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Running Reward Conditioned Place Preference Task
运动奖励条件性位置偏爱任务实验   

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Abstract

The conditioned place preference (CPP) test is a standard pre-clinical behavioral tool used to study the motivational effects of drugs and non-drug treatments in experimental animals. The basic characteristic of this task involves the association of a particular environment and contextual cues with a reward stimulus, followed by the association of a different environment with the absence of the reward stimulus (Prus and Rosecrans, 2009). Besides the motor component, voluntary wheel running exercise also has a rewarding component, and has been suggested as a strong natural reinforcer. Consistent with this notion, rodents will readily begin to run when a wheel is introduced (Eikelboom and Mills, 1988; Looy and Eikelboom, 1989), will work by lever pressing to gain access to a running wheel (Pierce et al., 1986), and spend more time in a place previously associated with the aftereffects of running (Lett et al., 2000; Lett et al., 2001). More recently, we underscored an important role for the adipocyte-derived hormone leptin in midbrain dopamine neurons in the modulation of running reward (Fernandes et al., 2015). Here, we describe a CPP protocol to measure the rewarding aftereffects of wheel running exercise in mice.

Keywords: Physical activity(体育活动), Motivation(动机), Behavior(行为), Animal model(动物模型), Wheel running(跑轮)

Materials and Reagents

Animals

  1. Floxed Stat3 mice (C57Bl6 background) [Osaka, Japan (Takeda et al., 1998)]
    Note: All experiments were carried out in accordance with the guidelines and approval of the Institutional Animal Care Committee of the CHUM Research Center. Floxed Stat3 mice (C57Bl6 background) in which loxP sites flank exon 22 of the Stat3 gene that encodes a tyrosine residue (tyr705) essential for Stat3 activation, were graciously provided by Dr. Shizuo Akira [Osaka, Japan (Takeda et al., 1998)]. Female mice homozygous for the floxed Stat3 allele were crossed with male mice heterozygous for the floxed Stat3 allele and heterozygous for the DAT-Cre transgene [B6.SJL-Slc6a3tm1.1(cre)Bkmn/J] (Backman et al., 2006) to generate DAT-Cre-Stat3fl/fl mice and littermate controls (Stat3fl/fl or Stat3fl). Male mice were weaned at P28 and housed in a temperature and humidity controlled room that was maintained on a 12:12 hour reverse light/dark cycle. CPP experiment (pre-test, conditioning trials and post-test) was conducted in the dark phase of the cycle.

Equipment

  1. Low-profile wireless running wheel for mouse (Med Associates Inc., catalog number: ENV-044 )
  2. Three-compartment automated mouse CPP chambers (Med Associates Inc., catalog number: ENV-3013 )

Software

  1. Wheel Manager software (Med Associates Inc., catalog number: SOF-860)

Procedure

  1. Running reward
    Singly-housed male mice (sample size of >10 mice, as rodents’ behavior tend to be variable; 11-14 weeks of age) had free access to home cage low-profile wireless running wheels for 3 weeks (Figure 1). Running wheels have a 15.5 cm (6.10 in) diameter and revolve on a bearing-mounted pin, which creates less friction. Wheel Manager software was used to collect and organize data. Following 3 weeks of ad libitum access to the wheels, mice were subjected to a CPP task using a three-compartment, automated mouse CPP apparatus (Figure 2). Each CPP apparatus consisted of three chambers: A black compartment with a stainless steel grid rod floor and a white compartment with a spaced stainless steel mesh floor, separated by a neutral center compartment with a grey finish and PVC floor.


    Figure 1. Single-housed mouse running on the wheel


    Figure 2. Illustrative schema of a three-compartment place preference chamber for mouse

  2. Pre-test
    Mice were confined to both the black and white compartments of the CPP apparatus for 5-min to permit habituation to each compartment. Mice were then placed along the midline formed by the joining of the two chambers and allowed to move freely in the apparatus for 15-min during which the amount of time spent in each compartment was recorded (Table S1). All mice returned to their home cages afterwards. Of note, a mouse was considered to be in a chamber only when all four paws were in that chamber.

  3. Conditioning trials
    Mice had 2-h access to a running wheel (paired trial) or a locked running wheel (unpaired trial) in their respective home cages, and were then confined to either the black or white compartment of the CPP apparatus for 30 min. Paired and unpaired conditioning trials took place in opposite compartments (counterbalanced across mice) on alternating days, thus each mouse ran every-other day. Conditioning trials were conducted for 14 days (Figure 3).

  4. Post-test
    The CPP post-test occurred on the day after the last conditioning trial. For the post-test, mice were placed back in the open CPP apparatus for 15 min. Please note that animals only have access to the wheels in their home cages-running wheels where not inserted into the CPP apparatus. As previously mentioned, at the start of the CPP test, the mouse was placed along the midline formed by the joining of the two chambers. The amount of time spent in each compartment was once again recorded and the proportion of time spent in the paired side was compared to that obtained during the pre-test (Table S2). Importantly, each mouse was tested in the same CPP apparatus where it was trained, and CPP chambers were wiped with 70% EtOH in between animals.


