所有的行为测试都是在德克萨斯大学医学科 (UTMB) 啮齿动物活体评估 (Dineley) 的核心, 由凯利博士指导, 并设在成瘾研究中心, 由凯瑟琳·坎宁安博士指导。对这项工作的支持来自于彼得 F. 美国国家环境健康科学研究所 UTMB (UL1TR001439)T32ES007254翻译科学研究所环境毒理学中心,米切尔神经退行性疾病中心和 UTMB 成瘾研究中心 (DA007287、DA070087 和试点研究基金)。

所有动物操作都按照《动物护理和使用指南》 (2011) 进行, 并得到机构动物护理和使用委员会的批准。
1. 动物
<ol><li>适应男性大鼠大约8-9 星期的年龄 (250-260 g) 为至少七天在殖民地房间维护在21-23 °c 和45-50% 湿气在 12 h 光黑暗的周期 (灯在 6:00-18:00 h)。</li>
	<li>房子鼠二个或笼子并且每日处理在整个研究期间。在整个研究阶段, 在家庭笼子里提供食物和水的广告随意。 注: 食品限制增加可卡因奖励的显著48, 增强可卡因诱发的运动活动49, 并作为一个压力恢复50 , 从而也可能影响提示反应性。</li>
	<li>将老鼠随机分配到生理盐水或可卡因治疗组。</li>
</ol>2. 手术
<ol><li>使用含有8.6 毫克/千克的甲苯噻嗪、1.5 毫克/千克乙酰丙嗪和43毫克/千克的氯胺酮在抑菌盐水中悬浮的鸡尾酒在麻醉下放置老鼠。通过捏脚趾和监测马达反射和呼吸来确认大鼠有足够的麻醉。将眼膏直接应用于眼部, 防止眼部干燥。 注: 如果老鼠没有足够的麻醉, 它将表现出马达对脚趾捏的反应, 而老鼠的呼吸速度可能会迅速增加。<ol>
<li>使用无菌技术, 而大鼠在麻醉下, 植入静脉导管进入颈静脉连接到背部安装与套管。术后植入, 允许大鼠恢复5-7 天28,36,37,51。 注: 可卡因自我管理的静脉注射路线在人导致脑子可卡因水平的快速的增量52,53。因此, 在它最常见的临床前变种之一, 老鼠被训练通过颈内导管自我管理可卡因。</li>
	</ol>
</li>
	<li>用0.1 毫升含肝素钠 (10 U/毫升), 链激酶 (0.67 毫克/毫升) 和卡西林二钠 (66.67 毫克/毫升) 溶液, 维持每日冲洗的导管通畅。在手术后的第二天开始导管冲洗, 并在自我管理疗程后每天继续。<ol>
<li>用溶液填充注射器, 通过聚乙烯油管将后座连接到注射器上冲洗。</li>
		<li>通过静脉注射10毫克/千克 methohexital 钠, 在整个实验中定期验证导管的正常功能。 注: 这是一个足够的剂量, 以简单地麻醉动物时, 只有静脉注射。</li>
	</ol>
</li>
</ol>3. 自我管理
<ol><li>使用软件编程生成4个单独的程序, 如下所述。<ol>
<li>代码两个程序为自我管理的固定比率 (FR) 1 附表, i. e., 其中一个积极的杠杆压力导致一个输液。对于程序 #1, 将左拉杆编码为活动拉杆;对于程序 #2, 将右杠杆编码为活动拉杆。构建两个程序的长度为180分钟, 并以下列方式提供可卡因刺激和离散提示复杂:<ol>
<li>有一个积极的杠杆压力导致同时照明的房子灯和刺激灯以上的主动杠杆。</li>
			<li>经过1秒的延迟, 有输液泵提供0.1 毫升的解决方案超过6秒。</li>
			<li>刺激灯在7秒后关闭 (在输液泵的同时灭失)。</li>
			<li>在27秒后, 把房子的灯关了, 最后20秒表示有一个超时期间, 在这期间, 主动杠杆压力没有预定的后果, 但这些仍然记录下来。</li>
			<li>对于数据输出, 收集输液, 主动杠杆压力, 非活动杠杆压力, 和滞后的第一主动杠杆压力机。</li>
		</ol>
</li>
