Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion

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May 2017



Pain is a complex experience. The aversive component of pain has been assessed through conditioned place aversion in rodents. However, this behavioral test does not allow the evaluation of the aversion of an acute pain stimulus. In Zhang et al. (2017), we provide an updated version of a Conditioned Place Aversion paradigm to address this challenge. In this protocol, a detailed version of this method is described.

Keywords: Conditioned Place Aversion (条件性位置厌恶), Acute pain (急性疼痛), Laser stimulus (激光刺激), Optogenetic stimulation (光遗传学刺激)


Pain is a multidimensional experience that includes sensory and affective components. As such, behavioral tests that assess both the sensory and the emotional aspects of pain are critical to understanding the whole pain experience. However, the majority of available behavioral tests depends on the measurement of nociceptive stimuli and relies on withdrawal thresholds, which are reflexive responses. Nociceptive withdrawal reflex behaviors are under supraspinal control and can occur in the absence of supraspinal inputs. On the other hand, higher order pain behaviors such as conditioned place aversion (CPA) assess some of the emotional component of pain. These experiments involve having the animal choosing to avoid or escape a pain-inducing behavior and/or treatments by moving to the other chamber of the box (LaBuda and Fuchs 2000; Ding et al., 2005; van der Kam et al., 2008; McNabb et al., 2012; He et al., 2012). Lesions of the anterior cingulate cortex (ACC) block the development of escape-avoidance pain behaviors (Johansen et al., 2001; LaGraize et al., 2004; Qu et al., 2011). However, those previous behavioral assays always implied an avoidance learning paradigm, and the conditioning lasts for consecutive days. These assays also require that pain stimulation be applied for prolonged periods of time during the multi-day conditioning phases, and thus most of these assays employed a persistent or chronic pain model. As a result, these assays do not assess the aversive response to an acute pain stimulus. Therefore, testing the aversion of acute pain stimulus is urgently needed to allow for the accurate and comprehensive assessment of acute pain and the screening of analgesics.

With this in mind, we modified the previously described conditioned place aversion test. We applied an acute nociceptive stimulus to the hind paw of the animals. This stimulus was short-lived, as a paw withdrawal removed it. To make sure of a behavioral response, we repeated this stimulus. However, to ensure that we are testing an acute pain response, we shortened the experiment to 3 consecutive sessions of 10 min, with 10 min total for the pain conditioning phase.

Through this assay, we can observe an avoidance behavior which is short lasting and easily regulated by optogenetic modulation of ACC neurons.

Materials and Reagents

  1. Male Sprague-Dawley rats, 250-300 g (Taconic Biosciences, catalog number: SD-M )
  2. 70% ethanol solution (Decon Labs, catalog number: 2716 )
  3. Nivea Lip Care A Kiss of Cherry Fruity Lip Care 0.17 oz. (Walgreen, Nivea, catalog number: 331781 )
  4. Nivea Lip Care A Kiss of Mint & Minerals Refreshing Lip Care 0.17 oz. (Walgreen, Nivea, catalog number: 443307 )
  5. Isoflurane (Piramal Critical care, NDC 66794-017-25)
  6. AAV1.CAMKII.ChR2-eYFP.WPRE.hGH (Penn vector Core, University of Pennsylvania)
  7. AAV1.CAMKII.NpHR-eYFP.WPRE.hGH (Penn vector Core, University of Pennsylvania)


