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May 2016

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Examining Cocaine Conditioning Place Preference in Mice
可卡因调节小鼠位置偏爱研究   

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Abstract

A key component of combating substance use disorders is understanding the neural mechanisms that support drug reward. Tasks such as self-administration assess the reinforcing properties of a drug using a learned behavior but require numerous training sessions and surgery. In comparison, the conditioned place preference (CPP) task assesses reward with little training, without costly surgeries, and confounds that accompany the use of anesthesia or pain-relieving drugs. The CPP task contains three phases: pretest, conditioning, and posttest. During the pretest, mice are allowed to explore a three-compartment apparatus. The two outer compartments contain unique olfactory, tactile, and visual cues whereas the middle compartment is used as an entrance and exit for the mice on test days. During conditioning, mice receive cocaine before being confined to one of the outer compartments. The following day, mice are given saline then confined to the other outer compartment. These pairings are then repeated once. At posttest, mice are permitted to freely explore all compartments in a drug-free state while the time spent in each compartment is recorded. A CPP score is calculated for both the pretest and posttest by comparing the time spent in the cocaine-paired and saline-paired compartments. Enhancements in the CPP score from the pretest to the posttest serve as a measure of the rewarding property of the cocaine. This task offers several notable advantages: 1) the simultaneous recording of locomotor activity and reward, which may utilize different neural mechanisms, 2) the three-compartment CPP setup removes the bias that can be observed in a two-compartment design, and 3) use of multimodal cues support the acquisition of a robust preference in a variety of mouse strains.

Keywords: Mouse (小鼠), Behavior (行为), Cocaine (可卡因), Reward (奖励), Conditioned place preference (条件性位置偏爱)

Background

The conditioned place preference (CPP) paradigm has been used for decades to examine the role of genes, histone modifications, signaling pathways, and brain regions in drug-associated memory and reward (see Tzschentke, 1998; Malvaez et al., 2009). It incorporates classical conditioning to assess the rewarding effects of contextual cues that have been associated with a drug (for reviews see Schechter and Calcagnetti, 1993; Everitt et al., 1999; Bardo and Bevin, 2000). There are various CPP chamber designs and configurations, depending on the specific research question being addressed (Bardo and Bevin, 2000). However, the most commonly used designs are the two-compartment or three-compartment chambers. Both chambers types rely on the ability of mice to differentiate between two compartments that contain distinct olfactory, tactile, and visual cues. The three-compartment design contains an additional compartment, between the two larger compartments, that does not contain olfactory or tactile cues, and is used only for the entry and removal of mice at test. Here, we describe the equipment setup, key parameters, and data collection in the three-compartment CPP paradigm using adult mice. The focus of this protocol is on cocaine CPP. However, this paradigm can be used for other drugs of abuse (e.g., other psychostimulants, opioids, and nicotine).

The three-compartment design allows mice to choose whether to enter either the cocaine or saline-paired compartments or remain in the center compartment. Conversely, the two-compartment chambers require mice to be placed in either the saline-paired or the cocaine-paired compartments to begin the test. This has the potential to introduce bias for the compartment in which the mice is place at post-test. Using tactile, olfactory and visual cues allows mice to form a memory for each context that does not rely on one sensory system. Therefore, the likelihood that mice with sensory deficits (e.g., poor vision) will fail to acquire a drug-associated preference is reduced. Other variations of the task (e.g., olfactory only) can require four cocaine pairings, which results in eight total injections when the saline injections are included. Acquisition in our task requires two cocaine pairings (four total injections) for robust cocaine CPP, which reduces the likelihood that stress and irritation at the site of injection will be significant factors in the experiment. Furthermore, if both the control and experimental groups equally acquire a preference, the posttest day of the CPP task can serve as the first day of an extinction experiment. This subsequent extinction experiment would allow the experimenter to examine the persistence of the acquired cocaine-associated memory. Overall, the CPP paradigm provides an adaptable, quick, and inexpensive method of assessing drug reward in adult mice.

Materials and Reagents

  1. Paper towels
  2. BD 1 ml Syringe slip tip with BD PrecisionGuideTM needle, 26G x 5/8 (0.45 mm x 16 mm; Fisher Scientific, catalog number: BD 309597 )
  3. Gloves (to be worn at all times that researchers will come into contact with mice)
  4. Handling sleeve (Ansell, catalog number: 19-120-3177 )
  5. Bedding: Pine shaving (P.J. Murphy Forest Products)
  6. Cedar bedding (PetsPick)
  7. Mice (8-15 weeks old) of either sex are housed individually beginning a week prior to behavioral testing
    Mice have free access to food and water in their homecage. Environmental enrichment such as nesting materials (e.g., nestlets) and a retreat are features of each homecage. Their homecage area is temperature-controlled (23 °C) with a 12:12 h. light/dark cycle. Behavioral testing is performed during the light portion of the cycle.
    Note: This protocol has been optimized using C57BL/6J mice but can be used for other strains as well.
  8. 70% (v/v) ethanol diluted from stock using water (Fisher Scientific, catalog number: A4094 )
  9. Sterile saline (Growcells, catalog number: msdw-1000 , Shelf life: 3 years, stored at room temperature)
  10. Cocaine hydrochloride (Sigma-Aldrich, CAS number: 53-21-4)

