Name: simultaneous long-term recordings at two neuronal processing stages in behaving honeybees
Uploaded: 2014-07-21T15:00:00.000+00:00
Duration: 13 min 55 s
Description: Watch this Scientific Journal Video about Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees at JoVE.com

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><tbody><tr><td>Paraffin oil</td><td>Fluka</td><td>76235</td><td></td></tr><tr><td>Odors</td><td>Sigma Aldrich&amp;nbsp;</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 Brill &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>Polyurethane-coated copper wire</td><td>Elektrisola</td><td>15 &amp;micro;m diameter &amp;amp; P155 insulation</td><td></td></tr><tr><td>Dental Wax&amp;nbsp;</td><td>Densply Detrey</td><td>64103015S1</td><td>moderate melting point</td></tr><tr><td>Dental Wax</td><td>Flexaponal&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>CED&amp;nbsp;</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&amp;nbsp;</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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

12.9K 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

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

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 ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

生物学

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

触觉空调和取样策略在蜜蜂触角的运动分析（<em&gt;蜜蜂</em&gt; L.）

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

同时监测无线电生理学和记忆行为测试作为研究海马神经发生的工具

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

行为学

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

自由移动鸽子穿越三个阶段学习记录单个神经元&#39;动作电位

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

在两个神经元处理阶段在做人蜜蜂同时长期录音

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此视频中的章节

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

从查明的神经元和取食行为的钙信号的同时记录果蝇

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

多单位记录方法来描述蝗虫的神经活动（

在体内的Ca ^{2 +} -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

触觉空调和取样策略在蜜蜂触角的运动分析（

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

同时监测无线电生理学和记忆行为测试作为研究海马神经发生的工具

自由移动鸽子穿越三个阶段学习记录单个神经元'动作电位

约束和自由飞行，蜜蜂的神经药理学操纵，