Consummatory Successive Negative Contrast in Rats
大鼠完成连续阴性对照实验   

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PLOS ONE
Oct 2016

 

Abstract

Using animal models in addiction and pain research is pivotal to unravel new pathways and mechanisms for the treatment of these disorders. Reward devaluation through a consummatory successive negative contrast (cSNC) task has shown the ability to reduce physical pain sensitivity (hypoalgesia) and increase oral ethanol consumption in rats. The procedure is based on exposing the experimental animals to a 32% sucrose solution during several sessions (preshift sessions) followed by a devaluation to 4% sucrose during the next few sessions (postshift sessions). The cSNC effect can be monitored by comparing the experimental group to an unshifted control that had access to 4% sucrose throughout the entire experiment (preshift and postshift sessions). The cSNC phenomenon is defined by lower consumption of sucrose in the downshifted group than in the unshifted group during postshfit sessions.

Keywords: Addiction (成瘾), Pain (疼痛), Rats (大鼠), Successive negative contrast (连续阴性对照), Frustration (沮丧), Reward loss (报酬损失), Psychological pain (心理疼痛), Emotion (情绪), Anxiety (焦虑)

Background

There is experimental evidence that emotional states (e.g., anxiety or frustration) influence sensitivity to physical pain and correlate with the consumption of psychoactive substances like alcohol (Wiech and Tracey, 2008; Xie et al., 2012; Manzo et al., 2015). Reward devaluation is a viable strategy to induce a negative emotion such as anxiety (see Flaherty, 1996; Papini et al., 2015). Reward devaluation can be implemented in different procedures, including appetitive extinction, partial reinforcement, and successive negative contrast (SNC). All of them imply unexpected changes in reward magnitude or quality. In appetitive extinction, the reward is withheld altogether, while for partial reinforcement the reward is withheld on some random number of trials. SNC implies not the elimination, but a devaluation of the magnitude or quality of the reward. In the consummatory successive negative contrast (cSNC) task, one type of SNC procedure, animals receive free access for 5 min to a high-value sucrose solution (usually 32% sucrose) during several daily sessions, followed by several sessions of access to a low-value sucrose solution (usually 4% sucrose) (Jiménez-García et al., 2016). Performance of downshifted animals is compared to the consummatory behavior of animals that have always received access to 4% sucrose (unshifted controls). The cSNC effect involves a suppression of consummatory behavior after the 32%-4% sucrose downshift followed by a recovery to the level of unshifted controls. The initial suppression (typically observed during the first devaluation session) and the recovery that follows (normally starting during the second devaluation session) are dissociable stages of the cSNC effect (Flaherty, 1996).

Several sources of evidence suggest that cSNC modulates and is also modulated by physical pain (Jiménez-García et al., 2016). Papini et al. (2015) suggested a connection between physical pain (tissue damage) and psychological pain (reward loss) that invites further comparisons between these two sets of phenomena. For example, opioid ligands known to modulate physical pain also modulate cSNC, either during the first downshift session ([D-Pen2,D-Pen5]enkephalin, a selective delta-receptor agonist; Wood et al., 2005), during the second downshift session (U50,488H, a selective kappa-receptor agonist; Wood et al., 2008), or during both sessions (morphine; Rowan and Flaherty, 1987). Two opioid-receptor antagonists enhance the cSNC effect either selectively during the first downshift session (naltrindole, a selective delta receptor antagonist) or during both the first and second downshift sessions (naloxone, a nonselective opioid receptor antagonist; Pellegrini et al., 2005). Similarly, lesions of brain areas involved in pain processing, such as the anterior cingulate cortex, also affect recovery from reward downshift from the second downshift session onward (Ortega et al., 2011).

Likewise, ethanol consumption is increased by cSNC. Reward devaluation produces an anxious state (frustration) that can be mitigated by the anxiolytic effects of ethanol. Manzo et al. (2015) showed an increase in ethanol consumption after reward devaluation (post-shift sessions).

