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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	<li>Koob, G. F., Volkow, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurocircuitry+of+addiction.">Neurocircuitry of addiction.</a> Neuropsychopharmacology. 35 (1), 217-238 (2010).</li><li>Kalivas, P. W., Peters, J., Knackstedt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+and+brain+circuits+in+drug+addiction.">Animal models and brain circuits in drug addiction.</a> Mol Interv. 6 (6), 339-344 (2006).</li><li>Bossert, J. M., Marchant, N. J., Calu, D. J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reinstatement+model+of+drug+relapse:+recent+neurobiological+findings,+emerging+research+topics,+and+translational+research.">The reinstatement model of drug relapse: recent neurobiological findings, emerging research topics, and translational research.</a> Psychopharmacology (Berl). 229 (3), 453-476 (2013).</li><li>Connor, E. C., Chapman, K., Butler, P., Mead, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+predictive+validity+of+the+rat+self-administration+model+for+abuse+liability).">The predictive validity of the rat self-administration model for abuse liability).</a> Neurosci Biobehav Rev. 35 (3), 912-938 (2011).</li><li>Watson, D. J., 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=Selective+blockade+of+dopamine+D3+receptors+enhances+while+D2+receptor+antagonism+impairs+social+novelty+discrimination+and+novel+object+recognition+in+rats:+a+key+role+for+the+prefrontal+cortex.">Selective blockade of dopamine D3 receptors enhances while D2 receptor antagonism impairs social novelty discrimination and novel object recognition in rats: a key role for the prefrontal cortex.</a> Neuropsychopharmacology. 37 (3), 770-786 (2012).</li><li>Takahashi, A., Shimamoto, A., Boyson, C. O., DeBold, J. F., Miczek, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(B)+receptor+modulation+of+serotonin+neurons+in+the+dorsal+raphe+nucleus+and+escalation+of+aggression+in+mice.">GABA(B) receptor modulation of serotonin neurons in the dorsal raphe nucleus and escalation of aggression in mice.</a> J Neurosci. 30 (35), 11771-11780 (2010).</li><li>Spencer, R. C., Klein, R. M., Berridge, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychostimulants+act+within+the+prefrontal+cortex+to+improve+cognitive+function.">Psychostimulants act within the prefrontal cortex to improve cognitive function.</a> Biol Psychiatry. 72 (3), 221-227 (2012).</li><li>Bowers, M. S., 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=Activator+of+G+protein+signaling+3:+a+gatekeeper+of+cocaine+sensitization+and+drug+seeking.">Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking.</a> Neuron. 42 (2), 269-281 (2004).</li><li>Abudara, V., 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=The+connexin43+mimetic+peptide+Gap19+inhibits+hemichannels+without+altering+gap+junctional+communication+in+astrocytes.">The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes.</a> Front Cell Neurosci. 8, 306 (2014).</li><li>Cahill, E., 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=D1R/GluN1+complexes+in+the+striatum+integrate+dopamine+and+glutamate+signalling+to+control+synaptic+plasticity+and+cocaine-induced+responses.">D1R/GluN1 complexes in the striatum integrate dopamine and glutamate signalling to control synaptic plasticity and cocaine-induced responses.</a> Mol Psychiatry. 19 (12), 1295-1304 (2014).</li><li>McFarland, K., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circuitry+mediating+cocaine-induced+reinstatement+of+drug-seeking+behavior.">The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior.</a> J Neurosci. 21 (21), 8655-8663 (2001).</li><li>Fuchs, R. A., Branham, R. K., See, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+neural+substrates+mediate+cocaine+seeking+after+abstinence+versus+extinction+training:+a+critical+role+for+the+dorsolateral+caudate-putamen.">Different neural substrates mediate cocaine seeking after abstinence versus extinction training: a critical role for the dorsolateral caudate-putamen.</a> J Neurosci. 26 (13), 3584-3588 (2006).</li><li>Ebersberger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+meningeal+innervation:+aspects+and+consequences+of+chemosensitivity+of+meningeal+nociceptors.">Physiology of meningeal innervation: aspects and consequences of chemosensitivity of meningeal nociceptors.</a> Microsc Res Tech. 53 (2), 138-146 (2001).