Name: behavioral assessment of manual dexterity in non-human primates
Uploaded: 2011-11-11T13:00:00
Duration: 16 min
Description: Watch this Scientific Journal Video about Behavioral Assessment of Manual Dexterity in Non-Human Primates at JoVE.com

作者要感谢ME的施瓦布教授，博士，体育弗氏，博士答韦斯，S ·巴希尔博士，博士答米尔，研究布洛赫博士，博士摩根富林明布吕，J博士Aebischer，博士A.梅萨罗斯，博士五Goetschman以前的实验和分析他们的贡献。安德烈盖拉德，陈智思Aebischer和洛朗Monney，构建了实验装置。在动物设施，猴子们划归专业动物管理员的监督：约瑟夫Corpataux，洛朗专横和雅克美拉德。行为测试和分析数据，以及组织学检查，进行了实验室技术员的非常宝贵的贡献：Moret韦罗妮克（网主），弗朗索瓦汤格利，恭Roulin，莫妮卡Bennefeld，克里斯蒂安马蒂和乔其纱菲舍尔。这项工作是由瑞士国家科学基金会的支持，赠款31-61857.00 310000-110005，31003A - 132465（EMR）的，310030-118357，31003A - 10406​​1（台湾），310030-120411（ABS）PZ00P3_121646（ES），诺华基金会的权限，国家研究中心在“神经可塑性和修复”（NCCR）。

