The authors would like to thank Isabelle Reus for establishment of tracing the electrode insertion side, Tobias Rosenbaum for LabView programming, Anneke Meyer for data analyzes and helpful discussions. We thank Randolf Menzel for discussion and practical help during early stage of electrode development. Furthermore we thank Brian Smith for postdoctoral association to MS-B. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SPP 1392, Ro1177/5-2) to WR.

1，电极建筑物（图1） <ol><li>生产适合商用的多声道功放系统1,2,25的电极接口板的电极适配器。 <ol><li>用一个小的有机玻璃板粘到一个18针的连接器基地。 </li><li>该基地3小段绝缘线来拧在有机玻璃板3个独立的焊接表耳（ 图1 A1〜A3）连接。 </li><li>插入槽插入的有机玻璃板，其中的玻璃毛细管可以很容易地移动，并通过螺钉保持在适当位置（ 图1 B1）。 </li><li>延长5毫米左右使用minutien针玻璃毛细管。 </li><li>装上微电极线沿minutien引脚和玻璃毛细管，以保证稳定和支持。 </li></ol></li><li>多通道微线材生产（从Ryuichy冈田32,33采纳） <ol><li>跨度3微线（聚氨酯铜线，15微米直径）的方式，它们彼此相邻放置（ 如图1所示B2）。 </li><li>使用一个12伏特的焊接针散布低熔点牙科用蜡的薄膜（50℃）部分地沿着电线将它们粘合在一起（电极头）（ 图1 B3）。离开脱胶几厘米（电极端），因为这部分将在后面用来与电极适配器连接微型导线。 </li></ol></li><li>多通道微线连接到电极适配器<ol><li>从支架上取下玻璃毛细管，并与minutien引脚附加到电极末端。使其位置平行于微电极（ 图1 B3）。 </li><li>胶电极尖端使用低熔点牙模的minutien针和切微电极的尖端，从minutien针和在电极末端突出2-3厘米（小箭头图1 B3）。 </li><li>滑毛细管SLightly放回电极适配器。用螺钉固定（ 图1 B4）。 </li><li>焊料的三根电线的端部松动，以用焊接枪以大约360℃的温度，以确保其绝缘熔化（ 图1 B4）的焊接凸块。焊接后，确保有足够的电接触（〜300欧姆）。 </li><li>安装电极（主）之一的探头的电极接口板和固定在一个单独的适配器的其它多通道电极（奴隶）。连接从站的信道的主电极（ 图1的C1）。此外，焊料的参考以及肌肉电极对主电极基座（图1中的C2）。 </li></ol></li></ol> 2，蜂下游（图2） 在这些所描述的实验中，蜜蜂（ 蜜蜂 ），它是一种无脊椎动物动物因此并不需要，用于使用特定的伦理许可证。 <ol><li>捉蜜蜂觅食（ 西方蜜蜂 ）在上午蜂巢入口如由他人34,35。 </li><li>放在冰上冷却，蜜蜂直到固定（5至10分钟），在某种程度上，该头部露出（ 图2 A）固定1在标准有机玻璃支架或金属管。用于最小化头部运动用的低熔点牙科用蜡（〜50℃），小心地固定头部周围的复眼和颈部的基础保持器。 </li><li>采用低熔点蜡固定天线的SCAPI头上胶囊（ 图2 B），而不触及鞭毛。需要天线的鞭毛要指出前进。确保蜜蜂可以自由移动它的长鼻。 </li><li>剃了头胶囊，以确保不受干扰的意见，并获得了头顶。 </li><li>有30％的蔗糖溶液喂养蜜蜂，直到saturati上，以确保脑组织和动物的良好生活力（ 图2 C）的充分润湿。 </li><li>周密垂直切口沿复眼的边界和水平以上触角基地，以及在单眼下方，取出一块松动的角质层（ 图2 D）。 </li><li>仔细预留下咽腺和取出气管，以确保一个明确的说法，并获得到电极插入（ 图2E，2F）前脑。 </li></ol> 3，电极插入在图2所示的示例的情况下，一个电极被定位瞄准升-ALT，另一个目标上的m ALT 2。使用特殊的标志性建筑，其他目标区域也是可能的，例如AL和MB的输出区域1。 <ol><li>利用显微操作在感兴趣的区域定位电极（ <stroNG&gt;图2G和3A）。瞄准M-ALT地方的AL和内侧从MB的垂直波瓣之间的电极。确保在插入位点是上方的正中侧面ALT的PN的分支点。突出电极放入大脑约180微米（ 图2E）的深度。对于L-ALT PN记录地方的电极下面的垂直波瓣和AL的中间的横向侧之间的假想线的中点的横向protocerebrum（在左侧）。插入所述电极具有大约300微米（ 图2E）的深度</li><li>将参考（银线，约25微米直径）到同侧复眼通过一个小切口在角质层。插入另一张银线到下面的侧单眼肌肉投影区域。注意：如果需要蜜蜂的学习行为能够以高时间精度通过记录肌肉M17，这是我参与监测n中蜂36的长鼻延伸反应（PER）25中所描述的。 </li><li>以牢固地锚定在电极的大脑和头部胶囊内，覆盖大脑上述整个空间具有两个组分的硅（ 图2H），这将防止大脑干燥。注：该记录可以持续几个小时到几天，蜜蜂可以例如古典调理过程（ 图2H）中进行记录或用不同气味的大面板的刺激。 </li></ol> 4，数据采集与预处理<ol><li>使用符合下列要求正确采集软件：抽检分钟25 kHz的速率;模拟1,25或电极通道之间的数字2的交叉分化;乐队从300赫兹低通滤波器到8000赫兹提取秒杀活动。 </li><li>使用有效穗整理软件来提取一个单位的活动，例如TEMPL吃匹配技术作为包含在的Spike2软件（ 图3）。 </li><li>用于进一步分析使用所提取的单元的时间标记来计算单个单元的平均气味的反应（ 图4）或计算人口向量的主成分分析（PCA）（ 图5），使用市售的软件。进一步分析单个单元和人口的响应延迟，请比较近期出版物1,2,25。 </li></ol> 5，相对电极的位置的可视化（图4） <ol><li>浸在电极尖端进入任一5％的溶液的Alexa酰肼568或5％的Alexa酰肼488将其溶于记录实验之前，0.5M的氯化钾溶液。 </li><li>实验后，小心地取出电极和覆盖硅，冲洗大脑与蜂林格液，去除腺体和气管，并插入tetramethylrhodami的微小晶体Ñ​​葡聚糖或插入解决了在1.0M的醋酸钾入AL到顺行标记低价竞标的5％溶液。执行在黑暗以下步骤。 </li><li>使染料被吸收并通过投射神经元沿其轴突束（30-45分钟）（ 图4A）输送，洗涤脑蜂林格氏溶液三次，另一个30-45分钟之前。 </li><li>寒意蜜蜂置于冰上，直到固定和小心地从头壳取出大脑。用含有4％多聚甲醛清洗它在0.1M的PBS溶液固定的脑和过夜保持在4℃。 </li><li>等待至少12小时洗完脑两次在0.1 M的PBS（每次10分钟）之前。 </li><li>在0.2％的洗脑三倍20分钟的Triton X-100进行脱水而在一个上升醇系列（30％，50％，70％，90％，95％，3×100％乙醇中，20分钟，然后稀释在0.1M PBS中各工序）。 </li><li>嵌入脱水大脑中水杨酸甲酯在显微镜载玻片和s有盖滑动EAL它。 </li><li>使用共聚焦激光扫描显微镜和使用谐波化合物计划复消色差物镜（10X 0.4 NA浸没）扫描大脑作为光学切片每2-5微米。利用激发568 nm波长处的tetramethylrhodamin右旋糖酐和488 nm处的电极位置的波长的组织。 </li><li>从重建的三维重建与软件（ 例如 ，AMIRA或斐济）（ 图4）的图像栈的染色的脑结构和电极路径。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

