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>Equipment</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&#34;x12&#34;, 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&#8482; 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 1.52mmOD 0.51mm thick x 152.4m</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>14-170-15B</td>
			<td>1/experiment</td>
		</tr>
		<tr>
			<td>Chemicals</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&#174;, 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 &#946;-hemolytic Streptococcus (Lancefield Group C) &#8805;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></td>
			<td></td>
			<td></td>
			<td>*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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

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.

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 ...

科研

神经科学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

在线经颅磁刺激测量与反应抑制相关的皮质生理

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

小鼠经颅光生物调节治疗方案

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.