionalized nerve conduit system was developed that integrated all strategies for the purpose of attaching, elongating, and guiding nervous tissue in vitro. Future directions of this work include silk conduit translation into a rat sciatic nerve model in vivo for the purpose of repairing long

  17. Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush

    PubMed Central

    Wong, Laura Elisabeth; Gibson, Molly E.; Arnold, H. Moore; Pepinsky, Blake; Frank, Eric

    2015-01-01

    Recovery after a spinal cord injury often requires that axons restore synaptic connectivity with denervated targets several centimeters from the site of injury. Here we report that systemic artemin (ARTN) treatment promotes the regeneration of sensory axons to the brainstem after brachial dorsal root crush in adult rats. ARTN not only stimulates robust regeneration of large, myelinated sensory axons to the brainstem, but also promotes functional reinnervation of the appropriate target region, the cuneate nucleus. ARTN signals primarily through the RET tyrosine kinase, an interaction that requires the nonsignaling coreceptor GDNF family receptor (GFRα3). Previous studies reported limited GFRα3 expression on large sensory neurons, but our findings demonstrate that ARTN promotes robust regeneration of large, myelinated sensory afferents. Using a cell sorting technique, we demonstrate that GFRα3 expression is similar in myelinated and unmyelinated adult sensory neurons, suggesting that ARTN likely induces long-distance regeneration by binding GFRα3 and RET. Although ARTN is delivered for just 2 wk, regeneration to the brainstem requires more than 3 mo, suggesting that brief trophic support may initiate intrinsic growth programs that remain active until targets are reached. Given its ability to promote targeted functional regeneration to the brainstem, ARTN may represent a promising therapy for restoring sensory function after spinal cord injury. PMID:25918373

  18. Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.

    PubMed

    Wong, Laura Elisabeth; Gibson, Molly E; Arnold, H Moore; Pepinsky, Blake; Frank, Eric

    2015-05-12

    Recovery after a spinal cord injury often requires that axons restore synaptic connectivity with denervated targets several centimeters from the site of injury. Here we report that systemic artemin (ARTN) treatment promotes the regeneration of sensory axons to the brainstem after brachial dorsal root crush in adult rats. ARTN not only stimulates robust regeneration of large, myelinated sensory axons to the brainstem, but also promotes functional reinnervation of the appropriate target region, the cuneate nucleus. ARTN signals primarily through the RET tyrosine kinase, an interaction that requires the nonsignaling coreceptor GDNF family receptor (GFRα3). Previous studies reported limited GFRα3 expression on large sensory neurons, but our findings demonstrate that ARTN promotes robust regeneration of large, myelinated sensory afferents. Using a cell sorting technique, we demonstrate that GFRα3 expression is similar in myelinated and unmyelinated adult sensory neurons, suggesting that ARTN likely induces long-distance regeneration by binding GFRα3 and RET. Although ARTN is delivered for just 2 wk, regeneration to the brainstem requires more than 3 mo, suggesting that brief trophic support may initiate intrinsic growth programs that remain active until targets are reached. Given its ability to promote targeted functional regeneration to the brainstem, ARTN may represent a promising therapy for restoring sensory function after spinal cord injury. PMID:25918373

  19. Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration.

    PubMed

    Collyer, E; Catenaccio, A; Lemaitre, D; Diaz, P; Valenzuela, V; Bronfman, F; Court, F A

    2014-11-01

    After an incomplete spinal cord injury (SCI), partial recovery of locomotion is accomplished with time. Previous studies have established a functional link between extension of axon collaterals from spared spinal tracts and locomotor recovery after SCI, but the tissular signals triggering collateral sprouting have not been identified. Here, we investigated whether axonal degeneration after SCI contributes to the sprouting of collaterals from axons spared after injury. To this end, we evaluated collateral sprouting from BDA-labeled uninjured corticospinal axons after spinal cord hemisection (SCI(H)) in wild type (WT) mouse and Wld(S) mouse strains, which shows a significant delay in Wallerian degeneration after injury. After SCI(H), spared fibers of WT mice extend collateral sprouts to both intact and denervated sides of the spinal cord distant from the injury site. On the contrary, in the Wld(S) mice collateral sprouting from spared fibers was greatly reduced after SCI(H). Consistent with a role for collateral sprouting in functional recovery after SCI, locomotor recovery after SCI(H) was impaired in Wld(S) mice compared to WT animals. In conclusion, our results identify axonal degeneration as one of the triggers for collateral sprouting from the contralesional uninjured fibers after an SCI(H). These results open the path for identifying molecular signals associated with tissular changes after SCI that promotes collateral sprouting and functional recovery. PMID:25079366

  20. Acute Reduction of Microglia Does Not Alter Axonal Injury in a Mouse Model of Repetitive Concussive Traumatic Brain Injury

    PubMed Central

    Bennett, Rachel E.

    2014-01-01

    Abstract The pathological processes that lead to long-term consequences of multiple concussions are unclear. Primary mechanical damage to axons during concussion is likely to contribute to dysfunction. Secondary damage has been hypothesized to be induced or exacerbated by inflammation. The main inflammatory cells in the brain are microglia, a type of macrophage. This research sought to determine the contribution of microglia to axon degeneration after repetitive closed-skull traumatic brain injury (rcTBI) using CD11b-TK (thymidine kinase) mice, a valganciclovir-inducible model of macrophage depletion. Low-dose (1 mg/mL) valganciclovir was found to reduce the microglial population in the corpus callosum and external capsule by 35% after rcTBI in CD11b-TK mice. At both acute (7 days) and subacute (21 days) time points after rcTBI, reduction of the microglial population did not alter the extent of axon injury as visualized by silver staining. Further reduction of the microglial population by 56%, using an intermediate dose (10 mg/mL), also did not alter the extent of silver staining, amyloid precursor protein accumulation, neurofilament labeling, or axon injury evident by electron microscopy at 7 days postinjury. Longer treatment of CD11b-TK mice with intermediate dose and treatment for 14 days with high-dose (50 mg/mL) valganciclovir were both found to be toxic in this injury model. Altogether, these data are most consistent with the idea that microglia do not contribute to acute axon degeneration after multiple concussive injuries. The possibility of longer-term effects on axon structure or function cannot be ruled out. Nonetheless, alternative strategies directly targeting injury to axons may be a more beneficial approach to concussion treatment than targeting secondary processes of microglial-driven inflammation. PMID:24797413

  1. Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression

    PubMed Central

    Fournier, Adam J.; Hogan, James D.; Rajbhandari, Labchan; Shrestha, Shiva; Venkatesan, Arun; Ramesh, K. T.

    2015-01-01

    Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism. PMID:26111004

  2. Regulation of Neurotrophin-Induced Axonal Responses via Rho GTPases

    PubMed Central

    HANDEÖZDINLER, P.; ERZURUMLU, REHA S.

    2014-01-01

    Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons. In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization. Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons. PMID:11559894

  3. Organization and maintenance of molecular domains in myelinated axons.

    PubMed

    Buttermore, Elizabeth D; Thaxton, Courtney L; Bhat, Manzoor A

    2013-05-01

    Over a century ago, Ramon y Cajal first proposed the idea of a directionality involved in nerve conduction and neuronal communication. Decades later, it was discovered that myelin, produced by glial cells, insulated axons with periodic breaks where nodes of Ranvier (nodes) form to allow for saltatory conduction. In the peripheral nervous system (PNS), Schwann cells are the glia that can either individually myelinate the axon from one neuron or ensheath axons of many neurons. In the central nervous system (CNS), oligodendrocytes are the glia that myelinate axons from different neurons. Review of more recent studies revealed that this myelination created polarized domains adjacent to the nodes. However, the molecular mechanisms responsible for the organization of axonal domains are only now beginning to be elucidated. The molecular domains in myelinated axons include the axon initial segment (AIS), where various ion channels are clustered and action potentials are initiated; the node, where sodium channels are clustered and action potentials are propagated; the paranode, where myelin loops contact with the axolemma; the juxtaparanode (JXP), where delayed-rectifier potassium channels are clustered; and the internode, where myelin is compactly wrapped. Each domain contains a unique subset of proteins critical for the domain's function. However, the roles of these proteins in axonal domain organization are not fully understood. In this review, we highlight recent advances on the molecular nature and functions of some of the components of each axonal domain and their roles in axonal domain organization and maintenance for proper neuronal communication. PMID:23404451

  4. GnRH neurons elaborate a long-range projection with shared axonal and dendritic functions.

    PubMed

    Herde, Michel K; Iremonger, Karl J; Constantin, Stephanie; Herbison, Allan E

    2013-07-31

    Information processing by neurons has been traditionally envisioned to occur in discrete neuronal compartments. Specifically, dendrites receive and integrate synaptic inputs while axons initiate and conduct spikes to distal neuronal targets. We report here in mice, using morphological reconstructions and electrophysiology, that the gonadotropin-releasing hormone (GnRH) neurons that control mammalian fertility do not conform to this stereotype and instead possess a single projection structure that functions simultaneously as an axon and dendrite. Specifically, we show that the GnRH neuron projection to the median eminence to control pituitary hormone secretion possesses a spike initiation site and conducts action potentials while also exhibiting spines and synaptic appositions along its entire length. Classical axonal or dendritic markers are not detectable in the projection process. Activation of ionotropic glutamate and/or GABA receptors along the GnRH neuron projection is capable of depolarizing the membrane potential and initiating action potentials. In addition, focal glutamate application to the projection is able to regulate the width of propagating spikes. These data demonstrate that GnRH neurons elaborate a previously uncharacterized neuronal projection that functions simultaneously as an axon and dendrite. This structure, termed a "dendron," greatly expands the dynamic control of GnRH secretion into the pituitary portal system to regulate fertility. PMID:23904605

  5. Insulin-like Signaling Promotes Glial Phagocytic Clearance of Degenerating Axons through Regulation of Draper.

    PubMed

    Musashe, Derek T; Purice, Maria D; Speese, Sean D; Doherty, Johnna; Logan, Mary A

    2016-08-16

    Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. Here, we find that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. We find that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity, it is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. We propose that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity. PMID:27498858

  6. Arrest of Myelination and Reduced Axon Growth when Schwann Cells Lack mTOR

    PubMed Central

    Sherman, Diane L.; Krols, Michiel; Wu, Lai-Man; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J.

    2014-01-01

    In developing peripheral nerves differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years there has been an increasing understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination together with a growing appreciation of some of the signalling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal post-natal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signalling in both the longitudinal and radial growth of the myelinating Schwann cell. PMID:22302821

  7. Arrest of myelination and reduced axon growth when Schwann cells lack mTOR.

    PubMed

    Sherman, Diane L; Krols, Michiel; Wu, Lai-Man N; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J

    2012-02-01

    In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell. PMID:22302821

  8. c-Jun in Schwann cells promotes axonal regeneration and motoneuron survival via paracrine signaling

    PubMed Central

    Fontana, Xavier; Hristova, Mariya; Da Costa, Clive; Patodia, Smriti; Thei, Laura; Makwana, Milan; Spencer-Dene, Bradley; Latouche, Morwena; Mirsky, Rhona; Jessen, Kristjan R.; Klein, Rüdiger

    2012-01-01

    The AP-1 transcription factor c-Jun is a master regulator of the axonal response in neurons. c-Jun also functions as a negative regulator of myelination in Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We demonstrate here that, after injury, the absence of c-Jun specifically in SCs caused impaired axonal regeneration and severely increased neuronal cell death. c-Jun deficiency resulted in decreased expression of several neurotrophic factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic inactivation of Ret specifically in neurons resulted in regeneration defects without affecting motoneuron survival and, conversely, administration of recombinant GDNF and Artemin protein substantially ameliorated impaired regeneration caused by c-Jun deficiency. These results reveal an unexpected function for c-Jun in SCs in response to axonal injury, and identify paracrine Ret signaling as an important mediator of c-Jun function in SCs during regeneration. PMID:22753894

  9. Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

    PubMed Central

    Elluru, R G; Bloom, G S; Brady, S T

    1995-01-01

    The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport. Images PMID:7538359

  10. DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function.

    PubMed

    Rabe Bernhardt, Nadine; Memic, Fatima; Gezelius, Henrik; Thiebes, Anja-Lena; Vallstedt, Anna; Kullander, Klas

    2012-06-15

    Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction. PMID:22521513

  11. Myelin regeneration in multiple sclerosis: targeting endogenous stem cells.

    PubMed

    Huang, Jeffrey K; Fancy, Stephen P J; Zhao, Chao; Rowitch, David H; Ffrench-Constant, Charles; Franklin, Robin J M

    2011-10-01

    Regeneration of myelin sheaths (remyelination) after central nervous system demyelination is important to restore saltatory conduction and to prevent axonal loss. In multiple sclerosis, the insufficiency of remyelination leads to the irreversible degeneration of axons and correlated clinical decline. Therefore, a regenerative strategy to encourage remyelination may protect axons and improve symptoms in multiple sclerosis. We highlight recent studies on factors that influence endogenous remyelination and potential promising pharmacological targets that may be considered for enhancing central nervous system remyelination. PMID:21904791

  12. Early ultrastructural defects of axons and axon-glia junctions in mice lacking expression of Cnp1.

    PubMed

    Edgar, Julia M; McLaughlin, Mark; Werner, Hauke B; McCulloch, Mailis C; Barrie, Jennifer A; Brown, Angus; Faichney, Andrew Blyth; Snaidero, Nicolas; Nave, Klaus-Armin; Griffiths, Ian R

    2009-12-01

    Most axons in the central nervous system (CNS) are surrounded by a multilayered myelin sheath that promotes fast, saltatory conduction of electrical impulses. By insulating the axon, myelin also shields the axoplasm from the extracellular milieu. In the CNS, oligodendrocytes provide support for the long-term maintenance of myelinated axons, independent of the myelin sheath. Here, we use electron microscopy and morphometric analyses to examine the evolution of axonal and oligodendroglial changes in mice deficient in 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and in mice deficient in both CNP and proteolipid protein (PLP/DM20). We show that CNP is necessary for the formation of a normal inner tongue process of oligodendrocytes that myelinate small diameter axons. We also show that axonal degeneration in Cnp1 null mice is present very early in postnatal life. Importantly, compact myelin formed by transplanted Cnp1 null oligodendrocytes induces the same degenerative changes in shiverer axons that normally are dysmyelinated but structurally intact. Mice deficient in both CNP and PLP develop a more severe axonal phenotype than either single mutant, indicating that the two oligodendroglial proteins serve distinct functions in supporting the myelinated axon. These observations support a model in which the trophic functions of oligodendrocytes serve to offset the physical shielding of axons by myelin membranes. PMID:19459211

  13. Focal axonal swellings and associated ultrastructural changes attenuate conduction velocity in central nervous system axons: a computer modeling study.