本文演示了生产和定制设计的多通道微线电极的使用。所描述的电极适用于记录单单元和人口活动，是为一个单一的样本内延迟测量和不同的神经元和不同neuropils其他时间响应特性尤其有用（详见1,2,25）。此外，我们还展示了如何实现永久微电极导线允许在行为的持续时间可达几天蜜蜂稳定的长期记录。 胞外多单元录音成为一个有利的工具来实现结合空间信息的高时间分辨率。在我们的例子中，这些要么是平行的神经传导通路2或两个不同的neuropils 1。多个神经元可以被记录和分析以并联的单神经元水平和高时间分辨率。多单元记录英格斯首次在哺乳动物38应用，后来又在昆虫39 - 41。与胞外多通道记录技术42,43的发展和改善，取得了实质性进展。这一点，例如，包括新的电极44或新颖的秒杀分类和聚类算法45的发展。胞外多单位记录技术的一般方法有很好的描述46 - 48。在这个视频中显示的自建电极还可以通过增加每个电极更微丝或微线可以被扭曲，以获得尖端之间的可测量的距离不断进行调整。这两个程序会然而，导致降低柔韧性和增加电极的厚度。 相较于矽探针常用于细胞外记录在像鹰蛾，蝗虫和蟑螂40,49大得多的昆虫- </SUP&gt; 51所描述的微线电极是小的，灵活的，可以与潜在的脑运动轻松应对，因此，能够可靠地在像蜜蜂和蚂蚁小的社会性昆虫，显示更广泛的行为库使用。大多数硅胶探头有柄锋利状结构切割轴突和神经组织沿其插入通道，而所述微线是圆的，灵活的和更小的，并因此危害较小周围组织这是一个明显的优势，如果我们的目标是研究长长期的可塑性在一个完整的和行为的动物。微电极丝的另一个优点是其低成本的生产和容易处理。而不是仔细清洗昂贵的有机硅探头的电极布线前脑插入新鲜切割的，因此，缺乏拥塞问题的。此外，有可能使用一个以上的微电极丝在同一制剂中不同neuropiles 1°或插入ř大片2，因为我们在这里展示。这种方法特别有利于分析和比较的时间方面，如响应延迟和相互作用在不同的神经处理水平。 我们都知道一个事实，即细胞外记录的信号并不能反映单个细胞的活动本身 。它始终是活动的电压的电极头周围的化合物。精确定位的一个电极内的两个相邻的微线信道的差总是计算出的信号源。因此，源用来提取单单位活动的尖峰信号总是非常接近一个或导致容易分辨的尖峰波形，另一个电极通道。从更远的信号，像邻近neuropils的肌肉活性或活性，达到两个电极同时唤起可比的形状和振幅，并将由该过程被丢弃。使用的Spike2的模板匹配手法，我们非常有信心获得一个单位的活动，这是不一样的，但非常接近单个神经元的活动。然而，就可以避免秒杀排序的问题用细胞内记录技术。 单细胞记录与任何锋利的电极或补丁移液器允许对单个神经元的生理特性深入了解。然而，由于昆虫神经元的体积小，它们的轴突（ 如 。，小于1微米的蜜蜂的PN 52）只有短期的录音是可以管理的。此外，细胞内记录可能是侵入性的，并可能损害其可能是另一个原因，时间限制的细 ​​胞， 在体内细胞内记录的昆虫很少拖垮一个小时。一时间窗口，它足以让马丁锤53的开创性工作谁从单一识别神经元，腹未成maxilar神经元＃1（VUMmx1）记录细胞内。他可以升墨其活性直接奖励的途径。朱莉安Mauelshagen 54细胞内登记的识别蘑菇体外在神经元的活动中，pedunculus外在神经元＃1（PE1）经典条件反射中。同样的神经元在门泽尔和曼茨55的焦点，当他们发现LTP凯尼恩细胞的电刺激后。不过，冈田和他的同事56可以使用在细胞内很好的特点扣球模式（双人间和三人间穗）为PE1的过程中细胞外记录的标识。后两种方法的所有组合，从确定神经元和细胞外长期录制细胞内的录音可能是一个强大的工具，为今后的调查。 但是，使用锋利的电极在不同的加工水平在许多小时到几天同时记录多个单元格（单元）来分析他们的时间响应关系和/或连塑改变我几乎是不可能的。 与第一钙成像方法在蜜蜂57,58使用钙敏感染料气味的反应空间格局的分析是访问的59 - 62。然而，在许多情况下，钙敏感染料具有被引入到通过侵入性的操作是再次限制了蜂的寿命，并且所分析的细胞的内在特性的脑组织。这一问题已被克服其他模式生物如使用基因引入钙传感器63,64的果蝇。然而，在一般情况下，钙的传感器可以引入其他的限制，因为它们可以作为钙缓冲，有可能影响到气味反应的时间特征。同时细胞内记录与钙结合影像学或计算方法可以证明成像的适当时间分辨率处理65,66。然而，在成像过程本身的时间分辨率是过早地ř有限。光学采集系统通常使用的CCD成像的5-20赫兹67时间分辨率，虽然2 -光子成像也许能获得更快的序列68。但是，增加采样率总是伴随着空间分辨率的损失。此外在蜜蜂中使用的钙敏感染料经受漂白，这也降低了捕获时间69。 相比于其他昆虫生理记录技术，我们的灵活的多通道微电极丝来确保单个单元和行为蜜蜂群体神经元活动的长期访问。 我们演示了如何使用其中的两个电极在不同加工阶段中相同的动物，这有利于不同记录点之间的时间编码方面的分析。依赖于研究问题和模型昆虫电极的建筑在这里证明的基本方法很容易扩展能和/或可以适应。例如可以设想使用超过三个单丝，以产生多通道电极。此外，记录点的个数可以被扩展和观察的两个以上的大片或neuropils时间方面是可行的。我们的希望是，这种方法会激发许多科学家和肯定，对复杂的神经元在处理小脑筋的理解作出贡献。

