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Sucrose Preference Test to Measure Stress-induced Anhedonia
蔗糖偏好实验测量小鼠的应激诱导快感缺乏   

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参见作者原研究论文

本实验方案简略版
The Journal of Neuroscience
Oct 2015

Abstract

The 2-bottle choice procedure for assessing sucrose preference is a useful test to investigate anhedonia (i.e., inability to feel pleasure) in laboratory rodents, particularly in stress-based models of depression. The 2-bottle choice procedure allows for a comparison between behavioral preference for sucrose solution in drinking water compared to water only. Preference is measured by volume and/or weight of liquid consumed daily, which is then converted to a percent preference compared to a water only baseline period. Sucrose preference is attenuated by a diversity of chronic stressors, including chronic mild and unpredictable stress (Willner et al., 1992; Willner, 1997; Pothion et al., 2004) and social defeat stress (Krishnan et al., 2007). It may also be susceptible to perturbation in mouse models of drug addiction because sucrose preference is altered in drug-dependent individuals (Kampov-Polevoy et al., 1997; Bogucka-Bonikowska et al., 2002; Janowsky et al., 2003). Both stress- and drug-induced alterations in sucrose preference may stem from maladaptations in the reward pathway, which consists of the dopaminergic neurons extending from the ventral tegmental area to the nucleus accumbens (NAc). Indeed, alterations in cyclic-AMP response element binding protein (CREB) activity in NAc underlie preference for sucrose (Barrot et al., 2002). Additionally, the transcription factor ΔFosB in NAc (Wallace et al., 2008), but not dorsal hippocampus (Eagle et al., 2015), regulates natural rewards, such as sucrose consumption. Therefore, the sucrose preference test described below provides a well-validated model to assess anhedonia and the function of specific brain regions and circuits.

Keywords: Anhedonia (快感缺失), Reward (奖励), Mouse (鼠标), Behavior (行为), Drinking (饮酒)

Materials and Reagents

  1. Sipper caps, composed of 2.5 cm straight stainless steel sipper tubes (VWR International, catalog number: 10718-330 ) inserted into rubber stoppers (VWR International, catalog number: 59581-287 )
  2. 50 ml conical centrifuge tubes (VWR International, catalog number: 89039-656 )
  3. Adult (7 weeks or older) mice (C57BL/6J) (The Jackson Laboratory)
    Note: Alternate strains and ages of mice may also be used. Mice are housed singly, in an environment with controlled temperature (around 23 °C) and humidity under a 12-12 h light-dark cycle with food and water ad libitum. See Animal considerations in Notes for more details.
  4. Sucrose (crystals)
  5. Reverse osmosis (RO) filtered water
    Note: Alternatively substitute with the mouse’s normal drinking water.

Equipment

  1. Mouse caging
    1. Polycarbonate tubs (Mouse Cage; 7 ½ in W x 11 ½ in L x 5 in H) (Ancare) with standard woodchip mouse bedding (NORTHEASTERN PRODUCTS CORP., Aspen Chip)
      Note: Standard cage changes are allowed during the acclimation period (see below), however it is recommended to avoid cage changes during the period of data collection (Procedure steps 2-5) to prevent leakage. Leakage should be avoided to reduce undesired variability.
    2. Stainless steel wire cage lid (Ancare, model: N10SSR Mouse Lid )

Procedure

  1. Single house mice in proper mouse caging (see Equipment) under controlled conditions (see Materials and Reagents). Conditions should comply with the Guide for the Care and Use of Laboratory Animals, 8th ed. (https://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf). Allow mice to acclimate for 7 d under these conditions.
  2. Setup 2-bottle choice test for baseline conditions (Figure 1).
    1. Fill 50 ml centrifuge tubes to ~40 ml with RO water and apply sipper cap.
    2. Invert tubes and allow air bubble in sipper tube to rise.
    3. Make sure the water line is below the conical tapered end when inverted.
    4. Label each tube (e.g., “A” & “B” in permanent marker) and mark location of water line using a permanent marker.
    5. Alternatively, the initial bottle weight can be recorded.
    6. Place the 2 inverted tubes into wire lid, with minimal shaking to avoid dripping.
    7. Both bottles should be placed on one side of the divided wire rack, leaving the other side for food.
    8. Make sure that the rubber stopper is flushed with caging and that steel sipper tubes extend below wire cage lid (Figure 1).
    9. Avoid water leakage from sipper tubes when replacing cage on rack.
    10. Put food chow on the other side of the wire lid.
    Note: Ensure that bedding is not in excess. Mice can pile the bedding when nesting or if stressed, and bedding touching the sipper tube can cause the contents of the tube to empty. Alternatively, you may also add a “drip cage” to use as a control [for an example of this method see (Hwa et al., 2011)]. The drip cage is an empty cage that gets the same cage change and bottles daily. This sample drip loss can be subtracted from final calculations as an estimate of typical leakage.


