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, 如持续时间和数量的调理疗程。关联的数量通常从一到六不等, 四是最常见的。根据药物的特点及其药代动力学, 建立了调节试验的持续时间。
CPP 研究通常涉及许多试验, 因此使用可减少所需时间的协议是很有用的。实现这一目标的一个方法是减少试验次数, 另一种方法是每天执行两次调理, 如本议定书所示。在这种情况下, 必须确保前一届会议的药物影响不会拖到第二届会议39, 这就是为什么选择可卡因-车辆会议之间间隔四小时的原因。例如, 在调节阶段可卡因的配对时间应为每24小时4天, 配对期为30分钟。相反, 乙醇的调理过程的时间过程应该是每48小时, 总共8天, 每天1次调理, 和交替的车辆和乙醇, 最后总共有4乙醇配对。乙醇配对应有短时间, 如5分钟, 因为更多的时间可以产生厌恶状态。有效剂量范围从 1-2 克/千克和阈剂量为0.75 克/千克40。启动诱导恢复应为前一剂量的一半, 该仪器应该是相同的。
然而, 每天选择做1或2次的疗程可能会影响某些特殊滥用药物的结果。例如, 在摇头丸的两个会话协议的情况下, 条件偏好的后处理没有观察到, 而这可以通过执行一个单一的协会每天30, 所以摇头丸也应管理每48小时, 但调节期应为30分钟。
有两种主要的灭绝方法可以在建立 CPP 后运行, 无论是在以前的药物和车辆条件舱41,42, 每天注射车辆限制动物,43, 或通过重复 CPP 测试, 让动物探索三个车厢没有断头台, 直到首选是不再观察7,18,30,38。在本议定书中, 我们使用了后者, 因为它是一种自发灭绝的类型, 没有任何与调理有关的药理或行为操作 (更类似于人类成瘾者), 但这两种方法都是可以接受的,有效的, 因为文学已经显示了多年。
技术的局限性:CPP 范式和恢复模式的最重要限制是对任何环境变化都非常敏感。因此, 在整个过程中, 房间和 vivarium 条件应保持稳定和恒定。应特别注意与异性伴侣住在同一个房间里的一些方面, 有像轮子这样的工件的笼子, 它们会发出特定的和恒定的噪音, 或者光线条件。除了控制这些变量, 研究人员的作用也很关键, 因为它们可以诱发动物的压力水平的增加, 并干扰结果。
对于现有方法的意义:至于其他方法, 在 CPP 版本的恢复模式, 研究员是谁管理的药物对动物, 对比的自律模式, 评估 animal´s 动机服用药物。重要的是我们要考虑到这一点。这意味着我们不是通过操作性反应或药物的主要增强作用来评估动机。正如我们提到的, CPP 是一个有用的模型, 用来评估环境线索对物质滥用疾病复发的关键作用, 这实际上是调节人类复发的主要因素。
未来应用:CPP 恢复模型的未来应用应面向所谓的渴求孵化, 它包括在药物禁欲后的提示反应性逐渐增加, 模仿人类的渴求。现在, 在 CPP 范式中很少知道这一点。几年前, 只有一项研究表明, CPP 也可以测量这个潜伏期44。最近, 评估了在 CPP 中与吗啡配对的环境线索是否会增加45戒断期的行为反应。这项研究发现, 随着时间的推移, cpp 车厢的输入数量不断增加, 这反映了渴望的孵化, 并表明在 cpp 范式中可以区分奖励记忆和药物渴求。
关键步骤: 协议中有几个关键步骤。为了确认动物恢复了他们的喜好, 灭绝数据应该反映出偏好的真正消亡, 在恢复试验前得到确认。从消光测试中得到的数据不应显示与 c 前测试有关的差异, 而是对 c 后测试的区别。
关于社会压力导致的恢复版本, 我们应该已经证实, 动物已经被击败, 因为一个积极的社会接触, 不结束在失败显示保护效果阻碍恢复的地方可卡因的偏爱。如果实验动物没有被击败, 他们将被排除在研究之外。我们建议记录和评估竞赛的遭遇, 测量攻击行为或提交行为的滞后时间、袭击次数以及这些行为所花费的时间。我们认为实验动物在收到至少2次攻击 (叮咬) 并显示提交信号 (如图 6所示) 时被击败。在所有情况下, 相遇持续10分钟。