Equipment

  1. Heavy-Duty Utility Cart (RubbermadeTM, catalog number: 11-926-75 )
  2. CPP chambers (custom-made; Figure 1)
  3. Four digital cameras mounted above the CPP chambers (e.g., monochrome IR GigE camera set; Noldus Information Technology)
  4. Overhead camera-mounting bracket
  5. Overhead lamps
  6. Stopwatch with a silent mode
  7. Lux meter (Fisher Scientific, catalog number: 0 666264 )
  8. Dell Precision desktop (e.g., Processing Unit Precision T5810)


    Figure 1. Two conditioned place preference apparatus. The outer compartments (Floor: 12.5 cm x 17 cm; Height: 32.5 cm) are separated by a smaller inner compartment (Floor: 12.5 cm x 11.5 cm; Height: 32.5 cm). The compartment with checkered contact paper has a grid floor with cedar shaving below. The compartment with white contact paper has bar flooring with pine chips below. The middle compartment has a solid gray floor, two gray walls, and two walls with either checkered or white contact paper to match the adjoining outer compartments. Guillotine doors separate the outer compartments from the middle compartment.

Software

  1. EthoVision XT tracking system (Noldus Information Technology, Inc. Leesburg, Virginia)
  2. Microsoft Excel (Microsoft)
  3. Statistical Software (i.e., SPSS, GraphPad Prism, R)

Procedure

  1. Experiment setup
    1. Place the CPP apparatus in a room dedicated to behavioral research. This room should be devoid of extraneous odors or sounds. The room’s house lights should be turned off throughout the experiment, as the overhead lights will be used to track mice during testing (Figure 2). A lux meter is placed in the middle of each chamber (i.e., on top of either the grid or bar floors shown in Figure 1) to ensure that the light intensity (100 lux) from the overhead lights is similar across all chambers of the CPP apparatus.
      Note: Rooms that are used for surgical procedures or animal husbandry are not suitable. Similarly, clothes that have fragrances and other strong odors should be changed prior to the start of the experiment.


      Figure 2. Conditioned place preference room setup with eight apparatus. Each CPP chamber is assigned a box ID (e.g., Box 1). The orientation of each box is antiparallel (rotated 180°) from the adjacent box. The location of each box is permanently fixed. The overhead lights are positioned to avoid shadows on the floor of the compartments. Each camera is positioned to record from two boxes.

    2. Create a table in Microsoft Excel that includes the mouse ID, Group Assignment, Box #, and Time spent in the checkered and white compartments.
    3. Assign each mouse to a dedicated behavioral box with assigned cocaine-paired and saline-paired compartments.
      Note: During this step, ensure that both control and experimental groups are equally represented during each trial. For each group, assign the white compartment as the cocaine-paired compartment for half of the mice. For the other half, assign the white compartment as the saline-paired compartment. It is best to ensure that across the entire experiment, each group is represented in each research box. Counterbalancing the injection-compartment assignments in this manner prevents chamber bias from confounding the CPP scores.
    4. House mice in individual cages one week prior to the start of the experiment. At this point, unique identification can be assigned to mice.
      Note: Experimenters should be blinded to the treatment group of each mouse.
    5. Weigh the mice before the start of the experiment.
    6. Prepare the desired cocaine dose for the experiment.
    7. Set recording software to record video as well as live tracking if possible.

  2. Handling the mice
    1. Turn on the overhead lights in the behavioral room and put on nitrile or latex gloves.
      Note: The same type of glove must be worn throughout the experiment.
    2. Remove the cages containing the mice from their animal rack and place them on the utility cart.
    3. Transfer the mice to the behavioral room using the utility cart.
      Note: It is important to avoid excess noise and rattling during this transfer. These sounds and motions can act as stressors for the mice.
    4. Clean gloves and handling sleeve using 70% ethanol but wait until the ethanol evaporates before handling the mice.
    5. Remove a mouse from its cage by quickly grabbing the base of its tail and placing it on the handling sleeve.
    6. Allow the mouse to freely explore the handling sleeve for 3 min before returning it to its cage (Figure 3).
    7. Clean your gloves and the handling sleeve after handling each mouse.
    8. Repeat the handling and glove cleaning steps until all mice have been handled.
    9. Use the utility cart to return mice to their housing room and animal rack.