Materials and Reagents

  1. Anti-drip bottles with a capacity of 150 ml (Classic Drinker de Luxe; Zooplus) (see Figure 1)


    Figure 1. Anti-drip bottles 

  2. Animals: Adult male Wistar rats (Envigo Laboratories, Barcelona, Spain) with 11 weeks of age and weighing 281.2 (±2.1) g were used
  3. Filtered and autoclaved water
  4. Sucrose (white sugar suitable for human consumption. Azucarera Española S.A.)
  5. 32% sucrose solution (see Recipes)
  6. 4% sucrose solution (see Recipes)

Equipment

  1. Timer and chronometer (Digital Onstart 100) (Figure 2A)
  2. Consumption chamber
    Rectangular (30 x 30 x 15 cm) Plexiglas boxes (Figure 2B)
  3. Weighing scale (WLC 1/A2 Precision Balance; Radwag©) (Figure 2C)
  4. Personal protective equipment (Laboratory coat, gloves, masks, etc.) (Figure 2D)


    Figure 2. Equipment. A. Timer and chronometer. B. Consumption chamber. C. Weighing scale. D. Personal protective equipment.

Procedure

See Figure 3 for the Timeline of daily experimental procedure.


Figure 3. Timeline of daily experimental procedure. *High/low value sucrose depending on each experimental group.

    1. Individualize each animal in polycarbonate standard cages with free access to water and maintain under controlled standard conditions (12 h dark/light cycles starting at 08:00 h, 24 °C temperature, and 50%-60% humidity). Deprive animals of food to 82%-85% of their ad lib weights throughout the experiment. Once animals reach the required weight, feed daily with 9 g (±1.5 g) of rat chow to maintain body weight. All manipulations must be carried out at the same time every day and preferably by the same person. Efforts must be made to minimize suffering and reduce the number of animals used in the experiments. To maintain the animal’s body weight as assessed before the start of the experiment, it is important to weigh each specimen every day and provide supplementary food no less than 30 min after the end of each session. Moreover, sucrose solution must be prepared fresh daily to avoid contamination. 
    2. cSNC training lasts 19 daily sessions: 16 preshift sessions followed by 3 postshift sessions. 
    3. In the cSNC task, at least two experimental groups are required. During the preshift sessions, one of them has access to a large reward (e.g., 32% sucrose solution) and the other to a small reward (e.g., 4% sucrose solution). During postshift sessions, both groups are exposed to the small reward (4% sucrose solution).
    4. Before each session, animal racks must be moved into a different room where the experiment will be held, allowing the rats 15 min in their home cages to settle down, before placing them the consumption chambers. 
    5. The contrast session lasts 5 min from the first contact with the sipper tube. The position of the bottle is counterbalanced daily to minimize the effects of side preference. The animal is placed back in its home cage at the end of the session. 
    6. Session duration for each box can be measured manually with digital clocks (Digital Onstart 100). 
    7. Sucrose consumption is recorded for each animal weighing the bottles before and after the session, and then transforming the weight to milliliters (having accounted for the density of each solution) using the formula: Grams of solution consumed/density (grams per milliliter). All animals receive training every day in groups of 4, with the order of groups counterbalanced across days. At the end of every session, contrast chambers must be wiped with a wet paper towel and feces removed as needed.
    8. Animals must be fed with the proper amount of rat chow to maintain body weight no less than 30 min after the end of each session.

    Data analysis

    In the cSNC situation, the dependent variable was the total amount of sucrose consumed in each session (in milliliters; Figure 4).
     

    Figure 4. Mean (±SEM) sucrose consumption (ml) during the last preshift session (16) and the three postshift sessions (17-19). 32-4: animals exposed to reward devaluation from 32% to 4% sucrose during postshift sessions. 4-4: animals exposed to an unshifted reward condition, always receiving access to 4% sucrose throughout the experiment. The asterisk reflects a significant difference between the downshifted and unshifted groups. (Figure modified from Jiménez-García et al., 2016.)

