Name: utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis
Uploaded: 2016-03-16T14:00:00
Description: Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

RO1-GM108970（SLG）とF31-NS087837（CW）：この作品は、国立衛生研究所によってサポートされていました。

研究倫理の声明 ：本明細書に詳述動物に関わる全ての実験は、動物管理上および実験動物の管理と使用のためのNIHの基準にルールやUNC委員会の規制の対象となっています。 1.準備と解離皮質ニューロンのメッキ<ol><li>頸椎脱臼に続いてCO 2吸入によって時限妊娠雌を安楽死させます。各半球から胎生15.5（E15.5）マウスと顕微解剖皮質の頭蓋骨から全...

<ol>
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Axon branching is a fundamental neurodevelopmental process and underpins the vast neuroconnectivity of the mammalian nervous system. Understanding the mechanisms involved in localized plasma membrane expansion is integral to our understanding of both normal and pathological neurodevelopment. The use of a multipronged approach incorporating both population level and single cell level methodologies enhances reproducibility and increases spatial and temporal resolution without compromising population level analysis. At the ...

Recent estimates suggest that the human brain contains 1011 neurons with 1014 synaptic connections1, highlighting the importance of axon branching in vivo. Extracellular axon guidance cues such as netrin-1 guide axons to appropriate synaptic partners and stimulate axonal branching, thereby increasing synaptic capacity2-5. Netrin-1-dependent axonal arborization involves substantial plasma membrane expansion6, which we hypothesized requires delivery of additional membrane components via SNARE complex dependent exocytic vesicle fusion7. 
Investigating the role of SNARE-mediated exocytosis in netrin-1 dependent axon branching is complicated by several factors. First, the heterogeneity of cortical neurons increases the sample size required to identify significant effects, complicating single cell techniques like imaging. Second, although biochemical techniques permit observation of changes that occur at the population level, they lack the temporal and spatial resolution necessary to localize plasma membrane expansion to the axon in the time frame of axon branching. Lastly, although axon branches form over hours, the cellular changes that contribute to axonal extension may begin within minutes and occur on the order of seconds, thus extending the temporal scope for experimental consideration. 
We outline a multi-technique approach that addresses these diverse temporal and spatial scales of exocytosis and axon branching, and thus enhances our understanding of the fundamental cellular mechanisms. Utilizing these approaches provides evidence that supports a critical role for SNARE-mediated exocytosis in axon branching.