</li><li>Netter, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Musculoskeletal System Part I: Anatomy; Physiology, and Metabolic Disorders. 8, (1987).</li><li>Ferguson, S. M., Eskenazi, D., Ishikawa, M., Wanat, M. J., Phillips, P. E., Dong, Y., Roth, B. L., Neumaier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+neuronal+inhibition+reveals+opposing+roles+of+indirect+and+direct+pathways+in+sensitization.">Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization.</a> Nat Neurosci. 14 (1), 22-24 (2011).</li><li>Gonzalez-Perez, O., Guerrero-Cazares, H., Quinones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs,+viral+vectors,+or+cell+transplants.">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> J Vis Exp. (46), (2010).</li><li>Inquimbert, P., Moll, M., Kohno, T., Scholz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+injection+of+a+viral+vector+for+conditional+gene+manipulation+in+the+mouse+spinal+cord.">Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord.</a> J Vis Exp. (73), (2013).</li><li>Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography.">Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.</a> J Vis Exp. (67), (2012).</li><li>Fornari, R. V., 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=Rodent+stereotaxic+surgery+and+animal+welfare+outcome+improvements+for+behavioral+neuroscience.">Rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience.</a> J Vis Exp. (59), e3528 (2012).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> J Vis Exp. (20), e880 (2008).</li><li>Richardson, N. R., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+schedules+in+drug+self-administration+studies+in+rats:+a+method+to+evaluate+reinforcing+efficacy.">Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy.</a> J Neurosci Methods. 66 (1), 1-11 (1996).</li><li>Cheeta, S., Brooks, S., Willner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reinforcer+sweetness+and+the+D2/D3+antagonist+raclopride+on+progressive+ratio+operant+performance.">Effects of reinforcer sweetness and the D2/D3 antagonist raclopride on progressive ratio operant performance.</a> Behav Pharmacol. 6 (2), 127-132 (1995).</li><li>Roberts, D. C., Morgan, D., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+rat+addicted+to+cocaine.">How to make a rat addicted to cocaine.</a> Prog Neuropsychopharmacol Biol Psychiatry. 31 (8), 1614-1624 (2007).</li><li>Arnold, J. M., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+fixed+and+progressive+ratio+schedules+used+to+examine+the+neural+substrates+of+drug+reinforcement.">A critique of fixed and progressive ratio schedules used to examine the neural substrates of drug reinforcement.</a> Pharmacol Biochem Behav. 57, 441-447 (1997).</li><li>Hodos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+as+a+measure+of+reward+strength.">Progressive ratio as a measure of reward strength.</a> Science. 134 (3483), 943-944 (1961).</li><li>Depoortere, R. Y., Li, D. H., Lane, J. D., Emmett-Oglesby, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+self-administration+of+cocaine+in+rats+under+a+progressive-ratio+schedule.">Parameters of self-administration of cocaine in rats under a progressive-ratio schedule.</a> Pharmacol Biochem Behav. 45 (3), 539-548 (1993).</li><li>Bowers, M. S., 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=Nucleus+accumbens+AGS3+expression+drives+ethanol+seeking+through+G+betagamma.">Nucleus accumbens AGS3 expression drives ethanol seeking through G betagamma.</a> Proc Natl Acad Sci U S A. 105 (34), 12533-12538 (2008).</li><li>Bull, C., 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=Rat+Nucleus+Accumbens+Core+Astrocytes+Modulate+Reward+and+the+Motivation+to+Self-Administer+Ethanol+after+Abstinence.">Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence.</a> Neuropsychopharmacology. 39 (12), 2835-2845 (2014).</li><li>Hopf, F. W., Chang, S. J., Sparta, D. R., Bowers, M. S., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motivation+for+alcohol+becomes+resistant+to+quinine+adulteration+after+3+to+4+months+of+intermittent+alcohol+self-administration.">Motivation for alcohol becomes resistant to quinine adulteration after 3 to 4 months of intermittent alcohol self-administration.</a> Alcohol Clin Exp Res. 34 (9), 1565-1573 (2010).</li><li>Hopf, F. 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=Reduced+nucleus+accumbens+SK+channel+activity+enhances+alcohol+seeking+during+abstinence.">Reduced nucleus accumbens SK channel activity enhances alcohol seeking during abstinence.