MSB is supported by the Alcohol Beverage Medical Research Foundation, a Center for Translational Research Award (UL1 TR000058), the National Institutes for Alcohol Abuse and Alcoholism (P50 AA022537), and startup funds provided by the Virginia Higher Education Equipment Trust Fund and the VCU School of Medicine.

涉及受试动物的程序已被批准的机构动物护理和使用委员会（IACUC）在弗吉尼亚联邦大学，并坚持美国国立卫生研究院指南实验动物的护理和使用。 1.准备实现了手术前和显微注射<ol><li>建立了材料准备： <ol><li>获得＆G套管管道，33克闭孔丝，33克显微注射针管，微量塑料管（PE20或等效物），两个中重直止血剂，实验室卷尺，直尺，永久标记，70％的乙醇（体积/体积），存储小瓶（例如，将20ml玻璃闪烁），超级胶，小称量皿，并且旋转工具配备有非重型截止光盘。 注：固定旋转工具来使用带或磁带枪头盒子是有帮助的，因为它提升了工具。戴眼睛的保护是使用旋转工具时避免损伤重要的。 </li><li>附上一张实验室或高压灭菌带了替补席上。 </li></ol></li><li>准备插管的： 注：至少有两个套管需要每鼠的双侧注射。一个附加套管将在后面的步骤中使用。 <ol><li>获得足够的长度套管管道的部分，以避免标记和切割过程中的弯曲。 </li><li>从用细尖笔1.1.2步上绘制带15毫米的基准线。这将作为对管连续15个毫米的部分，用锋利的永久性标记标记的导向。 </li><li>触摸套管管道，在标记的点，到旋转工具的慢速纺丝截止光盘以缺口管材。旋转套管180˚和缺口管材的相对侧。不要通过管子完全切断，因为这可能会导致阻塞。 </li><li>剧烈的弯曲管道，以便它分解成〜15毫米部分。 </li><li>握住套管的各切端垂直于截止拇指和食指之间光盘。旋转套管管在触摸尖端截止盘（ 即不是边缘）的大前锋表面。这将生成一个钝尖。注意：不要弯曲套管，为可能产生的异地注射。 </li><li>令与＆G斜口针（棕色毂）套管的两端，以确保所有的毛刺已被删除并且该套管是通畅。 </li><li>通过闭孔线切割样品到〜35毫米的通切插管，以确认不存在任何内部障碍物。 </li><li>拿起单独使用松开止血每个插管和衡量每完成插管用尺子来确保它的长度整整14毫米。 </li></ol></li><li>准备阻塞器的： <ol><li>夹紧一个插管在大约半长与重量适中，直止血。躺在长椅上锁定的止血钳。套管应垂直于替补席。 注：此套管不应该被用在外科手术被保持由锁定止血后，因为它很可能弯曲。 </li><li>订阅闭孔线的一端插入套管，直到它达到了替补席上。弹起丝在插管以确保毛刺不阻止它到达替补; 即 ，在到达套管的底部。正确的闭孔长度将防止堵塞和额外的组织疤痕。 </li><li>弯曲的线用手指捏，直到同时保持闭孔和替补之间的接触〜30˚角来实现的。确保弯曲的角度是这样的，它规定与头帽齐平，和过量的面向前方，从而使其更难大鼠除去。 </li><li>从套管中取出电线切断，在超出的部分约2.5毫米的小斜刀（线切割）的弯曲。 注意：至少有两个密闭装置将需要每只大鼠的双侧注射，因为每个植入插管需要一个插塞。创建通过闭孔额外〜35˚弯曲中途也将有助于防止去除动物。同时存储插管和含70％乙醇（体积/体积），直到使用在分开的小瓶中充填器（例如，20毫升玻璃闪烁），但至少O / N。 </li></ol></li><li>制备及microinjectors的测试： <ol><li>获取一块微量注射器针管的是约1米长。 </li><li>握住微量管的一端垂直地锁定直止血。 </li><li>扭动止血，同时保持张力的油管紧紧线圈它周围的止血钳的至少一个时间。 </li><li>测量从微量管的对面，宽松的端部32毫米，垂直地抓住这一点与连续第二个，中等重量止血并将其锁定。抓住微量管上的32毫米边止血标志最接近宽松的结束，而不是最接近止血一边用绕线。 </li><li>本D中的管来回，同时保持紧张油管直至其断裂。 注意：一个单一的，尖锐的向上和向下的弯曲会产生一个钝断裂，这是所希望确保显微注射发​​生在期望的大脑区域。 </li><li>检查微量管，以确保两端是直的并且其内径是通畅。 </li><li>获得套管管道将被用于制造微量衣领。 </li><li>测量并用永久性记号笔画和磁带上（步骤1.1.2）一个5毫米的基准线标记套管管连续5毫米部分。 注意：每个微量需要一个项圈。项圈是简单的短套管坚持以微量电线。 </li><li>触摸套管管，在标志点，以旋转刀具的缓慢旋转切断盘，以缺口管材。旋转套管180˚和缺口管材的相对侧。切割完全可能堵塞管道。 </li><li>一针见血弯曲微量领管，以它分解成〜5毫米部分。 </li><li>铰用＆G斜口针（棕色毂）的套环的两端的内径，以确保大部分毛刺已被删除。一些小毛刺会抢了微量电线，从而有助于粘合。 </li><li> 1环滑动到每个微量管〜2厘米的结束。 </li><li>小权衡船角落创建的强力胶一个小水池。 </li><li>使用废料套管管道切成〜25毫米到涂抹少量强力胶到微量管〜5毫米，从一端。接着，滑动套环移到该超级胶水珠，以便它是从微注射管的端部〜1毫米。覆盖用胶水颈圈的两端。 注意：避免强力胶大量堆积在衣领上，因为它会阻止塑料管从周围的微量密封。虽然，以避免强力胶的微量管的开口端，因为这会令是很重要的的微量不可用，这也是很重要的滑动轴环尽可能靠近端，以尽量减少空隙（未注射）的体积。 </li><li>精益完成的微量上的另一个小权衡与衣领的一面朝上船外，并晾干24小时。 </li><li>得到〜8厘米一块微量的塑料管（PE20或等效物），1毫升注射器填充有无菌水，和＆G斜口针（棕色毂）。 </li><li>附加26克（棕色集线器）针注射器滑动切管到针，确保不要刺破管。 </li><li>滑动塑料管在干的，完全微量的衣领结束。 </li><li>挤水填充针筒柱塞以测试通畅。 注：水宜喷很远又直出微量尖。如果流是不直，注射位置将不正确。横盘或弥漫性喷雾的来源是一个贫穷的突破（步骤1.4.5）。微量注射器不应该T表示，如果喷雾是横盘或弥漫性使用。盐水不应该被使用，因为它会阻塞微量或针。 </li><li>为了从微量去除水中拉出注射器回来。应贮存在干燥，密封的小瓶;例如，一个20毫升玻璃闪烁瓶中。 注：Microinjectors可以制造与大多数超级胶水，31后进行高压灭菌，但是这必须根据经验来确定。替代灭菌技术包括环氧乙烷和γ照射。 </li></ol></li></ol> 2.准备和执行显微注射<ol><li>获得完成microinjectors，微量塑料管（PE20或等效物），显微注射泵，两个小称量皿，无菌水，70％的乙醇（体积/体积），丙酮，二气密1μl的玻璃注射器的钝针（25 G）的棉签木喷头，备用密闭装置，1ml注射器，26 G（棕色集线器）的针，小弯钳（款式＃7推荐），和实验室W¯¯IPES。 </li><li>准备microinjectors注射： <ol><li>弯曲完成微量注射器从相对的套环端15毫米，使两者之间的角度为〜95˚。 注：精确的弯曲位置是刻不容缓的准确注射位置。使用＃7镊子（或类似）抓住微量和弯曲向上是非常有用的。始终重新测量进行显微注射​​前的长度。 </li><li>将塑料管（PE20）到〜70厘米，滑过微量衣领。 注意：添加磁带选项卡，其中的一个塑料筒近两个微量和适度宽松的结束完成注射时，注射两种不同的解决方案，特别是当是很有用的。标签不推荐用于管，因为他们成为在注射过程中繁琐。 </li></ol></li><li>准备泵显微注射： <ol><li>电源开泵。 </li><li>设置直径显微注射针的，所需的输注率，和目标注射量。 注意：输液鼠e和注射量取决于实验的要求。这是在讨论中阐述。有些泵有预装注射器表。如果不是这样，一个注射器表可以在制造网站（多个）中找到。 </li><li>将泵模式为“音量”，这样一个精确的音量自动发送。如果选择“泵”模式，请实验者必须手动停泵。 </li><li>调节泵逆止以允许注射器来填充到注射体积外加0.2微升。 </li></ol></li><li>准备显微注射的注射器和微量注射： <ol><li>锁每个气密显微注射注射器插入微量注射泵的齿条。 </li><li>放置气密注射器的尖端到包含在一个小的无菌水称重舟，轻轻扑柱塞以润滑内部导线。均匀地拉柱塞逆止泵，填补了水。 </li><li>滑动PE20管材是合作nnected到微量（步骤2.2）在连接到一个1ml注射器填充有无菌水26克（棕色集线器）针。通过微量喷雾无菌水，并检查喷雾形状和距离。 注：水宜喷很远又直出微量尖。如果流是不直，注射位置将不正确。 </li><li>将灭菌微量到无菌区。 </li><li>滑动管道落针，保持微量管直立，以防止水滴落出来，同时也降低了由针受损区域的下方的管道。 </li><li>推显微注射针筒柱塞稍微向前以创建在所述针的端部的液滴和滑动充水PE20管（被连接到微量）到针。 