实验的总体方案如图1所示。 1。动物准备和转让行为实验室<ol><li>在实验室里，准备行为设置：填充颗粒，作为奖励服务过程中的行为测试的不同测试板的井（测试1至3段）。 </li><li>转移组成转移笼屋室的猴子。猴子训练进入一个隧道，给访问的灵长类动物的椅子，与头部的后续定位。猴子的体重测量，转移到实验室在灵长类动物的椅子前。 </li></ol> 2。测试1：修改布林克曼板<ol><li>这个测试，修改，改编自4,5以前的报告，是职权行为的基本任务，每一个行为的会议上进行。上述设置的数码相机（放置2广告的可能性也主动与录像ditional相机，在每个电路板的一面）和放置在前面的布林克曼板的猴子。 </li><li>例如在右侧窗口打开的灵长类动物的椅子给右手的访问。用右手，猴子检索从50插槽（垂直25和25水平）的食物颗粒。 </li><li>测试完成后，关闭右边的窗口和填充用颗粒板。 </li><li>打开左边的窗口和重复测试左手。 </li><li>奖励的动物，在测试结束几个干葡萄干或杏仁，在每次测试结束保持在整个日常会话的动机反复的过程。 </li></ol>其他信息：三个数码摄像机用来记录离线处理的顺序，每个电路板的一面放在上面董事会和（精确地评估手指的位置，而抓）。在相同的日常会话，日发送猴子可以执行另一个任务（任务2和/或任务3和/或任务4;之间每周的不同天分布）。测试1，如果猴子的第1天的右手开始，开始用左手第2天等。 <ol start="6"><li>可选：测试执行上面的一个或分别允许左手右手（确定的“显性的手”）的性能进行比较，另一方面，它也可能在早期阶段的训练，让猴子用双手同时执行任务。如果一方面是比其他更常用把握颗粒，然后它可能会被视为“首选之手”。 </li></ol> 3。测试2：布林克曼盒（有或无视觉控制） <ol><li>布林克曼中包括20口井（10和10个垂直横）。测试1相比，猴子已控制在有限的空间，另一方面减少程度的自由，执行精度握运动。填充颗粒板和关闭框的上部面，以进行第一次在视觉控制的情况下，依靠触觉探索试验。 </li><li>布林克曼框前广场的猴子。打开左侧窗口中的灵长类动物的椅子视觉控制的情况下测试的左手。猴子尝试检索20颗粒，而序列是从下面的框中放置一台数码相机记录。 </li><li>关闭的灵长类动物的椅子的左侧窗口。与颗粒填充板。打开右侧窗口中的灵长类动物的椅子。猴子与视觉控制的情况下，右手重复测试。关闭的灵长类动物的椅子的右侧窗口。 </li><li>为了测试的把握能力在布林克曼框视觉控制下的颗粒，打开箱子的顶部面。 </li><li>笔芯一盒颗粒和猴进行测试，用右手。 </li><li>关闭的灵长类动物的椅子的右侧窗口。与颗粒填充框。 </l我&gt; <li>打开的灵长类动物的椅子的左侧窗口。猴子进行试验，使用左hand.Repeat步骤2.5。 </li></ol> 4。测试3：旋转布林克曼板<ol><li>这个测试是布林克曼委员会的任务（测试1）相媲美，除了板旋转，迫使猴子预测（顺时针）或其他（逆时针）方向板的位移。与颗粒的填充板。以上的设置（可能性也放置2个额外的摄像头，一个每边）放置一台数码相机记录的序列。 </li><li>打开右侧窗口中的灵长类动物的椅子。猴子从32口井，分布在四个同心行检索的颗粒，而董事会是顺时针旋转。 </li><li>关闭右边的窗口。与颗粒填充板。 </li><li>打开的灵长类动物的椅子的左侧窗口。猴子进行试验，用左手4.2。 </li><li>重复4.2至4.4分，而我的板小号逆时针旋转（一后另一方面）。重复步骤2.5。 </li></ol> 5。测试4：达到和把握抽屉任务<ol><li>要结合捏的能力，有能力产生力量，这种测试（6-11派生从以前的版本）的目的是使猴子第一个握力施加旋钮抽屉，打开抽屉，负载力打开抽屉，访问放在抽屉内的颗粒。沉淀检索用同样的手，而抽屉仍然开放，再次使用了精密的抓地力。数码相机被放置在抽屉上，记录脱线控制的数据（如错误的试验检测）试验。 </li><li>打开右侧窗口中的灵长类动物的椅子。猴子在5个不同层次的阻力每个执行的10项试验，用右手（有至少5个正确的试验，每一个阻力水平）。 </li><li>同日重复试验（50项试验）发送左手。重复步骤2.5。 </li></ol>其他信息：在未来的行为的日常会话，替代动物测试第一的上届会议上的手。 6。行为会议结束<ol><li>预见到这一天的测试完成后，除了在测试期间收到的颗粒饲料和奖励与食物的猴子。通常情况下，猴子收到谷物和水果。 </li><li>猴子是返回与队友组房屋房间。 </li></ol>书面协议的形式进行的各种测试的准确时间序列。 7。代表性的成果 （FI研究旨在调查颈髓损伤的功能恢复的背景下，说明了上述四种行为的测试（图1）已经在我们的实验室中广泛使用gure 2A）或运动皮层（ 图2B），在缺乏或存在的一种治疗应用，以提高自发恢复12-19。 分析测试1（ 修改布林克曼板 ），主要集中在两个参数（ 图3和图4A）：我）的得分检索猴子在第30秒，分别计算垂直插槽的颗粒数，和水平插槽（脱线重播录制的视频序列）;二）接触时间（CT），定义为手指和颗粒之间的接触时间（持续时间）（参见图6，底部的系列图片）。它的第一个插槽（通常是食指）手指触摸颗粒插入和检索的颗粒以及发病之间的时间间隔。测量的时间间隔是通过重播帧帧的视频序列。 CT是测量的第一个五年VERTIC针对人插槽和第一五横槽猴16-18。