		<li>再编写两个程序, 在 FR5 的时间表上进行自我管理, 其中五杆压力机导致一次输液。再次, 使一个程序与主动杠杆作为左, 和一个程序与积极杠杆作为权利。使两个程序180分钟的长度, 并以下列方式提供强化和离散提示复杂:<ol>
<li>有五个主动杠杆压力机导致房子光和刺激灯的同时照明在主动杠杆之上。</li>
			<li>重复步骤3.1.1.2通过3.1.1.5。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
自我管理培训
	<ol>
<li>每天在无菌0.9% 氯化钠中准备可卡因溶液, 剂量为0.75 毫克/千克/0.1 毫升。 注意: 在每180分钟的自我管理会话期间, 老鼠管理大约4-5 毫升可卡因溶液, 从而准备过剩, 预计每天大约8毫升/鼠。</li>
		<li>用至少8毫升可卡因或盐水溶液填充导管注射器 (10 毫升)。在确保可卡因/盐水溶液流动后, 将注射器插入输液泵中, 并连接到每个自管理会话开始时由金属弹簧皮带包裹的聚乙烯油管。仔细调整每个输液泵中的注射器, 使溶液在连接到每个导管之前均匀地分布在聚乙烯油管中。</li>
		<li>安置老鼠入各自标准操作的调理室安置在通风, 酣然衰减的隔间与风扇。始终将每只老鼠放在同一操作室中, 并在所有实验中保持同一侧的主动拉杆。大鼠左、右主动杆间的平衡分配。 注: 每个腔室均装有两个可伸缩反应杆两侧的颗粒贮器, 在每个反应杆上方有一个刺激灯, 而在杠杆的对面有一盏房子灯。可卡因/盐水注射器连接到一个23g 针 (归档, 以避免戳油管) 连接到聚乙烯油管包裹在金属弹簧皮带连接到每个鼠的导管和操作的液体旋转。注射器被放置在靠近隔间的输液泵中。</li>
		<li>在每日180分钟的时间内, 使用已建立的方法28、36、37、51, 训练大鼠将可卡因注入 (0.75 毫克/千克/0.1 毫升输液) 或盐水输液 (0.1 毫升) 的杠杆压力机。 注意: 这项训练是简单地把老鼠放入操作室, 并允许它杠杆压力;按下主动拉杆将导致收到离散提示复合物和可卡因输液, 而按下不活动的杠杆没有计划的后果。<ol>
<li>计划在有效杠杆上的完成结果在6秒的时间内将可卡因或生理盐水送到一起, 同时与照明的房子光和刺激灯以上的主动杠杆和激活输液泵 (这是离散提示复杂配对与可卡因交付);非活动拉杆压力机不会产生预定的结果。在强化 (可卡因) 交付之后, 刺激光和输液泵被灭活;房子的灯仍然为额外的20秒, 以表明在一个超时期间, 杠杆压力机没有计划的后果。 注: 有关如何设置此操作的说明, 请在步骤3.1 中找到. 1.1-3.1. 1.4
</li>
		</ol>
</li>
		<li>完成180分钟的疗程后, 将每只老鼠从其操作调理室中取出, 并冲洗其导管 (步骤 2.2)。在两个疗程之间仔细清洁房间, 用70% 乙醇溶液擦拭每个表面。 注意: 在自我管理期间接受可卡因的老鼠可能很难从分庭中取出, 而且更有可能有攻击性。使用缓慢, 仔细的议案, 以切断每只老鼠的绳索。</li>
		<li>训练大鼠在 FR1 的进度表上, 在达到七输液/小时的标准后, 在每节输液的总数量中, 连续三天的10% 的可变性。继续自我管理的会议, 直到老鼠已经达到了14天的自我管理培训。 注意: 这是操作性学习的一个变体;在整个实验过程中, 有些协议更倾向于让老鼠留在 FR1。固定比率计划从 FR1 到 FR5 的变化不会影响动物自我管理54,55的输液数量。然而, 增加固定比大小有助于保证大鼠反应的稳定性, 并可能增加奖励的显著56。此外, 如果需要在强迫禁欲期间进行治疗, 在达到稳定时, 应将老鼠伪随机分配给治疗组 (每次疗程至少连续3天不到10% 的变异性) 在FR5 计划。此外, 盐水大鼠不会从 FR1 到 FR5, 因为生理盐水的显著还不足以达到从 FR1 到 FR5 的进展所需的稳定性。</li>
	</ol>
</li>
</ol>4. 强迫禁欲