      Figure 3. Conditioned place preference schematic. A representative daily sequence for two mice in the same group are illustrated. Mice undergo handling for 3 min on three consecutive days. At pretest, mice are allowed to freely explore the three-compartment apparatus for 15 min. On days 5 and 7, cocaine is administered before the mice are confined to one of the larger compartments. They are then permitted to explore that compartment for 30 min. The experiment is counterbalanced to ensure that half of the mice have cocaine paired with the white compartment while the other half have the cocaine paired with the checkered compartment. On days 6 and 8, saline is administered before the mice are confined to the large compartment that has not been associated with cocaine. They are then permitted to explore that compartment for 30 minutes. Mice are given a day off on day 9 and remain in their home cages. On posttest, mice are allowed to freely explore the three-compartment apparatus in an identical manner to the pretest (15 min duration).

  3. Pretest
    1. Prepare the behavioral room by turning on the overhead lights.
    2. Clean each CPP chamber using 70% ethanol.
    3. Open the guillotine doors so that mice can freely move between each compartment.
    4. Turn on the computer and recording software.
    5. Transfer only the mice that will be run together to the behavioral room using the utility cart.
    6. Set the recording software to begin tracking when mice enter each chamber and to stop after 15 min have elapsed.
    7. Pick up each mouse by the base of their tail and transfer them to your palm.
    8. Place each mouse in the center compartment of their predetermined CPP box.
      Note: Ensure that as each mouse is lowered into the CPP apparatus, they are facing one of the gray walls of the center compartment. This will reduce any bias for a particular compartment.
    9. Allow mice to explore the CPP apparatus for 15 min (Figure 3).
    10. Remove the mice from the apparatus and transfer it to its cage. Start by confining the mice to the center compartment by closing the guillotine doors, then grab it by the base of its tail before immediately placing it in your palm then cage.
    11. Return all mice to their housing room using the utility cart.
    12. Open the guillotine doors and clean the CPP apparatus thoroughly using 70% ethanol. Pay special attention to areas that may accumulate pooled urine or defecation.
      Note: The bedding (i.e., pine and cedar) can remain in place for the entirety of the experiment unless there is an accumulation of urine or defecation.
    13. Set the computer to record the next set of mice.
    14. Clean gloves using 70% ethanol before retrieving the next set of mice.
    15. Repeat Steps C5-C14 until all mice are tested.

  4. Conditioning
    1. Prepare the saline and cocaine solutions.
      Note: A moderate cocaine dose (e.g., 10 mg/kg) is recommended for preliminary experiments–where either a memory impairment or enhancement may be observed.
    2. Measure out the amount of saline or cocaine to be administered to each mouse from the stock using a syringe.
    3. Place each filled syringe in front of the CPP chamber in which the injected mouse will be added.
    4. Clean each CPP chamber using 70% ethanol.
    5. Close the guillotine doors so that mice will be confined to the outer compartments.
    6. Transfer only the mice that will be run together to the behavioral room using the utility cart.
    7. Administer saline or cocaine via intraperitoneal injection before placing the mouse into the assigned outer compartment.
    8. Set a timer for 30 min. Allow mice to explore the assigned compartment (Figure 3).
      Note: Mice do not need to be recorded during conditioning sessions. However, the recording software can be activated if the activity during conditioning is of interest.
    9. After 30 min have elapsed, remove each mouse from their respective CPP box and transfer it to its homecage.
    10. Return all mice to their housing room using the utility cart.
    11. Clean gloves using 70% ethanol before retrieving the next set of mice.
    12. Repeat Steps D1-D10 until all mice are tested.

  5. Posttest
    Repeat the steps outlined in the pretest.

Data analysis

  1. Use your recording software to calculate the time spent in each compartment during the pretest.
  2. Calculate the CPP score for each mouse on the pretest. The CPP score is the time spent in the cocaine-paired compartment minus the time spent in the saline-paired compartment (Table 1).
    Note: Because the groups are arranged so that each group is represented in each box and that there are an equal number of mice that have cocaine paired with the checkered and white compartments, vigilance is required to accurately calculate the CPP scores of neighboring mice. For example, in Table 1, the CPP scores below for Mouse 1 (CPP Score = Checkered - White) and Mouse 2 (CPP Score = White - Checkered) are calculated differently.
  3. Organize the mice by the independent variable (e.g., genotype, treatment or sex). A representative table of an experiment with two groups is illustrated in Table 2.
  4. Calculate the CPP score for each mouse on the posttest (Table 3).
  5. Comparing the total distance traveled across on the pretest and posttest days can also be used as a behavioral control. Observing similar distance traveled between groups suggests that any potential differences in CPP scores from your experimental groups relative to your control group cannot be attributed to differences in locomotion.
  6. Organize the mice by the independent variable in a similar manner to the pretest (Table 4).

    Table 1. Representative CPP Data from the Pretest. Before the experiment, each mouse is assigned a CPP box, cocaine-paired and saline-paired compartments.


    Table 2. Representative Group Data from the Pretest. The mean CPP score of each mouse is reorganized by group to eliminate outliers and enable future analysis


    Table 3. Representative CPP Data from the Posttest. Mice that acquire a preference will show a positive CPP score at the posttest. The magnitude of the CPP score illustrates the strength of the learned cocaine association.


