Name: reinstatement of drug-seeking in mice using the conditioned place preference paradigm
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加泰罗尼亚自治区 Valenciana, PROMETEOII 2014/063;卫生部、社会服务和平等部。政府对国家药物计划、药物依赖性研究项目2014I007 的代表团;经济和竞争力部 (MINECO), 卫生研究所卡洛斯 III. 成瘾性疾病网络 RD12/0028/0005 和 RD16/0017/0007 和欧洲联盟, 菲德基金 "manera de hacer 欧罗巴", PSI2014-51847 R。

所有涉及小鼠及其照料的程序都符合国家、区域和地方法律和条例, 这些法规符合欧洲议会和2010年9月22日理事会关于保护使用动物的指示 2010/63/欧盟。科学目的。瓦伦西亚大学动物使用和护理委员会批准了本议定书。
1. 条件位置偏好的材料和设置:
<ol><li>处理老鼠3天前测试 1-2 分钟每。应该抓住尾部的底座。如果可能, 应支持该机构。这样做是为了尽量减少在处理动物时的疼痛和痛苦, 并防止干扰变量, 如压力。 注: 本实验采用1只雄性小鼠60天 (年轻成人) 重 35-40 克。药物诱发的恢复可以在男女小鼠身上进行。然而, 关于压力引起的恢复, 社会失败范式只为雄性啮齿动物设计, 因为雌性动物并没有在居住的老鼠身上引起积极的反应。</li>
	<li>对于地方调节, 使用相同的盒子, 两个相同的车厢 (30.7 厘米长 x 31.5 厘米宽 x 34.5 厘米高) 分离的一个较小的中央灰色区域 (13.8 长 x 31.5 厘米宽 x 34.5 高)。 注意: 隔间有不同的地板纹理和墙壁颜色 (在黑色的车厢里光滑的地板和白色的一层粗糙的地板)。CPP 箱的每个隔间都包含四红外线光束和六的中央隔间。这允许记录间隔之间的交叉点和动物的位置。</li>
	<li>每天设置房间灯 (关闭或降低)。使用红色灯 (推荐), 因为动物不知觉它并且它为实验者被减弱 (大约40莱克丝测量在 1 m 在地板水平之上)。</li>
	<li>设置程序, 注册15分钟前和后调理测试 (见软件材料表) (图 2)</li>
	<li>准备整个实验所需的可卡因溶液。将0.9% 氯化钠 (生理盐水) 中的盐酸盐溶于 0.1 mL/10 g 体重。漩涡混合物和存储在4°c 在一天结束。 注: 可卡因浓度可能会因研究目的而异。如果目的是评估阈剂量的可卡因, 可以使用1毫克/千克浓度, 这是无效的控制动物, 如上文所示的研究24,35。如果有效剂量是目的, 可以选择6毫克/千克浓度, 这是有效的, 但不诱发恢复24, 或25毫克/千克浓度, 这是有效的, 并促使恢复首选19。</li>
	<li>测试动物在他们的黑暗阶段, 并使用相同的箱子, 每只动物在几天。</li>
	<li>每天把动物带到他们的笼子里的测试室, 让他们不受干扰的15分钟作为一个适应期, 所有的测试相关的噪音。</li>
</ol>2. 测试
<ol><li>收购 注: 在初始自发偏好条件下, 放置调节的过程由三阶段组成: 前置调节、调理和后调理 (图 1)。<ol>
<li>
预调节 (前 C) (3 天) (图 2,图 3,图 4) 注意: 在这里, 将计算 CPP 框中的隔间的中性值。测量每隔间间隔的时间, 然后将其与在后处理测试中同一个隔间中的花费进行比较。<ol>
<li>把动物带到测试室, 让他们习惯15分钟。</li>
			<li>检查断头台是否从笼子中移除。</li>
			<li>设置计算机和程序。输入动物 id 并按 "试用" 和 "开始" 命令 (图 2)。</li>
			<li>将鼠标轻轻地放进中间房间 (灰色隔间), 并离开测试室的最低噪音。</li>
			<li>把所有的动物带回他们的笼子里, 在审判结束后保存数据。</li>
