这项工作是由欧洲项目BRIANBOW（FP7-年轻探险家的支持，笔者想感谢雅格布Tessadori博士对稿件有用的意见，和Silvia Chiappalone为她制作的视频所使用的图形帮助。

该程序是根据美国国立卫生研究院的护理标准和使用实验动物的完成，并批准了特拉维夫大学动物护理和使用委员会（许可证号 - L-14-019）。 1.准备仪器的PDMS <ol><li>准备晶片（ 材料的表 ，或从一个微细加工实验室的晶片），手术刀和镊子-灭菌是不需要的。 </li><li>根据以下条件使聚-D-赖氨酸（PDL）溶液：4毫克/毫升的0.1M硼酸盐缓冲液，pH 8，并储存在-20℃。 </li><li>根据以下程序制备的PDMS模具： <ol><li>由基和以10比上述固化剂混合在一起制备聚二甲基硅氧烷（PDMS，聚硅氧烷弹性体）：1，然后搅拌2物质为大约5分钟。 </li><li>每次放置在真空室中两次15分钟，并确认气泡消失。 </li><li>什么时候与PDMS就绪，准备旋涂器：开放氮旋钮，以允许气体使用，放置在旋涂器在硅晶片（ 图1A），并使用真空，以防止其移动。 </li><li>倾PDMS超过晶片并激活离心器1分钟以1,000rpm（创建大约100微米的高度）。 </li><li>放置晶片的热板上在100℃下进行30分钟。 </li><li>当PDMS已硬化，勾勒出模板的边界与PDMS，使用巴斯德吸管。放置晶片的热板上在100℃下进行30分钟。 </li><li>当PDMS边界已经硬化，用手术刀根据边框刻蜡板，并从晶圆剥离硅模板（含单一模式的平方PDMS）。 </li></ol></li></ol> 2.培养皿和盖玻片准备<ol><li>准备23毫米方形玻璃盖玻片，并清除它们按以下顺序： <ol><li>用蒸馏水，70％的乙醇和丙酮洗涤。 </li><html><li>用异丙醇和快速干燥氮气清洗。 </li></ol></li><li>放在盖玻片图案的PDMS模具，并轻轻按压，以确认它们牢固地附着在玻璃表面。放置在真空室15分钟。 </li><li>掉在模板1毫升PDL的。插入盖玻片进入真空室两次，每次20分钟，并留下的PDL O / N晾干。 </li><li>准备培养皿中创建一个支持蜂窝网络： <ol><li>覆盖培养皿（3.5厘米）用1毫升的PDL的2小时中的RT的表面上。除去用吸管将PDL下降。 </li><li>用蒸馏水冲洗，并留下来干。放置一个非常小的滴硅脂上盖玻片的每个角。 </li></ol></li><li>将盖玻片上的培养皿（ 即 PDMS应朝上），然后按中央轻轻核实附件。 </li><li>轻轻地用小镊子从盖玻片删除PDMS。灭菌，暴露于紫外线照射7分钟。 </li></ol>乐“&gt; 3。多电极阵列（MEA）的制备<ol><li>清洁MEA在这个顺序（ 表材料 ）： <ol><li>水在自来水和超声的集中，酶洗涤剂洗3次。 </li><li>声处理的蒸馏水的3倍。 </li><li>用蒸馏水（在引擎盖，1000微升尖）3次并置于紫外光下30分钟洗涤。 </li></ol></li><li>支持网络准备： <ol><li>准备设计支持网络的模具。 <ol><li>倾PDMS成一个12孔板（22毫米直径）。 </li><li>当与PDMS硬化取出模具并使用手术刀，切孔在中间以创建一个环状。 </li><li>放置在MEA（ 图2A）的中心的设计支持网络的模具中，盖上PDL中的表面的其余部分进行2小时，在室温。 </li></ol></li><li>使用移液管除去PDL和洗涤用蒸馏水。 </li><li>取出模具，并留下干燥（ 图2A）。&lt;/ li&gt; </ol></li><li>对准蜡纸对于MEA以下列方式： <ol><li>模板放置在指定的微机器人。使用倒置显微镜，以精确对准图案化结构的电极，并降低模版，直到它被放置在MEA表面（ 图2B）。 注：带好清洗MEA联系提供了PDMS和MEA自身之间的竞争附着力。 </li><li>解除显微和如果需要的话，可以使用镊子应用上述的PDMS少量的压力，以防止它从MEA分离。 </li><li>轻轻按下模板来MEA表面，并用显微镜才能验证它是否连接牢固，并且完全一致。 （ 图2C-D）。 </li></ol></li><li>放置的MEA在真空室15分钟;把1毫升滴PDL对模板。 </li><li>插入到真空腔室2次，每次20分钟。离开PDL干O / N在孵化器。 </li><li>电镀前，从多边环境协定取出PDMS模具和洗用无菌瓦特亚特。在紫外光照射下放置7分钟。 </li></ol> 4.解剖和文化<ol><li>在赫尔佐格等2011 21介绍准备文化。 </li></ol> 5.电镀<ol><li>计算所需要的电镀的细胞数，使用血细胞计数器（根据电路的作为结果讨论大小最佳密度）。 <ol><li>确保计数室（血球）是干净的，在其上放置盖玻片（使用酒精清洗）。 </li><li>稀释10微升的细胞悬浮在190微升电镀介质（稀释1:20）的。 </li><li>负载10微升稀释的细胞上的计数室的边缘，并慢慢地吸取细胞进行允许该腔室，以填补本身。 </li><li>使用倒置显微镜，可视化的血球网格。确定细胞中通过直接计数室中数（健康的细胞应该是圆形）。算上中大型广场的无覆盖细胞UT斯达康那些过路的边缘。 </li><li>计算细胞的浓度： 细胞的总数目/ 1000微升=计数×稀释倍数×10 4（血细胞计数仪的面积）的总细胞。 注意：例如，如果稀释因子为20和计数细胞总数分别为100： 100×20×10 4 = 0.1×10 7细胞/ 1,000微升或1×106/100微升。为了制版0.75×10 6个细胞，取75微升出细胞。 </li></ol></li><li>取所需的每单MEA或培养皿电镀单元的数量，悬浮细胞，以防止聚集，并镀它们在中心处，在patternate区域的上方（如果需要的话，也可以以稀释的细胞在培养基中） 。对于在MEA，放置在中间100微升下降。对于盖玻片，放置在中间1000微升下降。 </li><li>孵育细胞铺板在37℃下40分钟。加镀介质向镀细胞，高达1每毫升MEA和每盖玻片2毫升。 </li><li>保持在37℃和，每4天，以稀释富含0.5青霉素 - 链霉素，2％的B-27和0.75％Glutamax的新鲜生长培养基。 </li><li>从6/7 DIV，看到岛屿之间的连接后，稀释培养基10微升/毫升FUDR（25毫克脱氧尿苷+ 62.5毫克尿苷在12.5毫升MEM（最低必需培养基Eagle））或任何其他抗有丝分裂剂，以防止神经胶质过度生长。 </li></ol>