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], 因为所有的动物都在 c 天后, 比 c 前产生了偏爱。所有动物都有25毫克/千克可卡因的条件, 当灭绝达到时, 一组接受了12.5 毫克/千克可卡因的注射剂量, 以测试恢复 (图 7a), 另一个则受到社会互动, 其中实验动物被一个好斗的对手打败了 (图 7b)。两次事件后15分钟, 动物在相应的 CPP 盒中进行了测试。
启动诱导恢复 (图 7a) 是随后减少启动剂量 (前半), 直到启动剂量被证实是无效的。剂量为: 12.5, 6.25, 3.125, 和1.56 毫克/千克可卡因。Student´s t 检验显示, 对12.5 毫克/千克可卡因恢复与灭绝会议 (t 统计 (t) = 20.589, 自由度 (d.f.) = 17 的显著影响;p < 0.001) 和后续剂量 (6.25 毫克/千克: t = 18.356; 3.125 毫克/千克: t = 14.260; 1.56 毫克/千克: t = 16.934 d.f. = 17; p < 0.001)。启动与0.78 毫克/公斤没有导致恢复。
关于压力引起的恢复 (见图 7b), 暴露在一个侵略性的动物和击败15分钟之前的测试, 以恢复先前熄灭的偏好 (t = 26.810; d.f. = 15, p < 0.001)19.
其他环境操作, 如在 CPP 程序前遭受反复的社会失败, 修改阈剂量可卡因的奖赏效果 (图 7c)。对变量 "应力" 与变量 "天" 之间的混合方差分析进行了研究, 并对相互作用应力 x 天 (F (129) = 4.144; p < 0.05] 进行了显著的影响。仅社会上被击败的动物陈列了倾向于药物配对的室以阈药量可卡因, 在标准动物7被证明是无效的。另外, 在偏爱的绝种以后, 社会上被击败的动物恢复了他们的偏爱以仅0.5 毫克/公斤可卡因启动 (t = 25.484 d.f. = 17; p < 0.001)。
第二, 作为一个消极的结果, 我们可以看到如何恢复可以防止与积极的社会互动与非侵略性的同种 (图 8a)。因此, 重要的是要评估动物是否被击败或没有在一次社会接触37。
另一个防止恢复的消极结果的例子可以是营养操作, 例如高脂肪的饮食摄入。首先, 方差分析显示了可变天数的影响 [F (126) = 21.527; p < 0.001], 因为这两组都发展了药物配对室 (p < 0.01) 的首选 (图 8b)。灭绝后, 我们的结果表明, 动物转向高脂肪饮食后, C 后测试降低他们对药物启动诱导恢复的敏感性, 因为他们没有恢复首选与 6.25mg/千克可卡因剂量38。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56983/56983fig1.jpg"> 图 1: 被适应的地方偏爱的实验做法.它由三个阶段组成: 预调节、调节和后调理。有4个调理日, 该程序每天交替, 开始与可卡因在天1和3和车辆在天2和4。然后, 进行灭绝和恢复会话。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56983/56983fig2.jpg"> 图 2: 用于 c 前和 c 后测试的软件使用会话, 其中记录了每隔间的时间和条目数.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56983/56983fig3.jpg"> 图 3: 数据在 c 前和 c 后测试之后.在这里, 记录在测试期间获得的数据。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56983/56983fig4.jpg"> 图 4: 在下一阶段的条件下, 对动物进行无偏见的分配.在白色和黑色的隔间中, 取最后一个前 C 测试日的数据。一半的动物被分配接受药物或车辆在一个车厢 (黑色或白色), 另一半在其他车厢。排除显示强烈反感或偏爱的动物。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56983/56983fig5.jpg"> 图 5: 最后分配车辆和可卡因配对的会议.此图显示对配对顺序的交替。在1和3天, 一只动物 (例如, 1.1) 第一次接触到可卡因配对的隔间 (白色的), 在四小时后, 暴露在车辆配对的车厢 (黑色的)。在2和4天, 配对的顺序是颠倒的, 并且动物首先暴露在车配对 (黑) 隔间, 并且, 在四小时以后, 对可卡因配对 (白色) 隔间。由于这个程序是无偏和平衡的, 同时调节会话可以进行两个动物使用相同的箱子 (如动物1.1 和 2.4, 3.1 和 1.3), 这使我们能够减少专门用于实验程序。请注意, 这些对动物遵循的顺序配对通过调理阶段。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56983/56983fig6.jpg"> 图 6: 恢复的两种模式.(a) 启动性恢复和 (b) 应激导致的恢复。(a) 动物在非特遣队的房间接受药物注射, 并被带到 vivarium;15分钟后, 他们在 CPP 箱中进行测试。(b) 在非特遣队室中, 动物在竞赛相遇时被击败10分钟, 然后在 CPP 箱中进行测试。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56983/56983fig7.jpg"> 图 7: 代表积极的结果.启动 (a) 和应力诱发 (b) 恢复25毫克/千克可卡因 CPP。(c) 社会失败对阈剂量可卡因 (1 毫克/千克) 的奖励效应的影响。条形表示在预处理 (白色)、调理后 (黑色)、最后的消光疗程 (浅灰色) 和恢复 (暗灰色) 期间, 在药物配对车厢中所用秒数的平均时间。15分钟的恢复试验被评估后, 注射剂量的一半以前的剂量收到。* p < 0.05;** < 0.01;p < 0.001 显著差异与前 c (方差分析与内部变量天数 (比较前 c 和后 c) 或前消光会话 (学生的 t 测试)。这些数字已由7、19修订。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56983/56983fig8.jpg"> 图 8: 有代表性的负面结果.保护环境操作对可卡因奖励效果的影响。(a) 恢复试验前积极的社会互动的影响。(b) 在整个灭绝期和恢复期间高脂肪饮食的影响。对照组以标准饮食喂养 (13% 大卡脂肪, 67% 大卡从碳水化合物, 20% 大卡从蛋白质; 2.9 总大卡/克) 和高脂肪饮食组 (45% 大卡从脂肪, 36% 大卡从碳水化合物, 和19% 大卡从蛋白质; 4.6 总卡/克)。条形表示在预处理 (白色)、调理后 (黑色)、最后的消光疗程 (浅灰色) 和恢复 (暗灰色) 期间, 在药物配对车厢中所用秒数的平均时间。15分钟的恢复试验被评估后, 启动剂量的一半以前接受剂量。* p < 0.05;** < 0.01;显著差异与前 c (方差分析与内科目可变天数 (比较前 c 和后 c) 或前消光会话 (学生的 t 测试)。这些数字已由37、38修订。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56983/56983fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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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Pharmacotherapy of Depression and Anxiety Disorders