蜜蜂以及大多数其他昆虫倚重嗅觉。其中包括他们用嗅觉线索进行定向，交配，与同类沟通，和觅食。他们也阐述了嗅觉系统有助于与花香气味刺激的学习行为的丰富剧目。这些行为可以在受控的实验室条件下很容易地研究（综述见3 - 5）。他们的“小脑筋”（CP. 6）与他们的数量相对较少的神经元，使蜜蜂一个非常适合的模式生物研究嗅觉编码和监控神经活动的过程中学习。 嗅觉系统在昆虫以及哺乳动物中示出类似的组织，极大程度（有关综述见7,8）。在蜜蜂约80,000受体神经元9坐落在sensillae沿着天线10,11翻译环境的刺激气味进入neurONAL信号。从嗅觉受体神经元轴突支配触角叶（AL），其中有一个肾小球组织媲美脊椎动物嗅球。美联包括约164肾小球了约000名当地的interneurons（LN）的相互连接（综述见12）。特别是在蜜蜂它最近已表明，淋巴结提供片状横向连接及不同亚群具有元素和configural嗅觉编码属性13,14。对AL被证明是细分为腹侧和背侧半叶的内侧和外侧触角叶道（米-和l-ALT引起;以前称为米-和l-APT对于内侧 - 外侧和触角叶protocerebral道15 - 17）。在这里，通过最近的努力，对昆虫大脑的统一命名法引入了一个新的道术语将被使用18。既低价竞标（1 - 和m-ALT）结合或410（1-ALT）或510（M-ALT）uniglomerular PROJECTION神经元（PN），分别为15,16,19。两个大片的PN最近被证明在并行代码2气味（综述见17,20），两者大片形成突触连接的分歧与肯扬细胞（KC）的蘑菇体（MB）的主要神经元。每个MB包含172,000枯否21 - 23。该宏块是已知的能参与刺激整合，学习和记忆形成。角质形成的轴突树突形成蒂（蘑菇的茎），这主要有两个输出区域：Vertica的或α-叶和水平或β-叶22,24。该MB的输出收敛到只有约400外在的神经元（EN）24。 ENS负责嗅觉信息处理大多是受神经支配的垂直波瓣22的腹侧。最近，已经显示，记录在此区域登记护士进行编码的气味奖励关联25。 时间为昆虫和脊椎动物的嗅觉系统内有何看法已经成为一个潜在的编码原则26的一个重要和显著的方面- 29。为了能够从不同的站点在高时间分辨率的同时记录多个神经元，建立了利用介绍给不同的目标区域在蜜蜂的嗅觉系统定制的多通道电极丝双多单元记录技术。这种方法使我们在单个神经元和神经元之间要么平行嗅觉通路，双嗅觉通路2或不同的后续neuropils 1之间的人口水平，分析和比较的时间处理在蜜蜂的嗅觉系统。最近使用的电极不同的配置类似的实验方法在蝗虫嗅觉系统30能够分析时空编码机制的背景不变的气味识别31。钍我们所建立的双重录音启用收集有关神经元的同步活动的空间分布信息。 相比，从钙成像获得更广泛的空间抽样此方法允许记录从只有两个斑点。然而，其优点相比，钙成像技术是动作电位的记录，这是不能由任何常规的CCD成像或2光子成像采集提供了高的时间精度。这里所描述的外电极被永久植入和相对固定的脑和头壳避免电极漂移。这是一个明显的优势相比，锐细胞内电极的使用。相比，细胞内的录音和钙成像另一个优点是延长神经观察时间从几个小时到几天。这是考察学习和记忆的形成神经相关的重要前提。多额外的好处单元录音在讨论部分进一步概括。 在这种方法概述定制设计电极丝的生产过程将显示，改编自32,33和适合长期多单元记录在蜜蜂的大脑进行。此外，一个实施例如何将这些类型的电极被永久地植入在蜜蜂嗅觉系统内的两个不同位点的记录同时记录第l和第m-ALT以上的时间，以允许许多刺激方案长时间显示2。为了验证该记录位置的记录点​​的染色和后期录音可视化的例子和协议提供。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Paraffin oil</td>
			<td>Fluka</td>
			<td>76235</td>
			<td></td>
		</tr>
		<tr>
			<td>Odors</td>
			<td>Sigma Aldrich&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>4% Formaldehyde</td>
			<td>ThermoScientific</td>
			<td>28908</td>
			<td>Methanol free</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>BioChemica</td>
			<td>A1388</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylsalicylate</td>
			<td>Roth</td>
			<td>4529.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamin dextran, 10,000 MW (Microruby)</td>
			<td>Invitrogen</td>
			<td>D7162</td>
			<td>keep dark</td>
		</tr>
		<tr>
			<td>Alexa 488 hydrazide</td>
			<td>Invitrogen</td>
			<td>A-10436</td>
			<td>keep dark</td>
		</tr>
		<tr>
			<td>Alexa 568 hydrazide</td>
			<td>Invitrogen</td>
			<td>A-10437</td>