    Table 4. Representative Group Data from the Posttest. The mean CPP score of each mouse is reorganized by group to enable future analysis.


  7. Use your preferred statistical software (i.e., SPSS, GraphPad Prism, R) to analyze the CPP scores for the pretest and posttest using a two-way repeated-measures analysis of variance. If the data is normally distributed, Bonferroni post-hoc tests can be performed to compare difference in the means of the two groups. Alternatively, if the results are not normally distributed, non-parametric tests such as the Mann-Whitney test can be used. These statistical tests will indicate whether, within each group, significantly more time was spent in the cocaine-assigned chamber following cocaine conditioning. The results from the statistical tests will also indicate whether there are any significant changes in the amount of time in the cocaine paired chamber between groups. These specific comparisons can be made using Student’s t-tests with α-levels held at 0.05. Figure 4 illustrates representative data.
    Note: If your groups include both sexes, sex differences should be examined first or as a separate factor.


    Figure 4. Example graph comparing Groups A and B. Group A represents the control group (e.g., wildtype) whereas Group B represents the experimental group (e.g., mutant). Bars represents the mean CPP score (cocaine-paired minus saline-paired) ± S.E.M. for each group on pretest and posttest. Pretest data illustrates that neither group has a strong preference for either compartment prior to conditioning. Posttest data show that Group A mice developed a robust CPP score, whereas the CPP score of Group B is attenuated. A two-way repeated-measures analysis of variance (ANOVA) revealed main effects of group (F1,14 = 15.31, P = 0.0016) and conditioning (F1,14 = 25.61, P = 0.0002), as well as an interaction (F1,14 = 8.566, P = 0.0110).

Notes

This protocol has been optimized using C57BL/6J mice, which typically yield an individual CPP scores between -150 to 150 s at the pretest. However, this range may vary depending on the strain or treatment etc. Alterations can be made to the lighting and odors intensities to obtain similar results using various strains. Criteria for exclusion should be determined prior to the experiment. The features of the CPP setup (i.e., the odors, wall patterns and light intensity) were selected because they do not lead to CPP scores on the pretest that are significantly different from zero. Furthermore, mice with a CPP score outside of ± 250 s at the pretest are excluded.

Acknowledgments

This protocol was adapted from White et al., 2016. Previous work was supported by grants from the US National Institutes of Health (DA025922, DA036984 and MH101491), the National Institute of General Medical Sciences of the National Institutes of Health (GM055246), the Department of Education GAANN (P200A120165), the US National Institute of Drug Abuse (F31DA038505) and the National Institute on Aging T32 grant (AG000096-31). Recent work has been supported by Mount Holyoke College.

Competing interests

The authors have no conflicts of interest.

Ethics

All experimental procedures were approved by the Institutional Animal Care and Use Committees at the University of California Irvine (IACUC#: 2006-2620) and Mount Holyoke College (IACUC#: BR-56-1117) from the period 2011-2020.

References

  1. Bardo, M. T. and Bevins, R. A. (2000). Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology (Berl) 153(1): 31-43.
  2. Everitt, B. J., Parkinson, J. A., Olmstead, M. C., Arroyo, M., Robledo, P. and Robbins, T. W. (1999). Associative processes in addiction and reward. The role of amygdala-ventral striatal subsystems. Ann N Y Acad Sci 877: 412-438.
  3. Schechter, M. D. and Calcagnetti, D. J. (1993). Trends in place preference conditioning with a cross-indexed bibliography; 1957-1991. Neurosci Biobehav Rev 17(1): 21-41.
  4. Tzschentke, T. M. (1998). Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol 56(6): 613-672.
  5. White, A. O., Kramár, E. A., Lopez, A. J., Kwapis, J. L., Doan, J., Saldana, D., Davatolhagh, M. F., Alaghband, Y., Blurton-Jones, M., Matheos, D. P. and Wood, M.A. (2016). The role of neuron-specific nucleosome remodeling in cocaine-associated memories. Nat Commun 7: 11725.

简介

[摘要 ] 对抗药物滥用症的关键因素是了解支持药物奖励的神经机制。诸如自我管理之类的任务会通过学习行为来评估药物的增强特性,但需要进行大量的培训和手术。有条件的位置偏爱(CPP)任务无需培训就可以评估奖励,而无需进行昂贵的手术,并且在使用麻醉或痛苦镇痛时会感到困惑 CPP任务包含三个阶段:预测试,条件调整和后测试。在预测试期间,允许小鼠探索三格设备。两个外层格包含独特的嗅觉,触觉和视觉提示,中间格是在调节过程中,小鼠在被限制在一个外部隔室中之前接受可卡因。第二天,给小鼠注射生理盐水,然后将其限制在另一个外部隔室中,然后重复这些配对一次,在后测,小鼠被允许自由地探索所有舱室在无毒品国家虽然花费在每个隔间的时间记录。CPP的得分计算两者的检测前后通过比较花费在Cocai的时间NE-配对和盐水配对隔间从前测到后测的CPP分数的提高可以衡量可卡因的奖励特性。优点:1)同时记录运动活动和报酬,这可能利用不同的神经机制; 2)三室CPP设置消除了在两室设计中可以观察到的偏差,以及3)使用多模式提示支持在各种小鼠品系中获得强大的偏好。

