Name: rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection
Uploaded: 2017-06-13T14:00:00
Description: Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

我们要感谢宫原一郎多次有帮助的讨论和见解。 MA和PG承认NSERC的资金。

以下详细说明的所有步骤均由麦吉尔大学动物护理委员会批准，并符合加拿大动物护理委员会的指导原则。 使用微流控器件进行神经元培养的标准化：器件装配<ol><li>为所需实验选择合适的微流体装置。为了连接相同群体中的神经元，使用Neuro Devices（ 图1 ），并连接不同人群中的神经元使用共培养装置（ 图6 ）。 </li><li>清洁并?...

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使用标准显微操作和创新的微流体装置，开发了一种新技术，可以在很大距离上快速启动，延长和精确连接新的功能性神经元电路。移液管显微操作是大多数神经科学实验室4,13中的常用工具。实现可重复和可靠结果的真正挑战是通过开发微流体装置，以微米精度组织细胞培养，在实验期间（可能在几周内）将健康精确定位的神经元培养物标准化。高质量的细胞培养是数据验证的基石。这有助于更...

成人中枢神经系统（CNS）的损伤可能导致永久性残疾，由于多种机制限制轴突再生长1 。受伤后，许多CNS轴突不形成新的增长锥，不能建立有效的再生反应2 。此外，围绕CNS损伤的损伤和瘢痕组织显着抑制轴突生长1,2,3 。目前用于促进损伤后CNS再生的疗法主要集中在增强受损神经元的内在生长潜力，并掩盖与髓磷脂碎片和胶质瘢痕1,3相关的轴突延伸抑制剂。尽管如此，将长轴突再生到远处目标并形成适当的功能性突触的能力仍然受到严重限制4 ， </sup&gt; 5,6,7 。 在目前的工作中，微珠，移液管显微操作和微流体装置用于在长距离上快速启动，延长和精确连接新的功能性神经元电路。以前的工作已经显示聚-D-赖氨酸包被的珠粒（PDL珠）诱导膜粘附，然后突触囊泡复合物的聚集和功能突触前boutons的形成8 。还显示当PDL珠被突触前分化后机械地拉开时，突触蛋白簇跟随珠，引发新的神经突9 。以下程序利用这一事实以及使用聚二甲基硅氧烷（PDMS）微流体装置将大鼠的胚胎海马神经元培养成盖玻片上的有组织区域以将精神重新连接神经元电路。 这些PDMS微流体装置是无毒的，光学透明的，由通过微通道系统连接的两个室组成。一旦组装在盖玻片上，每个装置用作引导神经元生长的模具，并且在体外将精确模式维持健康的神经元培养超过4周。 在这里，提出了一个框架来研究新神经突的扩展和功能的限制。新的功能神经突被创建并定位成可控地（重新）连线的神经元网络。实现的扩展速率比毫米级距离快20微米/分钟，建立功能连接。这些结果意外地显示出这些神经突伸长的内在能力比以前想象的要快得多。这种提出的机械方法绕过了缓慢的化学策略，并实现了与特定目标的控制连接。钍是技术开创新的疗法的新途径，以恢复损伤后的神经元连通性的新疗法。它还使得神经元网络的操纵和重新布线能够在体外研究神经元信号处理和神经元功能的基本方面。