<table border="0" cellpadding="0" cellspacing="0" width="539">
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			<td height="40" style="height:40px;width:179px;">6-well tissue culture treated plates</td>
			<td style="width:99px;">Olympus Plastics</td>
			<td style="width:76px;">25-105</td>
			<td style="width:187px;"></td>
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			<td height="141" style="height:141px;width:179px;">glass coverslips</td>
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			<td style="width:187px;">12CIR-1.5;must be nitric acid treated for 24 hours, rinsed in DI H2O 2X, and dried prior to use. Must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Amaxa nucleofection solution</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:76px;">VPG-1001</td>
			<td style="width:187px;">100ml/transfection</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Amaxa Nucleofector/electroporator</td>
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			<td style="width:76px;"></td>
			<td style="width:187px;">program O-005</td>
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			<td height="81" style="height:81px;width:179px;">35mm Glass bottom live cell imaging dishes</td>
			<td style="width:99px;">Matek Corporation</td>
			<td style="width:76px;">p356-1.5-14-C</td>
			<td style="width:187px;">must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Olympus IX81-ZDC2 inverted microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
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			<td height="61" style="height:61px;width:179px;">Lambda LS xenon lamp</td>
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			<td height="41" style="height:41px;width:179px;">Environmental Stage top incubator</td>
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			<td height="41" style="height:41px;width:179px;">100x 1.49 NA TIRF objective</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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			<td height="21" style="height:21px;width:179px;">Andor iXon EM-CCD</td>
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			<td height="41" style="height:41px;width:179px;">Odyssey Licor Infrared Imaging System</td>
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			<td style="width:187px;">Used for scanning blots</td>
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			<td height="81" style="height:81px;width:179px;">Image studio software suite</td>
			<td style="width:99px;">LI-COR</td>
			<td style="width:76px;"></td>
			<td style="width:187px;">Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots</td>
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			<td height="61" style="height:61px;width:179px;">Metamorph for Olympus</td>
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			<td style="width:76px;">version 7.7.6.0</td>
			<td style="width:187px;">Software used for all imaging and the analysis of DIC timelapse</td>
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			<td height="41" style="height:41px;width:179px;">CELL TIRF control software</td>
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			<td style="width:187px;">Software used to control lasers for TIRF imaging</td>
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			<td height="61" style="height:61px;width:179px;">Fiji (Image J)</td>
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			<td height="41" style="height:41px;width:179px;">60x Plan Apochromat 1.4 NA objective</td>
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			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">40x 1.4 NA Plan Apochromat objective</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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			<td height="61" style="height:61px;width:179px;">Neurobasal media</td>
			<td style="width:99px;">GIBCO</td>
			<td style="width:76px;">21103-049</td>
			<td style="width:187px;">Base solution for both serum free and trypsin quenching media</td>
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			<td height="62" style="height:62px;width:179px;">Supplement B27</td>
			<td style="width:99px;">GIBCO</td>
			<td style="width:76px;">17504-044</td>
			<td style="width:187px;">500ml/50mLs Serum free media and Trypsin Quenching media</td>
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			<td height="41" style="height:41px;width:179px;">L-Glutamine</td>
			<td style="width:99px;"></td>
			<td>35050-061</td>
			<td style="width:187px;">1mL/50mLs Serum free media</td>
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			<td height="101" style="height:101px;width:179px;">Bovine serum albumin</td>
			<td style="width:99px;">Bio Basic Incorporated</td>
			<td style="width:76px;">9048-46-8</td>
			<td style="width:187px;">10% solution in 1XPBS for blocking coverslips; 5% solution in TBS-T for blocking nitrocellulose membranes.</td>
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			<td height="21" style="height:21px;width:179px;">10X&#160; trypsin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">59427C</td>
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			<td height="21" style="height:21px;width:179px;">HEPES</td>
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			<td height="61" style="height:61px;width:179px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)+ Ca + Mg</td>
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			<td colspan="2">21-030-cm</td>
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			<td height="41" style="height:41px;width:179px;">Fetal bovine serum</td>
			<td style="width:99px;">Corning/CELLGRO</td>
			<td style="width:76px;">35-010-CV</td>
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			<td height="41" style="height:41px;width:179px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
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			<td height="41" style="height:41px;width:179px;">NaCL</td>
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			<td height="41" style="height:41px;width:179px;">EGTA</td>
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			<td style="width:76px;">CAS67-42-5</td>
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			<td height="41" style="height:41px;width:179px;">MgCl2</td>
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			<td style="width:76px;">BP214-500</td>
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			<td style="width:76px;">T5941-500</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">TRIS base</td>
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			<td height="41" style="height:41px;width:179px;">N-Propyl Gallate</td>
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			<td height="41" style="height:41px;width:179px;">Glycerol Photometric grade</td>
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			<td style="width:76px;">18469-5000</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Glycerol (non optics grade)</td>
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			<td style="width:76px;">CAS56-81-5</td>
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			<td height="41" style="height:41px;width:179px;">B-mercaptoethonal</td>
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			<td style="width:76px;">BP176-100</td>
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			<td height="41" style="height:41px;width:179px;">SDS</td>
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			<td height="41" style="height:41px;width:179px;">Distilled Water&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Poly-D-Lysine</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">p-7886</td>
			<td style="width:187px;">Dissolved in sterile water at 1mg/mL</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Botulinum A toxin BoNTA</td>
			<td style="width:99px;">List Biological Laboratories</td>
			<td style="width:76px;">128-A</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Rabbit polyclonal anti human VAMP2</td>
			<td style="width:99px;">Cell signaling</td>
			<td style="width:76px;">11829</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Mouse monoclonal anti rat Syntaxin1A</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-12736</td>
			<td style="width:187px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Goat polyclonal anti human SNAP-25</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-7538</td>
			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse monoclonal anti human &#946;III-tubulin&#160;</td>
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			<td style="width:76px;">MMS-435P</td>
			<td style="width:187px;"></td>
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		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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		<tr height="121">
			<td height="121" style="height:121px;width:179px;">LI-COR IR-dye secondary antibodies</td>
			<td style="width:99px;">LI-COR</td>
			<td style="width:76px;">P/N 925-32212,P/N 925-68023, P/N 926-68022</td>
			<td style="width:187px;">800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:179px;">0.2um pore size nitrocellulose membrane</td>
			<td style="width:99px;">Biorad</td>
			<td style="width:76px;">9004-70-0</td>
			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Tween-20</td>
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			<td style="width:76px;">BP337-500</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">Methanol</td>
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			<td style="width:76px;">S25426A</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">Bromphenol Blue</td>
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			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Sucrose</td>
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			<td style="width:76px;">S6-212</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Paraformaldehyde</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">O-4042-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Triton-X100</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP151-500</td>
			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">TEMED</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP150-20</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">40% Bis-Acrylimide</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP1408-1</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Name</td>
			<td style="width:99px;">Company</td>
			<td style="width:76px;">Catalog Number</td>
			<td style="width:187px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Alternative Validated Antibodies</td>
			<td style="width:99px;"></td>
			<td style="width:76px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal Anti-Syntaxin HPC-1 clone</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:76px;">S0664</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">&#160;Mouse Monoclonal Synaptobrevin 2 (VAMP2)</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">104-211</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal SNAP25</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">111-011</td>
			<td style="width:187px;"></td>
		</tr>
	</tbody>
</table>

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.

神経細胞の集団におけるSDS耐性SNARE複合体の量を測定するためにインビトロ生化学的手法に活用。 図1は、SNAP-25、syntaxin1AとVAMP2についてプローブSDS耐性SNARE複合体アッセイの完了後、得られたウエスタンブロットを示しています。 基底細胞膜にTIRF顕微鏡法は、単一のセル内の個々のエキソサイト?...

Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

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

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 ...

Research

JoVE Journal

Neuroscience

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

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

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

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マイクロ流体文化·プラットフォーム（MCP）ベースの神経軸索とグリア共培養系におけるグリア相互作用へのニューロンのイメージング解析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

覚醒マウスにおける神経活性物質のMicroiontophoreticアプリケーションと組み合わせることで神経活動の細胞外記録

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

ショウジョウバエメラノガスターにおける軸索死における軸索とそのシナプスの形態学的および機能的評価

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...