311,833 research outputs found
Commissural Axon Kinetics and the Role of Netrin in Early Brain Circuitry Development
No full textAs neurons begin to differentiate, they send out processes called axons to initiate the formation of functional nerve connections. A specialized structure at the end of an axon called the growth cone is believed to possess the impressive navigational and target recognition ability crucial for this process. The goal of the research presented in this thesis was to understand the cellular and molecular mechanisms that shape axon growth and guidance in vivo during early brain development using a multifaceted experimental approach.
Towards this goal we employed the simple, well-characterized neuronal scaffold of the embryonic zebrafish brain in combination with cell labeling techniques and performed studies in three specific areas: (1) the dynamic behaviors of navigating growth cones to obtain information about their cellular interactions with each other and the local environment, (2) the action of specific proteins (netrin and its receptor DCC) known to be involved in axon guidance in order to determine their function in vivo, and (3) the mobility of GFP in growth cones as a way to gain insight into the dynamics of molecular species in these structures as they actively navigate.
Critical to our studies was a stable transgenic gata2::GFP zebrafish line in which we found high level of GFP expression in early forebrain neuronal clusters allowing in vivo timelapse study of the growth cones that pioneer the postoptic commissural (POC) axon tract. Following the development of the POC also allowed us to investigate how early commissural growth cones behave at the midline.
Timelapse analysis of POC axon kinetics revealed important insight into growth cone interactions with each other and their environment and showed that these have behavioral consequences. While it was known that commissural axons slow down while crossing the midline, our data showed that this is only true for the leader axons. Follower axons do not slow down unless the leader axon is ablated. Together this analysis revealed that in addition to specific molecular cues, axon-axon interactions are important for establishing early axon tract.
This characterization of POC axon kinetics and growth cone behavior in turn, provided us with an assay for studying the specific role of netrin, primarily a midline attractant for commissural axons in the spinal cord and its receptor, deleted in colorectal cancer (DCC). Loss- and gain-of-function experiments in combination with timelapse imaging uncovered a novel function for netrin as a positional repellent cue for POC axons.
Finally, prompted by the observed differences in leader and follower POC growth cones, we developed a new experimental approach to assay GFP mobility as a reporter for the diffusion rates of other molecular species inside growth cones in vivo. We found that diffusion rates in actively pioneering growth cones are significantly decreased compared to follower axons suggesting that diffusion rates might be linked to growth cone pathfinding.
Collectively, the findings presented in this thesis constitute a framework that allows for an integrative approach of studying growth cone navigation in vivo. Basic integrative knowledge of this sort is expected to aid the development of medical therapies related to nerve injury and repair.</p
Neuronal deletion of GSK3beta increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2
No full textBackgroundIn the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.ResultsFollowing a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3beta (GSK3beta) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3beta regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3beta activity in neurons. Following spinal cord injury, reduced GSK3beta levels or complete neuronal deletion of GSK3beta led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3beta substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth, of conditioned neurons and of GSK3beta-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3beta-CRMP-2 relevance during axon regeneration.ConclusionsOur work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3beta-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth
Development of the early axon scaffold in the rostral brain of the small spotted cat shark (<i>Scyliorhinus canicula</i>) embryo
No full textInternational audienceThe cat shark is increasingly used as a model for Chondrichthyes, an evolutionarily important sister group of the bony vertebrates that include teleosts and tetrapods. In the bony vertebrates, the first axon tracts form a highly conserved early axon scaffold. The corresponding structure has not been well characterised in cat shark and will prove a useful model for comparative studies. Using pan-neural markers, the early axon scaffold of the cat shark, Scyliorhinus canicula, was analysed. Like in other vertebrates, the medial longitudinal fascicle was the first axon tract to form from a small cluster of neurones in the ventral brain. Subsequently, additional neuronal clusters and axon tracts emerged which formed an array of longitudinal, transversal, and commissural axons tracts in the Scyliorhinus canicula embryonic brain. The first structures to appear after the medial longitudinal fascicle were the tract of the postoptic commissure, the dorsoventral diencephalic tract, and the descending tract of the mesencephalic nucleus of the trigeminal nerve. These results confirm that the early axon scaffold in the embryonic brain is highly conserved through vertebrate evolution
Inflammation contributes to axon reflex vasodilatation evoked by iontophoresis of an alpha-1 adrenoceptor agonist