<ol><li>将老鼠强迫禁欲30天;即, 不要给老鼠提供在30天内自我管理可卡因或生理盐水的机会。重要的是, 在此期间, 不要将老鼠送回操作室 (以前的自我管理的上下文)。然而, 从家庭笼子里取出老鼠作为日常的处理和称量, 作为衡量一般健康的措施。 注意: 强制禁欲的持续时间可以改变, 但重要的是要选择一个足以避免天花板和地板效果的持续时间。在选择强迫禁欲期的时间时, 要认识到的因素包括啮齿动物的应变、年龄、自我管理时间长短、药物、药物剂量和输液持续时间, 以及动物的性别。</li>
</ol>5. 可卡因提示反应性
<ol><li>为 FR1 计划中的提示反应性任务编写两个软件程序;一个程序与左杠杆作为主动杠杆, 和一个与右杠杆作为主动杠杆。构造每个程序收集杠杆压数据60分钟, 并提供离散提示复杂, 如下所述。<ol>
<li>重复步骤3.1. 1.1-3.1. 1.4
</li>
		<li>对于数据输出, 收集提示演示 (以前称为输液), 以前有活动的杠杆压力, 非活动杠杆压力, 并延迟到第一杠杆压力机。</li>
	</ol>
</li>
	<li>
可卡因提示反应性测试会话
	<ol>
<li>在60分钟的操作性测试会话中, 评估30天强迫禁欲的线索反应。错开开始时间的大鼠, 以促进快速斩首和脑部的收获。重要的是, 对于提示反应性测试, 不要将注射器放置在输液泵中, 这样输液泵的声音就可以作为听觉提示, 但不会提供可卡因增强。 注: 提示反应性可以评估在30天以外的强迫禁欲的天数, 但事实是, 对可卡因配对的线索的反应不同时间34应该记住, 当选择提示反应性测试的日期。</li>
		<li>把每只老鼠放在自己的管理室里, 在那里训练和拴在它的套管上, 就像在日常的自我管理会议中一样。在以前提供的可卡因或生理盐水的杠杆上的压力, 现在只加强了交付的离散线索复合体 (i., 刺激灯和房子被照亮, 输液泵被激活) 的 FR1 时间表 (即, 一个每个主动杠杆压力机显示离散提示复合体);在一个60分钟的会话中, 计算提示演示文稿和以前活动的杠杆压力, 并记录非活动杠杆压力机的数量。然而, 与自我管理一样, 不活跃的杠杆压力机不会产生预定的后果。 注: 另外一个控制选项是在没有预定结果的情况下记录以前活动的和非活动的杠杆压力 (i. e., 没有离散提示复杂强化)。</li>
		<li>当提示反应性测试会话完成后, 立即弄死大鼠捕获重新暴露于操作性调节室和离散提示复合体的生化效应。</li>
	</ol>
</li>
</ol>6. 数据收集、组织和分析
<ol><li>将数据分解为三个常规类别: 一般健康、自我管理和线索反应性。<ol>
<li>在整个实验过程中, 监测一般健康 (例如动物体重, 毛皮状况), 并在必要时为动物提供兽医护理, 或从研究中剔除, 如果杠杆压力出现在健康的影响下。</li>
		<li>在自我管理的14天内收集自我管理数据。使用不配对的双尾 t 检验来确定可卡因和生理盐水自我管理组在输液总数、主动杠杆压力机、非活动杠杆压下的统计差异, 以及第一主动杠杆压的潜伏期。 注: 在强迫禁欲之前, 需要对自我管理数据进行分析, 如果你想研究在强迫禁欲期间治疗对提示反应的影响 (因此, 小组可以有相同的平均输液次数, 以及活跃和不活跃杠杆压力机)。如果选择二次设计 (可卡因治疗, 生理盐水治疗), 应采用双向方差分析, 并进行适当的后处理, 以分析这些数据。</li>
		<li>为线索反应性数据收集和分析的总数量的提示演示, 以前主动杠杆压力, 不活跃的杠杆压力, 和滞后的第一杠杆压力。还要用一个配对的双尾 t 检验来分析这些数据, 以确定动物自我管理的可卡因和那些自我管理的盐水之间的差异。 注: 再次, 如果选择了两次设计 (可卡因的治疗, 盐水治疗), 一个双向方差分析和适当的后处理, 应用于分析这些数据。</li>
	</ol>
</li>
</ol>