    Notes

    1. The consumption chamber should be cleaned with sterile water to remove any animal odor. It’s necessary to cover the boxes’ floor with standard home cage sawdust. No access to food or water should be allowed.
    2. For the sucrose presentation, a graduated cylinder (in 0.01 ml units) with a metallic sipper tube can be used.
    3. cSNC training can be reduced to 15 daily sessions: 10 preshift sessions followed by 5 postshift sessions. This procedure is the typical protocol in cSNC experiments and, for that reason, it is advantageous too. Some experiments have reported a cSNC effect with minimal preshift experience (one session: Flaherty et al., 1983), but less than 10 preshift sessions is infrequently used. The final protocol chosen will depend on other requirements associated with the problem of interest.
    4. A recording system (i.e., camera) could be used optionally. Thus, place the recording system on the side of the chamber. Ensure that the oral region of the animal is clearly visible.
    5. A magnetic stirrer may be useful to properly dissolve sucrose.

    Recipes

    1. 32% sucrose solution
      32 g sucrose (commercial white sugar)
      68 g distilled water
    2. 4% sucrose solution
      4 g sucrose (commercial white sugar)
      96 g distilled water

    Acknowledgments

    This paper was partially supported by the Spanish Ministry of Economy and Competitiveness (grant PSI-2013-44945-P), the Junta de Andalucía (grant CTS109), FEDER funds and the Master’s program in Basic and Applied Neuroscience and Pain, University of Granada, Spain. MRP’s participation was supported by a Fulbright US Scholar Award. Animals were maintained according to the EU Directive 2010/63/EU for animal experiments. We thank the CTS430 group, especially M. Á. Zafra, for their help. This protocol and figures are adapted from our previous work (Jiménez-García et al., 2016).

    Competing interests

    The authors declare no conflicts of interest or competing interests.

    Ethics

    The experimental protocols were approved by the University of Granada Research Ethics Committee. Reference: SSA/SIS/MD/jv (valid from 31/03/2016 to 31/03/2021).

    References

    1. Flaherty, C. F. (1996). Incentive relativity. Cambridge University Press, UK: Cambridge.
    2. Flaherty, C. F., Becker, H. C., and Checke, S. (1983). Repeated successive contrast in consummatory behavior with repeated shifts in sucrose concentration. Anim Learn Behav 11: 407-414.
    3. Jiménez-García, A. M., Ruíz-Leyva, L., Cendán, C. M., Torres, C., Papini, M. R., and Moron, I. (2016). Hypoalgesia induced by reward devaluation in rats. PLoS One 11(10): e0164331.
    4. Manzo, L., Donaire, R., Sabariego, M., Papini, M. R., and Torres, C. (2015). Anti-anxiety self-medication in rats: oral consumption of chlordiazepoxide and ethanol after reward devaluation. Behav Brain Res 278: 90-97.
    5. Ortega, L. A., Uhelski, M., Fuchs, P. N. and Papini, M. R. (2011). Impairment of recovery from incentive downshift after lesions of the anterior cingulate cortex: emotional or cognitive deficits? Behav Neurosci 125: 988-995.
    6. Papini, M. R., Fuchs, P. N. and Torres, C. (2015). Behavioral neuroscience of psychological pain. Neurosci Biobehav Rev 48: 53-69.
    7. Pellegrini, S., Wood, M., Daniel, A. M. and Papini, M. R. (2005). Opioid receptors modulate recovery from consummatory successive negative contrast. Behav Brain Res 164(2): 239-249.
    8. Rowan, G. A. and Flaherty, C. F. (1987). The effects of morphine in the consummatory contrast paradigm. Psychopharmacology (Berl) 93: 51-58.
    9. Wiech, K. and Tracey, I. (2009). The influence of negative emotions on pain: behavioral effects and neural mechanisms. Neuroimage 47: 987-994.
    10. Wood, M., Daniel, A. M. and Papini, M. R. (2005). Selective effects of the δ-opioid receptor agonist DPDPE on consummatory successive negative contrast. Behav Neurosci 119: 446-454.
    11. Wood, M. D., Norris, J. N., Daniel, A. M. and Papini, M. R. (2008). Trial-selective effects of U50,488H, a κ-opioid receptor agonist, on consummatory successive negative contrast. Behav Brain Res 193: 28-36.
    12. Xie, J., Bi, Q., Li, W., Shang, W., Yan, M., Yang, Y., Miao, D. and Zhang, H. (2012). Positive and negative relationship between anxiety and depression of patients in pain: a bifactor model analysis. PLoS One 7: e47577.
  • 简介

