This work was supported by National Institutes of Health (National Institute of Neurological Disorders and Stroke) Grant 1R01NS060757-01 and by the E.L. Lindseth endowed chair to Dominique M. Durand. We thank Dr. Andrew M. Rollins&#8217; laboratory for the help on the OCT imaging.

注:动物实验方案进行了审查，并在大学批准的机构动物护理和使用委员会。 CD1小鼠任一性别的P10的年龄到P20被用于本研究。 1.解决方案手术和实验记录<ol><li>制备含有（mM计）正常的人工脑脊液（ACSF）缓冲液:氯化钠124，氯化钾3.75，KH 2 PO 4 1.25，MgSO 4干燥2， 碳酸氢钠 26，葡萄糖10，和氯化钙 2。在实验开始时使用该正常脑脊液解剖后组织的恢复，以及用于洗涤系统。 </li><li>备是海马解剖过程中使用，并含有蔗糖学联（mM计）:蔗糖220，氯化钾2.95，将NaH 2 PO 4 1.3，MgSO 4干燥2， 碳酸氢钠 26，葡萄糖10，和氯化钙 2。为了诱导癫痫样活动的完整海马组织制剂中，添加4-氨基吡啶（4-AP），以正常的脑脊液在为100μM的浓度。 </li></ol> 2.手术过程的完整海马准备<ol><li>滴异氟烷（1毫升）到腔室，在一个干燥器玻璃瓶（1号中的具体材料和设备）的底部，并使用常规的纸巾覆盖底部阶段的表面上，从而对动物没有得到与接触的液体。然后，将CD1鼠标放进瓶子里，并盖上盖子。保持在封闭罐的动物为约1分钟至2分钟。当呼吸频率为约1每秒，从罐取出鼠标。 </li><li>放置在鼠标上的手术阶段和斩首与合适的剪刀。立即斩首后，将头部放入冰冷（3-4℃），氧化蔗糖脑脊液约30秒。 </li><li>用细剪刀（第5号中的具体材料和设备），以去除皮肤上的头骨顶部和切割沿头部的中间线颅骨以及在接近速度两端RAL叶。使用钳子（6号中具体的材料和设备）剥离朝向头部的每一侧的切口颅骨以暴露脑。 </li><li>插入一个微型实验室压舌板（在特定的材料和设备第8号）到大脑和颅骨的顶叶之间的差距从头骨底部小心剥离脑，然后将其放到准备好的冰冷（3 -4°C）手术舞台铺上湿滤纸（第2号的具体材料和设备）。除去用冰 - 冷却的刀片小脑和通过切割脑的中线分开的两个半球。然后，将两个分离的半球入填充有冰冷蔗糖脑脊液通入95％O 2/5％CO 2的烧杯中。 </li><li>取一半的半球和地点上的冰冷滤纸阶段。将普通纸巾或滤纸周围脑吸多余的解决方案。使用两个火抛光的玻璃吸管工具（第10号中的材料和设备）与皮质从大脑的中心部分的其余部分分离。 </li><li>之后，海马从皮层内部暴露，换上了冰冷的阶段，将两三滴对组织冰冷的蔗糖学联然后取出海马有多余的解决方案。切断与皮层连接在海马的两端。然后，解剖海马出大脑与火抛光玻璃工具并取出大脑的剩余部分。 </li><li>分开整个海马与alveus面朝上，海马沟朝下。迅速下降两个或三个滴冰冷蔗糖脑脊液在组织再次并取出组织周围使用了一块纸巾的额外溶液。使用火抛光的玻璃的工具改过整个海马，露出沟（ 图1B，C）。 </li><li>在一个正常的光学显微镜，插入一个定制的玻璃针（11号在特殊材料 -ALS和设备）插入沟的一端，并切断光纤连接，从齿状回（DG）到下托或海马CA1领域，沿沟的方向（ 图1C）。适用的冰冷刀片如果需要修剪海马的间隔和时间结束。插入定制金属线环（第12号中的具体材料和设备）插入切沟，并从组织，同时保持了海马的下脚/ CA1端通过一火抛光的玻璃刀具拉过对DG远（ 图1D） 。 </li><li>按照上述的展开过程中，添加另外的两个或三滴的冰冷蔗糖脑脊液的到组织并移除组织周围的多余溶液。