作者没有披露的利益冲突。

暴露在药物配对的线索和生理变化响应这些线索16是与复发,11,16和可卡因线索反应性测试使用以上偶然提出可卡因配对线索在缺乏药物;因此, 以先前活动的杠杆压力机形式进行的药物寻求行为是一种复发脆弱性的度量。本文所描述的提示反应性协议是一种临床前的方法, 通过它可以测试复发修饰 pharmacotherapies, 并研究导致复发风险的遗传和环境因素。这是一项针对药物寻求行为的操作性任务, 它与人类吸毒和复发的许多性质相吻合, 许多出版物都优化了关键药物剂量和传递参数, 并采取了适当的控制措施来确定脸部的有效性和平移预测功率28,36,37,39,51。这一模式最近在米勒和al的研究中发表。调查使用过氧化物酶体核扩散激活受体伽玛 (PPARγ激动剂作为药物治疗, 以减弱可卡因提示反应性57。这项研究表明, 吡格列酮 (PPARγ激动剂) 降低了对可卡因配对线索的反应后, 强迫禁欲从可卡因自我管理。因为吡格列酮是 FDA 批准的, 米勒等。研究提示了双臂, 双重盲, 随机, 安慰剂控制的临床试验, 以检查 PPARγ agonism 在抑制可卡因提示反应性, 冲动和风险决策的有效性。鼠模型的平移优点和预测效度得到了研究的支持. 58发表在这项试验性研究中, 使用吡格列酮治疗的可卡因依赖对象表现为减少自我报告渴望的治疗 x 时间相互作用, 通过简单的物质渴求量表和视觉模拟量表测量渴望58。因此, 吡格列酮显示的承诺, 作为一种复发预防治疗的反刍, 符合米勒等 al。前期资料。
这个老鼠可卡因提示反应性测试在禁欲概括慢性人可卡因自我管理与间歇短 (例如, 21 小时) 和长 (例如, 30 天) 禁欲期间, 提供能力创造平行数据集在人和啮齿目动物模型之间。然而, 这项议定书不同于更普遍的灭绝恢复模式, 它测量了在灭绝过程中采取药物或寻求的时间, (通常) 禁欲期33,34,35 ,59。虽然消光恢复模型具有预测有效性33, 但也有显著的局限性。首先, 灭绝进程在没有药物增强60的情况下继续将盐水和可卡因自我管理小组重新暴露在自我管理的环境中, 因为药物相关的线索, 在人类中很少概括。包括情绪状态, 地点, 和人44-线索, 不能轻易复制在临床设置45,46,47。第二, 在恢复过程中, 老鼠表现出较低的反应水平, 造成地板效应, 抑制了人们检测干预措施对减少药物寻求行为的影响的能力33-这个问题是通过去除灭绝, 因为灭绝训练减少反应。有人可能会争辩说, 上面描述的60分钟的反应性测试会话可能会成为一个消光会话。然而, 该议定书包括非药物强化, 测试会话不重复, 和老鼠保持后续自我管理的动机后, 提示反应性测试表明, 这种测试设计不会导致灭绝36。
另一个问题是, 相对较短的自我管理会话长度 (180 分钟) 是否足以加强随后的自我管理。然而, 文献表明, 这种方案一贯的结果, 稳定模式的大鼠可卡因自我管理37,39,51,57,61, 提供较少的变异性在动物之间, 使实验者可以将科目分类到自我管理后的治疗组, 如果需要28,39,51,57。然而, 应当指出的是, 短时间和短会期程序并不完全概括人类的自我管理, 在这种情况下, 可卡因依赖的主题中出现了类似于狂欢的模式62。然而, 如果在啮齿动物模型中允许持续的自我管理, 死亡率就非常高63。
额外的变量要考虑的是可卡因剂量和输液率, 因为这些影响敏感性的主观奖励反应。更快的输液速度提高了对可卡因增强效果的认识, 而慢速则降低了64。由于可卡因自我管理的剂量依赖性曲线是一个倒 U 形和剂量影响每小时输液次数55;上述协议使用的剂量, 接近峰值的剂量-反应曲线的短期访问和长期准入程序61 , 以检测输液数量的变化。制衡杠杆是另一个重要的控制, 以确保老鼠不显示偏好的一个杠杆超过另一种。
图 1的代表性结果显示了雄性, 大大鼠的数据。历史上, 这些研究被限制在雄性大鼠, 因为发情周期对获得可卡因自我管理65和灭绝/恢复66。由于性别作为生物变量现在已被授权用于 NIH 资助的研究, 因此必须监测发情阶段和测试混合性研究中的性别差异, 因此, 所有实验组都应考虑统计能力。另一种说明是, 使用 outbred 鼠株, 如大, 是有益的, 因为它提供的遗传变异更可比人类人口-虽然即使 outbred 啮齿类动物菌株可能无法充分重述不同的遗传影响思想在人67的基础上上瘾象行为。最后, 初次接触可卡因的年龄是一个重要的考虑因素。在大鼠, 青春期接触可卡因导致一个条件性的地方偏好, 需要更多的灭绝试验, 以扑灭68, 这表明, 青春期鼠表明更持久的可卡因寻求行为。因此, 产前和产后早期接触可卡因可能会改变可卡因提示的反应性。
由于灭绝不是提示反应性测试的目标, 一个限制是任务不能重复或长时间的评估, 否则动物将扑灭可卡因 (主) 强化与离散线索复合体的配对 (条件强化)。因此, 在设计实验时必须注意, 以确保不会发生这种灭绝。然而, 这项任务的一个优点是, 接触可卡因配对的线索引起的精确生化变化可以用这个过程来阐明。对这项任务的第二个限制是, 它只适用于积极的强化, 尽管这是许多滥用药物模式的共同限制。该议定书的关键步骤包括维持导管通畅 (步骤 2.2), 获得自我管理 (步骤 3.2), 适当地将大鼠分配到治疗组 (如果需要), 在统计学上等同于输液和数量杠杆压力机 (步骤 3.2.6) 和适当的软件编程, 以确保在自我管理训练和线索反应性测试 (步骤 3.1, 5.1) 中呈现离散提示并与可卡因刺激配对。与其他复发脆弱性模型一样, 必须达到合理的禁欲长度, 以避免在提示性反应试验33、34 (步骤 4) 中对反应产生地板和天花板的影响。最可能遇到的并发症是 1) 导管通畅的丧失, 以防止获得自我管理, 2) 啮齿类动物在自我管理训练后和提示之前不正确地分配给治疗组反应性测试导致1型或2型错误和 3) 的禁欲不足以确定组间响应的差异。这些都可以通过每日自我管理绩效评估、常规导管冲洗、在自我管理绩效平衡后分配给后分析组, 以及确定适当禁欲的试点试验来解决。啮齿类动物的持续时间, 年龄, 自我管理疗程长度和持续时间, 药物, 药物剂量, 药物输液持续时间和性别。
此程序可扩展到许多啮齿动物菌株, 包括小鼠69, 以及任何模型, 其中包括配对的一个主要奖励强化与离散的线索 (条件强化)。替代初级强化包括其他药物类, 高脂肪食品和其他可口的奖励69,70。最后, 通过对经 FDA 批准的大鼠对反刍人体的干预实验, 证明了该模型的平移相关性, 从而减少了可卡因的渴求。因此, 可卡因的自我管理和禁欲后的古柯碱提示反应性试验将有望成为一种转化啮齿动物模型。