注：这将创建一个液 - 液连接，将允许适当的反压，可以赶走，可以形成以堵塞该微量的一角。 </li><li>推动柱塞完全向前，以备样量，并确保没有一丝酒精留在微量。 </li><li>通过触摸已经形成微量的尖端到一个干净的实验室擦拭多次水滴检查设置是否有泄漏。 </li></ol></li><li>注意：当接触到实验室擦拭，只有一个湿点应该形成。如果有更多的液滴，存在在系统中的泄漏。 </li><li>通过控制超级胶水的应用程序（步骤1.4.14），并通过调整PE20管材用锋利的剪刀或刀片的PE20管从任何连接删除的任何时间，避免泄漏。此外，重复步骤2.4.3至2.4.8，以弥补泄漏。 </li><li>拉注射器活塞回到0.2微升;创建无菌水在PE20管道/微量和溶液之间的气泡被注入。 </li><li>注意：此气泡可防止稀释注射液中，水的污染，并允许用于监测注射过程的，因为它会在注射过程中移动。一个单一的，坚实的泡沫应该产生。如果它坏了，存在泄漏或微量尖闭塞;这表明步2.4.8（和附带的注释）应反复。 </li><li>将在微量到试剂注入，然后慢慢将注射器柱塞，逆止器。 </li><li>动物准备注射： </li><li>抓住老鼠的胸部对实验者的胸部。用棉涂敷器浸泡在70％的乙醇清洗围绕插管的区域（体积/体积）。 </li><li>注意：使用适当的处理，老鼠可以轻轻地在杯形手的限制。否则，矿泉去油光可能需要。 </li><li>删除使用小镊子和地方密闭装置进入称重舟用70％乙醇（体积/体积）。 </li><li>执行注射： </li><li>将填充微量（步骤2.4.10）进入套管。按微量注射泵和监测气泡运动的开始。 </li><li>注意：マŸ需要稍微用力，以确保microinjectors都不抬。泡沫（形成步骤2.4.9）应统一推进，全面进入微量。 </li><li>除去从套管的microinjectors和替换密闭装置中，一旦注射完成和后喷射扩散周期已经过去。 </li><li>注意：典型后喷射的扩散时间是2 - 4分钟药理试剂，和2 - 10分钟的病毒。留在微量在后输液期间防止注射液从回流备份插管，而不是扩散到组织中。在乙醇填充权衡船一侧放置密闭装置，使乙醇重新插入到插管前排水。 </li></ol> 3.注射后清理<ol><li>获取一个小瓶的每一个：无菌水，70％乙醇和丙酮。 </li><li>清洗微量注射器和显微注射针筒： <ol><li>卸下玻璃注射器- 从玻璃注射泵的注射器架和注射管。 </li><li>从玻璃注射器断开微量管和附加1ml注射器经由＆G针（棕色毂）充满无菌水到微量管中。按1毫升注射器活塞前锋驱逐-0.3毫升接下来，触摸微量的尖端实验室擦拭和拉风回通过微量。 注意：微量和管道可以被重新用于类似的实验，如果认为谨慎。 </li><li>将玻璃注射器针头插入无菌水，并绘制活塞完全恢复。其次，推动柱塞完全向前冲。触摸玻璃注射器尖端实验室擦拭，以确保没有液体的存在。 </li><li>用无菌水，空气，70％乙醇和丙酮按以下顺序重复步骤3.2.3：无菌水，无菌水，空气，空气，70％乙醇，70％乙醇，空气，空气，丙酮，丙酮，空气，空气。执行所有这些清洁步骤将有助于维护显微注射针筒。 </li><li>放置微量注射器（S）为包装和拉柱塞回0.05微升。 注意：这是重要的，因为它允许在柱塞被推动或拉动，以便驱逐可能发生的应保存后的任何盐沉积物形成卡住柱塞。 </li></ol></li></ol> 4.编程动机测定<ol><li>登录到图形状态具有管理员凭据。 </li><li>选择或创建相关的数据库。 </li><li>创建渐进比强化程序： <ol><li>选择列表&gt;数据库级列表&gt;事件转换参数列表。 </li><li>选择“添加”。输入相应的列表号（L＃）和描述。 </li><li>选择“添加事件”。 </li><li>输入第一个强化程序的价值，并选择“添加”。重复。 注意：值可以通过使用其中渐进比时间表= 5E（j *（加强件挣得+ 1））-5一个公式确定7。为了调整此公式来正确测试该假说，值j必须凭经验确定，或从文献中获得。 </li><li>内的“选择订单”框中选择“为了&#39;。 </li><li>选择“保持值=为：____”中的“完成时”框。输入加固进度值到远远超过预期的响应文本框。这将是所有后续项目所需的努力，一旦名单已经用完。 </li></ol></li><li>创建一个实验方案： 注意：图形状态移动通过一系列状态被退出基于时间或性能标准的受试者。设置如下标记为“$”下检查，以适当地测试假设所有离散和上下文线索和参数。 <ol><li>选择文件&gt;新建实验方案。 </li><li>输入所需的协议号和描述成相应的文本框中。 </l我&gt; <li>选择“国家创作”。 </li><li>和“FIN - 完成国家” - 同时为“就绪状态RDY”将所有的刺激。 </li><li>选择“新的国家”，并命名为“S2 - PR响应。 </li><li>选择“新的国家”，并命名为“S3 - PR增强剂和提示” </li><li>选择“新的国家”，并命名为“S4 - 超时”。 </li><li>突出显示“S1”并将其命名为“S1 - 习惯化。” <ol><li>选择添加“时间定位。” </li><li>输入适当的时间（$）和单位（$）， 例如 ，分钟，距离为s下拉框中选择“S2”。时间单位“单位”是最初创建数据库时确定的。 注：创建的过渡时间应改为类似于[之后$分钟GO P = 100％，到S2 图 1是曾经创造了这个事件的例子。 </li></ol></li> &lt;/ OL&gt; </li></ol><img alt="图1" src="/files/ftp_upload/53018/53018fig1.jpg" /> 图1代表编程实例＃1中。在这种情况下一个时间分隔的状态示于其中$用户定义的值，在这里显示分钟，表示当目前的状态应退出到状态2（S2）。 <ol start="9"><li>突出“S2 - PR响应。 <ol><li>选择添加“活动回到”。 </li><li>输入列表名称包含在步骤4.3创建到第一个文本框为L $（ 例如，L1）的渐进比时间表，选择合适的operandum（例如，杆或鼻子戳）的第一个下拉框，选择“ S3“从送下拉框中。 注：创建的事件可以读取类似[如果L $ 1 - 主动GO P = 100％，达到S3。标识符1 - 主动数据库创建时被指定。在这里，1 - 活动指的是插入到交换机的一个operandum。 </li><li>选择“添加时间转到”。 </li><li>检查“R”框中，输入适当的时间和单位（$），并从送选“FIN”下拉框中。 注：创建时间事件可以读取类似，[R（选中），如果$分钟GO P = 100％，达到FIN。这将确保该会议将结束，如果有在后的设定阈值operandum任何活动; 即会话将超时动物隐瞒的强化物配对operandum了设定的时间段响应后。 </li><li>选择添加“活动回到”。 </li><li>检查“R”框中，输入1到第一个文本框，选择适当的operandum从第一个下拉框中的释放，而从送下拉框中选择“S2”。 注：创建的事件可以读取类似，[R（检查）后$ [1]主动GO P = 100％，到S2。此步骤确保于operandum释放（方括号如上所述），这个状态重新进入，以此来重置会话超时阈值（4.4.9.4）。此时，状态将退出当包含在列表中的渐进比完成时（步骤4.4.9.2）或会话超时（步骤4.4.9.4）。因此，每当该operandum被激活时，它计数为逐步比。 ，而且，每次的operandum失活的时间; 如在杆被释放或鼻子戳退出，会话超时阈值（步骤4.4.9.4）复位图2表示编程“S2所有过渡-公关回应。 “ </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53018/53018fig2.jpg" /> 图2.代表性的编程实例＃2。在这里，状态在以下两种条件退出。大号$是4.3步创建的渐进比时间表。经日程COMPLET离子，国家退出到状态3（S3）。注意，该标准不具有R，其允许日程表前进，而不是再次开始在每次重新进入这个状态。国家还可以经过“$”分离开来说明FIN。这是预定的超时阈值后，如果没有响应该窗口中发生的会话将终止。这将在讨论进一步的解释。最后，会话超时阈值后，每operandum释放复位。因此，“[1]”表示operandum释放&#39;1&#39;。 <ol start="9"><li>突出“S3 - PR增强剂和提示。” <ol><li>选择“添加时间去&#39; </li><li>输入适当的时间和单位从送下拉框中提供的增强剂（$），然后选择“S4”。 注：创建时间事件可以读取类似[之后$秒钟GO P = 100％，至S4。 </li></ol></li><li>突出“S4 - 超时。” <ol&gt; <li>选择添加“时间转到&#39; </li><li>输入适当的时间和单位仍然处于非活动状态（$）以下增强剂交付，并从送下拉框中选择“S2”。 注：创建时间事件可以读取类似[之后$秒钟GO P = 100％，到S2。状态&#39;S4 - 超时&#39;可以不需要所有设计。 </li></ol></li><li>选择解决国家。 注：此协议是现在就可以运行。 </li></ol>