然后，修剪展开海马用冰冷刀片的边缘（ 图1E）和在制备用刮刀放入填充有正常脑脊液和通入95％O 2/5％的CO 2在室温下将回收室（约25℃）。离开展开海马组织，以将其放置到录音室之前收回约1小时。 </li><li>采取其他脑半球并用它要经过的步骤，从2.5到2.9。通常，需要约1至2分钟，以完成整个过程展开单个海马和连续地滴冰冷蔗糖脑脊液，以保持氧化和水合的组织。 </li></ol> 3.实验系统设置<ol><li>胶上的引脚栅格阵列（PGA）封装的定制制造的PMEA，并使用微引线接合到从一个单一的微小电极连接每个垫的包（ 图3B）的销的垫。然后，将包插入一个特制的电路板13的插座中。 </li><li>每个单独的微电极与带通截止频率为1赫兹的100上的定制的电路板13的增益连接到它的过滤器，以4千赫和放大器。由A / D转换数字化从电路板输出的模拟耳听系统，并获取和存储数据的计算机上。 </li><li>胶与入口和出口管，用于将溶液的流动（ 图4A）的阵列周围一个特制的塑料记录室中。使用至少两个瓶子，以保持不同的解决方案，在系统中，并加入并通过三阀控制瓶子的输出管道。连接三阀的一个管件系统的输出与静脉滴注室被引导溶液到记录室中。 </li><li>三阀和录音室的入口之间，加电加热器来加热该溶液到受控温度（35℃）之前的溶液被引导到记录室中。附着在记录室的出口到一个真空管收集溶液成真空管连接烧瓶中。不回收任何实验在这项研究中的任何解决方案。 </li></ol> 4.放置展开海马到PMEA录制的神经活动</p&gt; <ol><li>要准备的实验装置，填补一瓶正常脑脊液和另一瓶与4-AP学联。在这两个瓶，泡沫的95％ 的 O 2/5％CO 2，从各实验的开始。使用三 - 阀连接器，以控制该溶液将在实验中进行选择。连接一个真空管，在该腔室的出口到泵将溶液到贮尘容器。递送它到记录室之前加热该管道，并保持溶液在受控的温度水平（35℃）。 </li><li>当记录室的入口和出口被关闭，使用一个定制玻璃吸管滴管转移并放置展开海马到记录室中。在显微镜下，使用常规的小漆刷而组织处于漂浮于溶液定位展开海马。将展开与海马的alveus面朝下，CA3区背向和CA1场指向研究员。 </l我&gt; <li>使用真空吸管从记录室的边缘，以降低该溶液的水平，直至所述腔室进行干燥，所述组织被降低到阵列小心吸走在腔室中的溶液中。然后，小心地放置在组织上的一个顶部定制组织锚定（ 图4）（在具体的材料和设备第14号），以保持展开的海马到阵列上。放几滴溶液到记录室重新填充它，并逐渐打开入口和出口，以调节流动速率，在第IV滴注室每秒大约2滴。 </li><li>孵育录音室与正常脑脊液中的组织约1分钟，回收，然后切换所述溶液供应到4-AP溶于脑脊液和适当调整流速。孵育所述组织在4-AP溶于脑脊液为约5至10分钟，然后研究人员可以启动软件时，会出现自发性活动来记录信号。 </li></ol> 5.删除组织从PMEA的实验后<ol><li>控制的组织锚定由一个显微和逐步解除从记录室中的组织。关闭入口和出口停止录音室中的流动。录音室一应具全用溶液或加几滴解决方案，填补了腔，如果录音室没有满。 </li><li>用小画笔解除所述组织的各角。如果组织未漂浮在溶液中，然后使用真空管来与组织仍然坐在阵列上仔细干燥室中。然后小心地打开进，逐步填充室并关闭入口停止流动时的录音室已满。应用小漆刷再次抬起组织的每个角落。 </li><li>重复步骤5.2，直到组织被从阵列中分离出来并漂浮在溶液中。如果组织被漂浮在溶液中，然后用真空管吸组织消失。打开吨他流在入口和打开出口处的真空。用蒸馏水清洗系统和干出来。 </li></ol>

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The authors have nothing to disclose.