可卡因使用紊乱 (反刍) 遵循重复自我管理的轨迹, 此前中性刺激获得奖励价值1。提示反应性是对先前与药物服用经验相关的暗示的敏感性, 它在人类渴求2、3、4、5中起着重要作用。人们认为, 对药物相关线索6、7敏感的个体来说, 进步到反刍的风险以及禁欲期间的复发是更高的。环境背景 (如人、建筑物、音乐流派) 和与药物相关的离散刺激 (例如, 用具) 与可卡因奖励有关;接触这些线索可以触发周围生理学的变化 (例如心率, 皮肤温度, 皮肤阻力), 大脑可塑性, 和大脑功能连接2,8,9 ,10。换言之, 再次接触可卡因相关的线索激活边缘 corticostriatal 电路唤起条件的生理和主观反应, 推动食欲方法 (药物寻求) 行为11,12 ,13,14,15。
用功能性脑成像分析测量的线索反应性是对反刍16患者复发易感性的预测。啮齿动物模型中的线索反应性测量作为复发风险的替代措施, 可用于转化研究。因此, 减少啮齿类动物的提示反应性药物治疗可以作为人类临床试验中的复发预防疗法进行。具有必要的转化优点和预测有效性的临床前模型尤其重要, 因为目前尚无 FDA 批准的 pharmacotherapies17。
啮齿动物自我管理程序是对人体药物服用18具有预测有效性的金本位、转化模型, 对了解反刍的分子和生理过程至关重要。与反应相关的可卡因暴露, 与反应无关的可卡因的传递导致不同的行为、分子和神经化学效应;e., 与反应无关的可卡因交付引起的死亡率显著高于19。此外, 从反应依赖的可卡因自我管理中禁欲的神经化学后果有别于从与反应无关的可卡因交付20的禁欲引发的影响, 21. 因此, 在评估线索反应性和相关的行动机制时, 基于反应依赖的可卡因提供的反刍模型是优越的平移模型。
在下面概述的议定书中, 可卡因是通过留置的颈内导管静脉注射的。然而, 已开发出通过口服和吸入途径自我管理药物的替代方法。重要的是, 啮齿类动物通过操作性反应控制药物的交付, 类似人类。因此, 由啮齿类动物和人类22之间的药物自我管理有很高的一致性。下面的临床前药物自我管理程序采用杠杆压制, 加强了药物传递, 以刺激反应率高于车辆控制。寻找毒品的行为是通过配对最初的 "中性" 暗示 (例如, 刺激的光或音调, 以及可卡因自我管理发生的上下文环境) 来训练可卡因注入;这些提示成为条件强化 (审查: 坎宁安 & Anastasio, 201423)。随后再次暴露于可卡因相关的线索引发啮齿类动物的寻毒行为 (即试图通过按压先前活跃的杠杆提供可卡因) 以及反刍科目24的渴求和复发,25,26,27。
通常情况下, 在可卡因自我管理之后进行的药物寻求行为的前啮齿动物研究利用在药物相关环境28、29中进行的灭绝训练和/或药物恢复,30,31,32. 在没有药物和/或提示传递的情况下, 按先前活动的杠杆, 通常构成在灭绝33、34、35之后恢复的措施。相反, 提示反应性药物寻求行为是评估后, 强迫禁欲, 没有事先灭绝训练28,36,37,38,39.
结果措施和实验变量被仔细地选择和验证, 以解剖不同的方面的神经生物学的寻求和复发的行为, 并建立了良好的 neuroadaptations 不同的模型与没有灭绝训练40,41,42,43。此外, 从平移的角度看, 由于药物相关的线索包括情绪状态、地点和人44, 啮齿类动物灭绝训练没有反映在反刍的临床环境中;这些线索的独特组合可能无法在临床环境45,46,47。因此, 这里所描述的啮齿动物模型比目前现有的许多模型更能与人类的状况相平行。
以下描述了一个验证的可卡因自我管理训练, 强迫禁欲和线索反应性测试协议的大鼠。简单地说, 老鼠被植入颈内导管, 通过 ' 主动 ' 杠杆压力训练自我管理可卡因或生理盐水, 接收可卡因或生理盐水刺激与离散的光和声音信号配对, 作为条件强化。经过14天的可卡因自我管理, 老鼠受到30天的强迫禁欲和随后60分钟的线索反应性测试, 其中杠杆压测量。提示反应性试验是可卡因复发易感性的替代措施。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Catheter Tubing: 0.50mm ID x 0.94mm OD x 0.2mm width</td><td>Fisher Scientific, Hampton, NH, USA</td><td>11-189-15A</td><td>1/experiment</td></tr><tr><td>Cue Light</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-229M</td><td>2/operant chamber</td></tr><tr><td>Guide Cannulae (22 gauge, pedestal size-8mm, cut length 11 mm, 5 mm above the pedestal)</td><td>Plastics One, Roanoke, VA, USA</td><td>8IC313G5UPXC</td><td>1/rat</td></tr><tr><td>House Light</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-227M</td><td>1/operant chamber</td></tr><tr><td>Infusion Pump</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>PHM-100</td><td>1/operant chamber</td></tr><tr><td>Levers</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-110M</td><td>2/operant chamber</td></tr><tr><td>Liquid Swivels</td><td>Instech, Plymouth Meeting, PA, USA</td><td>375/22</td><td>1/operant chamber</td></tr><tr><td>MED-PC Package with Infusion Pump Software</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>SOF-735 (infusions software SOF-700RA-10 version 1.04)</td><td>1</td></tr><tr><td>Metal Spring Leash</td><td>Plastics One, Roanoke, VA, USA</td><td>C313CS/SPC</td><td>1/operant chamber</td></tr><tr><td>Needle (23g, 1 in)</td><td>Becton Dickinson, Franklin