比分的图表说明了在最初的培训阶段，前期病变高原的得分大幅下降后，立即病变（通常为零），渐进（自发）对病变后高原的功能恢复。功能的恢复表示病变后，在高原的中位数中位数前病变的得分（高原）划分得分的比例（％）* 100（图3和图4A）。对于CT，增加反映了赤字，％表示功能恢复的比例中位数中位数后病变的CT前病变的CT分在高原* 100（高原） 。治疗后病变的效果，证明测试1的基础上，在本实验室14,15,17先前的报告中详细说明。进一步的分析可以解决的战略问题，即时间序列插槽参观了由Monkey（ 图4B）。 对于测试2（ 布林克曼盒 ），虽然比分还可以测试1，建立一个更有意义的参数是“总时间”，在第一个插槽之间的颗粒采摘的时间间隔和采摘定义在昨晚（20 日 ）插槽颗粒（ 图5）。一个功能的恢复，可以计算和以％表示。这是计算总时间中位数前病变（高原训练阶段后）除以中位数在高原* 100的总时间后病变。 （ 旋转布林克曼板 ）， 测试3的得分虽然也可作为上述（1试验）建立，一个敏感的参数是接触的时间（CT，定义为在上述测试1），首十个月插槽来衡量（图6 ）。 对于测试4（。 达到和把握抽屉任务 ），设置彗星omprises几个探测器，录音开始试验（手中断光束摆在前面的面板），用手触摸旋钮，拉抽屉的发病，抽屉开放，一方面进入插槽（接机时间，如离散事件），另一方面从插槽（接送时间）撤出。旋钮上的力传感器可以测量握力（拇指和食指按压旋钮施加）和负载力（施加拉的抽屉里，以抵消抽屉开放施加不同的阻力水平）。反对抽屉开放的阻力不同层次获得通过改变电流强度，适用于一个回转的电磁马达连接到抽屉背面。设置的目的是应用在抽屉开放的方向平行的阻力。抽屉里设置的详细计划，要求通讯作者。所有这些数据收集与接口ND软件穗2并显示在图7所示。 以前reported1 4,15，这些任务代表行为的基础上，以调查是否从颈髓损伤的自发恢复，可能与旨在促进轴突再生（ 图 8）的一个具体的治疗增强。 检索得分和接触时间参数反映手巧的不同组成部分：第一个包括整个电机序列（伸手，抓，撤回手，交通口的沉淀），而第二个是集中在只抓阶段。这两个参数修改和旋转布林克曼板任务，特别是准确和互补。鉴于得分很大程度上是多余的，在这两个任务的信息，在对比的接触时间提供了更加具体任务的信息，由于插槽的位置在旋转布林克曼委员会的任务的变化相比，他们在修改后的布林克曼委员会的任务的静态位置。布林克曼盒的任务不同于上述两个任务中，手的动作自由程度是有限的封闭空间。因此，布林克曼方块内的主板上的不同插槽位置在难度方面发挥了重要作用，执行手动捏，由于框exiguity。例如，抓手，从位于禁区右侧的插槽右侧壁干扰。 因此，在修改后的布林克曼板有限检索评分为30秒的评估，将根据实际访问的猴子，在此期间限制的时间内，产生很大的变异性，从一个会议到另一个插槽的位置上有失偏颇。出于这个原因，它是比较合适的，以invo绿萼所有插槽（N = 20），因此被选为总时间参数。在修改后的布林克曼板，总时间是不考虑，猴子可能在某些情况下松的动机（例如病变后）由于要进行大量的插槽（N = 50）。在同一直线上，接触时间分析会株连考虑的布林克曼框中的所有插槽（选定插槽的位置考虑，而修改布林克曼板中的前五个插槽为小，如果没有此参数的影响）。因此，布林克曼框，我们建议在第一种方法来确定的总时间，因为它是观察到的是一个高度相关的信息参数，至少在我们的研究与猴子电机皮质病变（无可用数据脊髓损毁的猴子）。然而，接触的时间可能会被认为是为布林克曼框，但在第二个步骤，包括所有二十个插槽，分别然而水平和垂直插槽（未显示）。 <img alt="图1" src="/files/ftp_upload/3258/3258fig1.jpg" /> 图1实验的总体方案。动物是从动物设施转移到灵长类动物的椅子上，然后运送行为实验室。在每天的会议，猴进行测试1。隔日，猴子，然后执行测试2，和/或测试3，和/或测试4。一些猴子（尤其是动机）可​​能会执行相同的日常会话中的所有测试。干的香蕉或葡萄糖粉，这是一个直径约4毫米的圆形压缩食品颗粒（用于奖励）。在我们所有的行为测试，我们使用BioServ，一街8号，一室，Frenchtown，新泽西州08825，美国提供的无尘精密球（45毫克）。 修改布林克曼板的尺寸为240毫米长，宽140毫米，而插槽的尺寸是长15毫米，8毫米WIDE和6毫米深。董事会回转布林克曼板的直径是114毫米。 <img alt="图2" src="/files/ftp_upload/3258/3258fig2.jpg" /> 图2代表（手术）的颈髓病变（A组，从15修改）和（化学）的运动皮层的病变（B组，从16修改），派生相应的组织切片，处理， SMI - 32染色。颈髓损伤最大程度地已连续矢状段脊髓（A组），而重建的程度和运动皮层病变的地位已经从大脑和重新定位的连续正面部分重建相应的大脑半球侧面观（在B组的红点）。颈髓损伤导致从手术刀片横断水平的C7 - C8，中断单方面的主要CS道组件中的一个子半切造成新界东北的背外侧索，控制手部肌肉的运动神经元 14,15,20以上。鹅膏蕈氨酸13,16,17输液生产永久皮质病变，涵盖的手表示，以前使用intracortical微刺激（ICMS， 见 16,21,22）建立的网站。皮质病变区出现突然中断III和V层神经元在中央沟喙银行（同在B组的虚线划定区），SMI - 32染色。 <img alt="图3" src="/files/ftp_upload/3258/3258fig3.jpg" /> 图3。