No full textIontophoresis of aradrenoceptor agonists in the human forearm evoke axon reflex vasodilatation, possibly due to an accumulation of inflammatory agents at the site of iontophoresis. To investigate this possibility, skin sites in the forearm of healthy participants were treated with an anti-inflammatory gel containing ibuprofen 5% before the iontophoresis of the aradrenoceptor agonist phenylephrine (350 mu A for 3 min). Red cell flux was measured with laser Doppler flowmetry at the site of iontophoresis and 8 mm away in the region of axon reflex vasodilatation. In additional experiments, skin sites were treated with the vasodilator sodium nitroprusside (to counteract vasoconstriction and disperse inflammatory mediators produced during the iontophoresis of phenylephrine); local anaesthetic agent (to determine whether the axon reflex to phenylephrine was neurally-mediated); or the alpha(2)-adrenoceptor agonist clonidine (to investigate the specificity of the adrenergic axon reflex). Phenylephrine evoked marked vasodilatation 8 mm from the site of iontophoresis whereas clonidine and saline-control did not (mean flux increase+/-S.E. 485+/-132% for phenylephrine; 44+/-24% for clonidine; 39+/-19% for saline-control; p<0.05 for phenylephrine versus control). Axon reflex vasodilatation to phenylephrine was unaffected by variations in blood flow at the site of phenylephrine iontophoresis, but was reduced by ibuprofen pretreatment and abolished by local anaesthetic pretreatment. These findings suggest that prostaglandin synthesis at the site of iontophoresis contributes to but does not account entirely for axon reflex vasodilatation to phenylephrine. Alpha-1 adrenoceptor mediation of axon reflexes could play a role in aberrant sensory-sympathetic coupling in neuro-inflammatory diseases
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Neu3 sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons
No full textThis work was supported by the Medical Research Council, the Christopher and Dana Reeve Foundation, the John and Lucille van Geest Foundation, the Henry Smith Charity, the Commonwealth and Overseas scholarships, and the Hinduja Cambridge Trust.PNS axons have a high intrinsic regenerative ability, whereas most CNS axons show little regenerative response. We show that activation of Neu3 sialidase, also known as Neuraminidase-3, causing conversion of GD1a and GT1b to GM1 ganglioside, is an essential step in regeneration occurring in PNS (sensory) but not CNS (retinal) axons in adult rat. In PNS axons, axotomy activates Neu3 sialidase, increasing the ratio of GM1/GD1a and GM1/GT1b gangliosides immediately after injury in vitro and in vivo. No change in the GM1/GD1a ratio after axotomy was observed in retinal axons (in vitro and in vivo), despite the presence of Neu3 sialidase. Externally applied sialidase converted GD1a ganglioside to GM1 and rescued axon regeneration in CNS axons and in PNS axons after Neu3 sialidase blockade. Neu3 sialidase activation in DRGs is initiated by an influx of extracellular calcium, activating P38MAPK and then Neu3 sialidase. Ganglioside conversion by Neu3 sialidase further activates the ERK pathway. In CNS axons, P38MAPK and Neu3 sialidase were not activated by axotomy.Peer reviewe
Análise e modelagem do potencial de repouso em porção de axônio utilizando o método Lattice Boltzman
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Mecânica, 2014Na ausência de excitação, neurônios permanecem em repouso. O estado de repouso é caracterizado pela existência de diferenças de potencial elétrico e concentrações iônicas através da membrana do axônio. No entanto, os mecanismos responsáveis e a magnitude dos fluxos durante o estabelecimento do estado de repouso, após uma perturbação externa, não estão ainda completamente elucidados. O estado de repouso não é um estado de equilíbrio, mas um estado dinâmico estacionário. Sabe-se da existência de proteínas, classificadas genericamente como bombas e canais, que transportam íons através da membrana modificando o potencial através da mesma. No entanto, há várias hipóteses contraditórias sobre a magnitude e relação entre os fluxos provocados por tais proteínas e sobre a importância de outros fatores, como, por exemplo, a existência de cargas fixas. Um dos motivos para a dificuldade em escolher as hipóteses que tornam a modelagem mais consistente reside nas escalas de espaço e tempo diminutas dos fenômenos envolvidos o que dificulta a obtenção de medições locais e controladas. Nesse sentido, o desenvolvimento de modelos consistentes permite a avaliação das características e dos efeitos das várias hipóteses e contribui para o estabelecimento de modelos mais confiáveis. Nesse trabalho, utiliza-se, o método lattice Boltzmann para tratar o transporte iônio e o campo elétrico nos meios intra e extra celulares nas vizinhanças da membrana celular de um axônio. Uma caracterização físico-química da região vizinha à membrana de um axônio é feita baseada nos dados experimentais existentes. De posse dessa caracterização, utiliza-se o método numérico para a simulação da eletrodifusão iônica de uma porção de axônio quando uma perturbação a partir do estado de repouso é introduzida. Várias hipóteses são usadas, as soluções numéricas são comparadas com soluções existentes na literatura e com medições e, com base nas simulações, conclui-se quais hipóteses são mais adequadas para se explicar a manutenção do repouso. Além disso, também se observa que são necessários mais experimentos para obter maior confiabilidade sobre os modelos constitutivos de fluxos através dos canais e, principalmente, das bombas.Abstract: In the absence of excitation, neurons remain in a state called the resting state. The resting state is characterized by differences in electrical potential and ionic concentrations across the axon membrane. However, there is still a lacking of a complete understanding of the local mechanisms responsible and the magnitudes of the