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None of the authors have competing or conflicting interests.

这里介绍的方法是制造显微注射插管和microinjectors将阐明的动机的行为的分子底物辅助的有效手段。此方法提供了几个优点。首先，通过制造自己的植入物和microinjectors，新颖的实验参数可以迅速地优化;也就是，一个不需要等待定制组件到达。第二，由于小直径的套管，多个套管可以同时注入。这缩短了所需的手术时间，从而可提高生存性，而且还允许每只动物的多个植入物。第三，用于控制操作室的软件容易地适应渐进比时间表自一个固定的比率范例可以迅速地通过简单地施加含有所需强化程序的事件过渡参数列表转换为渐进比范例。 成为广泛有用的，但是一般的显微注射过程被提出，应该是广泛地适用于现有的几乎任何试剂的显微注射。因此，我们预期，这种技术将继续以类似的高实用程序在将来有轻微变形。通过只改变几个变量，这种方法可以应用到试剂的宽数。这将最常被操纵参数包括该微量从套管伸出的长度，注射的体积，以及注射速度。例如，人们可能想要喷射器，以进一步从套管末端突出，以避免神经胶质疤痕，通常形成围绕慢性植入物。此外，人们可能希望注入量较大。为纹状体病毒显微注射，1微升的体积中通常使用这种卷典型地注入在一段较长的时间（通常10 - 10分钟加3 - 10分钟额外扩散时间）相比，使用d表示药物试剂（通常为0.3 - 0.5微升了2 - 3分钟加1 - 3分钟附加的扩散时间）。用户应该查阅文献和/或凭经验确定最适合自己需要的参数。无论如何，这个过程的成功是关键取决于4个变量：1）套管的长度，2）微量长度，3）微量喷雾图案的注射前的质量，以及4）系统的完整性。因为显微注射位置取决于该微量从套管伸出的深度，当务之急是两插管（步骤1.2.8）和微量长度（后弯曲，步骤2.2.1）都精确所有受试者之间已知的和均匀的。这可以容易地通过容易地拒绝任何实施不是在最终重新测量所需的长度来控制。此外，注射位置只能如果它立即发生的引导套管下方预测。因此，任何微量能源部在测试不是喷长，细流（步骤2.4.6），应予以拒绝。甲质量注射还涉及该系统的完整性在注射前。如果分配所有的水从注入后（装填试剂之前）多点是在实验室观察擦拭，然后泄漏需要纠正（请注意：步骤2.4.8）。此外，如果气泡（步骤2.4.9），其从水在PE20管分离所述药物是不是一个，单泡（填充试剂的微量之后），则喷射器部分堵塞。这种堵塞既可以防止或转移注射。这也可以很容易地补救（注上的步骤2.4.8）。 如果一个希望microinject而动物是在立体框架有三个备选方案。首先，人们可以增加微量颈圈，使得它可以通过立体定向操纵器牢固地保持并延伸得足够远以允许连接到PE20管道的长度。第二，上Ë可以暂时植入插管，并使用标准的微量这里介绍。第三，人们可以使用拉伸和抛光玻璃吸管16,17 这里提出的步骤的一个显著限制是，最好是在良好的处理大鼠中所熟悉的方法进行。因为同样的调查处理的大鼠，每天超过2个月的用于在结果部分中描述的数据大鼠不需要特别的处理程序。这包括每日观察手术植入物和操纵至少2周。然而，大鼠可迅速通过多种技术，用于前预脉冲抑制试验，这可受应力习惯于。这些特殊习惯化技术已被很好地先前详述。43除了这些程序，可取的做法是可以的大鼠习惯于其中缩短microinjectors使用杜显微注射过程环&#39;假&#39;注射。在这些假注射剂，可以认为微量不会突出到组织，以限制组织损伤是至关重要的。换句话说，该微量应不超过14毫米弯曲。因此，需要对这种技术的最佳实施彻底的习惯可能被视为一种限制。 虽然若干行为范例存在测量动机，所述进行比通常用于量化该受试者愿意施加以获得加强件的工作。渐进比范例产生被称为断点的量度，其通常被定义为在最后一个完整比杠杆压力机的最大数目; 即 ，最大响应生成一个加强件21的渐进比是敏感的加强件大小。例如，更高的可卡因（或蔗糖）的剂量产生较高的断点和低级可卡因（或蔗糖）的剂量得到降低breakpoint。21,22因此，断点为动机和/或增强功效的常规使用的代理。21,23-26由于断点的意图是确定当动物停止响应，逐步比范例的一个重要参数是会议的长度。有限会话长度可以穿上断点值的假帽，这可以通过预治疗，异常降低的自我管理，或增加后加固暂停的速度加剧。这变乱可以通过任何数目的方法来克服。 例如 ，会话终止时动物已经版主由平均内输注间隔的某一倍数响应44这种方法的一个更普遍应用于变型是终止会话一次响应具有被扣缴一些保持跨学科常凭经验确定的值。我们已经提供了应用这种方法的步骤4.4.9.11的方法。

