可卡因诱导行为敏和小鼠位置偏爱评估

摘要

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.

摘要

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.

引言

Research aimed at understanding drug addiction using animal models must take a variety of approaches to address each of the assorted components that obstruct treatment success, including reward/reinforcement/motivation and withdrawal and relapse, as well as the general persistence that further complicates these issues in addiction. Since rewarding experiences associated with taking a drug of abuse are thought to motivate subsequent use, studies focusing on drug-context associations may be particularly useful for understanding brain mechanisms that contribute to drug taking and seeking. One such assay, conditioned place preference (CPP) is a high-throughput method for comparing group differences in reward sensitivity. The traditional interpretation of the task involves classical, or Pavlovian, conditioning, where a conditioned stimulus (CS) is paired with an unconditioned stimulus (UCS), and after multiple pairings, the CS elicits the same behavior as the UCS (however, see^39,40). Theoretically, animals learn to associate an interoceptive state (reward or aversion) with contextual cues. The relative aversive or appetitive intensity of the interoceptive state is then assessed by then determining the animal's preference for the contextual cues. The use of place conditioning to measure drug-reward associations dates back to at least 1957, to a study using morphine on rats in a Y-maze^3,4. Over the past several decades, variations on this method have been widely used to study place preference and aversion in rodents to various stimuli, and it remains particularly useful in the study of associations induced by drugs of abuse. In drug-addiction research, the assay has been used to assess the rewarding properties of a number of drugs and the contribution of different brain systems and proteins to drug reward (for reviews, see^5-7,44). While there are superior methods of assessing factors that contribute to drug addiction, namely drug self-administration, CPP is a simple and much more accessible approach to measuring reward function.

Most current protocols for conditioned place preference and aversion (CPA) use an apparatus that allows rodents to have access to two distinct chambers, either via a doorway or smaller connecting chamber. Distinctions between the two chambers are often based, at a minimum, on visual and tactile cues, including wall color and floor texture, but sometimes include other elements, such as olfactory cues. "Biased" designs typically attempt to reverse a pre-existing, innate preference for one chamber over the other, such as the one that rodents generally show for a black chamber over white. "Unbiased" designs aim to create a preference to one of two chambers that were initially equally appealing by randomly counterbalancing assignment to either chamber within a group. A "balanced" design is used when animals show small preferences, but do not, as a group, favor the same chamber. Goals of this latter design are to produce 1) pre-test preference scores for the (eventual) cocaine-paired chamber that are not significantly different between experimental groups and 2) negligible preference for the cocaine-paired chamber at pre-test, either positive or negative⁸. The balanced design is ideal for use with the described chambers, which utilize contradicting biases for wall color (black over white) and flooring (wire over bar), resulting in a roughly equal distribution of small preferences for both the black and white sides in different animals. Balancing calculations are described in further detail below.

During conditioning, animals are exposed to a drug and quickly placed into one of these two environments for a limited time period. Exposure is typically via intraperitoneal (i.p.) or sometimes subcutaneous (s.c.) injection, although paradigms for intravenous (i.v.) self-administration⁹, and intracranial infusions³⁸ in a place preference apparatus have also been developed. These pairings are complemented by non-drug (vehicle) pairings of the same length conducted in the opposite chamber, which can take place on the same day as drug pairings or on separate days. In general, when allowed to explore the apparatus after conditioning, animals will spend more time where they received a rewarding drug (i.e., one that humans and animals will voluntarily self-administer), while they will avoid a place where they were given a drug that induced illness (e.g., lithium chloride). Several studies have been dedicated to optimizing the conditions for place preference to different drugs of abuse (for review, see⁷). Cocaine doses (i.p.) for mice generally range from 1 to 20 mg/kg, with doses less than 5 mg/kg often used to parse high sensitivity in one group. Two or more drug pairings are typically required for adult mice¹⁰, and the length of these pairings is an important consideration. Very low doses of cocaine require an immediate and brief conditioning, likely because this method captures the most rewarding period of the exposure. Delayed or very long conditioning periods can result in no preference, or may even induce aversion^11,12. Here is presented a basic method for obtaining conditioned place preference to cocaine in adult mice.