[背景 ] 条件位置偏爱(CPP)范式已用于研究基因,组蛋白修饰,信号传导途径和大脑区域在与药物相关的记忆和奖赏中的作用(参见Tzschentke ,1998;Malvaez 等,2009)。 )。它结合了经典条件来评估与药物相关联的上下文线索的奖励作用(有关评论,请参阅Schechter和Calc agnetti ,1993; Everitt 等人,1999; Bardo和Bevin,2000)。隔室的设计和配置,取决于要解决的具体研究问题(Bardo和Bevin,2000年),但是,最常用的设计是两室或三室的隔室,两种隔室的类型都取决于小鼠的区分能力在三间隔间的设计中,在两个较大的隔间之间有一个额外的隔间,其中不包含嗅觉,触觉和视觉提示。嗅觉或触觉提示,仅用于测试中小鼠的进入和取出。在此,我们描述了使用成年小鼠的三室CPP范例中的设备设置,关键参数和数据收集。然而,这种范例可以用于其他滥用药物(例如其他精神兴奋剂,阿片类药物和尼古丁)。

三室设计允许小鼠选择进入可卡因或盐水配对隔室还是留在中间隔室中。减少了,两室隔室要求将小鼠置于盐水配对或可卡因隔室中。配对隔间开始测试,这可能会在测试后对放置小鼠的隔间产生偏见,使用触觉,嗅觉和视觉提示可以使小鼠在不依赖任何环境的情况下形成记忆系统感觉。因此,可能老鼠感官赤字(例如,视力不良)将无法获得药物相关的偏好降低,其他变化的任务(例如, 仅嗅觉)可能需要四次可卡因配对,包括盐水注射在内,总共需要注射八次。我们的任务中的采集需要两对可卡因配对(共四次注射)才能获得稳定的可卡因CPP,从而降低了在工作时产生压力和刺激的可能性。注射部位将是实验中的重要因素。如果对照组和实验组均获得了偏爱,则CPP任务的测试后日期可以作为灭绝实验的第一天。随后的灭绝实验将允许总体而言,CPP范例提供了一种评估成年小鼠药物奖励的适应性,快速且廉价的方法。

关键字:小鼠, 行为, 可卡因, 奖励, 条件性位置偏爱

材料和试剂


 


1. 纸巾      


2. BD 1ml注射器滑动与BD尖端PrecisionGuide TM 针,26G X 5/8(0.45毫米×16毫米;费舍尔施恩IFIC,目录号的:BD一百三个和九个千五个一百和九十-七)      


3. 手套(研究人员与小鼠接触时应始终佩戴的手套)      


4. 处理套(Ansell,目录号:19-120-3177)      


5. 床上用品:剃须(PJ墨菲林产品)      


6. 雪松床上用品(PetsPick )      


7. 进行行为测试之前,我们先将一个性别的小鼠(8-15周大)单独收容。       


可以免费使用小鼠食物和水在他们的居住笼。环境富集这样的作为筑巢的材料(例如,Nestlets)一退特点是每个居住笼。他们的居住笼区进行温度控制(23°C)对于12:12 ^ h 亮/暗循环。在循环的亮部分进行行为测试。


注意:此方案已使用C57BL / 6J小鼠进行了优化,但也可用于其他菌株。


8. 70 Pasento(V / V)乙醇稀释从库存使用水(费舍尔小号系统求解,目录号:A4094)      


9. 无菌盐水(Growcells ,目录号:msdw-1000,保质期:3年,在室温下保存)      


10. 盐酸可卡因(Sigma-Aldrich,CAS编号:53-21-4)   


 


配套设备


 


重型手推车(Rubbermade TM ,目录号:11-926-75)
CPP腔室(定制;图1)
在CPP腔上方安装了四个数码相机(例如,单色IR GigE相机;Noldus 信息技术)
高架摄像机安装支架
高架灯
静音模式秒表
照度计(Fisher Scientific,目录号:0666264)
Dell Precision台式机(例如,处理单元Precision T5810)
 


D:\重新格式化\ 2020-3-2 \ 1902986--1358安德烈·怀特856412 \图jpg \图1.JPG


图1. 两个有条件的位置偏爱设备。外部隔间(地板:12.5厘米x 17 厘米;高度:32.5厘米)被较小的内部隔间(地板:12.5厘米x 11.5厘米;高度:32.5厘米)隔开。带有方格纸的隔层在下面带有雪松木屑的网格地板;带有白色纸面的隔间在下面带有松木屑的条形地板;中间的隔层为纯灰色地板,两个灰色壁和两个带有方格或白色触点的壁断头台将分隔开的隔层与中间的隔层分隔开来,以匹配相邻的隔层。