    Figure 3. Running reward CPP task


    Figure 4. Illustrative graphs showing how to represent CPP data

Acknowledgments

This protocol was designed by S. F. and M. F. A. F., and was implemented in (Fernandes et al., 2015). This work was supported by an operating grant (MOP-123280) and New Investigator award to S. F. from the Canadian Institutes of Health Research, and doctoral fellowships to M. F. A. F. from the Canadian Diabetes Association and from the Department of Physiology, Universite de Montreal. We thank Shizuo Akira for the Stat3-floxed mice.

References

  1. Backman, C. M., Malik, N., Zhang, Y., Shan, L., Grinberg, A., Hoffer, B. J., Westphal, H. and Tomac, A. C. (2006). Characterization of a mouse strain expressing Cre recombinase from the 3' untranslated region of the dopamine transporter locus. Genesis 44(8): 383-390.
  2. Buccafusco, J. J. (2009). Conditioned Place Preference. In: Prus, A. J., James, J. R., Rosecrans, J. A. (eds). Methods of behavior analysis in neuroscience 2nd frontiers in neuroscience. Augusta CRC Press.
  3. Eikelboom, R. and Mills, R. (1988). A microanalysis of wheel running in male and female rats. Physiol Behav 43(5): 625-630.
  4. Fernandes, M. F., Matthys, D., Hryhorczuk, C., Sharma, S., Mogra, S., Alquier, T. and Fulton, S. (2015). Leptin suppresses the rewarding effects of running via STAT3 signaling in dopamine neurons. Cell Metab 22(4): 741-749.
  5. Lett, B. T., Grant, V. L. and Koh, M. T. (2001). Naloxone attenuates the conditioned place preference induced by wheel running in rats. Physiol Behav 72(3): 355-358.
  6. Lett, B. T., Grant, V. L., Byrne, M. J. and Koh, M. T. (2000). Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. Appetite 34(1): 87-94.
  7. Looy, H. and Eikelboom, R. (1989). Wheel running, food intake, and body weight in male rats. Physiol Behav 45(2): 403-405.
  8. Pierce, W. D., Epling, W. F. and Boer, D. P. (1986). Deprivation and satiation: The interrelations between food and wheel running. J Exp Anal Behav 46(2): 199-210.
  9. Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T. and Akira, S. (1998). Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J Immunol 161(9): 4652-4660.

简介

条件性位置偏好(CPP)测试是用于研究药物和非药物治疗在实验动物中的激励作用的标准临床前行为工具。这个任务的基本特征涉及将特定环境和情境线索与奖励刺激相关联,然后将不同环境与没有奖励刺激相联系(Prus和Rosecrans,2009)。除了电机部件,自愿的车轮运行运动也有一个回报的成分,并已被建议作为一个强大的自然增强。与这个概念一致,当引入轮子时,啮齿动物将容易开始跑动(Eikelboom和Mills,1988; Looy和Eikelboom,1989),将通过杠杆按压来获得进入运行的轮子(Pierce等人, ,1986),并且在之前与运行的后果相关的地方花费更多的时间(Lett等人,2000; Lett等人,2001年) )。最近,我们强调了脂肪细胞衍生激素瘦素在中脑多巴胺神经元中在调节运行奖励中的重要作用(Fernandes等人,2015)。在这里,我们描述了一种CPP协议,以测量在小鼠中车轮运行运动的奖励后遗症。

关键字:体育活动, 动机, 行为, 动物模型, 跑轮

材料和试剂

动物

  1. Floxed Stat3小鼠(C57B16背景)[Osaka,Japan(Takeda等人,1998)]
    注意: 所有实验均按照CHUM研究中心的机构动物管理委员会的指导和批准进行。 Floxed Stat3小鼠(C57B16背景),其中loxP位点侧翼Stat3基因的外显子22侧翼编码Stat3激活必需的酪氨酸残基(tyr705),由Shizuo Akira博士[日本大阪(Takeda et al。 ,1998)]。将对于floxed Stat3等位基因纯合的雌性小鼠与对于floxed Stat3等位基因杂合的雄性小鼠杂交,对于DAT-Cre转基因杂交的雌性小鼠杂交[B6.SJL-Slc6a3tm1.1(cre)Bkmn/J](Backman >等人 ,2006)以产生DAT-Cre-Stat3 小鼠和同窝小鼠Stat3 fl/fl 或者Stat3 。将雄性小鼠在P28断奶,并置于温度和湿度控制的室中,保持在12:12小时的逆光/暗循环。在周期的黑暗阶段进行CPP实验(预测试,条件测试和后测试)。

设备

  1. 小型低调无线运行轮(Med Associates Inc.,目录号:ENV-044)
  2. 三室自动化小鼠CPP室(Med Associates Inc.,目录号:ENV-3013)