While the CPP assay is an ideal method for assessing reward-related learning and memory of drug-context associations, behavioral sensitization is arguably easier to perform and allows the assessment of changes that develop over repeated treatment. Also known as reverse-tolerance, behaviors undergoing sensitization are incrementally enhanced over repeated exposures to a particular drug of abuse, especially psychostimulants, and cross-sensitization is known to occur between some, but not all, of these drugs. One of the first assessments of cocaine-induced locomotor sensitization, in particular, in rodents was published in 1976¹³. A number of labs have shown that sensitized locomotion is detectable long after drug cessation, depending on the original length, location and dose of exposure^14-17, and the current protocol has been used to detect sensitization as long as 10 months following seven days (30 mg/kg) of cocaine treatment in mice¹⁸. The test can be performed using either photobeam or video-tracking technologies, in apparatuses of differing sizes and shapes, making it simple for many labs to perform. The robust nature, simplicity and persistence of locomotor sensitization makes its assessment an ideal part of examining basic mechanisms of long-lasting changes in drug-induced behavior.

As is expanded upon in the discussion, an important consideration when performing the locomotor sensitization assay is whether drug is given in the home- or test-cage environment. To take advantage of the robust sensitization that occurs when drug administration occurs outside of the home cage, this protocol employs this method. However, it has been observed that when animals are not adequately habituated to a new environment before drug exposure, a novelty-induced ceiling effect occurs on Day 1, which can partially or fully mask the progressive nature of sensitization. It is likely that this represents synergistic locomotor-activating effects of the drug together with novelty, and while the mechanisms underlying such effects may be interesting, the method described is designed to reduce the role of novelty and allow the effects of the drug to be measured more independently. While it is expected this method will be useful in the assessment of other locomotor-sensitizing drugs, it has primarily evaluated its effectiveness with cocaine in C57BL/6 mice.

研究方案

所有的实验程序已经批准麦克林医院机构动物护理和使用委员会。注:以下协议描述了一个方法，CPP和运动致敏，很多细节的其它成功的协议 （例如，光与暗阶段测试中，连续与间断给药等）有所不同。新手可能希望与这些协议开始，或者干脆用它们作为指导，从基于手头的实验问题（S）的文学适应变化。自动测量方法的描述;然而，也可以使用非自动化装置，用于每次测定（即，录像，手得分）。