    Figure 1. Picture of 2-bottle choice procedure setup in home cages. A. Sipper bottles separated out into its components consisting of metal sipper tube, rubber stopper (which the sipper tube is placed into via a hole in the center), and 50 ml conical centrifuge tube. B. Picture of 2-bottle setup in a home cage. The bottles are filled with solutions and place atop the wire mesh cage lid, with the metal sippers facing into the cage.

  3. Assess baseline water consumption daily for 4 d. To assess consumption, weigh bottle or mark volume level.
    1. If conducting weight measurements, record pre- and post-consumption weights of the tube (1 g = 1 ml of water).
    2. For volume measurements, record volume levels on tube using a permanent marker.
    3. Different colored markers for each day are recommended.
    4. Do not invert tubes (where sipper cap is upright) when marking volume, as this allows air into the sipper and can cause dripping.
    5. Monitor for any problems with liquid flow and observe for signs of spillage, e.g., wet bedding, below the sipper tube.
    6. Switch positions (left vs right) of tubes daily.
  4. Setup 2-bottle choice test for sucrose preference.
    1. Dissolve sucrose in RO water.
    2. Determination of sucrose concentration (% w/v; Figure 2) will require preliminary experiments using an initial dose-response curve (0.25-2%) to ascertain optimal sucrose preference necessary based on any a priori hypothesis.
    3.  For example, if your hypothesized outcome indicates a likely reduction in preference, a greater concentration is recommended. However, if you are expecting an increase in preference, lesser concentrations may be more appropriate.
    4. Fill one tube (e.g., one marked “A”) with fresh RO water and the other (“B”) with the sucrose solution. Place tubes on wire lid as previously described.


    Figure 2. An illustration of preference for sucrose in the 2-bottle choice test. A. Sucrose preference measured by raw volume consumed; B. Sucrose preference measured by % preference for sucrose.

  5. Assess water and sucrose solution consumption daily for 4 d. Record weights and/or volume of each tube. Monitor for any problems with liquid flow or spillage, e.g., wet bedding, below the sipper tube. Switch positions (left vs right) of tubes daily.
  6. Apply volume formula (see Notes) to determine consumption. Assess raw weight and/or volume consumed, and % preference for the sucrose-containing solution.

Notes

  1. Side bias
    It is likely that some mice will develop bias for one side or the other, resulting in an increased consumption for one tube. Therefore, bottle positions within each cage are switched daily to avoid false sucrose preference resulting from side bias. Alternatively, if bias is a problem, tubes can be moved to the middle of the wire top and food chow placed on either side.
  2. Volume formula
    Volume of liquid consumed can be determined based on vertical markings on the side of the cylinder. This derives from the tube dimensions and distance between daily marks, using the volume of a cylinder equation:
    V = π⋅ r2h
    Where V = volume (in cm3, which is equivalent to ml), π ≈ 3.1416, r = radius (half the interior diameter in cm), and h = distance between daily markings (in cm). It is important to utilize only the cylindrical portions of the 50 ml tube, and avoid any fluid in the conical portion, as the volume formula differs there.
  3. Animal considerations
    It is important to conduct preliminary studies in order to characterize the behavioral phenotype with particular equipment, environment, and animals. This is especially true in the case of varying mouse strains, either inbred or outbred, or if mice have been surgically or behaviorally manipulated prior to sucrose preference testing. All of these factors can affect baseline preference. In addition, some labs prefer to pre-handle mice for 5-7 d and singly house the animals for a period of 5-7 d prior to preference testing.