			<td>keep dark</td>
		</tr>
		<tr>
			<td>Bee Ringer Solution</td>
			<td>see 2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane-coated copper wire</td>
			<td>Elektrisola</td>
			<td>15&#181;m diameter &#38; P155 insulation</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Wax&#160;</td>
			<td>Densply Detrey</td>
			<td>64103015S1</td>
			<td>moderate melting point</td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Flexaponal&#160;</td>
			<td>124-202-00</td>
			<td>low-melting Wax</td>
		</tr>
		<tr>
			<td>KWIK SIl</td>
			<td>WPI</td>
			<td>03L</td>
			<td></td>
		</tr>
		<tr>
			<td>18 Pin Socket</td>
			<td>Conrad Electronic</td>
			<td>189634-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot melting glue</td>
			<td>Conrad Electronic</td>
			<td>827673</td>
			<td></td>
		</tr>
		<tr>
			<td>soldering needle</td>
			<td>Conrad Electronics</td>
			<td>830283</td>
			<td>12 V</td>
		</tr>
		<tr>
			<td>Soldering terminal lug&#160;</td>
			<td>Conrad Electronic</td>
			<td>531901</td>
			<td></td>
		</tr>
		<tr>
			<td>Glaselectrodes</td>
			<td>WPI</td>
			<td>1B100F-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td>V2A 0.2 x 12 mm</td>
		</tr>
		<tr>
			<td>switchable headstage</td>
			<td>Tucer Davis Technologies</td>
			<td>SH16</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstage connection module</td>
			<td>NPI</td>
			<td>INT-03M</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier Module</td>
			<td>NPI</td>
			<td>PDA-2F</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition boards</td>
			<td>National Instruments</td>
			<td>NI-6123, Ni-6143</td>
			<td></td>
		</tr>
		<tr>
			<td>Acquisition Software</td>
			<td>National Instruments</td>
			<td>Lab View 8.2</td>
			<td>custom design</td>
		</tr>
		<tr>
			<td>Spike-Sorting&#160;</td>
			<td>CED&#160;</td>
			<td>Spike 2 v7.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>R2008B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Leitz</td>
			<td></td>
			<td>manual</td>
		</tr>
		<tr>
			<td>AG-wires</td>
			<td>WPI</td>
			<td>AGT05100</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP2 AOBS</td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA</td>
			<td>Mercury Computer Systems&#160;</td>
			<td>2/5/2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous long-term recordings at two neuronal processing stages in behaving honeybees