1.位置偏爱

1. 设备和房间布置:
   1. 处理实验小鼠每天1-3分钟，在试验前的至少3-5天。
      注意:千万不要被处理的小鼠可能会​​发现从该室STRE去除ssful，其可干扰或改变调理。
   2. 获得四个或更多三腔CPP设备，最好配备photobeams用于自动数据收集^18。确保每个腔室具​​有经由可以提高门连接到一个较小的，中性室两个较大，visually-和触觉不同的腔室/降低来控制访问。每个室盖应打开插入/删除小鼠，并安装具有体积小，可单独控制（调光）灯（每室一个）。
      注:CPP室设计各不相同，可以从市场上购买或建造的研究人员。对于"平衡"的设计，推荐方案与白色的墙壁和线栅地板一个容量较大的室和其他与黑的墙壁和地板吧。中间室应该有灰色的墙壁和固体灰有机玻璃地板。为解释的目的，这些室将被称为"白色"，"黑色"和"中间"。盖子应该是透明的有机玻璃。备用舱构型（单或双室）也是可能的，并讨论了其他地方（见讨论）。
   3. 安装程序，它会在测试期间房间:关闭或设置架空灯最暗的设置并关闭大门。使用测光表各腔室内，并设置盖子灯光，使中间​​室比，以劝阻消费时间有老鼠黑白室（6-10勒克斯）稍亮（15-20勒克斯）。
      注:如果在中间花费的平均时间是一样多或比在黑色或白色的腔室以上，进一步增加在步骤1.1.3中描述的照明的对比度。另外，使用不太吸引人地板的中间。 ，但要避免使该室厌恶。试点任何改动，以确保在中间时间减少不是由于总的探索，以减少（口岸）（例如，〜220直径0.5厘米的孔，均匀地涂抹在6×3.5×¼"有机玻璃隔开） 。
   4. Program自动数据采集（"过程"， 图1A）或根据下列参数手动收集数据。集试验，以对在任调理室头梁断裂开始。设置"测试"会话是在长度为20分钟，以跟踪每个室所花费的时间和光束中断。设置"调理"会话是30分钟长和（任选）以测量在每个室光束中断。将所有的灯室审讯期间照亮。
   5. 准备全卷为实验手头需要可卡因的解决方案。溶解可卡因HCl的0.9％氯化钠（盐水），基础终浓度0.1毫升/ 10g体重的注射量（例如，对于剂量为5毫克/公斤，溶液浓度为0.5毫克/毫升）。涡流混合30-45秒，无菌过滤器（0.2微米的注射器过滤器），并存储在室温。
2. 测试:
   注:CPP一般的时间表是PRE-TEST，空调和然后检测后。预测试可能会通过调理1-3天分开;然而，空调和测试后每天应采取连续数天（图2A）的地方。该定时应保持在同一实验中的所有队列是相同的。
   1. 在动物使用跨天每个动物在同一设备轻相测试。
   2. 每个试验日（即，测试和调理天），将小鼠行为接待室，让他们在自己的笼子里静置试验前1-1.5小时。选择在哪里小鼠能够迅速从它们的笼移动到的设备中，以最小的中断，在试验开始时的位置。打开所有设备，使得与测试（例如，设备风扇）相关联的任何噪声都存在。
   3. 彻底清洗每个装置（内壁，地板和托盘）轻度，醇，乙二醇醚或基于氨的消毒擦拭物之前和之后各小鼠（使用SAM整个实验吸尘器的e型）。不要忽视地板底面。
   4. 检查房间的灯都在每一天的开始适当设置（关或灰色）。
   5. 入手下面根据其是否是一个"测试"或"空调"的一天:
      1. 在审判日1和6（即前，后测试），放置室间的门处于打开位置（图2B）。
      2. 加载"测试"的电脑程序，并输入动物标识（ 图1A），然后发出启动指令（图1B），如果适用。
      3. 轻轻地降低每个鼠标到其指定的设备中间室，对着后墙，轻轻地盖上盖子。一旦所有腔装，离开考场，并最大限度地降低噪音。