    PubMed

    Kolaric, Katarina V; Thomson, Gemma; Edgar, Julia M; Brown, Angus M

    2013-08-01

    The constancy of action potential conduction in the central nervous system (CNS) relies on uniform axon diameter coupled with fidelity of the overlying myelin providing high-resistance, low capacitance insulation. Whereas the effects of demyelination on conduction have been extensively studied/modeled, equivalent studies on the repercussions for conduction of axon swelling, a common early pathological feature of (potentially reversible) axonal injury, are lacking. The recent description of experimentally acquired morphological and electrical properties of small CNS axons and oligodendrocytes prompted us to incorporate these data into a computer model, with the aim of simulating the effects of focal axon swelling on action potential conduction. A single swelling on an otherwise intact axon, as occurs in optic nerve axons of Cnp1 null mice caused a small decrease in conduction velocity. The presence of single swellings on multiple contiguous internodal regions (INR), as likely occurs in advanced disease, caused qualitatively similar results, except the dimensions of the swellings required to produce equivalent attenuation of conduction were significantly decreased. Our simulations of the consequences of metabolic insult to axons, namely, the appearance of multiple swollen regions, accompanied by perturbation of overlying myelin and increased axolemmal permeability, contained within a single INR, revealed that conduction block occurred when the dimensions of the simulated swellings were within the limits of those measured experimentally, suggesting that multiple swellings on a single axon could contribute to axonal dysfunction, and that increased axolemmal permeability is the decisive factor that promotes conduction block. PMID:24303138

  14. Focal axonal swellings and associated ultrastructural changes attenuate conduction velocity in central nervous system axons: a computer modeling study

    PubMed Central

    Kolaric, Katarina V; Thomson, Gemma; Edgar, Julia M; Brown, Angus M

    2013-01-01

    The constancy of action potential conduction in the central nervous system (CNS) relies on uniform axon diameter coupled with fidelity of the overlying myelin providing high-resistance, low capacitance insulation. Whereas the effects of demyelination on conduction have been extensively studied/modeled, equivalent studies on the repercussions for conduction of axon swelling, a common early pathological feature of (potentially reversible) axonal injury, are lacking. The recent description of experimentally acquired morphological and electrical properties of small CNS axons and oligodendrocytes prompted us to incorporate these data into a computer model, with the aim of simulating the effects of focal axon swelling on action potential conduction. A single swelling on an otherwise intact axon, as occurs in optic nerve axons of Cnp1 null mice caused a small decrease in conduction velocity. The presence of single swellings on multiple contiguous internodal regions (INR), as likely occurs in advanced disease, caused qualitatively similar results, except the dimensions of the swellings required to produce equivalent attenuation of conduction were significantly decreased. Our simulations of the consequences of metabolic insult to axons, namely, the appearance of multiple swollen regions, accompanied by perturbation of overlying myelin and increased axolemmal permeability, contained within a single INR, revealed that conduction block occurred when the dimensions of the simulated swellings were within the limits of those measured experimentally, suggesting that multiple swellings on a single axon could contribute to axonal dysfunction, and that increased axolemmal permeability is the decisive factor that promotes conduction block. PMID:24303138

  15. Proton hopping: a proposed mechanism for myelinated axon nerve impulses.

    PubMed

    Kier, Lemont B; Tombes, Robert M

    2013-04-01

    Myelinated axon nerve impulses travel 100 times more rapidly than impulses in non-myelinated axons. Increased speed is currently believed to be due to 'hopping' or 'saltatory propagation' along the axon, but the mechanism by which impulses flow has never been adequately explained. We have used modeling approaches to simulate a role for proton hopping in the space between the plasma membrane and myelin sheath as the mechanism of nerve action-potential flow. PMID:23576345

  16. Glial regulation of the axonal membrane at nodes of Ranvier.

    PubMed

    Schafer, Dorothy P; Rasband, Matthew N

    2006-10-01

    Action potential conduction in myelinated nerve fibers depends on a polarized axonal membrane. Voltage-gated Na(+) and K(+) channels are clustered at nodes of Ranvier and mediate the transmembrane currents necessary for rapid saltatory conduction. Paranodal junctions flank nodes and function as attachment sites for myelin and as paracellular and membrane protein diffusion barriers. Common molecular mechanisms, directed by myelinating glia, are used to establish these axonal membrane domains. Initially, heterophilic interactions between glial and axonal cell adhesion molecules define the locations where nodes or paranodes form. Subsequently, within each domain, axonal cell adhesion molecules are stabilized and retained through interactions with cytoskeletal and scaffolding proteins, including ankyrins and spectrins. PMID:16945520

  17. IRE-1/XBP-1 pathway of the unfolded protein response is required for properly localizing neuronal UNC-6/Netrin for axon guidance in C. elegans.

    PubMed

    Asakura, Taro; Ogura, Ken-ichi; Goshima, Yoshio

    2015-03-01

    During developing nervous system, neurons project axons to their targets precisely. In this process, axon guidance molecules provide positional information to the axons. Therefore, the spatially and temporally controlled localization of the axon guidance molecules is required for the proper structure formation of the complex nervous system. In C. elegans, UNC-6/Netrin is a secreted protein that elicits both attractive and repulsive response in axon guidance. UNC-6/Netrin secreted from ventral cells may establish a concentration gradient from the ventral to the dorsal side of the animal, thus providing dorso-ventral positional information. However, the mechanisms specifying positional information of UNC-6/Netrin are largely unknown. Here, we show that the ire-1/xbp-1 pathway of the unfolded protein response (UPR) is required for axonal distribution of UNC-6/Netrin in the ventral neurons. In addition, the ire-1/xbp-1 pathway is also required for dorso-ventral axon guidance mediated by UNC-6/Netrin. Our results suggest that the ire-1/xbp-1 pathway of the UPR is crucial for establishing positional information of UNC-6/Netrin. We propose that the proper secretion of UNC-6/Netrin from the ventral neurons requires the activity of IRE-1. PMID:25469499

  18. Effects of Deltamethrin on crayfish motor axon activity and neuromuscular transmission.

    PubMed

    Meng, Linlin; Meyer, Pierre-Francois N R; Leary, Meghan L; Mohammed, Yussef F; Ferber, Shelbi D; Lin, Jen-Wei

    2016-03-23

    Deltamethrin (DM) is a widely used pesticide known to target sodium channels. Although this compound has been studied extensively at molecular and behavioral levels, the detailed action of DM on cellular and synaptic function is less well documented. In this report, we show that DM at nanomolar concentrations can silence tonic motor output of the crayfish ventral superficial flexor (VSF) within ∼10 min. Action potential (AP) amplitude was consistently reduced before silencing occurred, whereas AP duration and AP firing frequency did not change. In some synapses EPSP amplitude and synaptic delay were modified by DM, but the direction of change was not consistent. In order to better understand these diverse effects, intracellular recordings from motor axons of the crayfish opener were used for a detailed analysis. DM caused an initial, slow depolarization of resting membrane potential (Vm), which was accompanied by reduced AP amplitude but not AP duration. Resting Vm then underwent a step depolarization of ∼20 mV, which we propose corresponds to the onset of the depolarization block. In addition, DM shifted the AP initiation site in some opener axons during prolonged firing. This shift occurred concomitantly with a reduction in synaptic delay. A similar reduction in synaptic delay was also detected at some VSF axons, and can be attributed to the same mechanism. Results reported here suggest that DM at low concentrations result in: (i) depolarization block of motor axons before changes in network output can be detected, (ii) variable effects on synaptic transmission, with this variability presumably due to the diverse morphology and excitability of motor axons. PMID:26861201

  19. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  20. Collateral sprouting of sensory axons after end-to-side nerve coaptation--a longitudinal study in the rat.

    PubMed

    Kovacic, Uros; Tomsic, Martin; Sketelj, Janez; Bajrović, Fajko F

    2007-02-01

    The end-to-side nerve coaptation is able to induce collateral sprouting of axons from the donor nerve and to provide functional reinnervation of the target tissue. Sensory axon sprouting and its effects on the donor nerve up to 9 months after the end-to-side nerve coaptation were studied in the rat. Peroneal, tibial and saphenous nerves were transected and ligated, and the distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. The average skin area of the residual sensitivity to pinch due to the axons sprouting through the recipient peroneal nerve did not change statistically significantly between 4 and 9 months after surgery. Axon counting, measurements of compound action potentials and retrograde neuron labeling indicate that the sprouting of the myelinated sensory axons and unmyelinated axons through the recipient nerve was largely completed by 2 months and 4 months after the end-to-side nerve coaptation, respectively, and remained stable thereafter for at least 9 months. A decrease in the amplitude and area of the CAP of myelinated fibers, observed in the donor nerve up to 4 months after surgery, was probably due to mild degeneration of nerve fibers and a tendency of the diameter of myelinated axons to decline. However, no significant changes in functional, electrophysiological or morphological properties of the donor nerve could be observed at the end of the observational period, indicating that end-to-side nerve coaptation has no detrimental effect on the donor nerve on a long-term scale. PMID:17045263

  1. Axonal Velocity Distributions in Neural Field Equations

    PubMed Central

    Bojak, Ingo; Liley, David T. J.

    2010-01-01

    By modelling the average activity of large neuronal populations, continuum mean field models (MFMs) have become an increasingly important theoretical tool for understanding the emergent activity of cortical tissue. In order to be computationally tractable, long-range propagation of activity in MFMs is often approximated with partial differential equations (PDEs). However, PDE approximations in current use correspond to underlying axonal velocity distributions incompatible with experimental measurements. In order to rectify this deficiency, we here introduce novel propagation PDEs that give rise to smooth unimodal distributions of axonal conduction velocities. We also argue that velocities estimated from fibre diameters in slice and from latency measurements, respectively, relate quite differently to such distributions, a significant point for any phenomenological description. Our PDEs are then successfully fit to fibre diameter data from human corpus callosum and rat subcortical white matter. This allows for the first time to simulate long-range conduction in the mammalian brain with realistic, convenient PDEs. Furthermore, the obtained results suggest that the propagation of activity in rat and human differs significantly beyond mere scaling. The dynamical consequences of our new formulation are investigated in the context of a well known neural field model. On the basis of Turing instability analyses, we conclude that pattern formation is more easily initiated using our more realistic propagator. By increasing characteristic conduction velocities, a smooth transition can occur from self-sustaining bulk oscillations to travelling waves of various wavelengths, which may influence axonal growth during development. Our analytic results are also corroborated numerically using simulations on a large spatial grid. Thus we provide here a comprehensive analysis of empirically constrained activity propagation in the context of MFMs, which will allow more realistic studies

  2. Disrupted Axonal Fiber Connectivity in Schizophrenia

    PubMed Central

    Zalesky, Andrew; Fornito, Alex; Seal, Marc L.; Cocchi, Luca; Westin, Carl-Fredrik; Bullmore, Edward T.; Egan, Gary F.; Pantelis, Christos

    2016-01-01

    Background Schizophrenia is believed to result from abnormal functional integration of neural processes thought to arise from aberrant brain connectivity. However, evidence for anatomical dysconnectivity has been equivocal, and few studies have examined axonal fiber connectivity in schizophrenia at the level of whole-brain networks. Methods Cortico-cortical anatomical connectivity at the scale of axonal fiber bundles was modeled as a network. Eighty-two network nodes demarcated functionally specific cortical regions. Sixty-four direction diffusion tensor-imaging coupled with whole-brain tractography was performed to map the architecture via which network nodes were interconnected in each of 74 patients with schizophrenia and 32 age- and gender-matched control subjects. Testing was performed to identify pairs of nodes between which connectivity was impaired in the patient group. The connectional architecture of patients was tested for changes in five network attributes: nodal degree, small-worldness, efficiency, path length, and clustering. Results Impaired connectivity in the patient group was found to involve a distributed network of nodes comprising medial frontal, parietal/occipital, and the left temporal lobe. Although small-world attributes were conserved in schizophrenia, the cortex was interconnected more sparsely and up to 20% less efficiently in patients. Intellectual performance was found to be associated with brain efficiency in control subjects but not in patients. Conclusions This study presents evidence of widespread dysconnectivity in white-matter connectional architecture in a large sample of patients with schizophrenia. When considered from the perspective of recent evidence for impaired synaptic plasticity, this study points to a multifaceted pathophysiology in schizophrenia encompassing axonal as well as putative synaptic mechanisms. PMID:21035793

  3. Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain.

    PubMed

    Larsen, Camilla; Shy, Diana; Spindler, Shana R; Fung, Siaumin; Pereanu, Wayne; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2009-11-15

    The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are "scaffolded" by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments. PMID:19538956

  4. Axonal maintenance, glia, exosomes, and heat shock proteins

    PubMed Central

    Tytell, Michael; Lasek, Raymond J.; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  5. Axonal maintenance, glia, exosomes, and heat shock proteins.

    PubMed

    Tytell, Michael; Lasek, Raymond J; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  6. Spinophilin Facilitates PP1-Mediated Dephosphorylation of PSer297 Doublecortin in Microtubule Bundling at the Axonal “Wrist”

    PubMed Central

    Bielas, Stephanie L.; Serneo, Finley F.; Chechlacz, Magdalena; Deerinck, Thomas J.; Perkins, Guy A.; Allen, Patrick B.; Ellisman, Mark H.; Gleeson, Joseph G.

    2007-01-01

    The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the “wrist” of growing axons, leading to activation. Loss of activity at the “wrist” is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton. PMID:17482550

  7. L-carnitine enhances axonal plasticity and improves white-matter lesions after chronic hypoperfusion in rat brain

    PubMed Central

    Ueno, Yuji; Koike, Masato; Shimada, Yoshiaki; Shimura, Hideki; Hira, Kenichiro; Tanaka, Ryota; Uchiyama, Yasuo; Hattori, Nobutaka; Urabe, Takao

    2015-01-01

    Chronic cerebral hypoperfusion causes white-matter lesions (WMLs) with oxidative stress and cognitive impairment. However, the biologic mechanisms that regulate axonal plasticity under chronic cerebral hypoperfusion have not been fully investigated. Here, we investigated whether L-carnitine, an antioxidant agent, enhances axonal plasticity and oligodendrocyte expression, and explored the signaling pathways that mediate axonal plasticity in a rat chronic hypoperfusion model. Adult male Wistar rats subjected to ligation of the bilateral common carotid arteries (LBCCA) were treated with or without L-carnitine. L-carnitine-treated rats exhibited significantly reduced escape latency in the Morris water maze task at 28 days after chronic hypoperfusion. Western blot analysis indicated that L-carnitine increased levels of phosphorylated high-molecular weight neurofilament (pNFH), concurrent with a reduction in phosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN), and increased phosphorylated Akt and mammalian target of rapamycin (mTOR) at 28 days after chronic hypoperfusion. L-carnitine reduced lipid peroxidation and oxidative DNA damage, and enhanced oligodendrocyte marker expression and myelin sheath thickness after chronic hypoperfusion. L-carnitine regulates the PTEN/Akt/mTOR signaling pathway, and enhances axonal plasticity while concurrently ameliorating oxidative stress and increasing oligodendrocyte myelination of axons, thereby improving WMLs and cognitive impairment in a rat chronic hypoperfusion model. PMID:25465043

  8. PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1

    NASA Astrophysics Data System (ADS)

    Saijilafu; Hur, Eun-Mi; Liu, Chang-Mei; Jiao, Zhongxian; Xu, Wen-Lin; Zhou, Feng-Quan

    2013-10-01

    In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.

