这项工作是由神经科学的研究基金会和圣地亚哥基金会和G。哈罗德和莱拉华马瑟斯慈善基金会Blasker玫瑰Miah基金的拨款支持。

系统概述该系统包括三个主要部分组成：（1）双层隔声室（工业声学公司，布朗克斯，纽约）;（2）多通道电生理记录系统（Neuralynx，博兹曼，吨）;（3）完全自动化，从地中海Associates公司（VT），圣奥尔本斯，定制行为测试系统。 图1A所示，操作性室位于里面的隔音室。一个用于连接电缆从探头的电生理记录系统（Figure1A）换向器（型号SL-36，蜻蜓的研究与发展公司，Ridgeley，西弗吉尼亚州），并监测和记录动物行为摄像机安装以上操作性室（Figure1A-B）。 定制设计的操作性商会自定义设计，声学透明operanţ室（Figure1A-D）由三个声学透明的墙壁和一个模块化操作面板（ 图1B）。三个扬声器（笼高音，ENV_224BM，MED公司）安装在中间的顶部和两个侧面板用于发光听觉线索。听觉信号产生一个校准，可编程音频信号发生器（ANL-926）。刺激光（ENV_221M）和两个三重刺激LED显示屏（ENV_222M），分别位于中间和侧面板。这些刺激灯，可用于多感官的听觉，视觉行为测试。与三色LED灯（ENV_114M）的鼻子戳设备被安装在中间面板的底部。鼻子戳单元内安装一个红外探测器，用于信号的鼻子戳和持有期。可用于训练洞内的鼻子保持鼻子戳单元内的LED灯。操作性面板两侧安装在一个可移动的响应杆（ENV_112CM）。在mobilitŸ这些杠杆，可以灵活控制的杠杆，它可以有效地用于为初始任务，培训和研究的几个重要的认知功能的大脑（见下文）的存在。四对红外线光束源和1 ​​4通道红外控制器（ENV_253）控制探测器（EVN_253SD）被置于腔的底部，以既表明动物的位置和控制其他设备基于动物的位置（ 图1A- E）。两个球掌柜的每一个内置在的红外哨兵（ENV_203M-45IR）分配到颗粒插座（ 图1A-C）的奖励。红外哨兵用于监测颗粒机的故障，并提供预警信号。 系统配置 如图2所示的系统连接和硬件组件的概述。在功能上，有两个平行的，互动个子系统：一个行为训练和电生理记录等。同步通过的MED-PC IV软件平台（SOF - 735）两个子系统。计算机发送命令行为的设备和TTL脉冲神经录音系统（信号流图2中的红色箭头表示），并接收由动物的反应和神经活动（信号流量显示绿色和蓝色的箭头，分别产生的信号，在图2）。这些并行，交互式系统启用同步行为/神经元的数据记录，并允许操纵动物的行为基于神经活动，或反之亦然 。 行为训练子系统一个SmartCtrl连接面板（SG-716B）作为一种双向沟通面板：即发送控制信号（ 图2中的红色箭头）从计算机的行为装置（红色方框中列出），TRansmit动物的响应信号（ 图2中的绿色箭头）回电脑。四通道IR控制器（ENV_253）的输出也路由到连接面板。通信接口卡（SmartCtrl接口卡，DIG-716B）和解码卡（辛-700F）在电脑中安装的PCI卡的连接面板（辛-704PCI）的信号。听觉线索所产生的刺激发生器（ANL-926），这也是控制的MED-PC IV通过解码卡软件（辛-700F）。正如图2所示，所有的接口卡在桌面接口柜（SG-6080D）托管。这个内阁还提供所有的行为设备的权力。 瞬间电生理记录子系统动物的反应，由计算机接收到的信号发送到神经录音系统通过的SuperPort的TTL卡（辛-726）和猎豹数字接口盒（不适用euralynx，博兹曼，吨）（ 图2）。这些行为的事件时间标记，并同时记录神经活动。神经尖峰检测线上从Neuralynx录音系统，可以作为输入信号行为控制子系统，操纵或干扰动物的行为。相反，操纵或干扰神经元活动与电动或optogenetic刺激技术相结合时，触发器可以被用来作为动物的响应信号。这些方法将是有价值的阐明神经活动和行为之间的因果关系。 编程和数据处理行为的控制程序被写入与跨IV软件（托马斯·A·TATHAM和MED的联营公司），并用Pascal编译器编译。每个训练步骤实现自动化，MED-PC IV软件装入跨四程序。也可以在线调整，通过培训瓦特训练参数类型改为，MED-PC IV软件的运行。横贯IV代码需要特定的系统设置和行为任务。但是，标准的培训方案，从MED Associates的自由，可以修改，以满足个别实验室的特定需求。在我们的设置使用的培训计划也要求免费提供。 行为数据自动保存，MED-PC IV软件。保存的数据可以转化为Microsoft Excel文件，使用的MED-PC的Excel程序（MPC2XL，托马斯·A·TATHAM和MED协会）。