      4. 互留装置内部的小鼠，直到所有小鼠试验已关闭/结束（图1C）。导出试验数据。
   <利>只需之前或之后的预试验中，称重动物。空调在使用这些权重剂量计算。
   6. 为了调理试验做准备，减去另外在每一项花费（即"黑减白"与"白减去黑"的时间计算黑色和白色室每只小鼠的试验前的"偏爱"，见图3中，列9和10）。
   7. 由于具有较强的初始喜好小鼠做平衡困难，建立偏好分数可接受的限度（例如，<总试用时间的33％），并排除老鼠，从计算超过它。使用自由的限制（<总试用时间的66％），以最大限度地包容，当磨损很可能测试完成后（ 例如，由于脱靶的手术操作）。
      注:小鼠超过限制仍然可以进行测试，以试图保持自己的预测试的喜好平衡。后来，THESE小鼠可以从分析中排除，必要时，以平衡预测试分数。如果极端的预试验的偏见（ 即> 800秒）严重影响一个群体，可以考虑改变空调的环境和/或使用其他检测。
   8. 选择黑色或白色作为室内与毒品配对室每只小鼠，同时总结每组记住以下优先事项范围内相应的预测试偏好分数:
      注:请注意每个队列内 以及跨以往任何同伙平衡群体之间的分数。
      1. 让所有群体等同尽可能的款项。
      2. 使尽可能接近零越好（即，选择对某些小鼠和人非优选侧上的优选的侧）的款项。如果接近零的总和为任何给定组，是不可能的重新调整所有其他为限制组的最佳可获得得分紧密匹配，有利于轻微的负组总结以上正。
      3. 尽可能可行的，保持的黑色分配与白色的和优选的与非优选的腔室，即使在每个组药物配对。
      4. 因为它并不总是可能的，以满足上述目标，校正通过在稍后的同伙相对平衡的考虑任何偏差;但是，尽量避免与完全不同的平均预测试的偏好产生同伙。
   9. 上调理天2-5，放置室间的门处于关闭位置（图2C）。
   10. 制备单独的注射器与可卡因（天2和4）或盐水如步骤1.1.5描述并根据在预先试验测定体重（天3和5）的溶液。
       注:剂量应就实验的预期，在引言中提到的注意事项，以及潜在的地板和天花板效应进行选择。通常最好进行使用至少两种不同剂量的独立的实验。
   11. 加载"Conditioning"计算机程序，输入动物标识（ 图1A），然后发出启动指令（图1B），如果适用。
   12. 浮渣并注入每只小鼠（IP），立即将其降低到所面临的后墙分配给他们的设备的适当的黑色或白色室，然后轻轻盖上盖子。一旦所有腔装，离开考场，并最大限度地降低噪音。
   13. 卸下他们的室老鼠接近整整30分钟可行（ 即第一老鼠从它们的腔移除，而其他老鼠仍在调节）。取出的动物尽可能平静，没有引入噪声。
3. 统计分析:
   1. 选择分析的方法。无论是从时间的考验后期间花费在盐水配对侧减法时间后测试（可卡因 - 盐水，秒）期间在可卡因成对的侧花或使用在检测后药物的成对腔室中花费的时间减去测试前的时间花在了毒品配对室。
      注意:如果使用第一种方法，也的平均时间曲线图线图中的前和后试验中的每个组的中间，生理盐水和伴药室花费。相比于预试验中，试验后的应显示增加的时间在药物配对侧和减少在盐水配对侧（参照图6，底部和探讨说明）所花费的时间。
   2. 根据不同的性质和组数进行比较，采用t检验，一个或两个方差分析，适当，可能与​​事后分析，分析无论是上面给出的减法分数。
      注:可卡因偏好分数往往是可变的，并且此外，可以是负的（ 即 ，表示所厌恶的药物-成对的侧）。小鼠表现出厌恶不应该被删除（除非他们是统计离群值），因为这个结果是正常的，可能重要的是要确定克之间的差异oups。期望需要12至30个动物的样本大小每组，这取决于治疗的效果的大小。

