我々は感謝してこの方法論の開発で博士ロバートE. Buxbaumの重要な貢献を認識する。

<ol>
	<li>Zheng, J., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+growth+cone+adhesion+to+culture+surfaces+by+micromanipulation.">Measurements of growth cone adhesion to culture surfaces by micromanipulation.</a> J Cell Biol. 127, 2049-2060 (1994).</li><li>Chada, S., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+neurite+outgrowth+from+chick+brain+neurons.">Cytomechanics of neurite outgrowth from chick brain neurons.</a> J Cell Sci. 110, 1179-1186 (1997).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E., Haynes, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neuron in Tissue Culture. , 105-119 (1999).</li><li>Lamoureux, P., Altun-Gultekin, Z. F., Lin, C., Wagner, J. A., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+is+required+for+growth+cone+function+but+not+neurite+assembly.">Rac is required for growth cone function but not neurite assembly.</a> J Cell Sci. 110, 635-641 (1997).</li><li>Lamoureux, P., Ruthel, G., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+can+specify+axonal+fate+in+hippocampal+neurons.">Mechanical tension can specify axonal fate in hippocampal neurons.</a> J Cell Biol. 159, 499-508 (2002).</li><li>Lamoureux, P., Heidemann, S. R., Martzke, N. R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Dennerll, T. J., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytomechanics+of+axonal+elongation+and+retraction.">The cytomechanics of axonal elongation and retraction.</a> J Cell Biol. 109, 3073-3083 (1989).</li><li>Heidemann, S. R., Kaech, S., Buxbaum, R. E., Matus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+mechanical+behaviors+of+the+cytoskeleton+in+living+fibroblasts.">Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.</a> J Cell Biol. 145, 109-122 (1999).</li><li>Yoneda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Exerted+by+a+Single+Cilium+of+Mytilus-Edulis+.1.">Force Exerted by a Single Cilium of Mytilus-Edulis .1.</a> Journal of Experimental Biology. 37, (1960).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Tension+Produced+by+Nerve-Cells+in+Tissue-Culture.">Mechanical Tension Produced by Nerve-Cells in Tissue-Culture.</a> Journal of Cell Science. 37, 391-410 (1979).</li><li>Pfister, B. J., Iwata, A., Meaney, D. F., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Fass, J. N., Odde, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+force-dependent+neurite+elicitation+via+anti-beta1+integrin+antibody-coated+magnetic+beads.">Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.</a> Biophys J. 85, 623-636 (2003).</li><li>Yang, S., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Force+Sensors+and+Their+Applications+in+the+Study+of+Cell+Mechanical+Response.">Microfabricated Force Sensors and Their Applications in the Study of Cell Mechanical Response.</a> Exp Mech. 49, 135-151 (2009).</li><li>Bernal, R., Melo, F., Pullarkat, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drag+Force+as+a+Tool+to+Test+the+Active+Mechanical+Response+of+PC12+Neurites.">Drag Force as a Tool to Test the Active Mechanical Response of PC12 Neurites.</a> Biophysical Journal. 98, 515-523 (2010).</li><li>Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+growth+cones+pull.">Direct evidence that growth cones pull.</a> Nature. 340, 159-162 (1989).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+behavior+and+production+of+traction+force.">Growth cone behavior and production of traction force.</a> J Cell Biol. 111, 1949-1957 (1990).</li><li>O'Toole, M., Lamoureux, P., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Heidemann, S. R., Lamoureux, P., Ngo, K., Reynolds, M., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-dish+incubator+for+live+cell+imaging+with+an+inverted+microscope.">Open-dish incubator for live cell imaging with an inverted microscope.</a> Biotechniques. 35, 708-708 (2003).</li></ol>

利害の衝突は宣言されません。

携帯電話の力を適用し、測定する技術は長い歴史9持っている。我々の方法は、もともと私たちと電動油圧装置10を用いて一定速度で&quot;けん引&quot;神経細胞に似たガラスの針を使用デニスブレイ、の作業が動機とされた。ステッピングモータ11、磁気ビーズ12、微細加工ビーム13と流体の流れ14：含まれる細胞に力を適用することの多くの代替?...