翻译的Excel文件，然后可以导入和分析，在MATLAB环境（MathWorks公司，Natic​​k市，马萨诸塞州）。随着猎豹软件（猎豹5，Neuralynx，博兹曼，吨）的行为事件记录时间戳的神经数据也可以导入MATLAB进行分析。 训练为了说明这个系统的运作，我们在这里描述两个替代选择间距歧​​视任务，旨在探讨大鼠的频率歧视门槛。一个任务的示意图如图3所示。 1。前培训<ol><li>天真的成年男性，只SD大鼠，年龄〜60天开始。 </li><li>训练前，限制食物摄入量，直到动物的体重〜90％基线广告libidum重量。 </li></ol> 2。笼驯化<ol><li>驯化的准备室，收回的杠杆和阻止与橡胶瓶塞（60毫升注射器的柱塞）激活鼻子戳设备，以防止动物的鼻子戳洞。 </li><li>地方〜20充分的营养颗粒，每个颗粒插座（食品杯）（45毫克，产品＃F0021 BioServ，Frenchtown，新泽西州）。 </li><li>放置一个天真的动物在驯化室。老鼠将很快开始探索食物的杯子和吃颗粒。 </li><l我当这两种食物杯是空的，使用该软件控制单个颗粒分配到每个托盘。这样做，大鼠，将学习与世界杯关联的食品。 </li><li>迫使大鼠室双方将通过随机分配到每个食品杯颗粒。一个30分钟的会议通常是不够的，建立食品杯关联。在一个30分钟的会议，老鼠通常会获得200 - 300粒，这是足以维持他们的体重在一个基线〜90％的水平不变。 </li></ol> 3。杠杆推动培训<ol><li>在一个新的会话，延伸到会议厅的两个杠杆和离开食品杯空。 </li><li>下一步，放入驯化大鼠室。当动物进入附近的一个杠杆，免除手动弹丸通过MED-PC IV软件。还提供奖励时，老鼠显示了利率杠杆，如嗅，触，或攀爬，。一个偶然的杠杆推也应该触发奖励automatically通过该方案。 </li><li>鼓励杠杆推动和强制两个杠杆的探索，允许动物，以推动各杆连续的次数有限。当达到限制时，收回的杠杆。当两杆缩回，延长他们重复的过程。 </li><li>逐渐减少，直到杆缩回每次推的时间限制。一到两个30分钟的会议，通常是不够的，建立杠杆推 - 食物奖励协会。 </li></ol> 4。鼻捅培训的<ol><li>在一个新的会话，收回的杠杆，从鼻子戳橡胶瓶塞。将几个小球，鼻子里捅，鼓励老鼠的鼻子戳设备在探索的兴趣。 </li><li>重新引入动物室。随机的两个杠杆，当老鼠嗅着食物颗粒的鼻子戳孔扩展之一。 </li><li>见此延长杆，老鼠会接近和推动的杠杆来获取食物PEL让。按下杠杆和奖励被免除后，收回的杠杆，鼓励探索鼻子戳设备的大鼠。它通常需要大约20至30分钟，学习任务序列： 鼻子戳 → 杠杆扩展 → 杠杆推 → 奖励 。 </li></ol> 5。暗示训练<ol><li>在一个新的会话，发挥听觉线索后，有一个短暂的延迟（100到250毫秒）的鼻子捅事件。延长每个听觉线索演示后，左，右杠杆短期内（100毫秒）。 </li><li>奖励的老鼠，只有当它推听觉线索表明杆。动物会逐渐了解联想到一个特定的听觉线索，一杆。动物，然后开始一个新的序列审判： 鼻戳 → 线索 → 杆推 → 奖励/奖励 （ 图3）。高误报率，因为在最初的学习会，补充FO外径应保持在每次训练后的体重。 </li><li>在未来数30分钟的会议，让老鼠实践的新学到的任务，直到达到一致的性能水平（见一个典型的学习曲线图3A）。一旦任务被掌握，一只老鼠可以得到约200 - 300颗粒每30分钟的会议，这是足以维持其体重。 </li></ol> 6。代表结果按照上述协议，我们训练大鼠承认纯音脉冲两种不同的模式训练6个短音组成的具有相同的频率（F，升F，男，女，男，女），或不同频率（F，升F-ΔF ，F的F-ΔF，F-ΔF）1-5。每个音PIP持续时间为200毫秒的音点的时间间隔为400毫秒。在本研究中，F的设置为10千赫和ΔF1〜50％的F（ 图3）。通常情况下，ΔF成立在比较大的价值，在培训期间：5千赫，4 kHz时，3千赫，2千赫，1千赫，促进了培训。每个纯音ΔF值不同的脉冲序列，提出了随机在一个给定的会话。 如图3所示的两种替代选择间距辨别任务。大鼠进行了培训，他们的鼻子捅到鼻孔（ 图1B和图3，底部）开始试用。鼻子捅触发的听觉线索广播。识别的线索后，大鼠室的正确运行，接近杆，杆延伸到室等待，然后在一定时间内窗口（杠杆活动时间推杠杆，1至2秒），以获得奖励（ 图3）。命中率计算每个ΔF值除以每一个人ΔF值的总试验数的命中试验。