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的协议种植2D模块化神经网络的体外功能上相互连接组成的电路描述。该过程是基于图案的蜂窝的粘合剂层。图案形成用的PDMS模具再现期望网络体系结构的负特性来实现的。 PDMS模板定义，其中所述蜂窝粘合剂层沉积领域。一旦细胞被铺板，它们自发地装配到涂覆岛屿和自组织成活性间连接的电路。利用多边环境协定和钙成像记录功能模块化的网络录音提交。 所提出的...

实验和理论的证据支持，大脑运作通过的单元组件1-5协调活化，这可以被视为动态功能单元瞬时彼此，整形和下层不同大脑状态相互作用的可能性。官能模块化还依赖于与大脑电路6,7的结构的模块化组织关联。怎样的功能和大脑回路的结构互相塑造彼此仍是主要的开放性问题中的神经之一。以提供对这个问题更深入的了解，来识别最佳实验框架，其中能够解决，至少部分地，这些问题是重要的。由于控制操作的神经元网络的时空动力 ​​学在体内实验是具有挑战性的， 在体外的神经元网络模型的开发是显著感兴趣的，因为它们容易ACCessibility，监控，处理和建模8,9。在最近几年， 在由先进衬底图案化的方法支持体外技术已经允许诱导神经元网络开发一系列预定义的模块化结构体3，并与所施加的拓扑结构10学习网络的功能特性。特别是，方法进行了最近使用过的征收物理约束4,11组织网络。的确，以研究在神经元网络的结构和功能之间的联系，并提供相互作用的神经元组件的简化，但可能的表述， 在体外系统应提供相互连接的神经元亚群。广泛的研究二维均匀的神经细胞也没有对电路的自组织紧急连线任何空间的限制。因此，一个可能的方法来塑造人为地相互连接的电池组件被定位在不同的吐口水的神经元群体ially不同的领域。这些区域之间的距离不阻止除装配连接。这种方法，同时保证有相当的控制网络的复杂性，已经显示出，以提供同步模型6,7,12的更丰富的剧目。 为了方便的模块化神经元组件的可重复培养，协议组装的自组织网络到由轴突和树突连接的神经元集群的呈现和描述。聚合物结构对神经元培养物的物理约束已创建从polydimtheylsiloxane（PDMS）。 PDMS是一种弹性体广泛用于由于其生物相容性，透明性和透气性13生物医学应用。将PDMS制备并排除在微加工的SU8 2075 14,15的结构，通过旋涂液态PDMS上如杰克曼等人以前描述了“主”。16吨他实现了图形化的神经网络是由不同大小的相互连接的模块和他们两个盖玻片和微电极阵列（多边环境协定）17-20成功获得。在模块之间连接的密度可以改变它的网络同步的功能，从一个完全同步的网络中，典型的均一培养，以同步的瞬时状态模块之间。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PDMS, Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Vacuum Chamber</td>
			<td>Thermo</td>
			<td>5305-0609</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine PDL</td>
			<td>Sigma</td>
			<td>P7886</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell - Technologies Corporation</td>
			<td>WS-650-23</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well culture plate</td>
			<td>Sigma</td>
			<td>CLS3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8217;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA1060-Inv-BC</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Biological Industries Beit Haemek</td>
			<td>03-033-1c</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Minimum Essential Medium-Eagle</td>
			<td>Biological Industries Beit Haemek</td>
			<td>01-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Electrode Arrays 4Q</td>
			<td>Multi Channel Systems</td>
			<td>60-4QMEA1000iR-Ti-pr</td>
			<td>cleaning manual: http://www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>silicon wafer</td>
			<td>microchem</td>
			<td>SU8-2075</td>
			<td>Preparation protocol: www.microchem.com</td>
		</tr>
	</tbody>
</table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

大脑工作通过协调激活和神经元组件的动态通信。一个主要的开放问题是如何动态图案​​的巨大的剧目，这背后最多样化的大脑功能，可以浮现出的大脑回路的固定拓扑和模块化的组织。相比对神经元回路的体内研究，其呈现固有的实验的困难， 在体外制剂提供了更大的可能性，以操纵和探针实验神经系统的结构，动力学和化学性质。这项工作描述了在体外实验方法，它允许增长了空间层次分明，功能上相互联系的神经元组成的组件模块化网络。该协议允许控制神经网络的二维（2D）结构在不同级别的复杂性拓扑。 所期望的网络图案可定期盖玻片和基材嵌入式微电极阵列来实现两者。微机械结构被压印在硅晶片上，并用于创建生物相容性聚合物模具，其包含所述期望的网络架构的负特征。刻花模板与一个分子层用于促进细胞粘附的表面涂层过程期间放置在培养底物。除去模板后，神经元被镀和它们自发地重定向到涂覆区域。通过减小隔室间的距离，因此能够获得任何分离的或互相连接的神经元回路。促进细胞存活，细胞共培养，其位于培养皿的周围的支撑神经元网络。分别通过使用基板嵌入式微电极阵列和钙成像获得模块化网络的活动的电和光学记录被呈现。虽然每个模块显示SPONTaneous全球同步，对模块间同步的发生是由电路之间连接的密度调节。

甲SU8-2075模具的硅晶片具有约100μm的厚度的功能上是用来塑造的PDMS。的图案是由多个维度的平方，具有200至700微米（ 图1B）的侧的长度和距离变化。被选为广场的大小以适合的观点10X的字段（岛屿与一个边长&lt;800微米）和20X目标（对于岛屿边长&lt;400微米）。三个参数，电路之间即细胞铺板密度，距离，电路&#39;的大小是决定用于得到单层或聚集电路以及强加和形状的连接。然而，这?...

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

这手稿描述了一个协议， 在体外模块化网络包括空间上的限制，在功能上相互连接的神经回路的增长。的聚合物掩模被用来图案的蛋白质层，以促进通过培养基底细胞粘附。镀神经元生长在涂层区域自发建立连接并显示出电活性。

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

design-surface-treatment-cellular-plating-culturing-modular-neuronal

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Tel-Aviv University. 这手稿描述了一个协议， 在体外模块化网络包括空间上的限制，在功能上相互连接的神经回路的增长。的聚合物掩模被用来图案的蛋白质层，以促进通过培养基底细胞粘附。镀神经元生长在涂层区域自发建立连接并显示出电活性。 

视频: Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

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 ...

科研

神经科学

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

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 ...

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.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

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

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

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.

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 ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving 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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

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 ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...