<table><tbody><tr><td>R-6 cap. Tube</td><td>Drummond Scientific</td><td>9-000-3111</td><td>R-6 glass OD 0.9mm, ID 0.6 mm, 8&quot;</td></tr><tr><td>BB-CH puller</td><td>Mecanex S.A., Geneva, Switzerland</td><td>BB-CH puller</td><td>Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000</td></tr><tr><td>0.001&quot; Chromel wire</td><td>Omega Engineering, Inc.</td><td>SPCH-001-50</td><td>unsheathed, themocouple wire, 50ft spool now called Chromega</td></tr><tr><td>0.003&quot; Constatan wire</td><td>Omega Engineering, Inc.</td><td>SPCI-003-50</td><td>unsheathed, themocouple wire, 50 ft spool</td></tr><tr><td>fine forceps</td><td>Fine Science Tools</td><td>91150-20</td><td>Dumont Inox #5</td></tr><tr><td>universal microscope boom stand</td><td>Nikon Instruments</td><td>76135 or 90430</td><td>most brands or types of boom stand will work for this use</td></tr><tr><td>mechanical micromanipulator</td><td>Narishige International</td><td>M-152</td><td>three-axis direct-drive coarse micromanipulator</td></tr><tr><td>hydraulic micromanipulator</td><td>Narishige International</td><td>MO-203</td><td>now available as MMO-203, three movable axis type</td></tr><tr><td>needle holder</td><td>Leica Microsystems</td><td>11520145</td><td>set of 3</td></tr><tr><td>single instrument holder</td><td>Leica Microsystems</td><td>11520142</td><td></td></tr><tr><td>double instrument holder</td><td>Leica Microsystems</td><td>11520143</td><td></td></tr><tr><td>mechanical micromanipulator</td><td>Leica Microsystems</td><td>39430001</td><td>post mount,1 prob holder, RH Model 430001</td></tr><tr><td>joystick mech. micromanipulator</td><td>Leica Microsystems</td><td>11520137</td><td></td></tr><tr><td>Leica DM IRB</td><td>Leica Microsystems</td><td></td><td>inverted microscope</td></tr><tr><td>Vibraplane isolation table</td><td>Kinetic Systems</td><td>1200 series</td><td>ours is model 1201-02-12</td></tr><tr><td>Ringcubator</td><td>Home made (see reference 19)</td><td></td><td>reference 19, requires updated controller listed below</td></tr><tr><td>programable temperature controller</td><td>Instrumart.com</td><td>Fuji Electric PXR3</td><td>replaces the retired PXV3 temperature controller</td></tr><tr><td>Nikon Diaphot TMD</td><td>Nikon Instruments</td><td></td><td>inverted microscope, circa 1980</td></tr><tr><td>Nikon SMZ-10 binocular dissecting </td><td>Nikon Instruments</td><td></td><td>other dissecting microscopes will work</td></tr></tbody></table>

mechanical manipulation of neurons to control axonal development

セルの操作と神経軸索の延長は、10から1000μdyne範囲の1,2の力を測定し、適用可能な校正済みガラスのマイクロファイバーを使用して行うことができます。力の測定は、直接、経験的な方法3によって校正されているガラス針の曲げHookeanの観察を通して得られる。 、製造校正、治療、および細胞に針を使用するための機器の要件と手順が詳しく説明されています。力は、これらの技術は方法論の柔軟性を示すが適用されていると、将来の検討4-6の例として与えられるために以前に使用し、異なる細胞型の制度。技術的な利点は、操作によって生成される力の継続的な&quot;可視化&quot;し、直接携帯電話のさまざまなイベントに介入できることです。と同様に剥離し、培養細胞8の任意のタイプの機械的な測定は、これらは直接的な刺激や軸索の成長と収縮7の規定が含まれています。

Watch this Scientific Journal Video about Mechanical Manipulation of Neurons to Control Axonal Development at JoVE.com

Mechanical Manipulation of Neurons to Control Axonal Development

アプリケーションと2000から1000 microdyneの範囲内のニューロンに作用する力の直接測定は、校正済みのガラス針を用いて高精度で実現されています。この方法論は、軸索の開始、軸索張力、軸索伸長の速度、および力ベクトルを含む、軸索の開発のいくつかの側面を、制御および測定に使用することができます。

軸索の開発を制御する神経細胞の機械的な操作

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

mechanical-manipulation-of-neurons-to-control-axonal-development

Research

JoVE Journal

Neuroscience

10.4K ビュー.  Michigan State University, East Lansing. アプリケーションと2000から1000 microdyneの範囲内のニューロンに作用する力の直接測定は、校正済みのガラス針を用いて高精度で実現されています。この方法論は、軸索の開始、軸索張力、軸索伸長の速度、および力ベクトルを含む、軸索の開発のいくつかの側面を、制御および測定に使用することができます。

ビデオ: Mechanical Manipulation of Neurons to Control Axonal Development

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

軸索のストレッチの成長：神経成長のメカノトランスダ

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

生物工学

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神経形態形成の研究のための方法：<em&gt;エキソビボ</em胎児マウス大脳皮質&gt; RNAiのエレクトロポレーション

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

神経科学

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

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.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脳軸索のレーザーNanosurgery<em&gt;インビボ</em

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 ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

後根神経節ニューロンのアライメントや髄鞘形成を強化するためのアプローチ

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

神経系の再構築、変調、モデリングのための解剖学的にインスパイアされた3次元マイクロ組織工学ニューラルネットワーク

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

ニューロン回路の再学習：速い神経突起伸長と機能的ニューロン結合の新しい方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

<meta charset="utf8"></head><body>正確な神経突起伸長測定のための高度なライブイメージング技術

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

<meta charset="utf8"></head><body>著者スポットライト:痛みの知覚と神経因性疼痛の分子基盤を明らかにする

Neurite outgrowth assays provide a quantitative value about regenerative neuronal processes. The advantage of this semi-automatic software is that it segments cell bodies and neurites separately by creating a mask and measures various parameters such as neurite length, number of branch points, cell-body cluster area, and number of cell clusters.

神経突起伸長測定のための新しい半自動ソフトウェアの標準化 

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of ...

軸索の開発を制御する神経細胞の機械的な操作

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