一个标准的75％命中率被用来表明，老鼠已经学会的任务。一个典型的学习曲线，一只老鼠在图4A。每个颜色的线描绘了不同的ΔF（ΔF0代表恒定的脉冲序列），每个脉冲串的学习进度。平均而言，它花了约七培训班（从听觉线索介绍，5步骤提示训练的第一届会议开始）将达到75％的命中率标准。 该系统还允许在执行任务实验者设计动物的行为的定量表征。三个动物行为学研究中被广泛使用的测量， 如图4所示，B - ð反应时间，主要反映动物的注意力的任务，作为测量时间的推移发病之间的听觉线索和杠杆推动。审间的时间间隔，反映如何犯了一个动物的任务是在发起每个试验动物虚假审判，并没有超时处罚，被绘制在图4C。在一个会话中的表现时空变化，代表动物的整体性能的动态模式，反映改进/适应可能发生在一个单一的会议，绘制图4D。每届会议分为初，中，晚期的每个阶段（10分钟）。在这种测量被用来奖励在每个阶段的累计数字。 几个（ 图5A，并见讨论）认知行为的神经基础，也可以解决这个系统通过记录执行任务的动物的神经活动。同时记录在基底核（NB）和大鼠脑腹侧被盖区（VTA）的神经活动的例子表明在神经元的点火时间戳记，涉及到每个审判的事件（如图5B和C。鼻子戳，杆推，听觉线索演示和识别，实际收到的奖励），并在分析这些行为的任务事件。阐明各种认知行为的神经基础，结合行为和神经活动记录本系统的结果将是富有成效的。 <img alt="图1" src="/files/ftp_upload/3685/3685fig1.jpg" /> 图1系统的主要组成部分（A）和定制的听觉操作性室（二） 答操作性室的示意图坐落在双层隔音的房间。整个房间的墙壁，以消除声散射和挠度安装消声泡沫楔：换向器路由电线神经元活动的录音系统; B：摄像机用于监视和记录动物的行为; C：佩莱掌柜;操作性室E：红外线接收器。B.操作性室，包括三个声学透明的墙壁和一个模块化操作面板。看到详细的说明文字。 <img alt="图2" src="/files/ftp_upload/3685/3685fig2.jpg" /> 图2系统原理概述。该系统包括两个子系统：行为训练和神经活动录音系统。通过TTL脉冲（有关详细信息，请参阅系统配置 ）两个子系统交互方式互相沟通。红色箭头表示命令和/或行为的事件，从计算机发送的绿色箭头表示动物的响应信号反馈到计算机;和蓝色箭头表示信号输入神经穗事件检测在线的Neuralynx录音系统。 <img alt="图3" src="/files/ftp_upload/3685/3685fig3.jpg" /> 图3。双另类通道OICE频率歧视任务。框图 ​​显示了基本的任务序列。 底 ，主体行为的行动示意图描述。绿色箭头表示任务的顺序流。 <img alt="图4" src="/files/ftp_upload/3685/3685fig4.jpg" /> 代表结果的两种替代选择频率歧视任务A.学习曲线： 图4。每个颜色的线代表了大鼠的学习每个频率的变化（ΔFs）歧视的进展。暗线代表平均频率变化的学习曲线。B.反应时间的分布，提示发病时间杠杆推圈测量。 三审间的时间间隔分布。 四时空性能的动态范围内测量会议在早，中，后期阶段的会议取得的报酬积累。一将B中的数据 - 在最后阶段时，老鼠的表现超过75％的命中率是D的。 <img alt="图5" src="/files/ftp_upload/3685/3685fig5.jpg" /> 图5。认知行为和大脑功能，可以使用该系统的研究。认知行为的例子。顶端的标题描述一审判序列中的每一个动作。底部字幕显示，可以研究的认知行为。请注意，这两个杠杆在所有图片中提取除了在 D杠杆是在处理延伸到室。B.射击老鼠NB记录神经元模式执行两个选择听觉任务。光栅图像在每个试验射击的情节。每个彩色矩形代表了细胞的射击和发射率编码的颜色。 底 ，围甚至ţ直方图射速。烧制的建设的行动之前，请注意（零微秒由红色虚线垂直线表示，时间推杆）和逐步分散的射击后的行动。C.射击记录中的腹侧被盖区神经元模式老鼠执行两个选择听觉任务。一个单一的腹侧被盖区神经元在每个审判射击，光栅图像阴谋。每个彩色矩形代表的腹侧被盖区神经元发射，其发射率是由彩色编码。 底 ，围事件的发射率的直方图显示光栅图像。推拉杆的动作之前，请注意稀疏射击，马上（零微秒，垂直的红色虚线表示），在一段时间内，当老鼠获得奖励和大力射击。这种神经元的活性，这两个动作之间几乎是寂静。在大脑中植入的的钨stereotrodes被用于REC奥德从NB和腹侧被盖区神经元活动时，同时大鼠执行任务。穗排序进行脱机使用SpikeSort 3D软件（Neuralynx，博兹曼，吨）。