  9. High-resolution live imaging reveals axon-glia interactions during peripheral nerve injury and repair in zebrafish.

    PubMed

    Xiao, Yan; Faucherre, Adèle; Pola-Morell, Laura; Heddleston, John M; Liu, Tsung-Li; Chew, Teng-Leong; Sato, Fuminori; Sehara-Fujisawa, Atsuko; Kawakami, Koichi; López-Schier, Hernán

    2015-06-01

    Neural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of Büngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation. PMID:26035865

  10. High-resolution live imaging reveals axon-glia interactions during peripheral nerve injury and repair in zebrafish

    PubMed Central

    Xiao, Yan; Faucherre, Adèle; Pola-Morell, Laura; Heddleston, John M.; Liu, Tsung-Li; Chew, Teng-Leong; Sato, Fuminori; Sehara-Fujisawa, Atsuko; Kawakami, Koichi; López-Schier, Hernán

    2015-01-01

    ABSTRACT Neural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of Büngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation. PMID:26035865

  11. L-carnitine enhances axonal plasticity and improves white-matter lesions after chronic hypoperfusion in rat brain.

    PubMed

    Ueno, Yuji; Koike, Masato; Shimada, Yoshiaki; Shimura, Hideki; Hira, Kenichiro; Tanaka, Ryota; Uchiyama, Yasuo; Hattori, Nobutaka; Urabe, Takao

    2015-03-01

    Chronic cerebral hypoperfusion causes white-matter lesions (WMLs) with oxidative stress and cognitive impairment. However, the biologic mechanisms that regulate axonal plasticity under chronic cerebral hypoperfusion have not been fully investigated. Here, we investigated whether L-carnitine, an antioxidant agent, enhances axonal plasticity and oligodendrocyte expression, and explored the signaling pathways that mediate axonal plasticity in a rat chronic hypoperfusion model. Adult male Wistar rats subjected to ligation of the bilateral common carotid arteries (LBCCA) were treated with or without L-carnitine. L-carnitine-treated rats exhibited significantly reduced escape latency in the Morris water maze task at 28 days after chronic hypoperfusion. Western blot analysis indicated that L-carnitine increased levels of phosphorylated high-molecular weight neurofilament (pNFH), concurrent with a reduction in phosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN), and increased phosphorylated Akt and mammalian target of rapamycin (mTOR) at 28 days after chronic hypoperfusion. L-carnitine reduced lipid peroxidation and oxidative DNA damage, and enhanced oligodendrocyte marker expression and myelin sheath thickness after chronic hypoperfusion. L-carnitine regulates the PTEN/Akt/mTOR signaling pathway, and enhances axonal plasticity while concurrently ameliorating oxidative stress and increasing oligodendrocyte myelination of axons, thereby improving WMLs and cognitive impairment in a rat chronic hypoperfusion model. PMID:25465043

  12. Differential extraction of axonally transported proteoglycans

    SciTech Connect

    Elam, J.S. )

    1990-10-01

    Axonally transported proteoglycans were differentially solubilized by a sequence of extractions designed to infer their relationship to nerve terminal membranes. Groups of goldfish were injected unilaterally with 35SO4 and contralateral optic tecta containing axonally transported molecules were removed 16 h later. Tecta were homogenized in isotonic buffer and centrifuged at 100,000 g for 60 min to create a total supernatant fraction. Subsequent homogenizations followed by recentrifugation were with hypotonic buffer (lysis extract), 1 M NaCl, Triton X-100 or alternatively Triton-1 M NaCl. Populations of proteoglycans in each extract were isolated on DEAE ion exchange columns and evaluated for content of glycosaminoglycans (GAGs). Results show the distribution of transported proteoglycans to be 26.3% total soluble, 13.7% lysis extract, 13.8% NaCl extract, 12.2% Triton extract, and 46.2% Triton-NaCl extract. Proteoglycans from all fractions contained heparan sulfate as the predominant GAG, with lesser amounts of chondroitin (4 or 6) sulfate. The possible localizations of transported proteoglycans suggested by the extraction results are discussed.

  13. Mitochondrial biogenesis is required for axonal growth.

    PubMed

    Vaarmann, Annika; Mandel, Merle; Zeb, Akbar; Wareski, Przemyslaw; Liiv, Joanna; Kuum, Malle; Antsov, Eva; Liiv, Mailis; Cagalinec, Michal; Choubey, Vinay; Kaasik, Allen

    2016-06-01

    During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKβ, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized. PMID:27122166

  14. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

    PubMed

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  15. Quantitative Analysis of Axonal Branch Dynamics in the Developing Nervous System

    PubMed Central

    Scott, Ethan K.; Goodhill, Geoffrey J.

    2016-01-01

    Branching is an important mechanism by which axons navigate to their targets during neural development. For instance, in the developing zebrafish retinotectal system, selective branching plays a critical role during both initial pathfinding and subsequent arborisation once the target zone has been reached. Here we show how quantitative methods can help extract new information from time-lapse imaging about the nature of the underlying branch dynamics. First, we introduce Dynamic Time Warping to this domain as a method for automatically matching branches between frames, replacing the effort required for manual matching. Second, we model branch dynamics as a birth-death process, i.e. a special case of a continuous-time Markov process. This reveals that the birth rate for branches from zebrafish retinotectal axons, as they navigate across the tectum, increased over time. We observed no significant change in the death rate for branches over this time period. However, blocking neuronal activity with TTX slightly increased the death rate, without a detectable change in the birth rate. Third, we show how the extraction of these rates allows computational simulations of branch dynamics whose statistics closely match the data. Together these results reveal new aspects of the biology of retinotectal pathfinding, and introduce computational techniques which are applicable to the study of axon branching more generally. PMID:26998842

  16. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury

    PubMed Central

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A.

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  17. Synaptic connectivity of the cholinergic axons in the olfactory bulb of the cynomolgus monkey

    PubMed Central

    Liberia, Teresa; Blasco-Ibáñez, José Miguel; Nácher, Juan; Varea, Emilio; Lanciego, José Luis; Crespo, Carlos

    2015-01-01

    The olfactory bulb (OB) of mammals receives cholinergic afferents from the horizontal limb of the diagonal band of Broca (HDB). At present, the synaptic connectivity of the cholinergic axons on the circuits of the OB has only been investigated in the rat. In this report, we analyze the synaptic connectivity of the cholinergic axons in the OB of the cynomolgus monkey (Macaca fascicularis). Our aim is to investigate whether the cholinergic innervation of the bulbar circuits is phylogenetically conserved between macrosmatic and microsmatic mammals. Our results demonstrate that the cholinergic axons form synaptic contacts on interneurons. In the glomerular layer, their main targets are the periglomerular cells, which receive axo-somatic and axo-dendritic synapses. In the inframitral region, their main targets are the granule cells, which receive synaptic contacts on their dendritic shafts and spines. Although the cholinergic boutons were frequently found in close vicinity of the dendrites of principal cells, we have not found synaptic contacts on them. From a comparative perspective, our data indicate that the synaptic connectivity of the cholinergic circuits is highly preserved in the OB of macrosmatic and microsmatic mammals. PMID:25852490

  18. Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

    PubMed Central

    Dean, Camin; Dunning, F. Mark; Liu, Huisheng; Bomba-Warczak, Ewa; Martens, Henrik; Bharat, Vinita; Ahmed, Saheeb; Chapman, Edwin R.

    2012-01-01

    The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function. PMID:22398727

  19. Identification of Palmitoyltransferase and Thioesterase Enzymes That Control the Subcellular Localization of Axon Survival Factor Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2)*

    PubMed Central

    Milde, Stefan; Coleman, Michael P.

    2014-01-01

    The NAD-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is a critical survival factor for axons and its constant supply from neuronal cell bodies into axons is required for axon survival in primary culture neurites and axon extension in vivo. Recently, we showed that palmitoylation is necessary to target NMNAT2 to post-Golgi vesicles, thereby influencing its protein turnover and axon protective capacity. Here we find that NMNAT2 is a substrate for cytosolic thioesterases APT1 and APT2 and that palmitoylation/depalmitoylation dynamics are on a time scale similar to its short half-life. Interestingly, however, depalmitoylation does not release NMNAT2 from membranes. The mechanism of palmitoylation-independent membrane attachment appears to be mediated by the same minimal domain required for palmitoylation itself. Furthermore, we identify several zDHHC palmitoyltransferases that influence NMNAT2 palmitoylation and subcellular localization, among which a role for zDHHC17 (HIP14) in neuronal NMNAT2 palmitoylation is best supported by our data. These findings shed light on the enzymatic regulation of NMNAT2 palmitoylation and highlight individual thioesterases and palmitoyltransferases as potential targets to modulate NMNAT2-dependent axon survival. PMID:25271157

  20. Canonical wnt signaling is required for commissural axon guidance

    PubMed Central

    Avilés, Evelyn C.

    2015-01-01

    ABSTRACT Morphogens have been identified as guidance cues for postcrossing commissural axons in the spinal cord. Shh has a dual effect on postcrossing commissural axons: a direct repellent effect mediated by Hhip as a receptor, and an indirect effect by shaping a Wnt activity gradient. Wnts were shown to be attractants for postcrossing commissural axons in both chicken and mouse embryos. In mouse, the effects of Wnts on axon guidance were concluded to depend on the planar cell polarity (PCP) pathway. Canonical Wnt signaling was excluded based on the absence of axon guidance defects in mice lacking Lrp6 which is an obligatory coreceptor for Fzd in canonical Wnt signaling. In the loss‐of‐function studies reported here, we confirmed a role for the PCP pathway in postcrossing commissural axon guidance also in the chicken embryo. However, taking advantage of the precise temporal control of gene silencing provided by in ovo RNAi, we demonstrate that canonical Wnt signaling is also required for proper guidance of postcrossing commissural axons in the developing spinal cord. Thus, axon guidance does not seem to depend on any one of the classical Wnt signaling pathways but rather involve a network of Wnt receptors and downstream components. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 190–208, 2016 PMID:26014644

  1. The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration.

    PubMed

    Grill, Brock; Murphey, Rodney K; Borgen, Melissa A

    2016-01-01

    During development, a coordinated and integrated series of events must be accomplished in order to generate functional neural circuits. Axons must navigate toward target cells, build synaptic connections, and terminate outgrowth. The PHR proteins (consisting of mammalian Phr1/MYCBP2, Drosophila Highwire and C. elegans RPM-1) function in each of these events in development. Here, we review PHR function across species, as well as the myriad of signaling pathways PHR proteins regulate. These findings collectively suggest that the PHR proteins are intracellular signaling hubs, a concept we explore in depth. Consistent with prominent developmental functions, genetic links have begun to emerge between PHR signaling networks and neurodevelopmental disorders, such as autism, schizophrenia and intellectual disability. Finally, we discuss the recent and important finding that PHR proteins regulate axon degeneration, which has further heightened interest in this fascinating group of molecules. PMID:27008623

  2. Mechanisms of disease: a molecular genetic update on hereditary axonal neuropathies.

    PubMed

    Züchner, Stephan; Vance, Jeffery M

    2006-01-01

    Hereditary axonal peripheral neuropathies comprise a genetically heterogeneous group of disorders that are clinically subsumed under the name of Charcot-Marie-Tooth (CMT) disease type 2 (CMT2). Historically, two classes of CMT have been differentiated: demyelinating forms of CMT (CMT1), in which nerve conduction velocities are decreased, and the axonal CMT2 forms, in which nerve conduction velocities are preserved. Recently, a number of genes that are defective in patients with the main forms of CMT2 have been identified. The molecular dissection of cellular functions of the related gene products has only just begun, and detailed pathophysiological models are still lacking. The known CMT2-related genes represent key players in these pathways, however, and are likely to provide powerful tools for identifying targets for future therapeutic intervention. PMID:16932520

  3. Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth

    PubMed Central

    Endo, M.; Hattori, M.; Toriyabe, H.; Ohno, H.; Kamiguchi, H.; Iino, Y.; Ozawa, T.

    2016-01-01

    Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue. PMID:27052670

  4. Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

    PubMed

    Saab, Aiman S; Tzvetavona, Iva D; Trevisiol, Andrea; Baltan, Selva; Dibaj, Payam; Kusch, Kathrin; Möbius, Wiebke; Goetze, Bianka; Jahn, Hannah M; Huang, Wenhui; Steffens, Heinz; Schomburg, Eike D; Pérez-Samartín, Alberto; Pérez-Cerdá, Fernando; Bakhtiari, Davood; Matute, Carlos; Löwel, Siegrid; Griesinger, Christian; Hirrlinger, Johannes; Kirchhoff, Frank; Nave, Klaus-Armin

    2016-07-01

    Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons. PMID:27292539

  5. Journey to the skin: Somatosensory peripheral axon guidance and morphogenesis.

    PubMed

    Wang, Fang; Julien, Donald P; Sagasti, Alvaro

    2013-01-01

    The peripheral axons of vertebrate tactile somatosensory neurons travel long distances from ganglia just outside the central nervous system to the skin. Once in the skin these axons form elaborate terminals whose organization must be regionally patterned to detect and accurately localize different kinds of touch stimuli. This review describes key studies that identified choice points for somatosensory axon growth cones and the extrinsic molecular cues that function at each of those steps. While much has been learned in the past 20 years about the guidance of these axons, there is still much to be learned about how the peripheral axons of different kinds of somatosensory neurons adopt different trajectories and form specific terminal structures. PMID:23670092

  6. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  7. Restoration of Visual Function by Enhancing Conduction in Regenerated Axons.

    PubMed

    Bei, Fengfeng; Lee, Henry Hing Cheong; Liu, Xuefeng; Gunner, Georgia; Jin, Hai; Ma, Long; Wang, Chen; Hou, Lijun; Hensch, Takao K; Frank, Eric; Sanes, Joshua R; Chen, Chinfei; Fagiolini, Michela; He, Zhigang

    2016-01-14

    Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury. PMID:26771493

  8. Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons.

    PubMed

    Adalbert, R; Morreale, G; Paizs, M; Conforti, L; Walker, S A; Roderick, H L; Bootman, M D; Siklós, L; Coleman, M P

    2012-12-01

    Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later

  9. Cat hindlimb motoneurons during locomotion. I. Destination, axonal conduction velocity, and recruitment threshold.

    PubMed

    Hoffer, J A; Loeb, G E; Marks, W B; O'Donovan, M J; Pratt, C A; Sugano, N

    1987-02-01

    Fine flexible wire microelectrodes chronically implanted in the fifth lumbar ventral root (L5 VR) of 17 cats rendered stable records of the natural discharge patterns of 164 individual axons during locomotion on a treadmill. Fifty-one out of 164 axons were identified as motoneurons projecting to the anterior thigh muscle group. For these axons, the centrifugal propagation of action potentials was demonstrated by the technique of spike-triggered averaging using signals recorded from cuff electrodes implanted around the femoral nerve. The axonal conduction velocity was measured from the femoral nerve cuff records. For 43/51 motoneurons, the corresponding target muscle was identified by spike-triggered averaging of signals recorded from bipolar EMG electrodes implanted in each of the anterior thigh muscles: vastus intermedius, medialis and lateralis, sartorius anterior and medialis, and rectus femoris. For 32/51 motoneurons, the recruitment threshold during locomotion was determined from the mean value of the rectified digitally smoothed EMG of the target muscle measured at the time when the motoneuron fired its first spike for each step. The recruitment threshold of every motoneuron was relatively constant for a given speed of walking, but for some units there were small systematic variations as a function of treadmill speed (range: 0.1-1.3 m/s). Recruitment thresholds were standardized with respect to the mean value of peak EMG activity of the target muscle during 16 s of walking at 0.5 m/s. For 28/51 motoneurons recorded in nine cats, recruitment thresholds (range: 3-93% of peak target muscle EMG) were linearly correlated (r = 0.51, P less than 0.02) to axonal conduction velocities (range: 57-117 m/s). In addition, for seven recorded pairs of motoneurons that projected to the same muscle in the same cat, the recruitment thresholds were ordered by relative conduction velocities. Taken together, these results are consistent with the notion that, in normal cat

  10. Axon diameters and conduction velocities in the macaque pyramidal tract

    PubMed Central

    Firmin, L.; Field, P.; Maier, M. A.; Kraskov, A.; Kirkwood, P. A.; Nakajima, K.; Lemon, R. N.