</a> Neuron. 65 (5), 682-694 (2010).</li><li>Salerni, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Engineering Adhesives for Repeated Sterilization. , (2015).</li><li>Gundersen, H. J., Jensen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+systematic+sampling+in+stereology+and+its+prediction.">The efficiency of systematic sampling in stereology and its prediction.</a> J Microsc. 147 (Pt 3), 229-263 (1987).</li><li>Mastakov, M. Y., Baer, K., Xu, R., Fitzsimons, H., During, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+injection+of+rAAV+with+mannitol+enhances+gene+expression+in+the+rat+brain.">Combined injection of rAAV with mannitol enhances gene expression in the rat brain.</a> Mol Ther. 3 (2), 225-232 (2001).</li><li>Burger, C., Nguyen, F. N., Deng, J., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+mannitol-induced+hyperosmolality+amplifies+rAAV2-mediated+striatal+transduction+to+a+greater+extent+than+local+co-infusion.">Systemic mannitol-induced hyperosmolality amplifies rAAV2-mediated striatal transduction to a greater extent than local co-infusion.</a> Mol Ther. 11 (2), 327-331 (2005).</li><li>Agulhon, C., 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=Modulation+of+the+autonomic+nervous+system+by+acute+glial+cell+Gq-GPCR+activation+in+vivo.">Modulation of the autonomic nervous system by acute glial cell Gq-GPCR activation in vivo.</a> J Physiol. 591 (22), 5599-5609 (2013).</li><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G+protein-coupled+receptors+potently+activated+by+an+inert+ligand.">Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.</a> PNAS. 104 (12), 5163-5168 (2007).</li><li>Cruikshank, S. J., 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=Potent+block+of+Cx36+and+Cx50+gap+junction+channels+by+mefloquine.">Potent block of Cx36 and Cx50 gap junction channels by mefloquine.</a> PNAS. 101 (33), 12364-12369 (2004).</li><li>Miguel-Hidalgo, J., Shoyama, Y., Wanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+gliotoxins+or+a+gap+junction+blocker+in+the+prelimbic+cortex+increases+alcohol+preference+in+Wistar+rats.">Infusion of gliotoxins or a gap junction blocker in the prelimbic cortex increases alcohol preference in Wistar rats.</a> J Psychopharmacol. 23 (5), 550-557 (2008).</li><li>Bowers, M. S., Lake, R. W., Rubinchik, S., Dong, J. Y., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+Homer+1a+function+in+the+nucleus+accumbens+using+adenovirus+gene+transfer+technology.">Elucidation of Homer 1a function in the nucleus accumbens using adenovirus gene transfer technology.</a> Ann N Y Acad Sci. 1003, 419-421 (2003).</li><li>Mackler, S. A., 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=NAC-1+is+a+brain+POZ/BTB+protein+that+can+prevent+cocaine-induced+sensitization+in+the+rat.">NAC-1 is a brain POZ/BTB protein that can prevent cocaine-induced sensitization in the rat.</a> J Neurosci. 20 (16), 6210-6217 (2000).</li><li>Geyer, M. A., Swerdlow, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+startle+response,+prepulse+inhibition,+and+habituation.">Measurement of startle response, prepulse inhibition, and habituation.</a> Curr Protoc Neurosci. 8, Unit 8.7 (2001).</li><li>Bedford, J. A., Bailey, L. P., Wilson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+reinforced+progressive+ratio+performance+in+the+rhesus+monkey.">Cocaine reinforced progressive ratio performance in the rhesus monkey.</a> Pharmacol Biochem Behav. 9 (5), 631-638 (1978).</li></ol>

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><tbody><tr><td>Cannula Tubing</td><td>Amazon Supply/ Small Parts</td><td>HTXX-26T-60</td><td>26 G, 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&quot; x 0.025&quot;</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&quot;/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

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

14.3K 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

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

科研

行为学

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 ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

转录动力学的脑组织样本在行为经验综合分析

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

神经科学

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

多功能神经科学技术的适应性角度立体定向方法

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

通过特洛多毒素微注射可逆灭活，揭开鼠脑离散区在排卵调节中的作用

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...