修改布林克曼板遭受脊髓病变（如图2A所示）一只猴子执行的任务（测试1）派生的代表数据。该图显示的得分（协调）的垂直插槽（蓝色符号）和水平插槽（红色符号），分别。黄色符号的总和纵向和横向的成绩，对一个给定的日常会话。在横坐标，时间是连续两天的行为会。垂直虚线红线（0天）是病变的一天。三个不同的时期突出：第一个（黑色虚线），对应于训练期间，第二个（蓝色虚线）表现高原前病变，而第三个（绿色虚线）高原恢复性能。从15修改的数据。 <img alt="图4A" src="/files/ftp_upload/3258/3258fig4A.jpg" /><img alt="图4B" src="/files/ftp_upload/3258/3258fig4B.jpg" /> 图4a，在图3（测试1）相同，但在一只猴子受到了运动皮层的病变（如图2b所示）。该图表显示的分数（A组;相同的约定，在图3）和接触时间（CT，B组）的数据。在横坐标，时间是连续两天的行为会。垂直虚线红线（0天）是病变的一天。在B组中，每个点对应的手指和一个插槽（每个方向5次试验为每个会话;灰色列中值）的颗粒之间的接触时间。请注意，为试验动物无法执行任务（后立即病变），CT出现饱和值在5秒。从16,17修改的数据。 图4B。contralesional手，前运动皮层（预）的病变，在恢复阶段（恢复）和高原（邮政）病变后，执行任务的修改布林克曼板通过战略分析。每个插槽的颜色显示在一个会话中的猴子（参观的第一个插槽是最黑暗的蓝色和最黑暗的红色参观的最后一个插槽所示）访问的插槽顺序。注意THA吨前期病变，猴子板的左侧开始向右扫描系统。在恢复过程中，按顺序被改变。高原病变后，所采取的战略前病变重新出现（系统扫描从左至右）。 <img alt="图5" src="/files/ftp_upload/3258/3258fig5.jpg" /> 图5。代表布林克曼盒（2）测试所得的数据，为视觉控制下执行任务的猴子。统筹所需的总时间为空沿20口井的日常会话（横坐标），运动皮层的病变（垂直虚线）之前和之后进行的。方框内的访问量的尺寸是1360厘米3（120毫米*110毫米*103毫米）。注意训练的初始阶段，由一个总的时间从一个会话到下一个较大的可变性的特点。皮质病变后，立即猴子是无法执行的任务（数据点，在200秒饱和）。 P值之间的差异在上届会议中位数的前病变（中间水平在左边的灰色矩形）的总时间和总时间中位数后病变统计学意义（在合适的水平灰色的矩形中间）。功能恢复的比例是89.6％，而运动皮层病变的体积为 41.8毫米3。 19日修改的数据。 <img alt="图6" src="/files/ftp_upload/3258/3258fig6.jpg" /> 代表回转布林克曼委员会的任务（ 测试3）所得的结果（前两个图形），接触的时间测量前病变和病变后的插图与图4A（B组）相同的约定，图 6。一只猴子运动皮层的病变（17日修改的数据）。上图是按顺时针（“CL”）RO<html>档的电路板，而底部图形逆时针（“C - Cl键”）董事会旋转。两个垂直的灰色箭头表示，接触的时间是无限长，在几个交易日后立即病变，猴子contralesional手无法执行任务。系列图底部的图片说明的方法来衡量的接触时间（修改布林克曼板和旋转布林克曼板有效）。最左边的图片显示含有颗粒（食指和颗粒之间的接触前100毫秒）接近槽的手。右边的下一帧对应的联系方式（0毫秒）的时间点。然后接触的时间被定义为的时间间隔运行，直到达到相应的时间点颗粒插槽帧（最右边）（MS）。在这里接触的时间是240毫秒。 58/3258fig7.jpg“/&gt; 图7。会议进行了一个猴子的覆盖面和把握抽屉任务（ 测试4）所得的代表数据。 A组：对应的三个参数的原始数据在审单收购在线：负载力，红色，蓝色的握力和位移的绿色抽屉。期间还收购了一些标记任务：采摘时间对应的时间抓奖励，全开放式的抽屉和旋钮触摸（TCH）的时间点，当动物首次接触旋钮全面开放。分析，7个游标放置在关键的时间点展开的任务（如在底部三个水平线的灰色游标）：1）时间锁定的动物旋钮触摸; 2）发作的握力; 3）负载力最大握力; 4）发病5）最大负载力; 6）时间锁定时完全打开抽屉; 7）时间锁定到采摘时间。 B组：握力（使用武力把握的食指和拇指之间的旋钮）和负载力在两个图（用来打开抽屉力）：任务期间录得的两个参数的定量结果表示：最大值（左图）和斜率值（从发病到最大;右图）。 四五个不同的阻力相对水平已经所示：R0（0牛顿），R3（1.4 N），R5（2.75 N）到R7（5 N）。旋钮抽屉有一个三角形和平面形状。附加抽屉的三角形的基措施20mm和顶部从基（15毫米），在一个直径7毫米的圆形轮廓。抽屉本身的尺寸如下：长= 50毫米，宽度= 27毫米，高度=45毫米。 <img alt="图8" src="/files/ftp_upload/3258/3258fig8.jpg" /> 图8。提醒探讨颈髓损伤的功能恢复的治疗可能产生的影响（反NOGO - A抗体） 的使用行为测试1（从15修改）。对于分数和接触时间，以及为两个槽方向，对照抗体组治疗的猴子（蓝色符号; N = 6）恢复手巧不如反NOGO - A抗体治疗的猴子组（红色符号N = 7），特别是大量的病变。 2组差异显着P = 0.035（A组），P = 0.022（B组），P = 0.035（C组）和P = 0.008（面板四）。