    2014-01-01

    Small axons far outnumber larger fibers in the corticospinal tract, but the function of these small axons remains poorly understood. This is because they are difficult to identify, and therefore their physiology remains obscure. To assess the extent of the mismatch between anatomic and physiological measures, we compared conduction time and velocity in a large number of macaque corticospinal neurons with the distribution of axon diameters at the level of the medullary pyramid, using both light and electron microscopy. At the electron microscopic level, a total of 4,172 axons were sampled from 2 adult male macaque monkeys. We confirmed that there were virtually no unmyelinated fibers in the pyramidal tract. About 14% of pyramidal tract axons had a diameter smaller than 0.50 μm (including myelin sheath), most of these remaining undetected using light microscopy, and 52% were smaller than 1 μm. In the electrophysiological study, we determined the distribution of antidromic latencies of pyramidal tract neurons, recorded in primary motor cortex, ventral premotor cortex, and supplementary motor area and identified by pyramidal tract stimulation (799 pyramidal tract neurons, 7 adult awake macaques) or orthodromically from corticospinal axons recorded at the mid-cervical spinal level (192 axons, 5 adult anesthetized macaques). The distribution of antidromic and orthodromic latencies of corticospinal neurons was strongly biased toward those with large, fast-conducting axons. Axons smaller than 3 μm and with a conduction velocity below 18 m/s were grossly underrepresented in our electrophysiological recordings, and those below 1 μm (6 m/s) were probably not represented at all. The identity, location, and function of the majority of corticospinal neurons with small, slowly conducting axons remains unknown. PMID:24872533

  11. Brain injury tolerance limit based on computation of axonal strain.

    PubMed

    Sahoo, Debasis; Deck, Caroline; Willinger, Rémy

    2016-07-01

    Traumatic brain injury (TBI) is the leading cause of death and permanent impairment over the last decades. In both the severe and mild TBIs, diffuse axonal injury (DAI) is the most common pathology and leads to axonal degeneration. Computation of axonal strain by using finite element head model in numerical simulation can enlighten the DAI mechanism and help to establish advanced head injury criteria. The main objective of this study is to develop a brain injury criterion based on computation of axonal strain. To achieve the objective a state-of-the-art finite element head model with enhanced brain and skull material laws, was used for numerical computation of real world head trauma. The implementation of new medical imaging data such as, fractional anisotropy and axonal fiber orientation from Diffusion Tensor Imaging (DTI) of 12 healthy patients into the finite element brain model was performed to improve the brain constitutive material law with more efficient heterogeneous anisotropic visco hyper-elastic material law. The brain behavior has been validated in terms of brain deformation against Hardy et al. (2001), Hardy et al. (2007), and in terms of brain pressure against Nahum et al. (1977) and Trosseille et al. (1992) experiments. Verification of model stability has been conducted as well. Further, 109 well-documented TBI cases were simulated and axonal strain computed to derive brain injury tolerance curve. Based on an in-depth statistical analysis of different intra-cerebral parameters (brain axonal strain rate, axonal strain, first principal strain, Von Mises strain, first principal stress, Von Mises stress, CSDM (0.10), CSDM (0.15) and CSDM (0.25)), it was shown that axonal strain was the most appropriate candidate parameter to predict DAI. The proposed brain injury tolerance limit for a 50% risk of DAI has been established at 14.65% of axonal strain. This study provides a key step for a realistic novel injury metric for DAI. PMID:27038501

  12. Glial cell line-derived neurotrophic factor defines the path of developing and regenerating axons in the lateral line system of zebrafish

    PubMed Central

    Schuster, Kevin; Dambly-Chaudière, Christine; Ghysen, Alain

    2010-01-01

    How the peripheral axons of sensory neurons are guided to distant target organs is not well understood. Here we examine this question in the case of the posterior lateral line (PLL) system of zebrafish, where sensory organs are deposited by a migrating primordium. Sensory neurites accompany this primordium during its migration and are thereby guided to their prospective target organs. We show that the inactivation of glial cell line-derived neurotrophic factor (GDNF) signaling leads to defects of innervation and that these defects are due to the inability of sensory axons to track the migrating primordium. GDNF signaling is also used as a guidance cue during axonal regeneration following nerve cut. We conclude that GDNF is a major determinant of directed neuritic growth and of target finding in this system, and we propose that GDNF acts by promoting local neurite outgrowth. PMID:20974953

  13. Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

    PubMed Central

    Larsen, Camilla; Shy, Diana; Spindler, Shana R.; Fung, Siaumin; Pereanu, Wayne; Younossi -Hartenstein, Amelia; Hartenstein, Volker

    2009-01-01

    The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100–150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30–40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are ‘scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments. PMID:19538956

  14. Axon initial segment dysfunction in epilepsy

    PubMed Central

    Wimmer, Verena C; Reid, Christopher A; So, Eva Y-W; Berkovic, Samuel F; Petrou, Steven

    2010-01-01

    The axon initial segment (AIS) contains the site of action potential initiation and plays a major role in neuronal excitability. AIS function relies on high concentrations of different ion channels and complex regulatory mechanisms that orchestrate molecular microarchitecture. We review recent evidence that a large number of ion channels associated with epilepsy are enriched at the AIS, making it a ‘hotspot’ for epileptogenesis. Furthermore, we present novel data on the clustering of GABRγ2 receptors in the AIS of cortical and hippocampal neurons in a knock in mouse model of a human genetic epilepsy. This article highlights the molecular coincidence of epilepsy mutations at the AIS and reviews pathogenic mechanisms converging at the AIS. PMID:20375142

  15. DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

    PubMed Central

    Schmidt, Hannes; Rathjen, Fritz G.

    2011-01-01

    Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1). The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however

  16. The Number of Alphaherpesvirus Particles Infecting Axons and the Axonal Protein Repertoire Determines the Outcome of Neuronal Infection

    PubMed Central

    Koyuncu, Orkide O.; Song, Ren; Greco, Todd M.; Cristea, Ileana M.

    2015-01-01

    ABSTRACT Infection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons. PMID:25805728

  17. Anti-glycan antibodies halt axon regeneration in a model of Guillain Barrè Syndrome axonal neuropathy by inducing microtubule disorganization via RhoA-ROCK-dependent inactivation of CRMP-2.

    PubMed

    Rozés Salvador, Victoria; Heredia, Florencia; Berardo, Andrés; Palandri, Anabela; Wojnacki, Jose; Vivinetto, Ana L; Sheikh, Kazim A; Caceres, Alfredo; Lopez, Pablo H H

    2016-04-01

    Several reports have linked the presence of high titers of anti-Gg Abs with delayed recovery/poor prognosis in GBS. In most cases, failure to recover is associated with halted/deficient axon regeneration. Previous work identified that monoclonal and patient-derived anti-Gg Abs can act as inhibitory factors in an animal model of axon regeneration. Further studies using primary dorsal root ganglion neuron (DRGn) cultures demonstrated that anti-Gg Abs can inhibit neurite outgrowth by targeting gangliosides via activation of the small GTPase RhoA and its associated kinase (ROCK), a signaling pathway common to other established inhibitors of axon regeneration. We aimed to study the molecular basis of the inhibitory effect of anti-Gg abs on neurite outgrowth by dissecting the molecular dynamics of growth cones (GC) cytoskeleton in relation to the spatial-temporal analysis of RhoA activity. We now report that axon growth inhibition in DRGn induced by a well characterized mAb targeting gangliosides GD1a/GT1b involves: i) an early RhoA/ROCK-independent collapse of lamellipodia; ii) a RhoA/ROCK-dependent shrinking of filopodia; and iii) alteration of GC microtubule organization/and presumably dynamics via RhoA/ROCK-dependent phosphorylation of CRMP-2 at threonine 555. Our results also show that mAb 1B7 inhibits peripheral axon regeneration in an animal model via phosphorylation/inactivation of CRMP-2 at threonine 555. Overall, our data may help to explain the molecular mechanisms underlying impaired nerve repair in GBS. Future work should define RhoA-independent pathway/s and effectors regulating actin cytoskeleton, thus providing an opportunity for the design of a successful therapy to guarantee an efficient target reinnervation. PMID:26804001

  18. Lamina-specific contribution of glutamatergic and GABAergic potentials to hippocampal sharp wave-ripple complexes

    PubMed Central

    Schönberger, Jan; Draguhn, Andreas; Both, Martin

    2014-01-01

    The mammalian hippocampus expresses highly organized patterns of neuronal activity which form a neuronal correlate of spatial memories. These memory-encoding neuronal ensembles form on top of different network oscillations which entrain neurons in a state- and experience-dependent manner. The mechanisms underlying activation, timing and selection of participating neurons are incompletely understood. Here we studied the synaptic mechanisms underlying one prominent network pattern called sharp wave-ripple complexes (SPW-R) which are involved in memory consolidation during sleep. We recorded SPW-R with extracellular electrodes along the different layers of area CA1 in mouse hippocampal slices. Contribution of glutamatergic excitation and GABAergic inhibition, respectively, was probed by local application of receptor antagonists into s. radiatum, pyramidale and oriens. Laminar profiles of field potentials show that GABAergic potentials contribute substantially to sharp waves and superimposed ripple oscillations in s. pyramidale. Inhibitory inputs to s. pyramidale and s. oriens are crucial for action potential timing by ripple oscillations, as revealed by multiunit-recordings in the pyramidal cell layer. Glutamatergic afferents, on the other hand, contribute to sharp waves in s. radiatum where they also evoke a fast oscillation at ~200 Hz. Surprisingly, field ripples in s. radiatum are slightly slower than ripples in s. pyramidale, resulting in a systematic shift between dendritic and somatic oscillations. This complex interplay between dendritic excitation and perisomatic inhibition may be responsible for the precise timing of discharge probability during the time course of SPW-R. Together, our data illustrate a complementary role of spatially confined excitatory and inhibitory transmission during highly ordered network patterns in the hippocampus. PMID:25202239

  19. A Microchip for Quantitative Analysis of CNS Axon Growth under Localized Biomolecular Treatments

    PubMed Central

    Park, Jaewon; Kim, Sunja; Park, Su Inn; Choe, Yoonsuck; Li, Jianrong; Han, Arum

    2013-01-01

    Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration. PMID:24161788

  20. Dynamics of axonal regeneration in adult and aging zebrafish reveal the promoting effect of a first lesion

    PubMed Central

    Graciarena, Mariana; Dambly-Chaudière, Christine; Ghysen, Alain

    2014-01-01

    Axonal regeneration is a major issue in the maintenance of adult nervous systems, both after nerve injuries and in neurodegenerative diseases. However, studying this process in vivo is difficult or even impossible in most vertebrates. Here we show that the posterior lateral line (PLL) of zebrafish is a suitable system to study axonal regeneration in vivo because of both the superficial location and reproducible spatial arrangement of neurons and targets, and the possibility of following reinnervation in live fish on a daily basis. Axonal regeneration after nerve cut has been demonstrated in this system during the first few days of life, leading to complete regeneration within 24 h. However, the potential for PLL nerve regeneration has not been tested yet beyond the early larval stage. We explore the regeneration potential and dynamics of the PLL nerve in adult zebrafish and report that regeneration occurs throughout adulthood. We observed that irregularities in the original branching pattern are faithfully reproduced after regeneration, suggesting that regenerating axons follow the path laid down by the original nerve branches. We quantified the extent of target reinnervation after a nerve cut and found that the latency before the nerve regenerates increases with age. This latency is reduced after a second nerve cut at all ages, suggesting that a regeneration-promoting factor induced by the first cut facilitates regeneration on a second cut. We provide evidence that this factor remains present at the site of the first lesion for several days and is intrinsic to the neurons. PMID:24474787

  1. The immunophilin ligand FK506 attenuates axonal injury in an impact-acceleration model of traumatic brain injury.

    PubMed

    Singleton, R H; Stone, J R; Okonkwo, D O; Pellicane, A J; Povlishock, J T

    2001-06-01

    The immunophilin ligand, cyclosporin A (CsA), is effective in reducing the axonal damage associated with traumatic brain injury (TBI). Based upon extensive ultrastructural and immunohistochemical studies, the neuroprotection afforded by CsA appeared to be mediated via mitochondrial protection, specifically, the prevention of mitochondrial swelling and inhibition of mitochondrial permeability transition (MPT). However, the potential that CsA could also be neuroprotective via the immunophilin-mediated inhibition of the protein phosphatase, calcineurin (CN) has not been directly assessed. To address this issue, the current study assessed the ability of FK506, another immunophilin ligand that inhibits CN with no effect on MPT, to attenuate axonal damage in a rat impact-acceleration model of TBI. Traumatic axonal injury (TAI), detected via an antibody against beta-amyloid precursor protein (APP), a specific marker of axonal injury, was significantly reduced at 24 hr postinjury in Sprague-Dawley rats receiving intravenous FK506 (2 mg/kg; n = 5) 30 min prior to injury compared to vehicle controls (n = 3). While not rejecting the established efficacy of CsA in providing neuroprotection via its targeting of MPT, this study does underscore the potential importance of CN in the progressive pathobiology of TAI, suggesting that CN may constitute another important therapeutic target. PMID:11437083

  2. Differential Phosphorylation of Smad1 Integrates BMP and Neurotrophin Pathways through Erk/Dusp in Axon Development

    PubMed Central

    Finelli, Mattéa J.; Murphy, Kevin J.; Chen, Lei; Zou, Hongyan

    2013-01-01

    SUMMARY Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2—two key neurotrophin effectors. Specifically, BMPs signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2, and constituting a negative feedback loop to prevent axon overgrowth. Together, BMP and neurotrophin pathways are integrated in a tightly regulated signaling network with balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. PMID:23665221

  3. Channelrhodopsin-2 Localised to the Axon Initial Segment

    PubMed Central

    Grubb, Matthew S.; Burrone, Juan

    2010-01-01

    The light-gated cation channel Channelrhodopsin-2 (ChR2) is a powerful and versatile tool for controlling neuronal activity. Currently available versions of ChR2 either distribute uniformly throughout the plasma membrane or are localised specifically to somatodendritic or synaptic domains. Localising ChR2 instead to the axon initial segment (AIS) could prove an extremely useful addition to the optogenetic repertoire, targeting the channel directly to the site of action potential initiation, and limiting depolarisation and associated calcium entry elsewhere in the neuron. Here, we describe a ChR2 construct that we localised specifically to the AIS by adding the ankyrinG-binding loop of voltage-gated sodium channels (NavII-III) to its intracellular terminus. Expression of ChR2-YFP-NavII-III did not significantly affect the passive or active electrical properties of cultured rat hippocampal neurons. However, the tiny ChR2 currents and small membrane depolarisations resulting from AIS targeting meant that optogenetic control of action potential firing with ChR2-YFP-NavII-III was unsuccessful in baseline conditions. We did succeed in stimulating action potentials with light in some ChR2-YFP-NavII-III-expressing neurons, but only when blocking KCNQ voltage-gated potassium channels. We discuss possible alternative approaches to obtaining precise control of neuronal spiking with AIS-targeted optogenetic constructs and propose potential uses for our ChR2-YFP-NavII-III probe where subthreshold modulation of action potential initiation is desirable. PMID:21048938

  4. Axonal Transport and Morphology: How Myelination gets Nerves into Shape

    NASA Astrophysics Data System (ADS)

    Jung, Peter; Zhao, Peng; Monsma, Paula; Brown, Tony

    2011-03-01

    The local caliber of mature axons is largely determined by neurofilament (NF) content. The axoskeleton, mainly consisting of NFs, however, is dynamic. NFs are assembled in the cell body and are transported by molecular motors on microtubule tracks along the axon at a slow rate of fractions of mm per day. We combine live cell fluorescent imaging techniques to access NF transport in myelinated and non-myelinated segments of axons with computational modeling of the active NF flow to show that a), myelination locally slows NF transport rates by regulating duty ratios and b), that the predicted increase in axon caliber agrees well with experiments. This study, for the first time, links NF kinetics directly to axonal morphology, providing a novel conceptual framework for the physical understanding of processes leading to the formation of axonal structures such as the ``Nodes of Ranvier'' as well as abnormal axonal swellings associated with neurodegenerative diseases like Amyotrophic lateral sclerosis (ALS). NSF grants # IOS-0818412(PJ) and IOS-0818653 (AB).