在哺乳动物和昆虫的神经元信息在不同的高，低阶大脑中枢处理。这些中心通过收敛和发散的解剖连接，包括前馈和反馈线路耦合。此外，同一来源的信息，部分通过并行的途径不同，有时在同一个脑区发送。理解的进化优势这些布线策略，尤其是它们在彼此的时间相依性以及计算的优势，有必要同时访问单一的神经元在相同的制备在高时间分辨率的不同束或neuropiles的。在这里，我们专注于蜜蜂通过展示一种独特的细胞外长期访问在随后的两个neuropiles 1，触角叶（AL），第一嗅觉处理阶段和蘑菇体（MB），高阶整合中心INVO记录多单位活动LVED在学习和记忆的形成，或两个平行的神经传导通路2连接AL与MB。后者被选为为例，在充分进行说明。在支持视频的建设和灵活的多通道电极丝永久插入被证实。的微电极丝通道的成对差动放大大大降低了噪音并验证该信号的来源密切相关，在电极前端的位置。所使用的线电极的机械灵活性允许稳定侵袭性的长期记录在许多小时到几天，这是一个明显的优点相比，传统的额外的和细胞内体内记录技术。

“本协议允许同时进行录音的个体蜜蜂在两个不同的加工阶段，还允许通过例如测试相关的学习和记忆的机制。，市盈率内敛蜜蜂内调节。”这是一种用于分析神经元的处理时间方面的一个先决条件。该方法很容易适应不同的科学方法解开蜜蜂的嗅觉系统的神经网络。例如，此方法用于（i）分析蜜蜂的双重嗅觉途径中，L-和m-ALT期票（ 图5）内的PN的瞬时处理。在图5A中的L-ALT PN同时记录与第m-ALT的PN的一个实例被给定为10试验平均并示出其反应强度和延迟就五种不同的气味浓度为颜色编码的热曲线。在11 L-13 mA时平均七蜜蜂LT的PN（ 图5B，5C）示出了这两种响应的强度以及响应延迟，可以在一定程度上反映了加臭剂的浓度。从而在这个例子中的PN增加其反应强度，同时具有增加的加臭剂浓度的响应延迟降低（ 图5B，5C）。这个结果是相当有限的，并只对所分析的加臭剂，但仍然是与对AL 37最近的计算模型是一致的。无论是在蜜蜂的AL臭气浓度编码的基础非线性计算或其他的基础编码性能仍然需要在未来进行分析。此外，该方法可以用于（ii）向在两个后续的处理阶段，对AL-和MB-的输出（ 图6）在人群中的活性进行比较的时间方面。主成分分析（PCA）示出了气味计算被延长，拖垮整个气味介绍在PN水平而在EN唯一的开启和关闭设置异味的代表出席了在人口活动（ 图6）。从而在EN人口达到其最大的活动已经在一个时间点当PN活动仍在发展（CP. 电影1）。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51750/51750fig1highres.jpg" width="400" /> 。图1的生产三通道微线电极A1）的三根电线的结尾被焊接到在位置11，13和16 A2）四个焊接凸片被拧到一个有机玻璃塑料基板的IC引脚连接器;一个minutien销插入到玻璃毛细管中，然后附着到塑料板上。A3）的塑料片是粘在IC引脚连接器和各导线的自由端的顶端的顶端被焊接到顶部焊接之一耳。 &lt;强&gt; B1）为使所述电极保持器与细铜线，毛细管具有要取出一次。B2）的保持器的底部，然后固定在一个定制的对准装置。三铜微导线沿槽的每一端对齐，并用胶布固定B3）的平行微导线与牙科用蜡粘在一起，并在毛细管被放回原处（长箭头）;胶合微导线，然后连接到用牙科用蜡和其端部被切断（短箭头）。B4）毛细管的铜导线的三个松散端焊接在3顶端的焊接凸块从而使它们与电接触集成电路引脚连接器。C1）两个完全装配好的电极可以彼此连接为一个单一的探头。C2），使用为此目的，一个电极（左，从站）的引脚都通过绝缘导线连接到另一电极（右，桅尔），其连接到所述头部的阶段;该探头连接电极也可以从肌肉（M17）和参比电极（参考）收集输入。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig2highres.jpg" width="500" /> 图2的制备及永久电极插入蜜蜂的大脑。A）蜜蜂插入有机玻璃支架固定在冰后。天线与鞭毛（FL）和Scapus（SC）表示。 