 


软体类


 


EthoVision XT跟踪系统(Noldus 信息技术有限公司,弗吉尼亚州里斯堡)
Microsoft Excel(Microsoft)
统计软件(即SPSS,GraphPad Prism,R)
 


程序


 


实验设置
将CPP仪器放置在专门用于行为研究的房间中。该房间应没有异味或声音。在整个实验过程中,应关闭房间的室内照明灯,因为过高的照明灯将在测试过程中用于跟踪小鼠(图2)。在每个腔室的中间放置一个照度计(即,在图1所示的网格或条形地板的顶部),以确保来自所有腔室的头顶灯的光强度(100 lux)相似CPP设备的
注意:用于外科手术或畜牧业的房间不适合使用,同样,在开始实验之前,应先更换有香味和其他强烈气味的衣服。


 


D:\重新格式化\ 2020-3-2 \ 1902986--1358安德烈·怀特856412 \图jpg \图2.JPG


图2. 有八个设备的有条件的场所优先房间设置。每个CPP腔室都分配了一个盒子ID(例如,盒子1)。每个盒子的方向与相邻盒子呈反平行(旋转180°)。每个盒子的位置为永久固定,顶灯的位置应避免在车厢地板上形成阴影,每个摄像机的位置应从两个盒子中进行记录。


 


在Microsoft Excel中创建一个表,其中包括鼠标ID,组分配,框号和在格子和白色隔室中花费的时间。
将每只鼠标分配到专用的行为盒,该行为盒带有可卡因配对和生理盐水配对的隔室。
注意:在此步骤中,确保在每个试验中对照组和实验组的代表均相同。对于每组,将白色隔室分配为一半小鼠的可卡因配对隔室,对于另一半,将白色隔室分配为以这种方式平衡注射室分配可防止室偏倚混淆CPP分数。盐水配对室。最好确保在整个实验中,每个研究小组代表每个小组。


在实验开始前一周将家鼠放在单独的笼子中,此时可以为小鼠分配唯一的标识。
注意:实验者应该对每只小鼠的治疗组视而不见。


在实验开始前称重小鼠。
准备实验所需的可卡因剂量。
设置录制软件以录制视频以及实时跟踪(如果可能)。
 


处理老鼠
打开行为室的头顶灯,戴上丁腈或乳胶手套。
注意:在整个实验过程中,必须戴相同类型的手套。


从它们的动物架子上取下装有老鼠的笼子,然后将它们放在多功能车上。
使用工具车将小鼠转移到行为室。
注意:避免在传输过程中产生过多的噪音和嘎嘎声非常重要,这些声音和动作可能会给小鼠带来压力。


使用70%的乙醇清洁手套和处理套筒,但要等到乙醇蒸发后再处理小鼠。
通过快速抓住鼠标尾巴的底部并将其放在处理套中,将鼠标从笼子中取出。
让鼠标自由滑动处理套筒3分钟,然后再将其放回笼中(图3 )。
处理每只鼠标后,请清洁手套和处理套。
重复处理和手套清洁步骤,直到所有老鼠都被处理完为止。
使用工具车将老鼠送回其饲养室和动物架子。
 


D:\重新格式化\ 2020-3-2 \ 1902986--1358安德烈·怀特856412 \图jpg \图3.jpg


图3.条件位置偏好示意图。说明了同一组中两只小鼠的代表性日序列。小鼠连续三天接受处理3分钟。在预测试中,允许小鼠自由探索三室设备15分钟在第5天和第7天,在将小鼠限制在其中一个较大的车厢内之前,先给予可卡因,然后允许他们探索该车厢30分钟,然后进行平衡实验,以确保有一半的老鼠与可卡因配对。在第6天和第8天,在将小鼠限制在没有可卡因的大隔间之前,先给予生理盐水,然后允许他们探索该隔间以30分钟,第9天休息一天,将小鼠关在笼子里。在测试后,让小鼠自由地探索三厢设备 进行预测试的方式(持续15分钟)。


 


预测
通过打开头顶的灯准备行为室。
使用70%乙醇清洁每个CPP腔室。
打开断头台的门,以便小鼠可以在每个隔间之间自由移动。
打开计算机和录音软件。
使用工具车仅将要一起奔跑的小鼠转移到行为室。
设置记录软件以开始跟踪小鼠何时进入每个隔室,并在经过15分钟后停止。
抓住每只老鼠尾巴的根部,然后将它们移到您的手掌上。
将每只鼠标放在其预定CPP框的中央隔室中。
注意:确保每只鼠标放到CPP设备中时,它们都面对中间隔室的灰色壁之一,这将减少特定隔室的任何偏斜。