展开的海马制备，其中海马的纵轴和横轴被保存在具有穿透微电极阵列结合的发展，提供了一个有力的工具，调查解剖连接或神经传播在海马7。此展开过程也适用于在成年小鼠研究海马。与此制剂最近的研究表明，4-AP诱发的癫痫样活动可与一个对角波前越过展开的海马的整个区域（ 图6）7,8-传播。这些研究表明，完整的未折叠海马提供了超过任一横向或纵向切片显?...

理解的神经传导的神经信号或传播是用于判定在两个正常功能和病理条件神经通信在大脑1-3的机制是至关重要的。海马是最广泛研究的结构在大脑中，因为它起着基本作用在几个脑功能，如存储器和空间跟踪并参与几个病理变化，可显着地影响行为，以及1,6-之一。虽然，海马表现出复杂的组织，其结构的不同元件可以容易地确定和切片制备4-6访问。在海马的横向方向，神经活动已知通过三-突触通路组成该齿状回（DG），CA3，CA1 andsubiculum 4,5-传播。据认为，突触传递和轴突传导中发挥重要作用的通信对于在这横电路4,6。然而，神经信号的传播发生在横向和纵向方向上4,6。这意味着，海马，不能充分利用切片制剂限制观察到传播4的特定方向的影响。纵向切片的开发是为了调查沿着纵向轴线5的轴索途径。研究人员已经观察到的行为的特定r和θ振荡主要沿着横向和纵向轴线分别为6。这些行为已分别研究，但同时访问两个方向关键是要了解这些行为。甚至与完整海马制备的发展，它是很难监测在整个组织中传播，由于海马4的折叠结构。展开海马提供访问打包元在一个平面二维细胞层7,8的一种形式。 通过展开齿状回（DG）（ 图1），海马采用扁平形状具有矩形结构，其中横向和纵向的连接保持不变与设置在一个含两CA3和CA1二维片锥体细胞层，留下一块平坦的，可用于研究神经传播神经组织（ 图2）8。神经活动然后可以与个别玻璃吸管，微电极阵列，刺激电极，以及电压敏感染料（VSD）3,7,8监测。另外，从转基因小鼠的基因编码的电压指示器可用于跟踪传播图9。 展开的海马网络的扁平结构非常适合于光学记录方法，而且对于一个微电极阵列。 M市售阵列OST的制作扁平或低调的电极，可在组织切片和培养的神经元10-12记录的神经活动。然而，当信号从一个完整的组织中获得，因为神经元的胞体均位于更深的组织中的信号 - 噪声比（SNR）减小。具有高纵横比的微电极的电极阵列，需要改善的信噪比。 为此，一个穿透微电极阵列（PMEA）已经制定在我们的实验室，并通过插入64尖峰的直径为20微米和200微米的高度成折叠海马7,13-提供直接探测到组织的能力。此微电极阵列具有更高的SNR相比，电压敏感性染料成像和信噪比实验7,13期间保持稳定。展开的海马制备及PMEA的结合提供了一个投资新方式IGATE过的二维平面上的神经传播。使用这种技术的实验已经产生了关于神经信号传播在海马机制显著结果，由此神经活性可以独立的突触或突触电动7传播。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>desiccator jar</td>
			<td>LABRECYCLERS Inc.</td>
			<td>5410</td>
			<td>Place regular paper towels at the bottome of the jar for animal anesthesia use.&#160;</td>
		</tr>
		<tr>
			<td>A blade and Custome made surgical stage for unfolding hippocampus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.&#160;</td>
		</tr>
		<tr>
			<td>Custom made tissue recovery chamber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.</td>
		</tr>
		<tr>
			<td>Straight Operating&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>S17336B&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Medco Instruments No.:81995&#160;</td>
			<td>This scissors is used to&#160;&#160; decapitate the mice.</td>
		</tr>
		<tr>
			<td>Integra Miltex Goldman-Fox&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>12-460-517&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MILTEX INC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; No.:5-SC-320</td>
			<td>This scissors is used to cut the skull of the mice.&#160;</td>
		</tr>
		<tr>
			<td>Miltex 
			Hysterectomy Forceps</td>
			<td>Claflin Medical equipment</td>
			<td>CESS-722033-00001</td>
			<td>This Forceps is used to peel the cut skull to expose the brain</td>
		</tr>
		<tr>
			<td>Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.&#160;</td>
		</tr>
		<tr>
			<td>Frey Scientific Stainless Steel Semi-Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.</td>
		</tr>
		<tr>
			<td>small paint brush</td>
			<td>Lowe&#39;s</td>
			<td>tem #:&#160;105657&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Model #:&#160;90219</td>
			<td>The one with the smallest size in a normal paint brush package</td>
		</tr>
		<tr>
			<td>Fire polished glass help tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass tool with a metal wire loop</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.</td>
		</tr>
		<tr>
			<td>Custom made glass solution dropper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was&#160; made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.</td>
		</tr>
		<tr>
			<td>Custom made tissue anchor</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.&#160;</td>
		</tr>
		<tr>
			<td>Custom fabricated microelectrode array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Custom made filter and amplifiers circuits for the array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Data acquisition processor 3400a</td>
			<td>Microstar Laboratories</td>
			<td>N/A</td>
			<td>This is a complete data acquisition system with A/D converter.</td>
		</tr>
	</tbody>
</table>

neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array

这个协议描述了用于制备新的体外平坦海马制备结合了微加工阵列映射神经活动在海马的方法。横向海马切片制备是最常见的组织制剂来研究海马电。纵向海马切片也被开发，以调查海马纵向连接。完整的小鼠海马也可以在体外保持，因为它的厚度允许足够的氧扩散。但是，这三个制剂不提供直接访问神经传播，因为一些组织是丢失或折叠。展开的完整海马提供横向和在一个平面配置为直接访问组织分析在体外海马信号传播的完全程度纵向连接。为了有效地监控从T的神经活动他细胞层，定制穿透微电极阵列（PMEA），制作并施加到展开的海马。为200微米的高度64的电极的PMEA可以记录的神经活动的小鼠海马深处。未折叠海马制备及PMEA的独特组合提供了一种新的体外工具来研究的速度和神经活动的传播方向在海马的二维CA1-CA3区具有高信噪比。

在这里图中所示的数据被记录在未折叠海马制剂与4-AP（100μM）脑脊液录音室中的组织的孵育期间在RT加入（25℃）。正常情况下活动开始不到5分钟，但在从旧有的动物海马组织中，可能需要更长的时间。与PMEA观察到的4-AP诱发的神经元放电是相同的如先前报道14,15。由于电极有200μm的高度，电极尖端刚好位于细胞层（ 图3C），因为电池层通常为250至300微米的海马alveus以上（ <str...

Watch this Scientific Journal Video about Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array at JoVE.com

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

我们已经开发了一种在体外展开海马其中保留神经元的CA1-CA3阵列。结合穿透微电极阵列，神经活性可以在纵向和横向取向来监测。这种方法提供优于海马切片制剂作为整个海马的传播可以同时记录。

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

neural-activity-propagation-an-unfolded-hippocampal-preparation-with

Research

JoVE Journal

Neuroscience

8.3K 次观看.  Case Western Reserve University. 我们已经开发了一种在体外展开海马其中保留神经元的CA1-CA3阵列。结合穿透微电极阵列，神经活性可以在纵向和横向取向来监测。这种方法提供优于海马切片制剂作为整个海马的传播可以同时记录。 

视频: Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人北极黄鼠海马神经干细胞的生长和分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

科研

神经科学

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

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

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

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

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

神经活动的传播的展开海马准备具有穿透微电极阵列

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