			<li>允许老鼠在连续3天内每天15分钟 (900s) 进入设备的两个车厢。</li>
			<li>在3天, 记下900s 期间在每个隔间中所花费的时间, 并将其保存在下一阶段中的间隔分配 (图 3)。</li>
			<li>在每个组中分配一半的动物, 以接收药物或车辆在一个车厢 (如, 黑色), 另一半在其他车厢 (如白色) (图 4)。 注意: 动物的最终分布必须确保一半的动物被分配到最初的首选隔间, 另一半用于非首选的隔间。另外, 一半动物在一个隔间接受了药物 (例如, 黑一个) 和另一半 (在这种情况下, 白色一个)。</li>
			<li>在两个车厢内以及跨组的组中平衡分数 (测试组差异与方差分析)。作为近似的指导, 值应该在 C 前测试中的 360 370s 左右 (图 4)。注: 在药物配对和车配对的间隔时间内, 在 C 前试验期间, 不应发现明显的差异。</li>
			<li>排除对任何车厢有强烈反感或偏爱的动物。把厌恶看成是在车厢里花费的时间比33% 少, 并且考虑到当动物花了超过67% 的时间在那个车厢里时的偏好。</li>
		</ol>
</li>
		<li>
空调 (4 天) (图 5)
		<ol>
<li>权衡这些动物, 并使用这些重量的可卡因剂量在调节。</li>
			<li>根据体重, 用可卡因或生理盐水准备注射器。</li>
			<li>一次插入一个鼠标腹腔 (ip), 并立即把它轻轻地放到指定的黑色或白色车厢他们的盒子。</li>
			<li>在间隔4小时后, 立即注射药物剂量后, 鼠标被限制到药物配对的间隔为30分钟 (图 5)。</li>
			<li>每天交替这个过程, 开始与可卡因在天1和 3, 并且盐水在天2和4。</li>
			<li>检查在这两种情况下, 通过关闭分隔两个车厢的断头台门, 使中心区域无法进入的禁闭。 注意: 在这个阶段, 没有必要设置 PC 程序。</li>
		</ol>
</li>
		<li>
后调理 (后 C) (1 天)
		<ol>
<li>把动物带到测试室, 让他们习惯15分钟。</li>
			<li>拆卸两个隔间的断头台门。</li>
			<li>设置计算机和程序。输入动物 id 并按 "开始" 命令。</li>
			<li>记录在900s 观察期内每个车厢内未经处理的老鼠所花费的时间。</li>
			<li>当审判结束后, 把所有的动物带回他们的家庭笼子里, 保存数据。</li>
			<li>如果在药物配对的车厢内所用的时间比 c 预检期间大得多, 则认为该药物有诱发位置偏好。如果发生相反的情况, 请考虑对药物配对车厢的反感。</li>
		</ol>
</li>
	</ol></li>
	<li>灭绝
	<ol>
<li>在所有小组/动物中进行每周一次的灭绝会议, 开发出对药物配对车厢的偏爱。</li>
		<li>将动物放置在仪器中 (不断头台) 15 分钟 (与前、后 C 测试相同) (图 1)。 注: 如果在 CPP 中使用的剂量高 (25 毫克/千克), 每周执行1消光会话。如果使用阈剂量 (1 毫克/千克), 则每星期执行2次消光疗程 (例如星期一和星期四)。</li>
		<li>当消光测试中的数据不显示对预 c 测试的差异时, 请考虑该首选项熄灭, 但它们确实显示了 c 后测试的差异。</li>
		<li>重复测试24小时后, 以确认灭绝。</li>
	</ol></li>
	<li>恢复 CPP
	<ol>
<li>
启动诱导恢复
		<ol>
<li>评估在灭绝后注射可卡因24小时的效果。 注: 注射剂量是动物在调理过程中收到的前一剂量的一半。恢复性测试的程序与 c 前和 c 后测试中的相同, 只是动物在试验前15分钟内在一个非特遣队中获得可卡因剂量。在重复性消失/恢复经验23后, 药物启动在诱导恢复方面的功效可以提高, 尽管剂量的顺序似乎没有在获得36的结果中发挥作用。</li>
			<li>将动物从测试室 (非特遣队) 带到不同的房间 (图 6)。</li>