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11.4K 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

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

一致性的任务作为一种手段来获取目标行为的措施中的橡胶手错觉范式Crossmodal

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

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 ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

脂肪偏好:饮食行为的一种新模型大鼠

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

一个过程来观察大鼠巴甫洛夫空调酒精寻求行为的背景下引发的重建

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

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 ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

在打开场塔迷宫的地方，反应学习

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

使用仿引起范式检查召回内存在婴幼儿时期

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Using Eye Movements Recorded in the Visual World Paradigm to Explore the Online Processing of Spoken Language

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded via an eye tracker as the participant produces or comprehends a spoken language describing the concurrent visual world. This paradigm has high versatility, as it can be used in a wide range of populations, including those who cannot read and/or who cannot overtly give their behavioral responses, such as preliterate children, elderly adults, and patients. More importantly, the paradigm is extremely sensitive to fine grained manipulations of the speech signal, and it can be used to study the online processing of most topics in language comprehension at multiple levels, such as the fine grained acoustic phonetic features, the properties of words, and the linguistic structures. The protocol described in this article illustrates how a typical visual world eye tracking study is conducted, with an example showing how the online processing of some semantically complex statements can be explored with the visual world paradigm.

The visual world paradigm monitors participants' eye movements in the visual workspace as they are listening to or speaking a spoken language. This paradigm can be used to investigate the online processing of a wide range of psycholinguistic questions, including semantically complex statements, such as disjunctive statements.

利用视觉世界范式中的眼球运动探索口语的在线处理

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded ...

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 ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is currently a paucity of research exploring how preschool children with autism understand sentences using different types of cues. The mechanisms underlying sentence comprehension remains largely unclear. The present study presents a protocol to examine the sentence comprehension abilities of preschool children with autism. More specifically, a visual world paradigm of eye-tracking is used to explore the moment-to-moment sentence comprehension in the children. The paradigm has multiple advantages. First, it is sensitive to the time course of sentence comprehension and thus can provide rich information about how sentence comprehension unfolds over time. Second, it requires minimal task and communication demands, so it is ideal for testing children with autism. To further minimize the computational burden of children, the present study measures eye movements that arise as automatic responses to linguistic input rather than measuring eye movements that accompany conscious responses to spoken instructions.

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.