Acknowledgments

This behavioral procedure was adapted from previously published studies (Krishnan et al., 2007; Robison et al., 2014) and was performed by our group as described (Eagle et al., 2015). This work was supported by the Whitehall Foundation (AJR; 2013-08-43), the Multidisciplinary Training in Environmental Toxicology training grant (ALE; T32-ES007255), and a 2014 NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (ALE).

References

  1. Barrot, M., Olivier, J. D., Perrotti, L. I., DiLeone, R. J., Berton, O., Eisch, A. J., Impey, S., Storm, D. R., Neve, R. L., Yin, J. C., Zachariou, V. and Nestler, E. J. (2002). CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. Proc Natl Acad Sci U S A 99(17): 11435-11440.
  2. Bogucka-Bonikowska, A., Baran-Furga, H., Chmielewska, K., Habrat, B., Scinska, A., Kukwa, A., Koros, E., Kostowski, W., Polanowska, E. and Bienkowski, P. (2002). Taste function in methadone-maintained opioid-dependent men. Drug Alcohol Depend 68(1): 113-117.
  3. Eagle, A. L., Gajewski, P. A., Yang, M., Kechner, M. E., Al Masraf, B. S., Kennedy, P. J., Wang, H., Mazei-Robison, M. S. and Robison, A. J. (2015). Experience-dependent induction of hippocampal DeltaFosB controls learning. J Neurosci 35(40): 13773-13783.
  4. Hwa, L. S., Chu, A., Levinson, S. A., Kayyali, T. M., DeBold, J. F. and Miczek, K. A. (2011). Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol. Alcohol Clin Exp Res 35(11): 1938-1947.
  5. Institute of Laboratory Animal Resources (U.S.) (2011). Guide for the care and use of laboratory animals, 8th edition. Washington DC: National Academy of Sciences.
  6. Janowsky, D. S., Pucilowski, O. and Buyinza, M. (2003). Preference for higher sucrose concentrations in cocaine abusing-dependent patients. J Psychiatr Res 37(1): 35-41.
  7. Kampov-Polevoy, A., Garbutt, J. C. and Janowsky, D. (1997). Evidence of preference for a high-concentration sucrose solution in alcoholic men. Am J Psychiatry 154(2): 269-270.
  8. Krishnan, V., Han, M. H., Graham, D. L., Berton, O., Renthal, W., Russo, S. J., Laplant, Q., Graham, A., Lutter, M., Lagace, D. C., Ghose, S., Reister, R., Tannous, P., Green, T. A., Neve, R. L., Chakravarty, S., Kumar, A., Eisch, A. J., Self, D. W., Lee, F. S., Tamminga, C. A., Cooper, D. C., Gershenfeld, H. K. and Nestler, E. J. (2007). Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131(2): 391-404.
  9. Pothion, S., Bizot, J. C., Trovero, F. and Belzung, C. (2004). Strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress. Behav Brain Res 155(1): 135-146.
  10. Robison, A. J., Vialou, V., Sun, H. S., Labonte, B., Golden, S. A., Dias, C., Turecki, G., Tamminga, C., Russo, S., Mazei-Robison, M. and Nestler, E. J. (2014). Fluoxetine epigenetically alters the CaMKIIalpha promoter in nucleus accumbens to regulate DeltaFosB binding and antidepressant effects. Neuropsychopharmacology 39(5): 1178-1186.
  11. Wallace, D. L., Vialou, V., Rios, L., Carle-Florence, T. L., Chakravarty, S., Kumar, A., Graham, D. L., Green, T. A., Kirk, A., Iniguez, S. D., Perrotti, L. I., Barrot, M., DiLeone, R. J., Nestler, E. J. and Bolanos-Guzman, C. A. (2008). The influence of DeltaFosB in the nucleus accumbens on natural reward-related behavior. J Neurosci 28(41): 10272-10277.
  12. Willner, P. (1997). Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 134(4): 319-329.
  13. Willner, P., Muscat, R. and Papp, M. (1992). Chronic mild stress-induced anhedonia: a realistic animal model of depression. Neurosci Biobehav Rev 16(4): 525-534.