  5. Functional recovery of regenerating motor axons is delayed in mice heterozygously deficient for the myelin protein P(0) gene.

    PubMed

    Rosberg, Mette Romer; Alvarez, Susana; Krarup, Christian; Moldovan, Mihai

    2013-06-01

    Mice with a heterozygous knock-out of the myelin protein P0 gene (P0+/-) develop a neuropathy similar to human Charcot-Marie-Tooth disease. They are indistinguishable from wild-types (WT) at birth and develop a slowly progressing demyelinating neuropathy. The aim of this study was to investigate whether the regeneration capacity of early symptomatic P0+/- is impaired as compared to age matched WT. Right sciatic nerves were lesioned at the thigh in 7-8 months old mice. Tibial motor axons at ankle were investigated by conventional motor conduction studies and axon excitability studies using threshold tracking. To evaluate regeneration we monitored the recovery of motor function after crush, and then compared the fiber distribution by histology. The overall motor performance was investigated using Rotor-Rod. P0+/- had reduced compound motor action potential amplitudes and thinner myelinated axons with only a borderline impairment in conduction and Rotor-Rod. Plantar muscle reinnervation occurred within 21 days in all mice. Shortly after reinnervation the conduction of P0+/- regenerated axons was markedly slower than WT, however, this difference decayed with time. Nevertheless, after 1 month, regenerated P0+/- axons had longer strength-duration time constant, larger threshold changes during hyperpolarizing electrotonus and longer relative refractory period. Their performance at Rotor-Rod remained also markedly impaired. In contrast, the number and diameter distribution of regenerating myelinated fibers became similar to regenerated WT. Our data suggest that in the presence of heterozygously P0 deficient Schwann cells, regenerating motor axons retain their ability to reinnervate their targets and remyelinate, though their functional recovery is delayed. PMID:23564290

  6. Experimental subarachnoid haemorrhage results in multifocal axonal injury.

    PubMed

    Kummer, Terrance T; Magnoni, Sandra; MacDonald, Christine L; Dikranian, Krikor; Milner, Eric; Sorrell, James; Conte, Valeria; Benetatos, Joey J; Zipfel, Gregory J; Brody, David L

    2015-09-01

    The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising

  7. Experimental subarachnoid haemorrhage results in multifocal axonal injury

    PubMed Central

    Magnoni, Sandra; MacDonald, Christine L.; Dikranian, Krikor; Milner, Eric; Sorrell, James; Conte, Valeria; Benetatos, Joey J.; Zipfel, Gregory J.; Brody, David L.

    2015-01-01

    The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury—axonal injury—is also a functionally significant feature of subarachnoid haemorrhage

  8. Gαz regulates BDNF-induction of axon growth in cortical neurons

    PubMed Central

    Hultman, Rainbo; Kumari, Udhaya; Michel, Nadine; Casey, Patrick J.

    2014-01-01

    The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles - predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation - suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS. PMID:24321455

  9. The atypical homeoprotein Pbx1a participates in the axonal pathfinding of mesencephalic dopaminergic neurons

    PubMed Central

    2012-01-01

    Background The pre B-cell leukemia transcription factor 1 (Pbx1) genes belong to the three amino acid loop extension family of homeodomain proteins that form hetero-oligomeric complexes with other homeodomain transcription factors, thereby modulating target specificity, DNA binding affinity and transcriptional activity of their molecular associates. Results Here, we provide evidence that Pbx1 is expressed in mesencephalic dopaminergic neurons from embryonic day 11 into adulthood and determines some of the cellular properties of this neuronal population. In Pbx1-deficient mice, the mesencephalic dopaminergic axons stall during mid-gestation at the border between di- and telencephalon before entering the ganglionic eminence, leading to a loose organization of the axonal bundle and partial misrouting. In Pbx1-deficient dopaminergic neurons, the high affinity netrin-1 receptor, deleted in colon cancer (DCC), is down-regulated. Interestingly, we found several conserved Pbx1 binding sites in the first intron of DCC, suggesting a direct regulation of DCC transcription by Pbx1. Conclusions The expression of Pbx1 in dopaminergic neurons and its regulation of DCC expression make it an important player in defining the axonal guidance of the midbrain dopaminergic neurons, with possible implications for the normal physiology of the nigro-striatal system as well as processes related to the degeneration of neurons during the course of Parkinson’s disease. PMID:22748019

  10. Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.

    PubMed

    Zhong, Chongbo; Talmage, David A; Role, Lorna W

    2015-01-01

    Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)-accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro. PMID:26132461

  11. Intra-axonal myosin and actin in nerve regeneration.

    PubMed

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  12. An evolutionarily conserved mechanism for cAMP elicited axonal regeneration involves direct activation of the dual leucine zipper kinase DLK

    PubMed Central

    Hao, Yan; Frey, Erin; Yoon, Choya; Wong, Hetty; Nestorovski, Douglas; Holzman, Lawrence B; Giger, Roman J; DiAntonio, Aaron; Collins, Catherine

    2016-01-01

    A broadly known method to stimulate the growth potential of axons is to elevate intracellular levels of cAMP, however the cellular pathway(s) that mediate this are not known. Here we identify the Dual Leucine-zipper Kinase (DLK, Wnd in Drosophila) as a critical target and effector of cAMP in injured axons. DLK/Wnd is thought to function as an injury ‘sensor’, as it becomes activated after axonal damage. Our findings in both Drosophila and mammalian neurons indicate that the cAMP effector kinase PKA is a conserved and direct upstream activator of Wnd/DLK. PKA is required for the induction of Wnd signaling in injured axons, and DLK is essential for the regenerative effects of cAMP in mammalian DRG neurons. These findings link two important mediators of responses to axonal injury, DLK/Wnd and cAMP/PKA, into a unified and evolutionarily conserved molecular pathway for stimulating the regenerative potential of injured axons. DOI: http://dx.doi.org/10.7554/eLife.14048.001 PMID:27268300

  13. An evolutionarily conserved mechanism for cAMP elicited axonal regeneration involves direct activation of the dual leucine zipper kinase DLK.

    PubMed

    Hao, Yan; Frey, Erin; Yoon, Choya; Wong, Hetty; Nestorovski, Douglas; Holzman, Lawrence B; Giger, Roman J; DiAntonio, Aaron; Collins, Catherine

    2016-01-01

    A broadly known method to stimulate the growth potential of axons is to elevate intracellular levels of cAMP, however the cellular pathway(s) that mediate this are not known. Here we identify the Dual Leucine-zipper Kinase (DLK, Wnd in Drosophila) as a critical target and effector of cAMP in injured axons. DLK/Wnd is thought to function as an injury 'sensor', as it becomes activated after axonal damage. Our findings in both Drosophila and mammalian neurons indicate that the cAMP effector kinase PKA is a conserved and direct upstream activator of Wnd/DLK. PKA is required for the induction of Wnd signaling in injured axons, and DLK is essential for the regenerative effects of cAMP in mammalian DRG neurons. These findings link two important mediators of responses to axonal injury, DLK/Wnd and cAMP/PKA, into a unified and evolutionarily conserved molecular pathway for stimulating the regenerative potential of injured axons. PMID:27268300

  14. Action Potentials Initiate in the Axon Initial Segment and Propagate Through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

    PubMed Central

    Foust, Amanda; Popovic, Marko; Zecevic, Dejan; McCormick, David A.

    2010-01-01

    Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By utilizing a recent improvement in voltage sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than approximately 250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission. PMID:20484631

  15. Direct catecholaminergic innervation of spinal dorsal horn neurons with axons ascending the dorsal columns in cat.

    PubMed

    Doyle, C A; Maxwell, D J

    1993-05-15

    Previous ultrastructural studies have shown that catecholamine-containing nerve terminals in the spinal dorsal horn form synaptic junctions with dendrites and somata, but the identity of the neurons giving rise to these structures is largely unknown. In this study we have investigated the possibility that spinomedullary neurons, which project through the dorsal columns to the dorsal column nuclei, are synaptic targets for descending catecholaminergic axons. Neurons with axons ascending the dorsal columns were retrogradely labelled after uptake of horseradish peroxidase by their severed axons in the thoracic (T10-T12) or cervical (C2-C3) dorsal columns. After the retrogradely labelled neurons were visualized, the tissue was immunocytochemically stained with antisera raised against tyrosine hydroxylase or dopamine-beta-hydroxylase. Three hundred forty-three retrogradely labelled neurons within laminae III-V of the lumbosacral dorsal horn were examined under high power with the light microscope. In Triton X-100 treated material, over 60% of cells were found to have dopamine-beta-hydroxylase-immunoreactive varicosities closely apposed to their somata and proximal dendrites. The number of contacts per cell varied from 1 to 22, with a mean number of 4.5. Fewer cells (34%) received contacts from axons immunoreactive for tyrosine hydroxylase as a consequence of the weaker immunoreaction produced by this antiserum. Correlated light and electron microscopic analysis confirmed that many of these contacts were regions of synaptic specialization and that immunostained boutons contained pleomorphic (round to oval) agranular vesicles together with several dense core vesicles. These observations suggest that catecholamines regulate sensory transmission through this spinomedullary pathway by a direct postsynaptic action upon its cells of origin. Such an action would be predicted to suppress transmission generally through this pathway. PMID:8099918

  16. Analysis of YFP(J16)-R6/2 reporter mice and postmortem brains reveals early pathology and increased vulnerability of callosal axons in Huntington's disease.

    PubMed

    Gatto, Rodolfo G; Chu, Yaping; Ye, Allen Q; Price, Steven D; Tavassoli, Ehsan; Buenaventura, Andrea; Brady, Scott T; Magin, Richard L; Kordower, Jeffrey H; Morfini, Gerardo A

    2015-09-15

    Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity. PMID:26123489

  17. Npn-1 Contributes to Axon-Axon Interactions That Differentially Control Sensory and Motor Innervation of the Limb

    PubMed Central

    Bianchi, Elisa; Novitch, Bennett G.; Huber, Andrea B.

    2011-01-01

    The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs. PMID:21364975

  18. Rapid Hebbian axonal remodeling mediated by visual stimulation.

    PubMed

    Munz, Martin; Gobert, Delphine; Schohl, Anne; Poquérusse, Jessie; Podgorski, Kaspar; Spratt, Perry; Ruthazer, Edward S

    2014-05-23

    We examined how correlated firing controls axon remodeling, using in vivo time-lapse imaging and electrophysiological analysis of individual retinal ganglion cell (RGC) axons that were visually stimulated either synchronously or asynchronously relative to neighboring inputs in the Xenopus laevis optic tectum. RGCs stimulated out of synchrony rapidly lost the ability to drive tectal postsynaptic partners while their axons grew and added many new branches. In contrast, synchronously activated RGCs produced fewer new branches, but these were more stable. The effects of synchronous activation were prevented by the inhibition of neurotransmitter release and N-methyl-D-aspartate receptor (NMDAR) blockade, which is consistent with a role for synaptic NMDAR activation in the stabilization of axonal branches and suppression of further exploratory branch addition. PMID:24855269

  19. A frequency-dependent decoding mechanism for axonal length sensing

    PubMed Central

    Bressloff, Paul C.; Karamched, Bhargav R.

    2015-01-01

    We have recently developed a mathematical model of axonal length sensing in which a system of delay differential equations describe a chemical signaling network. We showed that chemical oscillations emerge due to delayed negative feedback via a Hopf bifurcation, resulting in a frequency that is a monotonically decreasing function of axonal length. In this paper, we explore how frequency-encoding of axonal length can be decoded by a frequency-modulated gene network. If the protein output were thresholded, then this could provide a mechanism for axonal length control. We analyze the robustness of such a mechanism in the presence of intrinsic noise due to finite copy numbers within the gene network. PMID:26257607

  20. Molecular disorganization of axons adjacent to human lacunar infarcts

    PubMed Central

    Lee, Monica D.; Tung, Spencer; Vinters, Harry V.; Carmichael, S. Thomas

    2015-01-01

    Cerebral microvascular disease predominantly affects brain white matter and deep grey matter, resulting in ischaemic damage that ranges from lacunar infarcts to white matter hyperintensities seen on magnetic resonance imaging. These lesions are common and result in both clinical stroke syndromes and accumulate over time, resulting in cognitive deficits and dementia. Magnetic resonance imaging studies suggest that these lesions progress over time, accumulate adjacent to prior lesions and have a penumbral region susceptible to further injury. The pathological correlates of this adjacent injury in surviving myelinated axons have not been previously defined. In this study, we sought to determine the molecular organization of axons in tissue adjacent to lacunar infarcts and in the regions surrounding microinfarcts, by determining critical elements in axonal function: the morphology and length of node of Ranvier segments and adjacent paranodal segments. We examined post-mortem brain tissue from six patients with lacunar infarcts and tissue from two patients with autosomal dominant retinal vasculopathy and cerebral leukoencephalopathy (previously known as hereditary endotheliopathy with retinopathy, nephropathy and stroke) who accumulate progressive white matter ischaemic lesions in the form of lacunar and microinfarcts. In axons adjacent to lacunar infarcts yet extending up to 150% of the infarct diameter away, both nodal and paranodal length increase by ∼20% and 80%, respectively, reflecting a loss of normal cell-cell adhesion and signalling between axons and oligodendrocytes. Using premorbid magnetic resonance images, brain regions from patients with retinal vasculopathy and cerebral leukoencephalopathy that harboured periventricular white matter hyperintensities were selected and the molecular organization of axons was determined within these regions. As in regions adjacent to lacunar infarcts, nodal and paranodal length in white matter of these patients is

  1. Apaf1-deficient cortical neurons exhibit defects in axonal outgrowth.

    PubMed

    De Zio, Daniela; Molinari, Francesca; Rizza, Salvatore; Gatta, Lucia; Ciotti, Maria Teresa; Salvatore, Anna Maria; Mathiassen, Søs Grønbæk; Cwetsch, Andrzej W; Filomeni, Giuseppe; Rosano, Giuseppe; Ferraro, Elisabetta

    2015-11-01

    The establishment of neuronal polarity and axonal outgrowth are key processes affecting neuronal migration and synapse formation, their impairment likely leading to cognitive deficits. Here we have found that the apoptotic protease activating factor 1 (Apaf1), apart from its canonical role in apoptosis, plays an additional function in cortical neurons, where its deficiency specifically impairs axonal growth. Given the central role played by centrosomes and microtubules in the polarized extension of the axon, our data suggest that Apaf1-deletion affects axonal outgrowth through an impairment of centrosome organization. In line with this, centrosomal protein expression, as well as their centrosomal localization proved to be altered upon Apaf1-deletion. Strikingly, we also found that Apaf1-loss affects trans-Golgi components and leads to a robust activation of AMP-dependent protein kinase (AMPK), this confirming the stressful conditions induced by Apaf1-deficiency. Since AMPK hyper-phosphorylation is known to impair a proper axon elongation, our finding contributes to explain the effect of Apaf1-deficiency on axogenesis. We also discovered that the signaling pathways mediating axonal growth and involving glycogen synthase kinase-3β, liver kinase B1, and collapsing-response mediator protein-2 are altered in Apaf1-KO neurons. Overall, our results reveal a novel non-apoptotic role for Apaf1 in axonal outgrowth, suggesting that the neuronal phenotype due to Apaf1-deletion could not only be fully ascribed to apoptosis inhibition, but might also be the result of defects in axogenesis. The discovery of new molecules involved in axonal elongation has a clinical relevance since it might help to explain neurological abnormalities occurring during early brain development. PMID:25975226