软件

  1. Wheel Manager软件(Med Associates Inc.,目录号:SOF-860)

程序

  1. 运行奖励
    单独饲养的雄性小鼠(> 10只小鼠的样本大小,因为啮齿类动物的行为倾向于是可变的; 11-14周龄)可自由进入家庭笼低剖面无线运行轮3周(图1)。运行轮直径为15.5厘米(6.10英寸),并且在轴承安装的销上旋转,这产生较小的摩擦。 Wheel Manager软件用于收集和组织数据。在3周后随意进入轮子,使用三室自动化小鼠CPP装置对小鼠进行CPP任务(图2)。每个CPP设备由三个室组成:具有不锈钢格栅杆地板的黑色隔室和具有间隔开的不锈钢网格地板的白色隔室,由具有灰色漆层和PVC地板的中性中心隔室隔开。

    图1.单轮鼠标在轮子上运行


    图2.鼠标
    的三室地点首选项室的说明性模式
  2. 预测试
    将小鼠限制在CPP装置的黑色和白色区室中5分钟以允许习惯到每个隔室。然后沿由两个室的连接形成的中线放置小鼠,并允许在装置中自由移动15分钟,在此期间记录在每个隔室中花费的时间量(表S1 )。所有小鼠返回到他们的家笼子。值得注意的是,只有当所有四只爪子都在这个房间中时,鼠被认为是在房间里
  3. 调节试验
    小鼠在其各自的家笼中对运行轮(成对试验)或锁定的运行轮(非成对试验)进行2小时,然后限制在CPP装置的黑色或白色隔室中30分钟。配对和非配对的调节试验在交替的天中在相对的隔室(平衡的小鼠)中进行,因此每只小鼠每隔一天运行。进行空调试验14天(图3)
  4. 后测试
    CPP后测试发生在最后一次条件试验的第二天。对于后测试,将小鼠放回开放的CPP装置中15分钟。请注意,动物只能进入他们的家笼中的轮子 - 轮子,不插入CPP设备。如前所述,在CPP测试开始时,沿着由两个室的接合形成的中线放置小鼠。再次记录在每个隔室中花费的时间量,并将在配对侧中花费的时间的比例与在预测试期间获得的时间比较(表S2 )。重要的是,每只小鼠在相同的CPP装置中进行测试,其中它被训练,并且CPP室用动物之间的70%EtOH擦拭。

    图3.运行奖励CPP任务


    图4.显示如何表示CPP数据的说明图

致谢

该协议由S.F.和M.F.A.F.设计,并且在(Fernandes等人,2015)中实现。这项工作得到了来自加拿大健康研究所的营业补助金(MOP-123280)和新研究员奖,以及来自加拿大糖尿病协会和蒙特利尔大学生理系的M. F. A. F.博士奖学金的支持。我们感谢Shizuo Akira的Stat3 floxed小鼠。

参考文献

  1. Backman,C.M.,Malik,N.,Zhang,Y.,Shan,L.,Grinberg,A.,Hoffer,B.J.,Westphal,H.and Tomac,A.C。(2006)。 从多巴胺转运蛋白基因座的3'非翻译区表达Cre重组酶的小鼠品系的表征。/a> 44(8):383-390。
  2. Buccafusco,J.J。(2009)。条件地方偏好。 In:Prus,A.J.,James,J.R.,Rosecrans,J.A。(eds)。神经科学行为分析方法神经科学的第二个边界。 Augusta CRC Press。
  3. Eikelboom,R。和Mills,R。(1988)。 对男性和女性大鼠的轮子运行进行微分析。 Physiol Behav 43(5):625-630。
  4. Fernandes,M.F.,Matthys,D.,Hryhorczuk,C.,Sharma,S.,Mogra,S.,Alquier,T.and Fulton,S。 Leptin抑制通过STAT3信号传导在多巴胺神经元中运行的有益效果。 Cell Metab 22(4):741-749。
  5. Lett,B.T.,Grant,V.L。和Koh,M.T。(2001)。 纳洛酮可减弱由大鼠中轮子运行所引起的条件性位置偏好。 Behav 72(3):355-358。
  6. Lett,B.T.,Grant,V.L.,Byrne,M.J.and Koh,M.T。(2000)。 独特房间与车轮后行效应的配对产生条件地点偏好 < em> Appetite 34(1):87-94。
  7. Looy,H。和Eikelboom,R。(1989)。 轮胎运行,食物摄入和雄性大鼠的体重 生理 Behav 45(2):403-405。
  8. Pierce,W.D.,Epling,W.F.and Boer,D.P。(1986)。 剥夺和满足:食物和车轮运行之间的相互关系。 Anal Behav。46(2):199-210。
  9. Takeda,K.,Kaisho,T.,Yoshida,N.,Takeda,J.,Kishimoto,T.and Akira,S。(1998)。 Stat3激活通过阻止凋亡负责IL-6依赖性T细胞增殖:生成和表征 T细胞特异性Stat3缺陷小鼠。 Immunol 161(9):4652-4660。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernandes, M. F. and Fulton, S. (2016). Running Reward Conditioned Place Preference Task. Bio-protocol 6(10): e1816. DOI: 10.21769/BioProtoc.1816.
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