fluxes involved during the changes towards the resting state after the neuron suffers an external disturbance. The resting state is most likely a dynamic state, notan equilibrium one. Known mechanisms rely on the action of proteins called generically pumps and channels. These biological devices selectively transport ionic charges across the membrane. Contradictory hypothesis on the magnitude and relations among the charge transfers and on the effect of other parameters, such as the existence of fixed charges, have been advanced. The main difficult in sorting out the validity of these hypothesis rests on the inherent difficult in obtaining local and controlled measurements. In this sense, a reliable and consistent theoretical formulation may come as an aid to evaluate the effects and features of each mechanism and underlying hypothesis. In this work, the Lattice Boltzmann method is used to simulate the transport of ions and the electrical field in the intra and extra cellular spaces in the surroundings of an axon membrane. A physical-chemical characterization of the system is made based on the experimental evidence available in the literature. This characterization is then used in the numerical simulation of the electro - diffusion problem in a section of an axon when a perturbation from the resting state is introduced. Many hypotheses are used and the numerical solutions are compared with solutions and measurements found in the literature. From the results, the validity of the different hypothesis is assessed. The need for additional measurements to increase the reliability of the models for the constitutive fluxes through channels and pumps is evidenced
Electrical activity suppresses intrinsic growth competence in adult primary sensory neurons
No full textThe ability of neurons to regenerate in the adult mammalian central nervous system (CNS) is often poor, leading to persistent deficits after injury. Failure of axon regeneration in the CNS has been attributed to the presence of an extrinsic inhibitory environment and to an intrinsic limitation to support growth. Remarkably, in adult primary sensory neurons of the dorsal root ganglia (DRG), a peripheral lesion primes neurons to grow and to override the inhibitory environment. Under this condition not only their peripheral axons regrow, but also their injured central axons coursing in the spinal cord regenerate. However, the nature of the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response is poorly understood.
This study started from the hypothesis that electrical silencing caused by peripheral deafferentiation is an important signal to trigger axon regrowth in adult DRG neurons. I first examined the effect of electrical activity on axon growth of cultured DRG neurons. I found that either chronic depolarization or electrical field stimulation strongly inhibits axon outgrowth in cultured DRG neurons. The inhibitory effect depends on Ca2+ influx through L-type voltage-gated calcium channels and involves transcriptional changes. Consistently, after a peripheral lesion, L-type current is diminished and the L-type pore-forming subunit Cav1.2 is downregulated. To determine whether the lack of L-type channels is sufficient to promote axon growth, mice lacking the pore-forming subunit of L-type channel, Cav1.2, in the nervous system were generated. Neurons isolated from adult Cav1.2 knockout (KO) mice grew more extensively than those from their control littermates.
Taken together, these data provide evidence that electrical activity is a limiting factor for axon growth in adult DRG neurons and that releasing this “brake” is sufficient to induce axon growth. My results further suggest that electrical silencing might promote axon regeneration in vivo. Consequently, I have attempted to apply this knowledge to a model of spinal cord injury. However, these in vivo experiments have been so far hampered by technical limitations. Further endeavors are currently in progress
Alpha-1 adrenoceptor stimulation triggers axon-reflex vasodilatation in human skin
No full textThe aim of this study was to determine whether pre-treatment of human skin with the α1-adrenoceptor antagonist terazosin would block vasoconstrictor responses and axon-reflex vasodilatation to the α1-adrenoceptor agonist methoxamine. Drugs were administered by iontophoresis into the skin of the forearm of 15 healthy participants, and skin blood flow was monitored with a laser Doppler flow probe at the site of methoxamine iontophoresis (to monitor direct vasoconstrictor responses) or 5–10 mm from the site of methoxamine iontophoresis (to monitor axon-reflex vasodilatation). Experimental sites were pre-treated with terazosin (administered by iontophoresis for 10 min at 200 µA), and the same current intensity was passed through 0.9% saline to control for the nonspecific effects of iontophoresis. Pre-treatment with terazosin blocked vasoconstrictor responses to increasing doses of methoxamine, and also blocked vasodilatation several mm from the site of terazosin and methoxamine administration. These findings support the view that α1-adrenoceptors play a role in generating axon-reflex vasodilatation, and thus might contribute to local vascular disturbances in acute and chronic inflammation
Applications of singularity theory to neurobiology
No full textThe nervous system of animals contains certain specialized
cells, called nerve cells or neurones, that are responsible for
the transmission of information within the animal's body. Those
cells consist of an enlarged part containing the nucleus, and
cytoplasmatic processes extending from if. The
processes are classified by hystological and physiological
criteria as axons or dendrites. Neurones of vertebrates
usually have only one axon, and it rarely branches except at
its termination
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