			<li>注射一半以前的可卡因剂量 (例如, 如果动物的条件是25毫克/千克可卡因, 启动剂量将是12.5 毫克/千克)。</li>
			<li>把他们带回他们的 vivarium 15 分钟。</li>
			<li>带他们进入测试室, 并立即将动物放在仪器 (没有断头台门分开的隔间) 15 分钟 (与前和 C 后测试相同)。</li>
			<li>考虑在最后一次消光疗程中, 药物配对间隔时间与恢复试验之间的差异, 以此来衡量药物诱导的恢复程度。</li>
			<li>每次减少剂量, 直到确认无效时, 重复消失恢复程序。 注: 盐水注射控制可用于控制可卡因启动 (而不是注射本身) 的能力, 以诱导恢复。</li>
		</ol>
</li>
		<li>
应力诱发恢复
		<ol>
<li>评估在消失后应激恢复24小时的效果 注: 恢复测试与 C 后 (免费移动15分钟) 中进行的检查相同, 只是在一次竞赛遭遇社会失败后, 动物被测试15分钟 (图 6)</li>
			<li>把动物带到 CPP 室的另一个房间去。</li>
			<li>将实验鼠标放在塑料保持架上, 与一个具有同等年龄和体重的好斗的对手性别匹配 (图 6)。 注: 竞赛遇到的是一个10分钟的测试, 在中性透明塑料笼 (23x13.5x13cm) 与失败的结果, 实验鼠标。为了准备攻击性的对手, 首先将不同年龄和体重的动物与实验性老鼠进行3周的分离。训练他们获得战斗经验, 并测试他们有高水平的侵略性的领土行为。为了获得积极的 conspecifics, 老鼠必须被隔离至少3周, 然后训练攻击和攻击行为。动物被训练在一个中性的环境, 面对他们成对。我们让他们咬对手2或3次, 并把他们从笼子里拿出来, 总是结束训练前的两只老鼠表现出的屈服, 使遭遇的体验强化了攻击性的行为。在遭受侵略之后 (从攻击性的同种的威胁和攻击), 实验鼠应该提出退避或出逃和防御/顺从行为。标准是采取一个特定的失败的姿势, 其特点是直立顺从的位置, 跛行的前爪, 向上倾斜的头, 和收回耳朵 (图 6)。根据伯克等人, 2016, 我们认为, 如果入侵者显示一个顺从的仰卧姿势 > 8 s 或如果发生了13攻击叮咬, 社会接触就会提前终止。另一种选择是筛查没有重大伤害危险的动物, 这可能是为了使动物在一个洞的网壳中加速, 而不是足够大的动物通过。 攻击可以被发现, 而不服从动物的直接攻击行为。 如果动物在直径1cm 和/或直接暴露肌肉的情况下受伤害, 它被排除在研究之外, 应该被安乐死。</li>
			<li>经过10分钟的竞赛遇到, 把老鼠带进试验室, 立即将动物放在 CPP 装置 (没有断头台门分隔隔间) 15 分钟 (与前和 C 测试相同)。</li>
		</ol>
</li>
	</ol></li>
</ol>3. 统计分析
注: 理想的样品大小应至少为每组15只动物。
<ol><li>用混合方差分析法和两个层次 (对照组, 实验组, 两组的例子在图 7c中) 对组药物配对间的平均时间进行定量分析, 其中一个在可变天数内,2级 (前 c 和后 c)。 注意: 在对照组, 动物接受药物, 但没有环境操作。另一方面, 在实验组中, 动物暴露在不同的环境条件下。比较后 c 天的药物配对室的时间, 并尊重 c 前测试, 看看是否有任何重大差异的药物敏感性。</li>
	<li>此外, 执行 Bonferroni 后的分析, 以检查任何一个重要的分数。</li>
	<li>在实验过程中, 通过 Student´s t 检验的方法, 分析与灭绝和恢复值相关的数据, 以继续进行消光恢复过程, 但在最后一刻, 分析它们在变量 (天数) 内具有与每个组不同的级别数的方差分析。 注: 当比较的数量增加时, 将会有显著几率的数量也会增加。因此, 为了避免获得误报 (1 型错误), 我们 ' 纠正 ' p 值, 从而使测试更保守的 Bonferroni 校正多 t 测试。然而, 对于恢复试验, 应在恢复试验和最后的消光试验之间进行比较。因此, 没有必要进行更正。</li>
</ol>