2.行为敏

1. 设备和房间布置
   1. 获得4×8（X X Y）光子束阵列（外部尺寸11.5"×20"）。构造由黑色有机玻璃的一个顶部开口的腔室（内部尺寸22 1/6"×13¾"×9 3/8"），以容纳所述阵列（ 图4A）。
   2. 准备测试室，使得红色光（吊顶或壁挂式）可以在测试过程中被使用。
   3. 准备日常习惯和注射试验分离的程序会话。
      1. 设置习惯试验是30-60分钟和注射试验之间是60-120分钟（ 图5B）之间。每个建议的长度是60分钟。保持试验长度相同的实验中在多个队列一致。
      2. 组试验开始RECO在启动信号被启动（图5B）后出现的第一光束破录制。对于注射试验，设置启动信号，以便能盒如果可能的话可以单独启动（不统一）。
      3. 要记录在用户定义的一组光束中断"箱"，优选5分钟每（图5B）。
   4. 准备全卷为实验手头需要可卡因的解决方案。溶解可卡因HCl的0.9％氯化钠（盐水），基础终浓度0.1毫升/ 10g体重的注射量（例如，对于剂量为5毫克/公斤，溶液浓度为0.5毫克/毫升）。涡流混合30-45秒，无菌过滤器（0.2微米的注射器过滤器），并存储在室温。
2. 测试
   注:测试的初始阶段，连续10-11天运行一次。小鼠接受每天两次试验:习惯（注免费）和注射。辖盐水前三到f我们的天（见讨论为盐水习惯的重要性），和可卡因为接下来的七个使用相同的剂量（例如，15毫克/公斤/天）。
   1. 每一天，适应在其家笼中的小鼠的行为接待室进行30分钟至1小时。
   2. 准备干净整洁的标准鼠标大小的透明的丙烯酸外壳笼极薄清新的床上用品（如松木芯片）层，以便不模糊photobeams，如果适用（图4C＆D）。
   3. 地方笼针对靠近一端的光子束阵列的Y轴，因此这五束沿着其长度均匀间隔开。 X轴束未在该试验中（图4C及D）的使用。
   4. 就拿老鼠从它们的笼子，以随机顺序，浮渣和权衡他们。通过他们的尾巴（的支持）和地点的基础上取下权衡船每只小鼠直接进入其指定的运动笼子。封面用标准的过滤器顶盖每个笼子。
   5. 准备个人注射器W¯¯第i盐水或可卡因溶液，如步骤2.1.7描述的，根据当前日的体重。用剂量为15或20mg / kg的开始，并考虑使用较高或较低剂量的第二实验（见探讨有关因素）。
   6. 一旦习惯试验已经结束了所有的笼子，装入注入程序。
   7. 一次，从他们的测试笼子，浮渣去除小鼠和给自己注射（IP）。鼠标返回到其测试前笼，迅速 ​​启动该笼（图5C，底部）的启动信号。
   8. 所有注射试验结束后，返回小鼠饲养笼和他们的住房空间。
   9. 如果需要的话，允许小鼠接受一系列停药期和药物的挑战，如下面的:相同剂量原，半剂量，双倍剂量，然后盐水，允许同一剂量攻击前七天，三至七个之前每个额外的挑战天。无论挑战的选择，maintai在实验中跨同伙ñ类似的停药期。
      注:如果原剂量较高 （例如，30毫克/公斤或以上），双剂量应跳过或以较低剂量取代。盐水挑战揭示由可卡因配对环境的任何空调自发活化单独，并在此过程中，这是可卡因依赖性致敏运动的量（见讨论）。
3. 统计分析
   1. 选择将被用于分析每个试验的（多个）部分。在啮齿类动物中最可卡因引起的运动的第一种药物注射15-30分钟后〜内发生。根据所涉及的变量，可以考虑分析累积运动多的时间窗口 （例如，第15，30，60和/或120分钟），或专注于审判独立的部分。
   2. 使用帧选择的时间，按天总结每个动物梁减免习惯和注射试验分别再verage的款项为每个组。情节是指在一个线图上所有天数的平均值（SEM）的标准误差。由于治疗可能会改变鼠标是如何应对的药物每天的早期试验和/或后期，也受到5分钟斌在每天的庭审过程中绘制均线组光束中断。
      注:绘制的习惯和注射试验日常活动的平均值（例如，前15分钟），使得它人为地出现了运动是由生理盐水浸湿，但请记住，活动有（最有可能）由习惯每天结束时下降到这个水平。
   3. 分别分析采用重复测量有（RM）方差分析的日期/时间的试内因素，包括任何主体间因子设计，适当的盐水连续和药物治疗天。使用t检验或单因素方差分析来研究可卡因（急性接触）的第1天的组间差异和多元方差分析的挑战。在适当的时候，跟着意义e为事后检验或单因素方差分析。

结果

从CPP测定代表性的结果示于图6中使用的野生型C57BL / 6N小鼠在约9周龄。研究设计是一个2×3的混合因子，以测试（前后）的主体内变量和试间治疗（生理盐水和可卡因5和10毫克/千克）的变量。一个RM方差分析结果表明检测及处理（F = _2,20 3.68，P <0.05），这是在代替这两个试验观察显著主效应的解释之间的互动显著（F = _1.20 9.86，P <0.01）治疗（F = _1.20<...

讨论

这个协议演示了条件性位置偏爱和运动致敏，其每一个可以被平均实验室用于评估药物诱导行为可塑性的各方面的方法。与大多数行为测试中，有超出基本协议的更多值得考虑。首先，每个这些技术可以被设想为具有两相，诱导和表达。 "感应"涵盖的行为，为CPP调节过程中出现它的发展，为致敏它是（通常是连续的）药物暴露初期。 "表达"为CPP是试验后的，而对于致敏它可以被定义为后停药或仅?...

材料

Name	Company	Catalog Number	Comments
Cocaine Hydrochloride USP	Mallinckrodt Pharmaceuticals	0406-1520	Purchase and use (Schedule II controlled substance) for research purposes requires compliance and licensure according to state and federal law.
Conditioned Place Preference,  Three Compartment Apparatus with Manual Doors and Lights for Mouse	Med-Associates Inc.	MED-CPP-MS & MED-CPP-3013	Our laboratory has used these boxes; however, many alternative boxes are available & acceptable.
PAS-Home Cage Activity Monitoring Photobeam Arrays	San Diego Instruments	2325-0223 & 7500-0221	Our lab houses these arrays inside of custom built chambers, as described in the text.  There are alternatives available.
Disposable Sani-Cloth disenfecting wipes	PDI	13872