Lakes, NJ, USA</td><td>305193</td><td>1/operant chamber</td></tr><tr><td>Nitex Mesh (6/6 woven mesh sheet, 12&quot;x12&quot;, 500 microns thick, 38% Open Area)</td><td>Amazon, Seattle, WA, USA</td><td>CMN-0500-C, B000FMUNE6</td><td>~1 sheet/100 rats</td></tr><tr><td>PCI Interface Package</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>DIG-700P2-R2, MED-SYST-16</td><td>1/16 operant chambers</td></tr><tr><td>Power Supply for Interface Modules</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>SG-6510D</td><td>1/16 operant chambers</td></tr><tr><td>Sound-attenuating Cubicle</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-018V</td><td>1/operant chamber</td></tr><tr><td>Syringes, 10 mL Luer-Lok&amp;trade; tip</td><td>Fisher Scientific, Hampton, NH, USA</td><td>14-827-52</td><td>1 case/experiment (1/operant chamber)</td></tr><tr><td>Tygon Tubing for flushes: 0.51mmID x 1.52mm OD 0.51mm width x 152.4m length</td><td>Fisher Scientific, Hampton, NH, USA</td><td>14-170-15B</td><td>1/experiment</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acepromazine (10mg/mL)</td><td>Henry Schein (Animal Health), Melville, NY, USA</td><td>003845</td><td>~0.5mg/rat*</td></tr><tr><td>Acraweld Repair Resin</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1013959</td><td>1/experiment</td></tr><tr><td>Altalube (ophthalmic ointment)</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>6050059</td><td>1/experiment</td></tr><tr><td>Cocaine</td><td>NIDA North Bethesda, MD, USA</td><td>N/A</td><td>~350mgs/rat for whole experiment*; requires DEA License</td></tr><tr><td>Heparin (10,000 USP units/10 mL)</td><td>Sagent Pharmaceuticals, Schaumburg, IL, USA</td><td>NDC 25021-400-10</td><td>1/experiment (~21 units/rat*)</td></tr><tr><td>Jet Liquid</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1256401</td><td>1/experiment</td></tr><tr><td>Ketamine (100mg/mL, 10mL)</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1049007</td><td>~15mg/rat*; requieres DEA license</td></tr><tr><td>Methohexital Sodium (Brevital&amp;reg;, 500 mg/50 mL)</td><td>Patterson Dental, Saint Paul, MN, USA</td><td>043-5461</td><td>1/experiment; requires DEA License</td></tr><tr><td>Saline (0.9%, USP)</td><td>Baxter, Deerfield, IL, USA</td><td>2B1307</td><td>1 case/experiment</td></tr><tr><td>Streptokinase from &amp;beta;-hemolytic Streptococcus (Lancefield Group C) &amp;ge;3,000 units/mg</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>S3134-250KU</td><td>1 vial/experiment (~1.5mg/rat/experiment*)</td></tr><tr><td>Ticarcillin Disodium Salt</td><td>Fisher Scientific, Hampton, NH, USA</td><td>50-213-695</td><td>~4 vials/exeriment or purchase the 25g vial cat.# 50-489-093 (~150mg/rat/experiment*)</td></tr><tr><td>Xylazine (100mg/mL)</td><td>Henry Schein (Animal Health), Melville, NY, USA</td><td>033198</td><td>~3mg/rat*</td></tr><tr><td colspan="4">*Assumes rat age is that described in the protocol, rats self-administer for 14 days, and flushes occur for 21 days.</td></tr></tbody></table>