  2. Axonal Noise as a Source of Synaptic Variability

    PubMed Central

    Neishabouri, Ali; Faisal, A. Aldo

    2014-01-01

    Post-synaptic potential (PSP) variability is typically attributed to mechanisms inside synapses, yet recent advances in experimental methods and biophysical understanding have led us to reconsider the role of axons as highly reliable transmission channels. We show that in many thin axons of our brain, the action potential (AP) waveform and thus the Ca++ signal controlling vesicle release at synapses will be significantly affected by the inherent variability of ion channel gating. We investigate how and to what extent fluctuations in the AP waveform explain observed PSP variability. Using both biophysical theory and stochastic simulations of central and peripheral nervous system axons from vertebrates and invertebrates, we show that channel noise in thin axons (<1 µm diameter) causes random fluctuations in AP waveforms. AP height and width, both experimentally characterised parameters of post-synaptic response amplitude, vary e.g. by up to 20 mV and 0.5 ms while a single AP propagates in C-fibre axons. We show how AP height and width variabilities increase with a ¾ power-law as diameter decreases and translate these fluctuations into post-synaptic response variability using biophysical data and models of synaptic transmission. We find for example that for mammalian unmyelinated axons with 0.2 µm diameter (matching cerebellar parallel fibres) axonal noise alone can explain half of the PSP variability in cerebellar synapses. We conclude that axonal variability may have considerable impact on synaptic response variability. Thus, in many experimental frameworks investigating synaptic transmission through paired-cell recordings or extracellular stimulation of presynaptic neurons, causes of variability may have been confounded. We thereby show how bottom-up aggregation of molecular noise sources contributes to our understanding of variability observed at higher levels of biological organisation. PMID:24809823

  3. Physiological properties of dystrophic mouse spinal root axons.

    PubMed

    Rasminsky, M

    1982-01-01

    In the spinal root axons of dystrophic mice conduction of nerve impulses is slow and either saltatory or continuous, presumably corresponding to areas of myelination and amyelination respectively. These abnormally myelinated axons contain foci of hyperexcitability manifested by spontaneous ectopic excitation, ephaptic excitation and autoexcitation. Similar phenomena in demyelinated central and peripheral nerve fibres may underly positive neurological symptomatology in human peripheral and central demyelinating diseases (Rasminsky 1981, 1982). PMID:6962064

  4. Molecular domains of myelinated axons in the peripheral nervous system.

    PubMed

    Salzer, James L; Brophy, Peter J; Peles, Elior

    2008-11-01

    Myelinated axons are organized into a series of specialized domains with distinct molecular compositions and functions. These domains, which include the node of Ranvier, the flanking paranodal junctions, the juxtaparanodes, and the internode, form as the result of interactions with myelinating Schwann cells. This domain organization is essential for action potential propagation by saltatory conduction and for the overall function and integrity of the axon. PMID:18803321

  5. Critical role of trkB receptors in reactive axonal sprouting and hyperexcitability after axonal injury

    PubMed Central

    Aungst, Stephanie; England, Pamela M.

    2013-01-01

    Traumatic brain injury (TBI) causes many long-term neurological complications. Some of these conditions, such as posttraumatic epilepsy, are characterized by increased excitability that typically arises after a latent period lasting from months to years, suggesting that slow injury-induced processes are critical. We tested the hypothesis that trkB activation promotes delayed injury-induced hyperexcitability in part by promoting reactive axonal sprouting. We modeled penetrative TBI with transection of the Schaffer collateral pathway in knock-in mice having an introduced mutation in the trkB receptor (trkBF616A) that renders it susceptible to inhibition by the novel small molecule 1NMPP1. We observed that trkB activation was increased in area CA3 1 day after injury and that expression of a marker of axonal growth, GAP43, was increased 7 days after lesion. Extracellular field potentials in stratum pyramidale of area CA3 in acute slices from sham-operated and lesioned mice were normal in control saline. Abnormal bursts of population spikes were observed under conditions that were mildly proconvulsive but only in slices taken from mice lesioned 7–21 days earlier and not in slices from control mice. trkB activation, GAP43 upregulation, and hyperexcitability were diminished by systemic administration of 1NMPP1 for 7 days after the lesion. Synaptic transmission from area CA3 to area CA1 recovered 7 days after lesion in untreated mice but not in mice treated with 1NMPP1. We conclude that trkB receptor activation and reactive axonal sprouting are critical factors in injury-induced hyperexcitability and may contribute to the neurological complications of TBI. PMID:23155176

  6. The Relationship between Dyslipidemia and Acute Axonal Function in Type 2 Diabetes Mellitus In Vivo

    PubMed Central

    Kwai, Natalie C. G.; Nigole, William; Poynten, Ann M.; Brown, Christopher; Krishnan, Arun V.

    2016-01-01

    Objectives Diabetic peripheral neuropathy (DPN) is a common and debilitating complication of diabetes mellitus. Treatment largely consists of symptom alleviation and there is a need to identify therapeutic targets for prevention and treatment of DPN. The objective of this study was to utilise novel neurophysiological techniques to investigate axonal function in patients with type 2 diabetes and to prospectively determine their relationship to serum lipids in type 2 diabetic patients. Methods Seventy-one patients with type 2 diabetes were consecutively recruited and tested. All patients underwent thorough clinical neurological assessments including nerve conduction studies, and median motor axonal excitability studies. Studies were also undertaken in age matched normal control subjects(n = 42). Biochemical studies, including serum lipid levels were obtained in all patients. Patient excitability data was compared to control data and linear regression analysis was performed to determine the relationship between serum triglycerides and low density lipoproteins and excitability parameters typically abnormal in type 2 diabetic patients. Results Patient mean age was 64.2±2.3 years, mean glycosylated haemoglobin (HbA1c%) was 7.8±0.3%, mean triglyceride concentration was 1.6±0.1 mmol/L and mean cholesterol concentration was 4.1±0.2mmol/L. Compared to age matched controls, median motor axonal excitability studies indicated axonal dysfunction in type 2 diabetic patients as a whole (T2DM) and in a subgroup of the patients without DPN (T2DM-NN). These included reduced percentage threshold change during threshold electrotonus at 10–20ms depolarising currents (TEd10–20ms)(controls 68.4±0.8, T2DM63.9±0.8, T2DM-NN64.8±1.6%,P<0.05) and superexcitability during the recovery cycle (controls-22.5±0.9, T2DM-17.5±0.8, T2DM-NN-17.3±1.6%,P<0.05). Linear regression analysis revealed no associations between changes in axonal function and either serum triglyceride or low density

  7. Parametric Probability Distribution Functions for Axon Diameters of Corpus Callosum

    PubMed Central

    Sepehrband, Farshid; Alexander, Daniel C.; Clark, Kristi A.; Kurniawan, Nyoman D.; Yang, Zhengyi; Reutens, David C.

    2016-01-01

    Axon diameter is an important neuroanatomical characteristic of the nervous system that alters in the course of neurological disorders such as multiple sclerosis. Axon diameters vary, even within a fiber bundle, and are not normally distributed. An accurate distribution function is therefore beneficial, either to describe axon diameters that are obtained from a direct measurement technique (e.g., microscopy), or to infer them indirectly (e.g., using diffusion-weighted MRI). The gamma distribution is a common choice for this purpose (particularly for the inferential approach) because it resembles the distribution profile of measured axon diameters which has been consistently shown to be non-negative and right-skewed. In this study we compared a wide range of parametric probability distribution functions against empirical data obtained from electron microscopy images. We observed that the gamma distribution fails to accurately describe the main characteristics of the axon diameter distribution, such as location and scale of the mode and the profile of distribution tails. We also found that the generalized extreme value distribution consistently fitted the measured distribution better than other distribution functions. This suggests that there may be distinct subpopulations of axons in the corpus callosum, each with their own distribution profiles. In addition, we observed that several other distributions outperformed the gamma distribution, yet had the same number of unknown parameters; these were the inverse Gaussian, log normal, log logistic and Birnbaum-Saunders distributions. PMID:27303273

  8. Glypican Is a Modulator of Netrin-Mediated Axon Guidance

    PubMed Central

    Blanchette, Cassandra R.; Perrat, Paola N.; Thackeray, Andrea; Bénard, Claire Y.

    2015-01-01

    Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration. PMID:26148345

  9. Myelin vs Axon Abnormalities in White Matter in Bipolar Disorder

    PubMed Central

    Lewandowski, Kathryn E; Ongür, Dost; Sperry, Sarah H; Cohen, Bruce M; Sehovic, Selma; Goldbach, Jacqueline R; Du, Fei

    2015-01-01

    White matter (WM) abnormalities are among the most commonly reported neuroimaging findings in bipolar disorder. Nonetheless, the specific nature and pathophysiology of these abnormalities remain unclear. Use of a combination of magnetization transfer ratio (MTR) and diffusion tensor spectroscopy (DTS) permits examination of myelin and axon abnormalities separately. We aimed to examine myelination and axon geometry in euthymic patients with bipolar disorder with psychosis (BDP) by combining these two complementary noninvasive MRI techniques. We applied a combined MRI approach using MTR to study myelin content and DTS to study metabolite (N-acetylaspartate, NAA) diffusion within axons in patients with BDP (n=21) and healthy controls (n=24). Data were collected from a 1 × 3 × 3-cm voxel within the right prefrontal cortex WM at 4 Tesla. Clinical and cognitive data were examined in association with MTR and DTS data. MTR was significantly reduced in BDP, suggesting reduced myelin content. The apparent diffusion coefficient of NAA did not differ from healthy controls, suggesting no changes in axon geometry in patients with BDP. These findings suggest that patients with BDP exhibit reduced myelin content, but no changes in axon geometry compared with controls. These findings are in contrast with our recent findings, using the same techniques, in patients with schizophrenia (SZ), which suggest both myelination and axon abnormalities in SZ. This difference may indicate that alterations in WM in BDP may have unique causes and may be less extensive than WM abnormalities seen in SZ. PMID:25409595

  10. Spinal irradiation does not inhibit distal axonal sprouting

    SciTech Connect

    Pamphlett, R.S.

    1988-05-01

    In an attempt to determine the relative importance of the nerve cell body and of the axon in initiating and controlling axonal regeneration, nerve cell bodies were irradiated and the ability of the distal axon to sprout was examined. Mice were subjected to either 25 or 50 Gray (Gy) of x-irradiation localized to the lumbar spinal cord. After times varying from 1 day to 6 months after irradiation, a sublethal dose of botulinum toxin (BoTx) was injected into the calf muscles of one leg. The soleus muscle was examined histologically after times varying from 1 week to 6 months after injection, and BoTx-induced ultraterminal axonal sprouting was assessed by the number of motor endplates showing sprouts, the length of the sprouts, and the long term endplate morphology. Apart from some irradiated subgroups having slightly shorter sprout lengths, no significant differences were found between irradiated and nonirradiated groups. The results suggest either that the processes in the nerve cell body responsible for initiating and supporting axonal growth are resistant to large doses of irradiation, or that growth regulatory mechanisms in the distal axon are under local control.

  11. Dynamics of signal propagation and collision in axons

    NASA Astrophysics Data System (ADS)

    Follmann, Rosangela; Rosa, Epaminondas; Stein, Wolfgang

    2015-09-01

    Long-range communication in the nervous system is usually carried out with the propagation of action potentials along the axon of nerve cells. While typically thought of as being unidirectional, it is not uncommon for axonal propagation of action potentials to happen in both directions. This is the case because action potentials can be initiated at multiple "ectopic" positions along the axon. Two ectopic action potentials generated at distinct sites, and traveling toward each other, will collide. As neuronal information is encoded in the frequency of action potentials, action potential collision and annihilation may affect the way in which neuronal information is received, processed, and transmitted. We investigate action potential propagation and collision using an axonal multicompartment model based on the Hodgkin-Huxley equations. We characterize propagation speed, refractory period, excitability, and action potential collision for slow (type I) and fast (type II) axons. In addition, our studies include experimental measurements of action potential propagation in axons of two biological systems. Both computational and experimental results unequivocally indicate that colliding action potentials do not pass each other; they are reciprocally annihilated.

  12. piggyBac-Based Mosaic Screen Identifies a Postmitotic Function for Cohesin in Regulating Developmental Axon Pruning

    PubMed Central

    Schuldiner, Oren; Berdnik, Daniela; Levy, Jonathan Ma; Wu, Joy Sing-Yi; Luginbuhl, David; Gontang, Allison Camille; Luo, Liqun

    2008-01-01

    Summary Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body γ neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as essential for axon pruning. The cohesin complex maintains sister chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1-/- clones is rescued by expressing SMC1 in neurons, revealing a new postmitotic function. SMC1-/- clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and enhanced by reduced dosage of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis. PMID:18267091

  13. piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning.

    PubMed

    Schuldiner, Oren; Berdnik, Daniela; Levy, Jonathan Ma; Wu, Joy S; Luginbuhl, David; Gontang, Allison Camille; Luo, Liqun

    2008-02-01

    Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis. PMID:18267091

  14. Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3.

    PubMed

    Jin, Duo; Liu, Yuanyuan; Sun, Fang; Wang, Xuhua; Liu, Xuefeng; He, Zhigang

    2015-01-01

    The limited rewiring of the corticospinal tract (CST) only partially compensates the lost functions after stroke, brain trauma and spinal cord injury. Therefore it is important to develop new therapies to enhance the compensatory circuitry mediated by spared CST axons. Here by using a unilateral pyramidotomy model, we find that deletion of cortical suppressor of cytokine signaling 3 (SOCS3), a negative regulator of cytokine-activated pathway, promotes sprouting of uninjured CST axons to the denervated spinal cord. A likely trigger of such sprouting is ciliary neurotrophic factor (CNTF) expressed in local spinal neurons. Such sprouting can be further enhanced by deletion of phosphatase and tensin homolog (PTEN), a mechanistic target of rapamycin (mTOR) negative regulator, resulting in significant recovery of skilled locomotion. Ablation of the corticospinal neurons with sprouting axons abolishes the improved behavioural performance. Furthermore, by optogenetics-based specific CST stimulation, we show a direct limb motor control by sprouting CST axons, providing direct evidence for the reformation of a functional circuit. PMID:26598325

  15. Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision

    NASA Astrophysics Data System (ADS)

    Ellis-Behnke, Rutledge G.; Liang, Yu-Xiang; You, Si-Wei; Tay, David K. C.; Zhang, Shuguang; So, Kwok-Fai; Schneider, Gerald E.

    2006-03-01

    Nanotechnology is often associated with materials fabrication, microelectronics, and microfluidics. Until now, the use of nanotechnology and molecular self assembly in biomedicine to repair injured brain structures has not been explored. To achieve axonal regeneration after injury in the CNS, several formidable barriers must be overcome, such as scar tissue formation after tissue injury, gaps in nervous tissue formed during phagocytosis of dying cells after injury, and the failure of many adult neurons to initiate axonal extension. Using the mammalian visual system as a model, we report that a designed self-assembling peptide nanofiber scaffold creates a permissive environment for axons not only to regenerate through the site of an acute injury but also to knit the brain tissue together. In experiments using a severed optic tract in the hamster, we show that regenerated axons reconnect to target tissues with sufficient density to promote functional return of vision, as evidenced by visually elicited orienting behavior. The peptide nanofiber scaffold not only represents a previously undiscovered nanobiomedical technology for tissue repair and restoration but also raises the possibility of effective treatment of CNS and other tissue or organ trauma. CNS regeneration | tissue repair | nanomedicine

  16. Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke.