让小鼠探索CPP仪器15分钟(图3 )。
将老鼠从设备中取出并转移到笼子中。首先,通过关闭断头台门将老鼠限制在中央隔室中,然后抓住其尾巴的底部,然后立即将其放在手掌然后放在笼子中。
使用工具车将所有老鼠送回其容纳室。
打开断头台的门,并用70%的乙醇彻底清洁CPP设备,特别注意可能积聚尿液或排便的区域。
注:床上用品(即,松树和雪松)可以留在地方,全部实验的,除非有一个积累的尿液或排便。


设置计算机以记录下一组小鼠。
在取出下一组小鼠之前,使用70%的乙醇清洁手套。
重复步骤C5-C14,直到所有小鼠都经过测试。
 


调理
准备盐水和可卡因溶液。
注意:建议在初步实验中使用适量的可卡因剂量(例如10 mg / kg)– 可能会观察到记忆力减退或增强。


出牛逼测量他量盐水或可卡因要给予每只小鼠从该股的使用注射器。
将每个已填充的注射器放在CPP腔室的前面,将在其中添加注入的鼠标。
使用70%乙醇清洁每个CPP腔室。
关闭断头台的门,以便将老鼠限制在外部隔间中。
使用工具车仅将要一起奔跑的小鼠转移到行为室。
在将鼠标放入指定的外部隔室之前,通过腹膜内注射施用盐水或可卡因。
设置一个30分钟的计时器,让小鼠探索指定的隔室(图3 )。
注意:在调节过程中无需记录小鼠,但是,如果需要调节过程中的活动,则可以激活记录软件。


30分钟过后,将每只鼠标从各自的CPP盒中取出,并将其转移到其笼子中。
使用工具车将所有老鼠送回其容纳室。
在取出下一组小鼠之前,使用70%的乙醇清洁手套。
重复步骤D1-D10,直到测试所有小鼠。
 


后测
重复预测试中概述的步骤。


 


资料分析


 


使用您的录音软件来计算在预测试期间每个隔间中花费的时间。
计算预测试中每只小鼠的CPP得分.CPP得分是在可卡因配对隔室中花费的时间减去在盐水配对隔室中花费的时间(表1)。
注意:由于各组的排列方式使得每个组在每个框中都有代表,并且有相等数量的可卡因与方格和白色隔室配对的小鼠,因此需要保持警惕,以准确计算相邻小鼠的CPP评分。例如,在表1中,以下计算鼠标1(CPP分数=白色格仔)和鼠标2(CPP分数=白色格仔)的CPP分数是不同的。


通过自变量(例如,基因型,治疗或性别)对小鼠进行组织。表2所示为两组实验的代表表。
计算后测中每只小鼠的CPP得分(表3)。
比较前测和测后几天的总距离也可以作为行为控制,观察各组之间相似的距离,则表明实验组相对于对照组的CPP评分的任何潜在差异都不能归因于运动。
通过自变量以与预测试类似的方式组织小鼠(表4)。




表1.来自预测试的代表性CPP数据实验前,每只小鼠被分配了一个CPP盒,可卡因配对和盐水配对隔室。


第一场


鼠标ID


组别


包装盒


方格的


白色


CPP分数


1个





1个


313.780


275.542


38.238


2





2


326.393


240.107


-86.286


3





3


336.336


204.004


132.332


4





4


257.591


196.463


-61.128


5





5


329.596


284.785


44.811


6





6


229.696


296.230


66.534


7





7


374.708


240.040


134.668


8





8


351.552


225.959


-125.593


会议2


 


 


 


 


 


9





1个


277.678


341.208


-63.53


10





2


272.206


261.261


-10.945


11





3


365.432


286.520


78.912


12





4


360.627


206.139


154.488


13





5


363.096


251.318


111.778


14





6


357.558


252.319


105.239


15





7


352.386


335.135


17.251


16





8


252.119


216.950


35.169


 


表2.预先测试的代表性组数据。每组小鼠的平均CPP得分按组进行重组,以消除异常值并进行未来分析


A组


B组


滑鼠


CPP分数


滑鼠


CPP分数


1个


38.238


3


132.332


2


-86.286


4


-61.128


5


44.811


7


134.668


6


66.534


8


-125.593


11


78.912


9


-63.530


12


54.488


10


-10.945


15


17.251


13


111.778


16


35.169


14


105.239


均值


31.139625


均值


27.852625


 






表3.来自后测的代表性CPP数据。获得偏好的小鼠在后测中将显示正CPP分数。CPP分数的大小说明了可卡因学会的强度。


第三场


鼠标ID


组别


包装盒


方格的


白色


CPP分数


1个





1个


433.233


225.359


207.874


2





2


214.414


401.802


187.388


3





3


406.273


302.069


104.204


4





4


232.499


369.57


137.071


5





5


448.048


136.003


312.045


6





6


269.069


331.131


62.062


7





7


431.565


383.05


48.515


8





8


227.895


315.249


87.354


会议4


9





1个


275.909


210.21


65.699


10





2


272.606


346.213


73.607


11





3


408.475


182.916


225.559


12





4


251.585


43.877


207.708


13





5


334.268


200.067


134.201


14





6


250.717


341.875


-91.158


15





7


465.532


180.914


284.618


16





8


485.752


287.888


197.864


 