    在成瘾和疼痛研究中使用动物模型是揭示治疗这些疾病的新途径和机制的关键。 通过完成连续负面对比(cSNC)任务的奖励贬值已经显示出降低大鼠的物理疼痛敏感性(痛觉减退)和增加口服乙醇消耗的能力。 该程序基于在几个阶段(预转换期)中将实验动物暴露于32%蔗糖溶液,然后在接下来的几个阶段(后移位期)中使4%蔗糖贬值。 cSNC效应可以通过将实验组与在整个实验中进行4%蔗糖的未移位对照(预移位和移位后期)进行比较来监测。 cSNC现象的定义是降档组中蔗糖的消耗量低于后配合期间未降低组中的蔗糖消耗量。
    【背景】有实验证据表明,情绪状态(例如,焦虑或沮丧)会影响对身体疼痛的敏感性,并与精神活性物质如酒精的消耗相关(Wiech和Tracey,2008; Xie et al。 ,2012; Manzo et al。,2015)。奖励贬值是诱发焦虑等消极情绪的可行策略(参见Flaherty,1996; Papini et al。,2015)。奖励贬值可以在不同的程序中实施,包括食欲消退,部分强化和连续负对比(SNC)。所有这些都意味着奖励幅度或质量的意外变化。在食欲的灭绝中,奖励被完全隐瞒,而对于部分强化,奖励在一些随机数量的试验中被扣留。 SNC意味着不是消除,而是奖励的幅度或质量的贬值。在完成的连续阴性对比(cSNC)任务中,一种类型的SNC程序,动物在几个日常会话期间可以自由进入5分钟到高价值的蔗糖溶液(通常是32%蔗糖),然后进行几次访问。低值蔗糖溶液(通常为4%蔗糖)(Jiménez-García et al。,2016)。将降低的动物的表现与总是接受4%蔗糖(未移位的对照)的动物的完成行为进行比较。 cSNC效应涉及在32%-4%蔗糖降档后恢复到未移位对照水平后抑制完成行为。最初的抑制(通常在第一次贬值会议期间观察到)和随后的恢复(通常在第二次贬值会议期间开始)是cSNC效应的可分离阶段(Flaherty,1996)。

    几个证据来源表明cSNC调节并且也受到身体疼痛的调节(Jiménez-García et al。,2016)。 Papini et al。(2015)提出了身体疼痛(组织损伤)和心理疼痛(奖励损失)之间的联系,这有助于进一步比较这两组现象。例如,已知调节身体疼痛的阿片类配体也会在第一次降档期间调节cSNC([D-Pen2,D-Pen5]脑啡肽,选择性δ-受体激动剂; Wood 等。在第二次降档期间(U50,488H,一种选择性κ-受体激动剂; Wood et al。,2008),或在两次会话期间(吗啡; Rowan和Flaherty,1987)。两种阿片受体拮抗剂在第一次降档期间选择性地增强cSNC效应(纳曲吲哚,选择性δ受体拮抗剂)或在第一次和第二次降档期间(纳洛酮,非选择性阿片受体拮抗剂; Pellegrini 等。 ,2005)。类似地,涉及疼痛处理的大脑区域的损伤,例如前扣带皮层,也影响从第二次降档会话后的奖励降档恢复(Ortega et al。,2011)。

