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）和F31-NS087837（CW）：这项工作是由美国国立卫生研究院的支持。

研究伦理的声明 ：所有涉及此详述动物实验是受规则和UNC委员会动物护理的法规和标准NIH为照顾和使用实验动物。 1.准备和游离皮层神经元的电镀<ol><li>通过CO安乐死定时怀孕女2吸入颈椎脱位。从每个半球胚胎天15.5（E15.5）小鼠和microdissect皮质的头骨取下整个大脑。有关胚胎小鼠皮层的显微请Viesselmann 等 8详细说明。 </li><li>放置在1ml​?...

<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 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>
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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 style="width:187px;">Used for scanning blots</td>
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			<td style="width:99px;">LI-COR</td>
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			<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>
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			<td height="41" style="height:41px;width:179px;">CELL TIRF control software</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 height="41" style="height:41px;width:179px;">40x 1.4 NA Plan Apochromat objective</td>
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			<td style="width:76px;"></td>
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			<td height="61" style="height:61px;width:179px;">Neurobasal media</td>
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			<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>
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			<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>
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			<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>
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			<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>
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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>
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			<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;">Glycerol (non optics grade)</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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			<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>
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		<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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			<td height="41" style="height:41px;width:179px;">Mouse monoclonal anti human &#946;III-tubulin&#160;</td>
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			<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 height="41" style="height:41px;width:179px;">Methanol</td>
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			<td style="width:76px;">S25426A</td>
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			<td height="41" style="height:41px;width:179px;">Bromphenol Blue</td>
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			<td height="41" style="height:41px;width:179px;">Sucrose</td>
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			<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>
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		<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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			<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>
			<td style="width:187px;"></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>
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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.

利用体外生物化学技术以测定神经元群体SDS抗性SNARE复合物的量。 图1示出探查SNAP-25，syntaxin1A和VAMP2的耐SDS SNARE复合测定的所得印迹以下完成。 在基底细胞膜TIRF显微镜提供了在单个细胞个体胞吐融合事件的高清晰度的图像。 图2A展示了用于识别VAMP2-phluorin介导的胞吐事件图像分析方法。插图显?...

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.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

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

Research

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

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

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

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