作者没有什么可透露的。

药物成瘾研究的重点是发展治疗, 减少渴求, 从而减少易复发的脆弱性。由于 CPP 范式的恢复模式, 有可能研究不同的程序和环境因素对复发的影响, 这是未来研究的重点。有一些重要的问题需要考虑, 因为 CPP 范式是一个对环境因素高度敏感的测试。
修改和疑难解答:恢复模式的 CPP 版本应根据感兴趣的药物进行修改。有不同的因素, 可能会影响到条件偏好的大小, 如...

条件位置偏好 (CPP) 范式提供了一个简单的方法来评估不同刺激1,2诱导的条件奖励, 并已广泛用于研究成瘾药物的条件奖励效果3.它是基于巴甫洛夫调节, 评估药物相关环境提示的动机价值, 以维持成瘾行为4。在这个模型中, 环境线索获得二次食欲属性 (条件奖励效果) 配对与一个初级强化3。例如, 最初中性的地方 (如 CPP 笼中一个隔间的颜色) 与在某些调理疗程5中滥用药物的具体效果成对, 而另一隔间则与注射车辆有关。根据调理, 如果动物花更多的时间在以前与药物相关的车厢里, 假设 CPP 已经发展了3。当动物对与药物有关的环境线索给予积极的价值时, 这种偏爱的确立就会实现, 这是主要的强化。因此, 动物将执行行为药物寻求反应, 以回应这些上下文线索6。CPP 模型允许对药物的阈剂量的奖励性质进行评估, 表明在特定条件下的动物 (例如, 以前遭受过社会失败) 是否更容易受到伤害, 并且对剂量敏感。不有效在天真的动物7。
CPP 模型也被用来评估灭绝/恢复作为一个动物模型研究复发3, 这是本议定书的目的。有三不同的阶段: 收购、灭绝和恢复 (图 1)。在 cpp 恢复模型中, 动物首先获得用于药物配对的舱的 cpp, 然后他们执行几个消光疗程。我们将灭绝定义为动物减少其行为反应的时间, 它的近似值被被去除的条件奖励刺激 (例如, 药物)8。在灭绝期间, 动物在没有药物的情况下探索隔间, 使获得的偏爱逐渐衰减9。需要考虑的一个重要问题是, 动物在灭绝期间所表现出的行为变化 (在药物配对的间隔时间逐渐减少) 可能是由于新的学习过程与先前所学的反应相竞争,或由于主题3的内部激励状态的减少。最后, 通过上下文或药物线索重建地方偏好将是我们恢复1的模式。
管理相关药物的启动注射可以恢复偏爱, 这被认为是对上下文线索的近似的重建。药物启动恢复发生的原因是持续记忆的药物的愉悦效果, 这导致渴望和激励动物寻求与奖励有关的环境线索。
CPP 恢复模型的一些优点是, 该程序是无侵入性的 (与自我管理相比, 需要手术), 成本低廉, 而且简单。此外, 这个模型有一个良好的标准有效性, 因为它模仿的人发生在10,11, 诱导恢复与刺激, 诱发复发, 如再次接触药物12,13或压力14。
还有其他技术, 如灭绝--静脉注射自我管理的恢复模式。在这里, 动物按一个杠杆来自我管理的药物, 允许评估的行为反应的动物, compulsivity, 和动机14,15,16。cpp 对自我管理程序的主要好处是, cpp 恢复被认为是反映了与药物配对的情境刺激的激励动机价值的重新激活, 包括这种方法的再现。行为到上下文17。此外, 非药物刺激, 如压力, 也可以诱发恢复18,19。例如, 一项自我管理研究表明, 在足部休克或克制压力20后, 对恢复海洛因摄入量没有影响。作者讨论说, 这是不成功的, 因为这些压力在自我管理室外测试在不同的背景下。相比之下, 在使用 cpp 恢复模型时, 吗啡诱导的 cpp 在使用相同的压力源后有明显的重建, 并在不同的环境下应用于 cpp 和不同时间 (0 和15分钟的压力暴露后)18.
文献中的几项研究显示了不同的药物和应力诱发恢复方法。一方面, 用吗啡5、21、22、23、可卡因24、25、安非他明26, 对大鼠和小鼠进行药物诱导恢复的报告, 27, 乙醇28,29, 34-Methylenedioxymethamphetamine (摇头丸)30。另一方面, 暴露于压力可能是易受药物滥用影响的一个决定性因素。压力是已知的增加奖励效果的药物7,31,32和他们的作用在复发是很好建立 33,34。例如, 在与同种恢复吗啡和可卡因 CPP18,19的社会互动中失败。此外, 遭受多次社会失败的动物更容易受到阈剂量可卡因的条件奖励效应的影响, 并恢复了极低剂量的可卡因7的偏爱。
CPP 恢复模型的应用是评估动物易复发性的一种有用和敏感的方法, 并允许对不同的微妙环境操作进行评估, 这些操作是威胁人类复发的主要诱因, 如药物或压力引起的恢复。