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没有利益冲突的声明。

虽然目前的行为任务被认为是迄今在与颈髓病变或病变的运动皮层的目的，以测试各种治疗方法的研究方面在我们的实验室 （ <a href="http://www.unifr.ch/neuro/rouiller">见</a> 14,15,17和呻<a href="http://www.unifr.ch/neuro/rouiller">/ /神经/ rouiller www.unifr.ch</a> &gt;在顶栏菜单中选择“研究”，然后“电机系统”&gt;“病灶后恢复”），他们可能也有一个更广泛的应用，手巧也是一个方面考虑在其他疾病，如帕金森病（MPTP的猴子）或感官afferentation去影响本体感觉和/或触觉（特别是视觉控制的情况下测试2）的情况下，。 这里提出的行为测试，适合调查的前肢远端运动的电机控制，手巧。测试的特殊性表现赤字的情况下，在不损害控制系统的相关组件的一个病变的情况下（几天除外）：的确，在病变的情况下放置超过控制手部肌肉的运动神经元的尾鳍，没有赤字。测试1的针对性，可病灶后恢复曲线，加强功能恢复，25％的不同，可能涉及到细胞疗法治疗17，在两个时间点的视频序列比较赞赏。 尽管一些初步的，相对较短的期初培训阶段（持续时间一般为2-3个月），这里提出的行为测试是相对“自然”和简单，因为比较复杂的任务（如条件）猴子训练可能需要近一年或一年以上。积极强化是基于对固体食物，这是比水剥夺伦理角度敏感，通常用于在多个COM复杂的任务23。有没有必要剥夺猴子从食物中获得稳定和一致的结果。任务期间收到的颗粒代表的食品行为会议（假设猴子在前面晚上不吃饭，但更多的粮食，可直到结束的前一天下午）当天的第一次访问。至关重要的是，每只猴子在一天的同一时间执行的行为的会议上，以及尊重形成一组在房屋室内不同猴子之间在同一顺序。由于猴子是敏感的外部令人不安的事件，任务的行为应进行背景音乐的存在，掩盖潜在令人不安的噪音来自隔壁房间或实验室。至关重要的是，在整个实验（从初步培训到最后的每天实验会议，几个月，如果不是几年内）的时间，一个给定的猴子每天放在同一实验者的监督下。 本行为测试，因为在我们的实验室几年用于量化手巧，在一定程度上与手巧的其他测试，最近文献报道24-28。然而，有一个关键的需要，在不同的实验室（为更好地进行比较），这是本报告的初步目标，以规范测试。根据需要，设置的详细属性说明这里1-4可以提供相应的作者，以复制他们的测试。除了再生医学的问题（从脊髓病变或大脑皮质恢复），目前的测试调色板可能是合适的，以解决在正常的非人类灵长类动物（如电机灵巧运动的发展历程）的发展问题，调查偏侧方面（手偏好/主导地位）和破译进化的问题，由Comparing不同品种的灵长类动物，包括人类受试者的运动能力。但是请注意，应根据数字的大小（厚度和长度）的灵长类动物适应的仪器的尺寸，因为它可能影响执行任务。在本研究中，对食蟹猴进行了测试，范围从2.5到8岁，体重2.5至8公斤。食指的长度（第一次使用操纵颗粒）的范围从32至35毫米，而食指末节（TIP）的周长是在22和25毫米的猴子在我们的研究包括之间。正如在以前的实验测试，同时抓测试，以及适合猕猴。 所有实验均按照“ 指南 ”中进行实验动物的关心和使用 （1996年），由当地兽医主管部门（瑞士）的批准。对猴子所有的实验程序S，以及在动物设施的拘留条件，从我们实验室最近的报告中详细描述：请参阅参考资料12-18。