二）头部和触角正在使用牙科用蜡固定。 三）头壳被剃光和蜜蜂喂糖水D）的头壳打开。 E）从大脑的顶部除去腺体和气管后，将不同neuropiles和主要地标可以很容易地加以区别。对低价竞标的轨迹表明，连同插入电极，其中一个标记。 （MB：蘑菇体，AL：触角叶，OL：光叶，α：阿尔法叶或垂直波瓣，ALT：触角叶道，E1：电极插入侧的M-ALT键录音，E2：电极插入侧的L-ALT记录）。F）基准（REF）和肌肉的电极（M17）通过在角质层或复眼G）的线电极在适当的站点·H插入到大脑）后的小孔插入头胶囊在固定用双组份硅地方的电极，蜜蜂仍显示市盈率及可调节（ 例如 ，用白糖水）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig3highres.jpg" width="500" /> 图3。细胞外记录在两个神经束和单独的单元提取（穗排序），一个 </str翁&gt;实验装置）示意图。蜜蜂被固定在有机玻璃支架。气味刺激经由玻璃管提供。两个电极柄正在录制从暴露的蜂脑B）从第l和m-ALT的PN（绿色和紫色的痕迹）表示向蜂蜜中的水溶液中的浓度为500毫秒刺激气味上都束兴奋性反应同时记录1:100在33°C。每个绘制线代表从A中列电极的焊接表耳的区别渠道颜色编码标签：50μVC）穗后整理程序单一动作电位进行排序和颜色编码。的排序单元叠加示出的波形中分离D）穗间隔的直方图表示的排序单元的分离质量足够穗分拣证明。注意，该单位的耐火时间内没有秒杀E）两种观点（E1，E2）从与主成分分析表明的排序单元彼此的距离排序的单元的三维聚类。圆圈表示2.5倍马氏距离它类似于SD在空间和指示集群中的主成分空间F）颜色编码单元描绘出动作电位在三个通道从一个道记录一个放大下可见的显著差异。 <a href="https://www.jove.com/files/ftp_upload/51750/51750fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig4highres.jpg" width="500" /> 图4。后记录的可视化和三维重构的记录位置的。 A）投影的邻切片视图沿z轴与安特罗的最大强度对齐和uniglomerular投射神经元从AL投射到MB和LH逆行回填。细胞内示踪剂Microruby（tetramethylrhodamin葡聚糖）被插入到AL记录实验之后。邻片沿z轴与从所述两个电极与Alexa的染色强度最大对齐的AL证明正确的PN染色B）投影视图内染色肾小球酰肼488指示电极放置于第m ALT（E1，箭头）和1 - ALT（E2，箭头）。示踪Alexa的酰肼488迁移到电极周围组织和污渍的电极插入位点。请注意，在AL突出染色是肤浅的加工过的食品的染色的靶细胞（PNS）的C）的三维重构和从A，B（右侧的电极插入位点）与蜜蜂嗅觉系统（的示意图一起左侧）与1 - 和m-ALT轨迹的指示。请注意，只有uniglomer显示ular PN大片。 AN：触角神经，AL：触角叶，LH：侧角，MB：蘑菇体，E1，E2：电极插入位点，M-ALT：内侧触角叶道，L-ALT：横向触角叶道，C：尾椎，记：喙，M：内侧，L：侧向<a href="https://www.jove.com/files/ftp_upload/51750/51750fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="3in" src="/files/ftp_upload/51750/51750fig5highres.jpg" width="300" /> 图5实施例的气味浓度编码的双重嗅觉路径内。甲 ）热曲线示出了单一的L-ALT（绿色的反应）和单米-ALT PN（紫色）购入同时从单个蜜蜂响应于所述加臭剂己醛是增加气味浓度（从1：10 -6至1:100）。每一行是一个平均的10审判STIMulation。的燃烧率被示为相对强度的变化也就是燃烧率就减去自发活动11升-和13间的PN ALT从7记录的蜜蜂。 乙）人口响应延迟。