<table><tbody><tr><td>MONPRE 2Z software</td><td>CIBERTEC S.A., Spain</td><td>N/A</td><td></td></tr><tr><td>Identical Plexiglas boxes with two equal sized compartments separated by a grey central area.</td><td>CIBERTEC S.A., Spain</td><td>N/A</td><td>The compartments have different colored walls (black vs white) and distinct floor textures (fine grid in the black compartment and wide grid in the white one). All boxes are equipped with four infrared light beams in each compartment of the box and six in the central area which allow the recording of the position of the animal and its crossings from one compartment to the other.</td></tr><tr><td>Cocaine hydrochloride</td><td>Laboratorios Alcaliber S.A., Madrid, Spain</td><td>N/A</td><td></td></tr><tr><td>Animals: mice of the OF1 outbred strain</td><td>Charles River, Barcelona Spain</td><td>N/A</td><td>Male mice who arrive to the laboratory on PND 42 and on PND 60 perform the CPP test. These animals are specially indicated for social defeat and aggression models.</td></tr><tr><td>Standard Diet- Teklad Global Diet 2014</td><td>Supplied by Harlan Laboratories Models, S. L. (Barcelona, Spain)</td><td></td><td>13 kcal % fat, 67 kcal % carbohydrates and 20% kcal protein; 2.9 kcal/g</td></tr><tr><td>High fat diet TD.06415</td><td></td><td></td><td>45 kcal % fat, 36 kcal % carbohydrates and 19% kcal protein; 4.6 kcal/g</td></tr></tbody></table>

reinstatement of drug-seeking in mice using the conditioned place preference paradigm

本议定书将条件性场所偏好 (CPP) 描述为吸毒成瘾复发的模型。在这个模型中, 动物首先被训练来获得在药物配对的车厢内的条件位置偏好, 并在后调理测试后, 他们执行几个疗程, 以扑灭既定的偏好。CPP 允许评估与环境线索有关的药物的条件奖励效果。然后, 被扑灭的 CPP 可以由非特遣队管理的药物的启动剂量, 并接触到压力刺激恢复。这两种方法都将在这里解释。当动物 reinitiates 行为反应时, 被认为已经发生了条件性奖励的恢复。
该协议的主要优点是它具有非侵入性、成本低廉、操作简单、有效性标准好等特点。此外, 它允许研究不同的环境操作, 如压力或饮食, 这可以调节复发的药物寻求行为。然而, 一个限制是, 如果研究人员的目的是探索药物的动机和主要的增强作用, 它应该辅以自我管理程序, 因为它们涉及操作性反应的动物。

首先, 在图 7中使用成年 OF1 雄性小鼠, 显示了启动和应力诱发恢复的代表性结果。
图 7a b中的数据表示在药物配对的前、后 c 试验中所花费的时间, 采用重复测量方差分析法, 在受试者可变天数 (比较前 c 和后 c)。结果显示, 可变日的显著影响 [F (134) = 51.179; p < 0.001...

Watch this Scientific Journal Video about Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm at JoVE.com

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

本协议将条件位置偏好 (CPP) 描述为复发的模型。这一程序允许测量实验动物的复发, 考虑到药物相关的环境暗示的影响, 因为戒瘾成瘾者的渴求和复发是目前药物滥用治疗方案的重点。

用条件位置偏好范式恢复小鼠药物寻毒作用

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

reinstatement-drug-seeking-mice-using-conditioned-place-preference

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11.5K 次观看.  Universitat de València. 本协议将条件位置偏好 (CPP) 描述为复发的模型。这一程序允许测量实验动物的复发, 考虑到药物相关的环境暗示的影响, 因为戒瘾成瘾者的渴求和复发是目前药物滥用治疗方案的重点。

视频: Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

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

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats 

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

神经科学

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

在鼠标的操作性感觉寻求

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

可卡因诱导行为敏和小鼠位置偏爱评估

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

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

评估大鼠蔗糖渴求育成的一个通用方法

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

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

一种用于大鼠渴望培养的条件场所偏好协议

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

研究压力诱导的海洛因复发的程序寻求惩罚强加的禁欲

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

可卡因使用障碍大鼠模型中提示反应性测定的一种协议

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.