简介

用于评估蔗糖偏爱的2瓶选择程序是对实验室啮齿动物(特别是基于压力的抑郁症模型)中调查麻醉(即不能感觉到快感)的有用测试。 2瓶选择程序允许比较饮用水中蔗糖溶液与仅水相比的行为偏好。偏好是通过每天消耗的液体的体积和/或重量来测量的,然后将其与仅基准水平相比转化为偏好百分比。蔗糖偏好由多种慢性压力因子减弱,包括慢性轻度和不可预测的压力(Willner等,1992; Willner,1997; Pothion等,2004)和社会失败压力(Krishnan等,2007)。在药物成瘾的小鼠模型中也可能会受到干扰,因为在药物依赖个体中蔗糖的偏好被改变(Kampov-Polevoy等,1997; Bogucka-Bonikowska等,2002; Janowsky等,2003)。应激和药物诱导的蔗糖偏好改变可能源于奖励途径中的适应不良,其由从腹侧被盖区域延伸到伏隔核(NAc)的多巴胺能神经元组成。实际上,NAc中的环AMP反应元件结合蛋白(CREB)活性的改变成为蔗糖的优先选择(Barrot等,2002)。另外,NAc中的转录因子ΔFosB(Wallace等人,2008),但不是背海马(Eagle et al。,2015),调节了天然的回报,如蔗糖消耗。因此,以下描述的蔗糖偏好测试提供了一个经过验证的模型,用于评估特异性脑区和电路的脱落和功能。

关键字:快感缺失, 奖励, 鼠标, 行为, 饮酒

材料和试剂

  1. 由插入橡胶塞(VWR International,目录号:59581-287)的2.5cm直的不锈钢吸管(VWR International,目录号:10718-330)组成的吸管盖
  2. 50ml锥形离心管(VWR International,目录号:89039-656)
  3. 成年(7周或更大)小鼠(C57BL/6J)(The Jackson Laboratory)
    注意:也可以使用小鼠的替代菌株和年龄。 将小鼠单独饲养在具有控制温度(约23℃)和湿度的环境中,在12-12小时光照 - 黑暗循环下,随意食物和水。 有关详情,请参阅备注中的动物注意事项。
  4. 蔗糖(晶体)
  5. 反渗透(RO)过滤水
    注意:或者用小鼠的正常饮用水代替。

设备

  1. 鼠标笼罩
    1. 聚碳酸酯盆(鼠笼; 71/2英寸×11/2英寸长×5英寸高) (Ancare)与标准木屑鼠标床上用品(NORTHEASTERN产品 CORP。,Aspen Chip)
      注意:允许在标准笼子更换 适应期(见下文),但是建议避免 在数据收集期间的笼子变化(过程步骤2-5) 以防止泄漏。 应避免泄漏以减少不需要的变异性。
    2. 不锈钢丝笼盖(Ancare,型号:N10SSR鼠标盖)

程序

  1. 在适当的小鼠笼中(参见设备)在受控条件下(参见材料和试剂)的单家鼠。 条件应符合实验动物的护理和使用指南,8 。 ( https://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf )。 允许小鼠在这些条件下适应7天。
  2. 为基线条件设置2瓶选择测试(图1)。
    1. 填充50ml离心管〜40毫升与RO水,并应用吸嘴帽。
    2. 倒置管,并允许吸管中的气泡上升。
    3. 确保水线在倒置时低于锥形锥形端。
    4. 在永久性标记中标记每个管(例如,,"A"和"B")并使用永久性标记标记水线的位置。
    5. 或者,可以记录初始瓶重量。
    6. 将2个倒置的管放入线盖,最小振动,以避免滴水。
    7. 两个瓶子应放置在分开的网架的一侧,留下另一侧的食物。
    8. 确保橡胶塞用笼子冲洗,并且钢筛管延伸到电线笼盖下方(图1)。
    9. 更换机架上的支架时,应避免从吸管中漏水。
    10. 把食物食物放在线盖的另一边。
    注意:确保床上用品不超额。小鼠可以在嵌套或如果受压时将床上用品堆叠,并且接触吸管的床垫可能会使管的内容物变空。或者,您也可以添加一个"滴灌笼"作为控制[关于这种方法的一个例子,参见(Hwa et al。,2011)]。滴水笼是一个空笼子,每天获得相同的笼子更换和瓶子。可以从最终计算中减去该样品滴漏损失作为典型泄漏的估计。


    图1.家庭保鲜盒中2瓶选择程序设置的图片。A.吸管瓶分成由金属吸管,橡胶塞组成的组件(吸管通过孔放入)在中心)和50ml锥形离心管。 B.在家笼中的2瓶装置的图片。 瓶子装满溶液,放在金属丝网笼盖上,金属杯子朝向笼子。