<table border="0" cellpadding="0" cellspacing="0" width="836">
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			<td height="41" style="height:41px;width:216px;">Co-culture devices</td>
			<td style="width:104px;">Ananda Devices</td>
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			<td style="width:397px;">Commercially available at http://www.anandadevices.com</td>
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			<td height="41" style="height:41px;width:216px;">Neuro devices</td>
			<td style="width:104px;">Ananda Devices</td>
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			<td>Commercially available at http://www.anandadevices.com</td>
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			<td height="41" style="height:41px;width:216px;">No. 1 Glass Coverslip 25 mm Round</td>
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			<td style="width:119px;">64-0705</td>
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			<td height="41" style="height:41px;width:216px;">35mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated</td>
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			<td height="41" style="height:41px;width:216px;">35mm cell culture dish, Non-Pyrogenic, Sterile</td>
			<td style="width:104px;">Corning Inc</td>
			<td style="width:119px;">430165</td>
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			<td height="41" style="height:41px;width:216px;">95mmx15mm Petri Dish, Slippable Lid, Sterile Polystyrene</td>
			<td style="width:104px;">Fisherbrand</td>
			<td>FB0875714G</td>
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			<td height="21" style="height:21px;">15mL Polypropylene Conical Tube, 17x120mm style, Non Pyrogenic, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
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			<td height="41" style="height:41px;width:216px;">Neurobasal Medium</td>
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			<td style="width:119px;">21103-049</td>
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			<td height="81" style="height:81px;width:216px;">B-27 Supplement (50X), serum free</td>
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			<td style="width:119px;">17504044</td>
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			<td height="41" style="height:41px;width:216px;">Pennicilin, Streptomyocin, Glutamine</td>
			<td style="width:104px;">Thermo Fisher Scientific&#160;</td>
			<td>11995-065</td>
			<td>Extracellular solution</td>
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			<td height="22" style="height:22px;width:216px;">200uL Pipettors</td>
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			<td height="21" style="height:21px;">BD Falcon 3mL Transfer Pipettes [Non-sterile]</td>
			<td>BD Falcon</td>
			<td>357524</td>
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			<td height="21" style="height:21px;width:216px;">Glucose</td>
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			<td style="width:119px;">15023-021</td>
			<td>Extracellular solution</td>
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			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">7365-45-9</td>
			<td>Extracellular solution/Beads</td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:104px;">Sigma-Aldrich</td>
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			<td style="width:119px;">7447-40-7</td>
			<td>Extracellular solution</td>
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			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">10043-52-4</td>
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			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">7786-30-3</td>
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			<td height="61" style="height:61px;width:216px;">#5 Dumont Dumostar Tweezers 11cm</td>
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			<td height="21" style="height:21px;">Dissection tools</td>
			<td>Braun, Aesculap</td>
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			<td height="21" style="height:21px;">Poly-D-lysine Hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
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			<td height="40" style="height:40px;width:216px;">Micro particles based on polystyrene, 10 um</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">72986</td>
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			<td height="40" style="height:40px;width:216px;">Borosilicate tubes</td>
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			<td height="40" style="height:40px;width:216px;">Horizontal Pipette Puller</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Micromanipulators, PCS-5000 Series</td>
			<td style="width:104px;">SD Instruments</td>
			<td style="width:119px;">MC7600R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml Syringe</td>
			<td style="width:104px;">BD Luer-Lok</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:104px;">Olympus&#160;</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">UIS2, LUCPLFLN 40X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD Camera</td>
			<td style="width:104px;">Photometrics</td>
			<td style="width:119px;">Cascade II: 512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s (1x) L-15 Medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typsin-EDTA (0.05%), Phenol red</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (1x) Dulbecco&#39;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (1x) Hank&#39;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td>
			<td>Life Technologies</td>
			<td>14170-112</td>
			<td>Rat Dissection</td>
		</tr>
	</tbody>
</table>

rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

脑和脊髓损伤可能导致永久性残疾和死亡，因为仍然不可能长距离再生神经元，并准确地将其与适当的目标重新连接。这里描述了一种快速启动，延长和精确连接长距离功能神经元电路的过程。实现的延伸率达到1.2mm / h以上，比来自周围神经系统（0.02至0.04mm / h）的生长最快的轴突的体内速率快30-60倍，比先前报道的速度快28倍和10倍早期发育阶段的神经元类型4 。首先，分离的大鼠海马神经元群体在微流体装置中生长2-3周以精确定位细胞，使得易于显微操作和实验重现性。接下来，将涂覆有聚-D-赖氨酸（PDL）的珠子放置在神经突上以形成粘合剂作用和移液管显微操作用于移动所得珠粒神经突复合体。当珠移动时，它拉出一个可以在数百微米上延伸的新神经突，并在不到1小时内与靶细胞功能连接。该过程使实验重现性和易于操作，同时绕过较慢的化学策略来诱导神经突生长。这里提出的初步测量表明，神经元生长速度远远超过生理因素。结合这些创新，可以以前所未有的程度来精确地建立文化中的神经元网络。这是一种新颖的方法，为神经元网络中的信号传输和通信开辟了大量的信息和洞察力，也是探索神经元生长极限的操场。潜在的应用和实验是广泛的，直接涉及旨在重新连接神经元的治疗创伤后或神经退行性疾病中的电路。

将胚胎大鼠海马神经元在微流体装置中培养以使细胞，PDL珠和显微操纵体的精确定位。第一步是将微流体装置适当地组装在玻璃盖玻片或盘子上。必须使微流体装置良好地附着到基底上以避免细胞离开腔室并移动到应密封的装置的部分（ 图1a ）。为了保持健康的文化数周，重要的是通过每2-3天检查细胞培养基并保留培养基的阳性弯液面来防止培养基...

Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

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

该程序描述了如何使用固定在引导神经突伸长的微量移液管上的聚-D-赖氨酸包被的珠快速启动，延伸和连接在微流体室中组织的神经突。

重建神经元电路：快速神经延伸和功能性神经元连接的新方法

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 ...

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

测绘激光扫描Photostimulation抑制神经回路

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...