在每一个蜜蜂从两个大片的期票被同时记录。人口响应延迟有减少等待时间由双方大片增加的PN臭气浓度。通过electroantennograms被记录的气味反应起始于触角为99毫秒，是从PN响应延迟中减去。注意，在最低浓度的反应是太弱延迟测量，并因此被排除在外。℃）人口响应射速从相同的PN如B.随着臭味浓度的响应强度正在增加。该L-ALT显示了强烈的反应强度。在B，C均值和SD给出。 <img alt="图6" fo:conten叔宽度=“5英寸”SRC =“/ files/ftp_upload/51750/51750fig6highres.jpg”宽度=“500”/&gt; 图6。在沿蜜蜂的嗅觉路径两个后续处理阶段比较人口活性。记录数据中20只动物的刺激与1 -己醇，2 -辛酮A）的每一行代表的假彩色编码的一个突起的平均放电率神经（PN）计算跨越的1 - 己醇10气味重复。气味演讲开始时间为0，持续了三秒钟。b）所示的相同，A），但对蘑菇体神经元外（EN）。 ）中A中所示的矩阵可以被看作是在刺激气味与1 - 己醇的PN人口向量。我们计算了相同种类的人口向量的过程中刺激气味与2 -辛酮和保持时间维度。C中使用的主成分分析的两种载体（PCA））的前三个主成分（PC1，2和3）分别为暗算对方，说明在触角叶输出的气味分离在PN合奏活动。气味发病前的时间被标记为黑色。在刺激与1 - 己醇三秒活性示于蓝色。刺激2 - 辛醇中的活性显示为红色。此外，我们显示1.5秒的活动（POST）组的1 - 己醇（淡蓝色）和2 - octanonen（粉红色）的气味之后。请注意，在PN合奏水平，既气味唤起非常明显的轨迹定居在一个“固定点”，这outlasts整个刺激气味的时期。只有在气味偏移的轨迹移动回到基线活动无刺激气味D）同样的分析做在EN合奏水平相当于在蘑菇体输出的活动。相较于PN活动气味唤起一个不太明显的轨迹。另外一个“固定点”是无法观测。最初气味诱发轨迹INTErmingle与基线活性虽然气味仍然存在。只有气味偏移引起的额外轨迹。 <a href="/files/ftp_upload/51750/Movie_R3_Brill_et_al._JoVE.avi" target="_blank">。电影1</a>解决时间后的PN人口向量（左）和EN人口向量的主成分分析的气味引起的轨迹进行评估。（右; CP图6）上部部件包括前三个主成分（PC1; 2和3）暗算对方。下部面板说明随着时间的推移PC1，2和3的评价。刺激气味的标志是灰色的吧。所有面板均同步。注意，在EN人口活动的PN人口活动之前稍微开始，这种现象似乎是contraintuitive但是可以由连接性和所涉及的层，这是早期1讨论的特性进行说明。

Watch this Scientific Journal Video about Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees at JoVE.com

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

从两个不同的大脑neuropiles或两个不同的解剖大片外同时长期记录成立于蜜蜂。这些录音允许神经元处理跨在单个神经元以及在一个动物行为的合唱水平不同脑区时间方面的调查。

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

simultaneous-long-term-recordings-at-two-neuronal-processing-stages

Research

JoVE Journal

Neuroscience

12.8K 次观看.  University of Würzburg. 从两个不同的大脑neuropiles或两个不同的解剖大片外同时长期记录成立于蜜蜂。这些录音允许神经元处理跨在单个神经元以及在一个动物行为的合唱水平不同脑区时间方面的调查。 

视频: Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...