    同样,cSNC增加了乙醇消耗。奖励贬值会产生焦虑状态(沮丧),可以通过乙醇的抗焦虑作用来缓解。 Manzo et al。(2015)显示奖励贬值后的乙醇消费增加(转换后会话)。

    关键字:成瘾, 疼痛, 大鼠, 连续阴性对照, 沮丧, 报酬损失, 心理疼痛, 情绪, 焦虑

    材料和试剂

    1. 容量为150毫升的防滴漏瓶(Classic Drinker de Luxe; Zooplus)(见图1)


      图1.防滴漏瓶 

    2. 动物:使用成年雄性Wistar大鼠(Envigo Laboratories,Barcelona,Spain),11周龄,体重281.2(±2.1)g
    3. 过滤和高压灭菌的水
    4. 蔗糖(适合人类食用的白糖.AzucareraEspañolaS.A。)
    5. 32%蔗糖溶液(见食谱)
    6. 4%蔗糖溶液(见食谱)

    设备

    1. 定时器和天文台(Digital Onstart 100)(图2A)
    2. 消费室
      矩形(30 x 30 x 15 cm)树脂玻璃盒(图2B)
    3. 称重秤(WLC 1 / A2精密天平; Radwag©)(图2C)
    4. 个人防护装备(实验室外套,手套,口罩,等。)(图2D)


      图2.设备。 A.计时器和计时器。 B.消费室。 C.称重秤。 D.个人防护装备。

    程序

      参见图3了解每日实验程序的时间表。

            
            图3.每日实验程序的时间表。 *高/低值蔗糖,取决于每个实验组。

    1. 在聚碳酸酯标准笼中使每只动物个体化,自由饮水并在受控的标准条件下维持(12小时黑暗/光照循环,从08:00开始,24℃温度和50%-60%湿度)。在整个实验过程中,将食物的动物剥夺到其自由重量的82%-85%。一旦动物达到所需的体重,每天喂食9克(±1.5克)大鼠食物以维持体重。所有操作必须每天在同一时间进行,并且最好由同一个人进行。必须努力减少痛苦并减少实验中使用的动物数量。为了在实验开始前评估动物的体重,重要的是每天称量每个样本并在每个疗程结束后不少于30分钟提供补充食物。此外,蔗糖溶液必须每天新鲜制备,以避免污染。 
    2. cSNC培训每天举行19次会议:16次转发前会议,然后是3次转职后会议。 
    3. 在cSNC任务中,至少需要两个实验组。在转移前的会议期间,其中一人可获得大额奖励(例如,32%蔗糖解决方案),另一人获得小奖励(例如,4%蔗糖解决方案)。在转换后的会议期间,两组都接受了小额奖励(4%蔗糖溶液)。
    4. 在每次训练之前,必须将动物架移动到进行实验的不同房间,让老鼠在他们的家笼中放置15分钟,然后将它们放置在消耗室中。 
    5. 对比期从第一次接触吸管开始持续5分钟。每天平衡瓶子的位置以最小化侧面偏好的影响。在会话结束时将动物放回家笼中。 
    6. 每个盒子的会话持续时间可以使用数字时钟手动测量(Digital Onstart 100)。 
    7. 记录每个动物在会话之前和之后称量瓶子的蔗糖消耗量,然后使用以下公式将重量转换为毫升(已考虑每种溶液的密度):消耗的溶液克数/密度(克/毫升)。所有动物每天以4人为一组接受训练,组的顺序在几天内抵消。在每次疗程结束时,必须用湿纸巾擦拭对比室,并根据需要去除粪便。
    8. 必须给动物喂食适量的大鼠食物,以在每次训练结束后不少于30分钟保持体重。

    数据分析

    在cSNC情况下,因变量是每个阶段消耗的蔗糖总量(以毫升计;图4)。
     
            
    的     图4.最后一次换水期(16)和三次换水期(17-19)期间的平均(±SEM)蔗糖消耗量(ml)。 32-4:暴露于奖励贬值的动物从32%降至4在移位后的会话中%蔗糖。 4-4:暴露于未移位的奖赏条件下的动物,在整个实验过程中始终接受4%蔗糖。星号反映了降档和未降档组之间的显着差异。 (图片改编自Jiménez-García等人,2016年。)