a protocol for measuring cue reactivity in a rat model of cocaine use disorder

可卡因使用紊乱 (反刍) 遵循重复自我管理的轨迹, 以前的中性刺激获得激励价值。提示反应性, 对先前与药物服用经验相关的线索的敏感性, 在禁欲期间对人类的渴求起着重要的作用。提示反应性可以评估为药物相关线索的注意方向, 这是可测量的食欲方法的行为, 在临床前和人类的研究。这里描述了对自我管理的可卡因训练大鼠的提示反应性的评估。可卡因自我管理与表现为条件强化的离散线索配对 (即, 房子光, 刺激光, 输液泵的声音)。在一段禁欲时期之后, 在可卡因自我管理环境中的杠杆压力和先前配对可卡因输液的离散提示被测量为提示反应性。这个模型是有用的探索神经生物学机制的基础上提示反应性过程, 并评估 pharmacotherapies 抑制提示反应性, 因此, 修改复发的弱点。该模型的优点包括其平移相关性、其表面和预测有效性。该模型的主要局限性是, 提示反应性任务只能很少执行, 只能在短时间内使用 (e., 1 小时), 否则老鼠将开始熄灭与可卡因刺激的离散线索配对。该模型可扩展到任何正向增强刺激与离散线索配对;虽然特别适用于滥用药物, 但这种模式可能会在肥胖等领域中进行未来的应用, 在这种情况下, 可口的食物奖励可以起到积极的增强刺激作用。