表4.来自Posttest的代表性组数据。每组小鼠的平均CPP得分按组进行重组,以进行将来的分析。


A组


B组


鼠标ID


CPP分数


鼠标ID


CPP分数


1个


207.874


3


104.204


2


187.388


4


137.071


5


312.045


7


48.515


6


62.062


8


87.354


11


225.559


9


65.699


12


-92.292


10


73.607


15


284.618


13


134.201


16


202.136


14


-91.158


均值


173.674


均值


69.937


 


使用您喜欢的统计软件(即,SPSS,的GraphPad Prism,R)来分析CPP成绩为检测前后采用双因素重复测量分析的遗传变异。如果数据是正态分布的,邦费罗尼事后测试可以进行比较两组均值的差异。此外,如果结果不是正态分布的,则可以使用非参数检验,例如曼恩·惠特尼检验(Mann-Whitney test),这些统计检验将表明在每个组内是否显着可卡因调理后在可卡因分配室中花费了更多时间。统计测试的结果还将表明各组之间可卡因配对室中的时间量是否有任何显着变化。这些特定的比较可以使用Student's α水平保持在0.05的t 检验。图4说明了代表性数据。
注意:如果您的小组中包括男女,则应首先检查性别差异或将其作为一个单独的因素。


 


D:\重新格式化\ 2020-3-2 \ 1902986--1358安德烈·怀特856412 \图jpg \图4.jpg


图4.比较A组和B 组的示例图。A 组代表对照组(例如,野生型)B组代表实验组(例如,突变体)。条形图表示平均CPP评分(可卡因配对减去盐水配对) ± 小号.E 。中号。对于每个组在检测前后。前测数据表明,无论是集团具有很强的偏好无论是车厢前调理。后测的数据显示,A组小鼠开发出一个强大的CPP得分,而CPP得分中B组衰减了。通过两次方差重复测量分析(ANOVA),揭示了组(F 1,14 = 15.31,P = 0.0016)和调节(F 1,14 = 25.61,P = 0.0002)的主要影响,以及相互作用(F 1,14 = 8.566 ,P = 0.0110)。


 


注意事项


 


该协议已使用C57BL / 6J小鼠进行了优化,通常在预测试时产生的CPP得分在-150到150 s之间。但是,此范围可能因劳损或治疗等而异。气味强度获得类似结果使用各种菌株。标准排除应确定在实验之前,的CPP设置的功能(即,气味,长城图案和光强)被选中是因为他们不会导致CPP比分在预测试属于西格Nificantly不同于零。此外,小鼠随着甲CPP评分之外±250秒。在预测试被排除在外。


致谢


 


该方案改编自White 等人,2016。先前的工作得到了美国国立卫生研究院(DA025922,DA036984和MH101491 ),国立卫生研究院的国立普通医学科学研究所(GM055246 )的资助,教育部GAANN(P200A120165),美国国立药物滥用研究所(F31DA038505)和国立衰老研究所T32补助金(AG000096-31)。最近的工作得到了Mount Holyoke学院的支持。


 


竞争利益


 


作者没有利益冲突。


 


道德规范


 


从2011 年至2020年,所有实验程序均获得了加州大学欧文分校(IACUC#:2006-2620 )和霍利奥克学院(IACUC#:BR-56-1117 )的机构动物护理和使用委员会的批准。


 


参考文献


 


Bardo,MT和Bevins,RA(2000)。有条件的就诊地点:它对我们对药物奖励的临床前了解增加了什么? Psychopharmacology(Berl )153(1):31-43。
Everitt ,BJ,Parkinson,JA,Olmstead,MC,Arroyo,M.,Robledo,P。和Robbins,TW(1999年),成瘾和奖励中的联想过程,杏仁核-腹侧纹状体子系统的作用,纽约州Acad Sci 877 :412-438。
谢克特,MD和Calcagnetti ,DJ(1993)。趋势位置偏爱调理用交索引参考书目; 1957年至1991年。神经科学Biobehav 版本17(1):21-41。
Tzschentke ,TM(1998)。测量报酬与位置偏爱范式:.综述药物影响,新进展,新问题 PROG 神经生物学56(6):613-672。
White,AO,Kramár ,EA,Lopez,AJ,Kwapis ,JL,Doan,J.,Saldana,D.,Davatolhagh ,MF,Alaghband ,Y.,Blurton -Jones,M.,Matheos ,DP和Wood,MA( 2016)。神经元特异性核小体重构在可卡因相关记忆中的作用。Nat Commun 7:11725。
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引用:Simkevich, M. J., Campbell, R. R. and White, A. O. (2020). Examining Cocaine Conditioning Place Preference in Mice. Bio-protocol 10(8): e3595. DOI: 10.21769/BioProtoc.3595.
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