Rodents and humans respond in remarkably similar ways to behaviorally relevant stimuli1-3. This suggests that rodents are appropriate subjects for elucidating the molecular substrates of behavior and complex psychiatric conditions4. Understanding the molecular substrates of complex behavioral processes, such as motivation, frequently requires brain microinjection. Both the brain microinjection technique and a primary motivation assay will be presented here. Rats will be used as subjects, but these procedures can readily be adapted to well-handled mice. Included herein are procedures for the manufacture of the required cannulae, obturators (dummy cannulae or stylets), and microinjectors. The method presented is significantly more flexible and more cost-efficient than prefabricated implements. This flexibility will prove valuable when optimizing conditions. Importantly, because the microinjection procedure can be used to test a myriad of hypotheses; the techniques presented here should be broadly applicable. For example, receptor ligands can be microinjected to understand neurochemistry3,5,6; cell-permeable peptides and small-molecules can be microinjected to understand intracellular signaling pathways7-10; toxins, ion channel blockers, or antagonist cocktails can be microinjected to understand circuitry1,11,12.
While the generic protocol presented here can be readily adapted by the user for their particular needs, the procedure is particularly well suited for behavioral assays since microinjection occurs in awake rodents that are only under mild hand restraint. No anesthesia or special restraints are required. This is possible because the brain itself lacks pain sensation. However, if anesthesia is not used, microinjection must occur through cannulae that were previously stereotaxically implanted. This is because nociceptors are present on the scalp, meninges,13 which are the membranes surrounding the brain, and the periosteum,14 which is the membrane covering the skull. It should be noted that microinjection under anesthesia is sometimes desirable. One example is when the virus is being injected, and one may wish to inject virus directly through either stainless steal needles15 or glass pipettes because this can reduce tissue damage and improve transduction efficiency.16,17 The microinjectors described below can be modified for this purpose and suggestions on how to do this can be found in the Discussion. Because other JoVE articles have demonstrated stereotaxic brain cannula implantation,18-20 these procedures will not be covered here.
We present these microinjection procedures together with an assay that quantifies motivation. Several rodent models of motivated behavior are currently in use, such as the runway box and barrier scaling. Here, we describe how to use an operant progressive ratio schedule of reinforcement to quantify motivation where operant responding is being maintained by a reinforcer. Responding on the progressive ratio is responsive to reinforcer magnitude.21,22 Accordingly, this assay is routinely used as a proxy for motivation and/or reinforcing efficacy. 21,23-30 Because several excellent reviews have covered this topic in detail,21,24 we will focus mainly on practical concerns.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cannula Tubing</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>HTXX-26T-60</td>
			<td>26 gauge, Hypotube S/S 316-TW 26GA</td>
		</tr>
		<tr>
			<td>Obturator</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>GWXX-0080-30-05</td>
			<td>33 gauge, Wire S/S 316LVM 0.008 IN</td>
		</tr>
		<tr>
			<td>Microinjector Wire</td>
			<td>MicroGroup</td>
			<td>33RW 304</td>
			<td>33 gauge</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Loctite</td>
			<td>3924AC</td>
			<td>Liquid, Non-gel, can be autoclaved</td>
		</tr>
		<tr>
			<td>Microinjector Plastic Tubing</td>
			<td>Becton Dickson</td>
			<td>427406</td>
			<td>PE20</td>
		</tr>
		<tr>
			<td>Medium Weight Hemostats</td>
			<td>World Precision Instruments</td>
			<td>501241-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher</td>
			<td>09-016</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>#7 Forceps</td>
			<td>Stoelting</td>
			<td>52100-77</td>
			<td>Dumont, Dumostar</td>
		</tr>
		<tr>
			<td>Rotary Tool</td>
			<td>Dremmel</td>
			<td>285</td>
			<td>Two-speeds</td>
		</tr>
		<tr>
			<td>Cut-off Disc</td>
			<td>McMaster Carr</td>
			<td>3602</td>
			<td>15/16&#34; x 0.025&#34;</td>
		</tr>
		<tr>
			<td>Microinjection Pump</td>
			<td>Harvard Apparatus</td>
			<td>PhD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ul Glass Syringe</td>
			<td>Hamilton</td>
			<td>7001KH</td>
			<td>Needle Style: 25s/2.75&#34;/3</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>Fisher</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Kimwipes</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Software</td>
			<td>Coulbourn</td>
			<td>Graphic State</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Chambers</td>
			<td>Coulborun</td>
			<td>Habitest</td>
			<td></td>
		</tr>
	</tbody>
</table>