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A., Thomas, F., Dinh, L., Atzori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+modulation+of+auditory+attentional+shift+in+the+rat.">Nicotinic modulation of auditory attentional shift in the rat.</a> Behavioural brain research. 210, 273-279 (2010).</li><li>Johnson, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+discrimination:+neural+processes+preceding+discrimination+decision.">Sensory discrimination: neural processes preceding discrimination decision.</a> J. Neurophysiol. 43, 1793-1815 (1980).</li><li>Johnson, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+discrimination:+decision+process.">Sensory discrimination: decision process.</a> J. Neurophysiol. 43, 1771-1792 (1980).</li><li>Fraser, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+anxiety-+and+locomotion-related+behaviours+in+mice:+a+new+way+of+using+old+tests.">Measuring anxiety- and locomotion-related behaviours in mice: a new way of using old tests.</a> Psychopharmacology (Berl). 211, 99-112 (2010).</li><li>Hui, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+tone+control+of+brain+reward+behavior+produces+highly+specific+representational+gain+in+the+primary+auditory+cortex.">Conditioned tone control of brain reward behavior produces highly specific representational gain in the primary auditory cortex.</a> Neurobiology of Learning and Memory. 92, 27-34 (2009).</li><li>Feduccia, A. A., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+stimuli+enhance+MDMA-conditioned+reward+and+MDMA-induced+nucleus+accumbens+dopamine,+serotonin+and+locomotor+responses.">Auditory stimuli enhance MDMA-conditioned reward and MDMA-induced nucleus accumbens dopamine, serotonin and locomotor responses.</a> Brain Res. Bull. 77, 189-196 (2008).</li><li>Kudoh, M., Shibuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+sequence+discrimination+learning+motivated+by+reward+requires+dopaminergic+D2+receptor+activation+in+the+rat+auditory+cortex.">Sound sequence discrimination learning motivated by reward requires dopaminergic D2 receptor activation in the rat auditory cortex.</a> Learning &amp; Memory. 13, 698-69 (2006).</li><li>Shiflett, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cue-elicited+reward-seeking+requires+extracellular+signal-regulated+kinase+activation+in+the+nucleus+accumbens.">Cue-elicited reward-seeking requires extracellular signal-regulated kinase activation in the nucleus accumbens.</a> J. Neurosci. 28, 1434-1443 (2008).</li></ol>