behavioral assessment of manual dexterity in non-human primates

皮质（CS）道解剖支持精湛的运动能力，熟练操作的小物件，一个主要的灵长类动物的特权。影响CS投影系统的病变，在其原产地（运动皮层区病变）或沿着它的轨迹（颈髓病变）的情况下，有一个巨大的损失（手巧的手麻痹），看到一些四肢瘫痪或偏瘫患者。虽然有一些自发等病变后的功能恢复，在成年后仍然非常有限。目前提出不同的治疗策略（如细胞疗法，中和抑制轴突生长分子，生长因子等应用），其中大多是在啮齿类动物中开发。然而，在临床应用中，它往往是建议测试的可行性，有效性和安全的治疗在非人类灵长类动物。这是尤其是当我们的目标是恢复后手巧中枢神经系统病变，是从灵长类动物1,2的不同组织的啮齿动物的电机系统。猕猴说明这里作为一个合适的行为在灵长类动物模型，以量化的手巧，反映运动皮层或例如颈髓病变造成的赤字，测量自发功能恢复的程度，治疗时，评估它可以提高多少的功能恢复。 手巧的行为评估是基于对四个不同的，互补，手动任务达到和把握（使用精密握把握颗粒）规定的成年猕猴的初步培训，。编制的动物展示，以及行为设置的定位。一个典型的猴子的表现是每个任务的说明。相关参数的收集和分析反映精确的手manipulaTION，以及力控制，解释和具有代表性的结果表明。然后这些数据被放在更广的范围内，展示了如何行为数据可以被利用来探讨脊髓病变或运动皮层和到什么程度的治疗可提高自发的功能恢复，通过比较病变的影响猴子的不同群体（例如治疗深水与处理）。行为测试的优点和局限性进行了讨论。目前的行为方式，是在以前的报告中强调的非人类灵长类动物模型在神经系统疾病2,3方面的针对性。

Watch this Scientific Journal Video about Behavioral Assessment of Manual Dexterity in Non-Human Primates at JoVE.com

Behavioral Assessment of Manual Dexterity in Non-Human Primates

由于手巧，是一个主要的灵长类动物的特权，行为的任务已在猕猴。 4个达到和把握捏任务，测量手操纵的能力和力量，允许建立后中枢神经系统病变的功能恢复及测试治疗效果。

在非人类灵长目动物行为评估手巧

The corticospinal (CS) tract is the anatomical support of the exquisite motor ability to skillfully manipulate small objects, a prerogative mainly of ...

behavioral-assessment-of-manual-dexterity-in-non-human-primates

Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

科研

神经科学

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, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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 ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...