rodent brain microinjection to study molecular substrates of motivated behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

在这里，我们示出了可与上述方法获得的结果的例子。所示的第一是受病毒载体（图3）转染的典型区域。在一般情况下，在纹状体组织中获得约1毫米3的转染体积。转染的区域的体积可以在使用卡瓦列的方法的连续切片进行定量32重要的是，转染的体积取决于许多因素，例如组织的转染的类型。病毒血清型;子;语速和音量注射;注入的粒子数;注射液pH值;和是否高渗试剂，例如甘露糖醇，使用了33,34典型地，我们microinject 10 12个粒子/ ml，1微升/侧，在10分钟，并允许额外的7分钟之前更换密闭装置。此外，注射液通常是约pH 8。接下来，我们表明，动机可以通过转基因表达被操作（图4）或药理学试剂的显微注射（图5）。 在图4中，我们表达了设计师受体由设计师药物（DREADDs）独家激活。所述DREADD受体编码区之后是一个内部核糖体进入位点和一个mCitrine盒。所述mCitrine允许转染的细胞的方便可视化。该DREADD偶联至异G蛋白Gαq。所述Gαq耦合DREADD的活化可刺激星形细胞，28,35和DREADD本身可以通过氯氮平-N-氧化物（CNO，3毫克/ kg，腹腔）的全身给药被激活。14,22,36训练大鼠杠杆回应乙醇加固，其中3杠杆压力机产生1机会在超过60个连续几天每天1小时的会议喝酒。其次，老鼠被迫禁欲，和Gαq偶联DREADDs表达的伏隔核核心的星形胶质细胞。经过3周禁欲的动机自我ADMI<html>尼斯特尔乙醇通过断点测量。21,27-30激活核伏隔核星形胶质细胞，经全身CNO管理，降低大鼠的积极性恢复乙醇的自我管理后禁欲相比，车辆。重要的是，CNO在这被表达绿色荧光蛋白代替DREADD同样受过训练队列没有影响。 <img alt="图3" src="/files/ftp_upload/53018/53018fig3.jpg" /> 通过显微注射病毒转染的图3代表腹侧纹状体区域。此图说明核的区域伏按照上述方法转染星形胶质细胞。数据从公牛等13重印版权持有人的许可。详情可在出版物和所附补充材料中找到。空白“&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53018/53018fig4.jpg" /> 图4.动机大鼠自己服用乙醇的减少通过激活转基因，这是过度表达于伏核的核心星形胶质细胞。病毒微量注射和通过断点测定动机允许一个星期的病毒表达后。转基因表达的是一个设计师受体由设计师药物（DREADDs）独家激活。甲单向ANOVA（F（2,30）= 3.29，p值= 0.04），随后是薛费事后表明活化DREADD的氯氮平-N-氧化物的全身施用（CNO，3毫克/ kg，腹腔）显著降低，而不是对DR大鼠自我管理乙醇戒断后的动机相比车辆CNO在这被表达绿色荧光蛋白（GFP同样受过训练队列没有影响）EADD。数据从公牛等13重印版权持有人的许可。详情可在出版物和所附补充材料中找到。 <img alt="图5" src="/files/ftp_upload/53018/53018fig5.jpg" /> 图5的显微注射的间隙连接阻滞剂增加大鼠的动机禁欲后自己服用乙醇的两个间隙连接阻滞剂进行了评价的大鼠上的动机它们的效果（通过断点测量）自己服用乙醇：甲氟喹和18- α-甘草次酸（18-α）。的双向方差分析表明，显微注射的间隙连接阻滞剂到伏隔核芯大鼠的动机增加自己服用禁欲后乙醇（F（3,40）= 5.56，p值= 0.003）。数据从公牛等13重印版权持有人的许可。细节可以在该出版物中找到并且附带的补充材料。

Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

啮齿动物脑显微注射学习动机行为的分子基板

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

rodent-brain-microinjection-to-study-molecular-substrates-motivated

Research

JoVE Journal

Behavior

14.2K 次观看.  Virginia Commonwealth University School of Medicine. Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented. 

视频: Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

立体定向显微注射病毒载体表达Cre重组酶研究的靶基因的作用在可卡因条件性位置偏爱

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

科研

行为学

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

揭开节拍耳聋：检测节律紊乱与同步手指敲击和感知定时任务

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond &#8216;yes&#8217; at the end of the sequence, otherwise participants would respond &#8216;no&#8217;. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.&#160;

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

的双任务过程结合快速系列视觉呈现到测试的注意闪烁Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

使用基于活动的厌食症啮齿动物模型来研究神经性厌食症的神经生物学基础

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

联合穿梭盒子训练与电的Cortex记录和刺激作为一种工具来研究感知和学习

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

鼠类精神运动警觉测试（rPVT）：一个在大鼠和小鼠的神经行为评估性能的方法

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Central and Divided Visual Field Presentation of Emotional Images to Measure Hemispheric Differences in Motivated Attention

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed by right frontal regions, while pleasant emotions are processed by left frontal regions. The other theory posits that the right hemisphere is more specialized for the processing of emotional information overall, particularly in posterior regions.
Assessing the different roles of the cerebral hemispheres in processing emotional information can be difficult without the use of neuroimaging methodologies, which are not accessible or affordable to all scientists. Divided visual field presentation of stimuli can allow for the investigation of lateralized processing of information without the use of neuroimaging technology. 
This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention. Future work will pair this paradigm with a more active behavioral task to explore the behavioral impacts on the attentional differences found.

This study compared central versus divided visual field presentations of emotional images to assess differences in motivated attention between the two hemispheres. The late positive potential (LPP) was recorded using electroencephalography (EEG) and event-related potentials (ERPs) methodologies to assess motivated attention.

中央和分割的视觉场表现的情绪图像, 以测量半球的差异, 激发注意

Two dominant theories on lateralized processing of emotional information exist in the literature. One theory posits that unpleasant emotions are processed ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

压力增强恐惧学习--创伤后应激障碍的健壮啮齿动物模型

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...