MED Associates公司赞助的生产和免费访问这个视频文章

在任何听觉行为的任务设计中的一个重要方面是消除散射和挠度测试环境造成的不必要的声音。穷人的声音控制，可以有一个显着的效果，被测试的行为，会产生误导甚至无法解释的结果。在系统中使用的行为腔形容这里是专门设计的声学透明，以避免室墙壁的声音偏转。事实上，当测量室中心，健全的偏转是有效地检测不到（数据未显示）。 虽然我们开发这个系统，主要用于研究听觉系统，它可以很容易地适应其他研究人员通过研究其他感官系统。修改可以很容易地对软件和硬件不同的任务，在不改变系统的整体配置。模块化的操作面板使得系统特别versati乐以允许除了和/或不同的设备替换为新的行为任务。例如，嗅觉行为的任务可以被植入到鼻子戳设备提供嗅觉刺激。五个选择照明鼻子与嗅觉刺激的MED（env-115A的）捅墙，可以很容易地安装，操作面板上的复杂的嗅觉任务。此外，所有操作性设备可以很容易地取代那些不改变系统配置的小鼠设计。 每个操作性的设备，精确的时间控制以及高分辨率记录单事件在给定的试验，使设备的准确操作，自定义行为的任​​务设计，以满足不同的认知脑功能（见下文）。当神经元的活动记录相结合，在神经科学领域的丰富多样的问题，可以研究这个系统。例如，在Auditory上述任务，对大脑认知功能有关的下列问题，可以在一个单一的试验研究： （1）。动机：由于每个审判是启动动物的“自我激励”的鼻子，戳行动（图5A-和图3）动机可以因此被评估通过测量在一个给定的会话的动物进行的试验总数的定量，或连续试验的数量进行6,7。 （2）。注意：获得奖励在审判的关键是要正确认识听觉线索。在约25％的大鼠，不能训练，执行任务，缺乏参加的听觉线索的主要因素。相反，在大鼠学习任务，短暂的停顿的行为是在听觉线索介绍明显（ 见图5A-B和图3）。通过使用这个系统，因此，它是可能为“关注DEFI（一）屏幕大鼠国旅总社“（二）重视学习的神经机制与神经元的录音时相结合而动物的听觉线索8-10出席。 （3）。决定：动物的听觉线索后确认，决定哪个方向，把接近正确的杠杆，在有限的时间窗口（ 图5A-C）。因此，它是研究决定，决策11,12的有效范式。 （4）。耐心：杆延伸的时机可以控制这种动物有等待杆后到达的地方的杠杆将延长（ 图5A-D）。通过改变等待的长度，可以测试和动物的忍耐程度，量化13。 （5）。奖励：任务的最终目标是获得的奖励（ 图5A-E和图3）。因此使用此系统的行为任务可以很容易地设计研究奖励的决定问题的许多方面和14-17大脑功能的价值体系。

a fully automated and highly versatile system for testing multi-cognitive functions and recording neuronal activities in rodents

我们已经开发出一种完全自动化的系统操作行为测试和神经元的活动记录由多个认知脑功能，可以在一个单一的任务序列研究。该系统的独特功能，是一个特制的声学透明的腔，消除了许多相关的问题与听觉线索在大部分市售室控制。与操作性的设备可以添加或更换的方便，使这个系统相当多才多艺，允许各种听觉，视觉和嗅觉行为任务的执行情况。自动化系统允许罚款的时间（10毫秒）控制和精确的时间标记，每个事件在预先设计的行为序列。当一个多通道电生理记录系统，多认知脑功能，如动机，注意力，决策，耐心，和奖励相结合，可以顺序或​​独立审查。

Watch this Scientific Journal Video about A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents at JoVE.com

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

在这份报告中，我们提出了一个完全自动化和高度灵活的系统，能够同时测试多认知行为和记录老鼠的神经元活动。

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

a-fully-automated-highly-versatile-system-for-testing-multi-cognitive

Research

JoVE Journal

Neuroscience

14.2K 次观看.  The Neurosciences Institute, San Diego, CA. 在这份报告中，我们提出了一个完全自动化和高度灵活的系统，能够同时测试多认知行为和记录老鼠的神经元活动。

视频: A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

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

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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

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

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

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

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

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 ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

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

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

大鼠慢性经颅电刺激及 Intracortical 记录

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

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...