  3. 每天评估基线用水量4天。 要评估消耗,称重瓶或标记音量。
    1. 如果进行体重测量,记录管的消耗前和消耗后的重量(1g = 1ml水)。
    2. 对于体积测量,使用永久标记记录管上的音量。
    3. 建议每天使用不同颜色的标记。
    4. 标记时,不要颠倒管(吸管盖直立) 体积,因为这允许空气进入吸入器并且可以导致滴水。
    5. 监测液体流动的任何问题,并观察溢出的迹象,例如,在吸管下方的潮湿垫料。
    6. 每天切换管的位置(左/右)。
  4. 设置2瓶选择测试蔗糖偏好。
    1. 将蔗糖溶解在RO水中。
    2. 蔗糖的测定 浓度(%w/v;图2)将需要初步实验 使用初始剂量反应曲线(0.25-2%)确定最佳 基于任何先验假设的蔗糖偏好。
    3.  例如,如果您的假设结果表明可能减少 优选地,推荐更高的浓度。 但是,如果你 期望增加偏好,可能较低的浓度 更合适。
    4. 填充一根管(例如,一个标有"A"),加上新鲜   RO水和另一种("B")与蔗糖溶液接触。 放置管 如上所述。


    图2.在2瓶选择测试中对蔗糖的偏好的说明。 A.通过消耗的原始体积测量蔗糖偏好; B.蔗糖偏好度通过蔗糖的%偏好度测量
  5. 每天评估水和蔗糖溶液消耗4天。记录每个管的重量和/或体积。监测液体流或溢出的任何问题,例如,在吸管下方的例如湿床上用品。每天切换管子的位置(左/右)。
  6. 应用体积公式(参见注释)以确定消耗量。评估原料重量和/或消耗的体积,以及含蔗糖溶液的%偏好。

笔记

  1. 侧偏差
    有可能一些小鼠将发展为一侧或另一侧的偏倚,导致一个管的消耗增加。因此,每个笼中的瓶位置每天切换以避免由侧偏压引起的假蔗糖偏好。或者,如果偏差是一个问题,管可以移动到电线顶部的中间和放置在任一侧的食物。
  2. 卷公式
    所消耗的液体体积可以基于圆筒侧面上的垂直标记来确定。这取决于管尺寸和每日标记之间的距离,使用柱体方程的体积:
    V =π⋅ r 2 ⋅ h
    其中V =体积(以cm 3表示,等于ml),π≈3.1416,r =半径(以cm为单位的内径的一半) h =每日标记之间的距离(以cm为单位)。重要的是仅使用50ml管的圆柱形部分,并且避免锥形部分中的任何流体,因为体积公式在那里不同。
  3. 动物考虑
    重要的是进行初步研究,以特定的行为表型与特定的设备,环境和动物。这在不同的小鼠品系(近交或远交)的情况下或者如果在蔗糖偏好测试之前已经通过手术或行为操纵小鼠的情况下尤其如此。所有这些因素都可能影响基线偏好。此外,一些实验室喜欢预处理小鼠5-7天,并在偏好测试之前将动物单独放置5-7天。

致谢

这种行为 程序改编自以前发表的研究(Krishnan等人, ,2007; Robison ,,2014),并由我们的团队进行 描述(Eagle等人,2015)。这项工作得到白厅的支持基础(AJR; 2013-08-43),多学科培训 环境毒理学培训补助金(ALE; T32-ES007255)和2014年 NARSAD年轻研究员奖从大脑和行为研究 基金会(ALE)。

参考文献

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Eagle, A. L., Mazei-Robison, M. and Robison, A. J. (2016). Sucrose Preference Test to Measure Stress-induced Anhedonia. Bio-protocol 6(11): e1822. DOI: 10.21769/BioProtoc.1822.
  2. Eagle, A. L., Gajewski, P. A., Yang, M., Kechner, M. E., Al Masraf, B. S., Kennedy, P. J., Wang, H., Mazei-Robison, M. S. and Robison, A. J. (2015). Experience-dependent induction of hippocampal DeltaFosB controls learning. J Neurosci 35(40): 13773-13783.
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