一个可卡因自我管理和禁欲实验的结果, 然后一个提示反应性测试从以前发表的研究57显示在图 1。研究时间线在图 1A中描述。
大鼠个体从 FR1 到 FR5, 因为它们符合标准。随着操作性调节在可卡因管理组中的作用, 大鼠逐渐增加输液数量直到达到高原 (图 1B)。不活跃的杠杆压力机在可卡因管理和盐水管理小组中保持一贯低;不活跃杠杆压力的差异表明动机或健康的变化。盐水自我管理作为控制由于盐水不加强;因此, 杠杆压力和输液不会升级从1天到14天 (没有图)。如果在可卡因管理组中的主动杠杆压力在1天和14天之间下降, 导管可能失去通畅 (也证明了步骤2.2.2 中描述的方法), 这种动物应该从研究中删除。作为统计异常值的动物 (用迪克森的 Q 测试计算) 也应该从研究中删除。
从可卡因线索反应性试验中收集了多种输出措施。例如,图 1C描述了先前活动拉杆上的杠杆压力机的数量。以前自制的可卡因的老鼠应该比自制生理盐水的大鼠压力大得多。而且, 如果治疗能有效降低可卡因的反应活性, 那么在接受可卡因的大鼠中, 前主动杠杆压力机的数量也应该大大减少, 而接受可卡因的老鼠和车 (没有图)。
其他测量包括第一杠杆压力机的滞后时间 (图 1D)、非活动拉杆的数量以及提示演示的数量。可观察到第一杠杆压力机的潜伏期变化;第一杠杆压力增加的潜伏期可能表明动物身体不适或者可卡因的动机减少。对运动行为的适当控制, 如 Rotarod 或开放的领域任务, 应用于区分身体疾病和动机的变化。
虽然人们可能认为, 提示演示的数量应该反映在以前的活动杠杆压力机的数量, 这是不经常的情况下。通常以前-主动杠杆压力比提示演示要大得多。大鼠经常显示 perseverative 以前主动的杠杆在超时期间紧迫, 表面上是为了应对未能获得预期的可卡因刺激。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55864/55864fig1.jpg"> 图 1.实验时间和代表性的自我管理和线索反应性结果。这个数字已经从米勒和al, 201657修改。(A) 议定书34、5节所述的试验时间线。(B) 日常活动杠杆压力机、非活动杠杆压和输液电镜进行自我管理培训;自管理会话每天180分钟, 连续14天。可卡因最初是在 FR1 时间表上交付的, 直到会议标准, 然后介绍了 FR5 时间表。(C) 前主动杠杆压力机上的平均杠杆按60分钟的反应性试验的 SEM;一个配对的双尾 t 检验显示可卡因和盐水自我管理大鼠之间的显著差异 (t (30) = 14.82, ** p < 0.0001)。(D) 60 分钟提示反应性试验的平均滞后时间为第一杠杆压电镜;无配对, 双尾 t 检验显示可卡因和生理盐水自我管理大鼠之间没有显著差异 (t (29) = 0.2758, p = 0.7847)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55864/55864fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder at JoVE.com

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

线索反应性被概念化为敏感的线索与药物服用的经验, 有助于渴望和复发的禁欲的人。提示反应性是通过测量药物相关线索的注意力取向来模拟大鼠的, 结果在自我管理和强迫禁欲后的提示反应性测试中食欲方法行为。

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 ...

a-protocol-for-measuring-cue-reactivity-rat-model-cocaine-use

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.5K 次观看.  University of Texas Medical Branch. 线索反应性被概念化为敏感的线索与药物服用的经验, 有助于渴望和复发的禁欲的人。提示反应性是通过测量药物相关线索的注意力取向来模拟大鼠的, 结果在自我管理和强迫禁欲后的提示反应性测试中食欲方法行为。

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

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

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

新型仪器和药物加固方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

神经科学

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

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 ...

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 ...

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

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 acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

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 ...

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 Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.