    PubMed

    Liu, Zhongwu; Li, Yi; Cui, Yisheng; Roberts, Cynthia; Lu, Mei; Wilhelmsson, Ulrika; Pekny, Milos; Chopp, Michael

    2014-12-01

    The functional role of reactive astrocytes after stroke is controversial. To elucidate whether reactive astrocytes contribute to neurological recovery, we compared behavioral outcome, axonal remodeling of the corticospinal tract (CST), and the spatio-temporal change of chondroitin sulfate proteoglycan (CSPG) expression between wild-type (WT) and glial fibrillary acidic protein/vimentin double knockout (GFAP(-/-) Vim(-/-) ) mice subjected to Rose Bengal induced cerebral cortical photothrombotic stroke in the right forelimb motor area. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the CST axons. Compared with WT mice, the motor functional recovery and BDA-positive CST axonal length in the denervated side of the cervical gray matter were significantly reduced in GFAP(-/-) Vim(-/-) mice (n = 10/group, P < 0.01). Immunohistological data showed that in GFAP(-/-) Vim(-/-) mice, in which astrocytic reactivity is attenuated, CSPG expression was significantly increased in the lesion remote areas in both hemispheres, but decreased in the ischemic lesion boundary zone, compared with WT mice (n = 12/group, P < 0.001). Our data suggest that attenuated astrocytic reactivity impairs or delays neurological recovery by reducing CST axonal remodeling in the denervated spinal cord. Thus, manipulation of astrocytic reactivity post stroke may represent a therapeutic target for neurorestorative strategies. PMID:25043249

  17. Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3

    PubMed Central

    Jin, Duo; Liu, Yuanyuan; Sun, Fang; Wang, Xuhua; Liu, Xuefeng; He, Zhigang

    2015-01-01

    The limited rewiring of the corticospinal tract (CST) only partially compensates the lost functions after stroke, brain trauma and spinal cord injury. Therefore it is important to develop new therapies to enhance the compensatory circuitry mediated by spared CST axons. Here by using a unilateral pyramidotomy model, we find that deletion of cortical suppressor of cytokine signaling 3 (SOCS3), a negative regulator of cytokine-activated pathway, promotes sprouting of uninjured CST axons to the denervated spinal cord. A likely trigger of such sprouting is ciliary neurotrophic factor (CNTF) expressed in local spinal neurons. Such sprouting can be further enhanced by deletion of phosphatase and tensin homolog (PTEN), a mechanistic target of rapamycin (mTOR) negative regulator, resulting in significant recovery of skilled locomotion. Ablation of the corticospinal neurons with sprouting axons abolishes the improved behavioural performance. Furthermore, by optogenetics-based specific CST stimulation, we show a direct limb motor control by sprouting CST axons, providing direct evidence for the reformation of a functional circuit. PMID:26598325

  18. PLR-1, a putative E3 ubiquitin ligase, controls cell polarity and axonal extensions in C. elegans.

    PubMed

    Bhat, Jaffar M; Pan, Jie; Hutter, Harald

    2015-02-01

    During embryonic development neurons differentiate and extend axons and dendrites that have to reach their appropriate targets. In Caenorhabditis elegans the AVG neuron is the first neuron to extend an axon during the establishment of the ventral nerve cord, the major longitudinal axon tract in the animal. In genetic screens we isolated alleles of plr-1, which caused polarity reversals of the AVG neuron as well as outgrowth and navigation defects of the AVG axon. In addition plr-1 mutants show outgrowth defects in several other classes of neurons as well as the posterior excretory canals. plr-1 is predicted to encode a transmembrane E3 ubiquitin ligase and is widely expressed in the animal including the AVG neuron and the excretory cell. plr-1 has recently been shown to negatively regulate Wnt signalling by removing Wnt receptors from the cell surface. We observed that mutations in a gene reducing Wnt signalling as well as mutations in unc-53/NAV2 and unc-73/Trio suppress the AVG polarity defects in plr-1 mutants, but not the defects seen in other cells. This places plr-1 in a Wnt regulation pathway, but also suggests that plr-1 has Wnt independent functions and interacts with unc-53 and unc-73 to control cell polarity. PMID:25448694

  19. Inhibition of TLR4 Signalling-Induced Inflammation Attenuates Secondary Injury after Diffuse Axonal Injury in Rats

    PubMed Central

    Zhao, Yonglin; Zhang, Ming; Zhao, Junjie; Ma, Xudong; Huang, Tingqin; Pang, Honggang

    2016-01-01

    Increasing evidence suggests that secondary injury after diffuse axonal injury (DAI) damages more axons than the initial insult, but the underlying mechanisms of this phenomenon are not fully understood. Recent studies show that toll-like receptor 4 (TLR4) plays a critical role in promoting adaptive immune responses and have been shown to be associated with brain damage. The purpose of this study was to investigate the role of the TLR4 signalling pathway in secondary axonal injury in the cortices of DAI rats. TLR4 was mainly localized in microglial cells and neurons, and the levels of TLR4 downstream signalling molecules, including TLR4, myeloid differentiation primary response gene 88, toll/IR-1-(TIR-) domain-containing adaptor protein inducing interferon-beta, interferon regulatory factor 3, interferon β, nuclear factor κB (NF-κB) p65, and phospho-NF-κB p65, significantly increased and peaked at 1 d after DAI. Inhibition of TLR4 by TAK-242 attenuated apoptosis, neuronal and axonal injury, and glial responses. The neuroprotective effects of TLR4 inhibition were associated with decreases in the levels of TLR4 downstream signalling molecules and inflammatory factors, including interleukin-1β, interleukin-6, and tumour necrosis factor-α. These results suggest that the TLR4 signalling pathway plays an important role in secondary injury and may be an important therapeutic target following DAI. PMID:27478307

  20. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy

    PubMed Central

    Lee, Sooyeon; Sato, Yutaka; Nixon, Ralph A.

    2012-01-01

    In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD. PMID:21613495

  1. Studies in the development of a bridging device for guiding regenerating axons

    NASA Astrophysics Data System (ADS)

    Wen, Xuejun

    At present there is no clinically effective treatment for injuries or pathological processes that disrupt the continuity of axons in the mature central nervous system. However, a number of studies suggest that a tremendous potential exists for developing therapies. In particular biomaterials in the form of bridging substrates been shown to support at least some level of axonal regeneration across the lesion site, but display a limited capacity for directing axons toward their targets. To influence the directionality of the regeneration process filaments and tubes appear promising but the technology is far from optimized. As a step toward optimization, we investigated various components of a tissue-engineered bridging device consisting of numerous filaments surrounded by a semipermeable biodegradable hollow fiber membrane (HFM). In the first part of the thesis, we studied the influence of filament diameter and various extracellular matrix coatings on neuron regeneration suing a dorsal root ganglion explant model. We found that laminin surface treated filaments that approached the size of spinal axons support significantly longer regenerative outgrowth than similarly treated filaments of larger diameter, and exceed outgrowth distance on similarly sized filaments treated with fibronectin. Such substrates also consistently supported the attachment and alignment of glial cells and directed the outgrowth of regenerating axons along the long axis of the filaments. In the last part of the thesis, biodegradable hollow fiber membranes were fabricated and their physical, chemical and degradation properties were analyzed. We found that it is possible to use phase inversion methods to fabricate hollow fiber membranes of widely varying properties that degrade of the course of several months. We then evaluated the biocompatibility of the new materials after implantation in the CNS using an adult rat model. We found that the implants were well tolerated and elicited a reaction

  2. Some factors influencing sodium extrusion by internally dialyzed squid axons.

    PubMed

    Mullins, L J; Brinley, F J

    1967-11-01

    Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm(2)sec when [Na](i) was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5-10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm(2)sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm(2)sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13 degrees C. PMID:4228931

  3. Full length talin stimulates integrin activation and axon regeneration

    PubMed Central

    Tan, Chin Lik; Kwok, Jessica C.F.; Heller, Janosch P.D.; Zhao, Rongrong; Eva, Richard; Fawcett, James W.

    2015-01-01

    Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. PMID:25771432

  4. Potassium channel distribution in spinal root axons of dystrophic mice.

    PubMed

    Bostock, H; Rasminsky, M

    1983-07-01

    We have used 4-aminopyridine (4AP), a potassium channel blocker, to assess the presence and distribution of potassium channels in the congenitally abnormally myelinated spinal root axons of dystrophic mice. 1 mM-4AP slightly depressed the amplitude but had no effect on the half-width of the monophasic action potential of normal A fibres, indicating the absence of a significant concentration of potassium channels at normal mouse nodes of Ranvier. By progressively increasing stimulus intensity it was possible to elicit three more or less discrete components of the compound action potential from dystrophic mouse spinal roots, presumably corresponding to myelinated fibres, large diameter bare axons and, in the case of dorsal roots, C fibres. The amplitude and duration of all three components were increased on exposure to 4AP, indicating the presence of potassium channels in all types of dystrophic mouse spinal root axons. Conduction in single fibres was studied using longitudinal current analysis. Both saltatory and continuous conduction were observed corresponding to the myelinated and bare portions of dystrophic mouse spinal root axons. Three types of 'nodal' membrane could be inferred from the membrane current recordings from myelinated dystrophic mouse axons: (1) pure sodium channel membrane, (2) membrane containing both sodium and potassium channels, and (3) membrane containing predominantly, if not exclusively, potassium channels. The large early outward currents at the latter two types of nodes suggested that these nodes were wider than normal. Recordings of continuous conduction indicated that potassium channels were also distributed irregularly along bare portions of the dystrophic mouse axons. These abnormalities of ion channel distribution are interpreted as reflecting failure of normal axon-Schwann cell communication in the dystrophic mouse spinal roots. PMID:6310095

  5. Gbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes.

    PubMed

    Chatterjee, Mallika; Li, Kairong; Chen, Li; Maisano, Xu; Guo, Qiuxia; Gan, Lin; Li, James Y H

    2012-12-01

    Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs. PMID:23136391

  6. Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

    PubMed Central

    Koch, Jan Christoph; Tönges, Lars; Michel, Uwe; Bähr, Mathias; Lingor, Paul

    2014-01-01

    The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush. PMID:25249936

  7. Retinal ganglion cell axons regenerate in the presence of intact sensory fibres.

    PubMed

    King, Carolyn; Bartlett, Carole; Sauvé, Yves; Lund, Ray; Dunlop, Sarah; Beazley, Lyn

    2006-02-01

    A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration. PMID:16407770

  8. Regrowth of Serotonin Axons in the Adult Mouse Brain Following Injury.

    PubMed

    Jin, Yunju; Dougherty, Sarah E; Wood, Kevin; Sun, Landy; Cudmore, Robert H; Abdalla, Aya; Kannan, Geetha; Pletnikov, Mikhail; Hashemi, Parastoo; Linden, David J

    2016-08-17

    It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests. PMID:27499084

  9. Regulation of Microtubule Dynamics in Axon Regeneration: Insights from C. elegans

    PubMed Central

    Tang, Ngang Heok; Chisholm, Andrew D.

    2016-01-01

    The capacity of an axon to regenerate is regulated by its external environment and by cell-intrinsic factors. Studies in a variety of organisms suggest that alterations in axonal microtubule (MT) dynamics have potent effects on axon regeneration. We review recent findings on the regulation of MT dynamics during axon regeneration, focusing on the nematode Caenorhabditis elegans. In C. elegans the dual leucine zipper kinase (DLK) promotes axon regeneration, whereas the exchange factor for Arf6 (EFA-6) inhibits axon regeneration. Both DLK and EFA-6 respond to injury and control axon regeneration in part via MT dynamics. How the DLK and EFA-6 pathways are related is a topic of active investigation, as is the mechanism by which EFA-6 responds to axonal injury. We evaluate potential candidates, such as the MT affinity-regulating kinase PAR-1/MARK, in regulation of EFA-6 and axonal MT dynamics in regeneration. PMID:27350865

  10. Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

    NASA Technical Reports Server (NTRS)

    Macias, M. Y.; Lehman, C. T.; Sanger, J. R.; Riley, D. A.

    1998-01-01

    Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.

  11. β3GnT2 Maintains Adenylyl Cyclase-3 Signaling and Axon Guidance Molecule Expression in the Olfactory Epithelium

    PubMed Central

    Faden, Ashley A.; Knott, Thomas K.

    2011-01-01

    In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase β3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the β3GnT2−/− OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80–90% in the β3GnT2−/− OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from β3GnT2−/− OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from β3GnT2−/− OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in β3GnT2−/− mice. PMID:21525298

  12. TGF-α increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice

    PubMed Central

    White, Robin E.; Yin, Feng Qin; Jakeman, Lyn B.

    2008-01-01

    Astrocytes respond to environmental cues and play a multifaceted role in the response to trauma in the central nervous system. As the most prevalent contributors to the glial scar, astrocytes are targeted as barriers to regeneration. However, there is also strong evidence that astrocytes are vital for neuroprotection and metabolic support after injury. In addition, consistent with their role during development, astrocytes may be capable of supporting the growth of injured axons. Therefore, we hypothesized that with appropriate stimulation, the reparative functions of endogenous astrocytes could be harnessed to promote axon growth and recovery after spinal cord injury. Transforming growth factor-α (TGF-α) is a mitogenic growth factor that is active on astrocytes and is poised to contribute to such a strategy. Recombinant TGF-α was administered intrathecally to adult C57BL/6 mice for two weeks following a moderate mid-thoracic spinal cord contusion. By three weeks post-injury, TGF-α infusion had not affected locomotor recovery, but promoted extensive axon growth and altered the composition of the lesion site. The center of the lesion in the treated mice contained greater numbers of new cells and increased astrocyte invasion. Despite the expression of inhibitory proteoglycans, there was a marked increase in axons expressing neurofilament and GAP-43 immunoreactivity, and the new axons were closely associated with increased laminin expression within and beyond the astrocyte matrix. The results demonstrate that astrocytes are dynamic players in the response to spinal cord injury, and the growth supportive role of these cells can be enhanced by TGF-α infusion. PMID:18647603

  13. The voltage dependence of Ih in human myelinated axons

    PubMed Central

    Howells, James; Trevillion, Louise; Bostock, Hugh; Burke, David

    2012-01-01

    HCN channels are responsible for Ih, a voltage-gated inwardly rectifying current activated by hyperpolarization. This current appears to be more active in human sensory axons than motor and may play a role in the determination of threshold. Differences in Ih are likely to be responsible for the high variability in accommodation to hyperpolarization seen in different subjects. The aim of this study was to characterise this current in human axons, both motor and sensory. Recordings of multiple axonal excitability properties were performed in 10 subjects, with a focus on the changes in threshold evoked by longer and stronger hyperpolarizing currents than normally studied. The findings confirm that accommodation to hyperpolarization is greater in sensory than motor axons in all subjects, but the variability between subjects was greater than the modality difference. An existing model of motor axons was modified to take into account the behaviour seen with longer and stronger hyperpolarization, and a mathematical model of human sensory axons was developed based on the data collected. The differences in behaviour of sensory and motor axons and the differences between different subjects are best explained by modulation of the voltage dependence, along with a modest increase of expression of the underlying conductance of Ih. Accommodation to hyperpolarization for the mean sensory data is fitted well with a value of −94.2 mV for the mid-point of activation (V0.5) of Ih as compared to −107.3 mV for the mean motor data. The variation in response to hyperpolarization between subjects is accounted for by varying this parameter for each modality (sensory: −89.2 to −104.2 mV; motor −87.3 to −127.3 mV). These voltage differences are within the range that has been described for physiological modulation of Ih function. The presence of slowly activated Ih isoforms on both motor and sensory axons was suggested by modelling a large internodal leak current and a masking of

  14. Cortical Interneuron Subtypes Vary in Their Axonal Action Potential Properties

    PubMed Central

    Casale, Amanda E.; Foust, Amanda J.; Bal, Thierry

    2015-01-01

    The role of interneurons in cortical microcircuits is strongly influenced by their passive and active electrical properties. Although different types of interneurons exhibit unique electrophysiological properties recorded at the soma, it is not yet clear whether these differences are also manifested in other neuronal compartments. To address this question, we have used voltage-sensitive dye to image the propagation of action potentials into the fine collaterals of axons and dendrites in two of the largest cortical interneuron subtypes in the mouse: fast-spiking interneurons, which are typically basket or chandelier neurons; and somatostatin containing interneurons, which are typically regular spiking Martinotti cells. We found that fast-spiking and somatostatin-expressing interneurons differed in their electrophysiological characteristics along their entire dendrosomatoaxonal extent. The action potentials generated in the somata and axons, including axon collaterals, of somatostatin-expressing interneurons are significantly broader than those generated in the same compartments of fast-spiking inhibitory interneurons. In addition, action potentials back-propagated into the dendrites of somatostatin-expressing interneurons much more readily than fast-spiking interneurons. Pharmacological investigations suggested that axonal action potential repolarization in both cell types depends critically upon Kv1 channels, whereas the axonal and somatic action potentials of somatostatin-expressing interneurons also depend on BK Ca2+-activated K+ channels. These results indicate that the two broad classes of interneurons studied here have expressly different subcellular physiological properties, allowing them to perform unique computational roles in cortical circuit operations. SIGNIFICANCE STATEMENT Neurons in the cerebral cortex are of two major types: excitatory and inhibitory. The proper balance of excitation and inhibition in the brain is critical for its operation. Neurons

  15. Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth.