    笔记

    1. 消毒室应使用无菌水清洗,以去除任何动物气味。有必要用标准的家用笼式锯末覆盖箱子的地板。不允许食用或饮用水。
    2. 对于蔗糖呈现,可以使用带有金属吸管的量筒(0.01ml单位)。
    3. cSNC培训可以减少到每天15次:10次预换班,然后是5次换班。该程序是cSNC实验中的典型方案,因此也是有利的。一些实验已经报道了cSNC效应,具有最小的升档前经验(一次会议:Flaherty 等人,,1983),但不常使用少于10次的预换档会话。选择的最终协议将取决于与感兴趣的问题相关的其他要求。
    4. 可以选择使用记录系统(即,相机)。因此,将记录系统放在腔室的侧面。确保动物的口腔区域清晰可见。
    5. 磁力搅拌器可用于适当溶解蔗糖。

    食谱

    1. 32%蔗糖溶液
      32克蔗糖(商业白糖)
      68克蒸馏水
    2. 4%蔗糖溶液
      4克蔗糖(商业白糖)
      96克蒸馏水

    致谢

    本文得到西班牙经济和竞争力部(授予PSI-2013-44945-P),JuntadeAndalucía(授予CTS109),FEDER基金以及格拉纳达大学基础与应用神经科学与疼痛硕士课程的部分支持。 ,西班牙。 MRP的参与得到了富布赖特美国学者奖的支持。根据欧盟指令2010/63 / EU维持动物用于动物实验。我们感谢CTS430小组,尤其是M.Á。萨弗拉,他们的帮助。该协议和数据改编自我们以前的工作(Jiménez-García et al。,2016)。

    利益争夺

    作者声明没有利益冲突或竞争利益。

    伦理

    实验方案经格拉纳达大学研究伦理委员会批准。参考:SSA / SIS / MD / jv(有效期为2016年3月31日至31/03/2021)。

    参考

    1. Flaherty,C。F.(1996)。 激励相对论。剑桥大学出版社,英国:剑桥。
    2. Flaherty,C.F.,Becker,H.C。和Checke,S。(1983)。 在蔗糖浓度反复变化的情况下,在完成性行为中反复连续对比。 Anim Learn Behav 11:407-414。
    3. Jiménez-García,A。M.,Ruíz-Leyva,L.,Cendán,C.M。,Torres,C.,Papini,M。R.和Moron,I。(2016)。 大鼠奖励贬值引起的痛觉过敏。 PLoS One 11(10):e0164331。
    4. Manzo,L.,Donaire,R.,Sabariego,M.,Papini,M.R。和Torres,C。(2015)。 大鼠抗焦虑自我药疗:奖励贬值后口服氯氮卓和乙醇。 Behav Brain Res 278:90-97。
    5. Ortega,L.A.,Uhelski,M.,Fuchs,P。N.和Papini,M。R.(2011)。 前扣带皮层病变后的激励降档恢复受损:情绪或认知缺陷? Behav Neurosci 125:988-995。
    6. Papini,M.R.,Fuchs,P。N.和Torres,C。(2015)。 心理痛苦的行为神经科学。 Neurosci Biobehav Rev 48 :53-69。
    7. Pellegrini,S.,Wood,M.,Daniel,A。M. and Papini,M。R.(2005)。 阿片受体调节从完全连续的阴性对比中恢复。 Behav Brain Res 164(2):239-249。
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    Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Jiménez-García, A. M., Ruiz-Leyva, L., Vázquez-Ágredos, A., Torres, C., Papini, M. R., Cendán, C. M. and Morón, I. (2019). Consummatory Successive Negative Contrast in Rats. Bio-protocol 9(7): e3201. DOI: 10.21769/BioProtoc.3201.
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