    PubMed

    Li, Hai; Kuwajima, Takaaki; Oakley, Derek; Nikulina, Elena; Hou, Jianwei; Yang, Wan Seok; Lowry, Emily Rhodes; Lamas, Nuno Jorge; Amoroso, Mackenzie Weygandt; Croft, Gist F; Hosur, Raghavendra; Wichterle, Hynek; Sebti, Said; Filbin, Marie T; Stockwell, Brent; Henderson, Christopher E

    2016-07-12

    Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT) and geranylgeranyl transferase I (PGGT-1). Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS). Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans. PMID:27373155

  16. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  17. A novel technique using hydrophilic polymers to promote axonal fusion

    PubMed Central

    Bamba, Ravinder; Riley, D. Colton; Kelm, Nathaniel D.; Does, Mark D.; Dortch, Richard D.; Thayer, Wesley P.

    2016-01-01

    The management of traumatic peripheral nerve injury remains a considerable concern for clinicians. With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury, outcomes of surgical intervention have been unpredictable. The inability to manipulate the pathophysiology of nerve injury (i.e., Wallerian degeneration) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration (~1 mm/day). When axons are severed, the endings undergo calcium-mediated plasmalemmal sealing, which limits the ability of the axon to be primarily repaired. Polythethylene glycol (PEG) in combination with a bioengineered process overcomes the inability to fuse axons. The mechanism for PEG axonal fusion is not clearly understood, but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion. The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur. This review highlights PEG fusion, its past and current studies, and future directions in PEG fusion. PMID:27212898

  18. Functional Complexity of the Axonal Growth Cone: A Proteomic Analysis

    PubMed Central

    Estrada-Bernal, Adriana; Sanford, Staci D.; Sosa, Lucas J.; Simon, Glenn C.; Hansen, Kirk C.; Pfenninger, Karl H.

    2012-01-01

    The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions. PMID:22384089

  19. Mechanisms of distal axonal degeneration in peripheral neuropathies.

    PubMed

    Cashman, Christopher R; Höke, Ahmet

    2015-06-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wld(S)) and Sarm knockout animal models. These studies have shown axonal degeneration to occur through a programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  20. Highly Effective Photonic Cue for Repulsive Axonal Guidance

    PubMed Central

    Black, Bryan J.; Gu, Ling; Mohanty, Samarendra K.

    2014-01-01

    In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm. PMID:24717339

  1. Highly effective photonic cue for repulsive axonal guidance.

    PubMed

    Black, Bryan J; Gu, Ling; Mohanty, Samarendra K

    2014-01-01

    In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm. PMID:24717339

  2. A novel technique using hydrophilic polymers to promote axonal fusion.

    PubMed

    Bamba, Ravinder; Riley, D Colton; Kelm, Nathaniel D; Does, Mark D; Dortch, Richard D; Thayer, Wesley P

    2016-04-01

    The management of traumatic peripheral nerve injury remains a considerable concern for clinicians. With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury, outcomes of surgical intervention have been unpredictable. The inability to manipulate the pathophysiology of nerve injury (i.e., Wallerian degeneration) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration (~1 mm/day). When axons are severed, the endings undergo calcium-mediated plasmalemmal sealing, which limits the ability of the axon to be primarily repaired. Polythethylene glycol (PEG) in combination with a bioengineered process overcomes the inability to fuse axons. The mechanism for PEG axonal fusion is not clearly understood, but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion. The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur. This review highlights PEG fusion, its past and current studies, and future directions in PEG fusion. PMID:27212898

  3. Mechanisms of Distal Axonal Degeneration in Peripheral Neuropathies

    PubMed Central

    Cashman, Christopher R.; Höke, Ahmet

    2015-01-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wlds) and Sarmknockout animal models. These studies have shown axonal degeneration to occur througha programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  4. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

    PubMed Central

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-01-01

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

  5. Axon Guidance in the Auditory System: Multiple Functions of Eph Receptors

    PubMed Central

    Cramer, Karina S.; Gabriele, Mark L.

    2014-01-01

    The neural pathways of the auditory system underlie our ability to detect sounds and to transform amplitude and frequency information into rich and meaningful perception. While it shares some organizational features with other sensory systems, the auditory system has some unique functions that impose special demands on precision in circuit assembly. In particular, the cochlear epithelium creates a frequency map rather than a space map, and specialized pathways extract information on interaural time and intensity differences to permit sound source localization. The assembly of auditory circuitry requires the coordinated function of multiple molecular cues. Eph receptors and their ephrin ligands constitute a large family of axon guidance molecules with developmentally regulated expression throughout the auditory system. Functional studies of Eph/ephrin signaling have revealed important roles at multiple levels of the auditory pathway, from the cochlea to the auditory cortex. These proteins provide graded cues used in establishing tonotopically ordered connections between auditory areas, as well as discrete cues that enable axons to form connections with appropriate postsynaptic partners within a target area. Throughout the auditory system, Eph proteins help to establish patterning in neural pathways during early development. This early targeting, which is further refined with neuronal activity, establishes the precision needed for auditory perception. PMID:25010398

  6. Fast and reliable identification of axons, axon initial segments and dendrites with local field potential recording

    PubMed Central

    Petersen, Anders V.; Johansen, Emil Ø.; Perrier, Jean-François

    2015-01-01

    The axon initial segment (AIS) is an essential neuronal compartment. It is usually where action potentials are initiated. Recent studies demonstrated that the AIS is a plastic structure that can be regulated by neuronal activity and by the activation of metabotropic receptors. Studying the AIS in live tissue can be difficult because its identification is not always reliable. Here we provide a new technique allowing a fast and reliable identification of the AIS in live brain slice preparations. By simultaneous recording of extracellular local field potentials and whole-cell patch-clamp recording of neurons, we can detect sinks caused by inward currents flowing across the membrane. We determine the location of the AIS by comparing the timing of these events with the action potential. We demonstrate that this method allows the unequivocal identification of the AIS of different types of neurons from the brain. PMID:26578887

  7. Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve.

    PubMed

    Taniguchi, Takazumi; Shimazawa, Masamitsu; Sasaoka, Masaaki; Shimazaki, Atsushi; Hara, Hideaki

    2006-05-01

    The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage. PMID:16719791

  8. Involvement of SARA in Axon and Dendrite Growth

    PubMed Central

    Arias, Cristina Isabel; Siri, Sebastián Omar; Conde, Cecilia

    2015-01-01

    SARA (Smad Anchor for Receptor Activation) plays a crucial role in Rab5-mediated endocytosis in cell lines localizing to early endosomes where it regulates morphology and function. Here, we analyzed the role of SARA during neuronal development and tested whether it functions as a regulator of endocytic trafficking of selected axonal and membrane proteins. Suppression of SARA perturbs the appearance of juxtanuclear endocytic recycling compartments and the neurons show long axons with large growth cones. Furthermore, surface distribution of the cell adhesion molecule L1 in axons and the fusion of vesicles containing transferring receptor (TfR) in dendrites were increased in neurons where SARA was silenced. Conversely, SARA overexpression generated large early endosomes and reduced neurite outgrowth. Taken together, our findings suggest a significant contribution of SARA to key aspects of neuronal development, including neurite formation. PMID:26405814

  9. Involvement of SARA in Axon and Dendrite Growth.

    PubMed

    Arias, Cristina Isabel; Siri, Sebastián Omar; Conde, Cecilia

    2015-01-01

    SARA (Smad Anchor for Receptor Activation) plays a crucial role in Rab5-mediated endocytosis in cell lines localizing to early endosomes where it regulates morphology and function. Here, we analyzed the role of SARA during neuronal development and tested whether it functions as a regulator of endocytic trafficking of selected axonal and membrane proteins. Suppression of SARA perturbs the appearance of juxtanuclear endocytic recycling compartments and the neurons show long axons with large growth cones. Furthermore, surface distribution of the cell adhesion molecule L1 in axons and the fusion of vesicles containing transferring receptor (TfR) in dendrites were increased in neurons where SARA was silenced. Conversely, SARA overexpression generated large early endosomes and reduced neurite outgrowth. Taken together, our findings suggest a significant contribution of SARA to key aspects of neuronal development, including neurite formation. PMID:26405814

  10. Chondroitin-4-sulfation negatively regulates axonal guidance and growth

    PubMed Central

    Wang, Hang; Katagiri, Yasuhiro; McCann, Thomas E.; Unsworth, Edward; Goldsmith, Paul; Yu, Zu-Xi; Tan, Fei; Santiago, Lizzie; Mills, Edward M.; Wang, Yu; Symes, Aviva J.; Geller, Herbert M.

    2008-01-01

    Summary Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition, and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate (CS) mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated CS GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings provide the evidence showing that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function. PMID:18768934

  11. Using quantum filters to process images of diffuse axonal injury

    NASA Astrophysics Data System (ADS)

    Pineda Osorio, Mateo

    2014-06-01

    Some images corresponding to a diffuse axonal injury (DAI) are processed using several quantum filters such as Hermite Weibull and Morse. Diffuse axonal injury is a particular, common and severe case of traumatic brain injury (TBI). DAI involves global damage on microscopic scale of brain tissue and causes serious neurologic abnormalities. New imaging techniques provide excellent images showing cellular damages related to DAI. Said images can be processed with quantum filters, which accomplish high resolutions of dendritic and axonal structures both in normal and pathological state. Using the Laplacian operators from the new quantum filters, excellent edge detectors for neurofiber resolution are obtained. Image quantum processing of DAI images is made using computer algebra, specifically Maple. Quantum filter plugins construction is proposed as a future research line, which can incorporated to the ImageJ software package, making its use simpler for medical personnel.

  12. Properties of postganglionic sympathetic neurons with axons in phrenic nerve.

    PubMed

    Bałkowiec, A; Szulczyk, P

    1992-06-01

    The aim of the study was to test the reflex and resting properties of postganglionic sympathetic neurons with axons located in the right phrenic nerve. The experiments have been performed on chloralose-anesthetized cats with both vago-aortic nerves cut. The somata or the postganglionic sympathetic neurons were located in the stellate ganglion. Axons of these neurons passed through the upper and lower phrenic nerve roots and through the phrenic nerve itself. The presence of cardiac and respiratory rhythmicities was detected in the activity of the phrenic postganglionic sympathetic neurons. Hyperventilation, which abolished burst discharges of the phrenic nerve, decreased the sympathetic activity by 14%. Systemic hypoxia (ventilating the animals for 2 min with 8% O2 in N2) increased the sympathetic activity threefold. The results of our experiments suggest that axons of the sympathetic neurons located in the right phrenic nerve could possibly be diaphragmatic muscle vasoconstrictors. PMID:1615229

  13. Optimization of the leak conductance in the squid giant axon

    NASA Astrophysics Data System (ADS)

    Seely, Jeffrey; Crotty, Patrick

    2010-08-01

    We report on a theoretical study showing that the leak conductance density, GL , in the squid giant axon appears to be optimal for the action potential firing frequency. More precisely, the standard assumption that the leak current is composed of chloride ions leads to the result that the experimental value for GL is very close to the optimal value in the Hodgkin-Huxley model, which minimizes the absolute refractory period of the action potential, thereby maximizing the maximum firing frequency under stimulation by sharp, brief input current spikes to one end of the axon. The measured value of GL also appears to be close to optimal for the frequency of repetitive firing caused by a constant current input to one end of the axon, especially when temperature variations are taken into account. If, by contrast, the leak current is assumed to be composed of separate voltage-independent sodium and potassium currents, then these optimizations are not observed.

  14. Neurofascins are required to establish axonal domains for saltatory conduction.

    PubMed

    Sherman, Diane L; Tait, Steven; Melrose, Shona; Johnson, Richard; Zonta, Barbara; Court, Felipe A; Macklin, Wendy B; Meek, Stephen; Smith, Andrew J H; Cottrell, David F; Brophy, Peter J

    2005-12-01

    Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves. PMID:16337912

  15. Clustered voltage-gated Na+ channels in Aplysia axons.

    PubMed

    Johnston, W L; Dyer, J R; Castellucci, V F; Dunn, R J

    1996-03-01

    Clustering of voltage-gated Na+ channels is critical for the fast saltatory conduction of action potentials in vertebrate myelinated axons. However, the mechanisms responsible for the generation and maintenance of Na+ channel clustering are not well understood. In this study we have raised an antibody against the cloned SCAP-1 voltage-gated Na+ channel of the marine invertebrate Aplysia californica and used it to examine Na+ channel localization in Aplysia ganglia and in cultured Aplysia sensory neurons. Our results show that there is a large cytoplasmic pool of Na+ channels in the soma of Aplysia neurons. Furthermore, we show that Na+ channels in Aplysia axons are not homogeneously distributed but, rather, are present in distinct clusters. Theoretical considerations indicate that Na+ channel clustering may enhance action potential conduction. We propose that clustered Na+ channels may be a fundamental property of many axons, and perhaps of many membranes that conduct Na(+)-dependent action potentials. PMID:8774441

  16. The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

    PubMed

    Godzik, Katharina; Coleman, Michael P

    2015-04-01

    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases. PMID:25352062

  17. Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

    PubMed

    McLean, Nikki A; Popescu, Bogdan F; Gordon, Tessa; Zochodne, Douglas W; Verge, Valerie M K

    2014-01-01

    Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies. PMID:25310564

  18. Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve.

    PubMed

    González, Carolina; Cánovas, José; Fresno, Javiera; Couve, Eduardo; Court, Felipe A; Couve, Andrés

    2016-02-16

    The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. PMID:26839409

  19. Reductions in kinesin expression are associated with nitric oxide-induced axonal damage

    PubMed Central

    Redondo, Juliana; Hares, Kelly; Wilkins, Alastair; Scolding, Neil; Kemp, Kevin

    2015-01-01

    Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra-axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time-dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO-exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. PMID:25639260

  20. Reductions in kinesin expression are associated with nitric oxide-induced