  1. Hamilton syringe, 75 ASN, 26 G, 5 µl (Hamilton, catalog number: 87989 )
  2. Two High Power blue DPSS Lasers HPL1 and HPL2, 1,000 mW, 473 nm (Shanghai Dreams Laser Technology, catalog number: SDL-473-1000T )
  3. Low Power blue DPSS Laser LPL, 200 mW, 473 nm (Shanghai Dream Lasers Technology, catalog number: SDL-473-10T )
  4. Low Power yellow DPSS Laser LPL, 50 mW, 589 nm (Ultralasers, model: MGL-III-589-50 )
  5. Digital Optical Power and Energy Meter (Thorlabs, model: PM100D )
  6. Standard Photodiode Power Sensor (Thorlabs, model: S121C )
  7. Mesh table (IITC Life Science, catalog number: 410 )
  8. USB 3.0 monochrome industrial camera (The Imaging Source, catalog number: DMK 23U618 )
  9. Varifocal manual iris lens (Computar, catalog number: T3Z3510CS )
  10. 1x2 fiber-optic rotary joint (Doric Lenses, model: FRJ_1x2i_FC-2FC )
  11. TTL pulse generator (Doric Lenses, model: OTPG_4 )
  12. Dohm sound machine (Marpac, catalog number: Marpac Dohm DS )
  13. Standard desktop computer or laptop
  14. Metal stand (Humboldt, catalog number: H-21330 )
  15. 2 chambered apparatus (designed in the laboratory, 16 x 7 x 13 cm) made from black (color number 2025) opaque acrylic, 1/4” (5.6 mm) thick (Canal Plastics Center, NY, USA)
  16. Mini Glue Gun, 15 Watt, 1/4 In (Stanley Black & Decker, model: GR10 , catalog number: 3NZW9Mfr)
  17. Clear Hot Melt Glue Stick, 1/4” Diameter, 4” Length, 24 PK (Stanley Black & Decker, model: GS10DT , catalog number: 2FDB9 Mfr.)
  18. Ceramic LC MM Ferrule 1.25 mm (Thorlabs, catalog number: CFLC230-10 )
  19. Ceramic split mating sleeve (Thorlabs, catalog number: ADAL1 )
  20. Fiber cable (Thorlabs, catalog number: M83L01 )
  21. Dental Cement Unifast Trad powder (Pearson Dental Supply, catalog number: G 05-12-24 ) and liquid (Pearson Dental Supply, catalog number: G 05-12-26 )


  1. ANY-maze tracking software (Stoelting Co., Illinois, USA,
  2. GraphPad Prism version 7 (GraphPad Software Inc, California, USA,
  3. OTPG4 software (Doric Lenses Inc, Quebec, Canada,


For the outline of this protocol, see Figure 1.

Figure 1. Flowchart of the basic steps of the protocol

  1. Hardware setup (detailed Figure 2)
    For these experiments, we used thermal stimuli applied via High Power Lasers (HPL), and modulate the neuronal activity using optogenetic stimulation. The set up described above, can be customized to fit another type of stimulus.

    Figure 2. Schematic of the complete set up used for performing a Conditioned Place Aversion experiment. CPA apparatus is placed on the mesh table. Animals are recorded via a camera connected to ANY-maze software. A High Power Laser (HPL) delivers a thermal stimulus applied by the experimenter, and a Low Power Laser (LPL) delivers optogenetic light stimulation. The LPL connects 2 fiber cables to the 2 optical fibers implanted in the head of the animal for bilateral optogenetic stimulation of the ACC. All lasers are controlled via a TTL pulse generator in the OPTG4 software to deliver the light stimuli synchronically.

    1. We used a modified escape-avoidance paradigm as previously published (LaBuda and Fuchs, 2000). The apparatus was made of Plexiglas with dimensions 16 x 7 x 13 cm and placed on top of a mesh table (Figure 3). The box was divided into two chambers. To allow the animal to discriminate between the 2 chambers, each one is paired with different cues (any kind of smell, visual, or tactile cues). In our experiment, we applied different smell cues inside each lateral side of the 2 chambers (right chamber = cherry fruity smell/left chamber = mint smell).

      Figure 3. Rat in the CPA apparatus. The rat is allowed to freely move between the two chambers during the 3 phases of the experiment. Its movements are recorded by the camera linked to the ANY-maze software, which calculates the time spent in each chamber.

    2. Install the camera on the Metal stand above the 2 chambered apparatus.
    3. Connect the lasers to the TTL pulse generator.
    4. Connect the TTL pulse generator to the computer.
    5. Turn on the Dohm sound machine.
    6. Turn On HPLs and LPL. Let them warm up for 15 min.
    7. Set HPLs to the desired power using the power meter
      HPL1 Non noxious Stimulus NS-50 mW, and HPL2 to High noxious Stimulus HS-250 mW;
      HPL1 Low noxious Stimulus LS-150 mW and HPL2 to High noxious Stimulus HS-250 mW;
      HPL1 Non noxious Stimulus NS-50 mW and HPL2 to Non noxious Stimulus NS-50 mW;
      HPL1 Low noxious Stimulus LS-150 mW and HPL2 to Low noxious Stimulus LS-150 mW;
      HPL1 High noxious Stimulus HS-250 mW and HPL2 to High noxious Stimulus HS-250 mW.
    8. Set LPL to the desired power using the power meter for optogenetic stimulation (power = 10 mW).

  2. Software set up
    Ensure that the number of the channel corresponds to the correct port on the TTL pulse generator. HPL1 connected to port entry #1 will be controlled in Channel 1, HPL2 connected to port entry #2 will be controlled in Channel 2, and LPL connected to port entry #3 will be controlled in Channel 3 (Figure 4).
    Set the parameters in OTPG4 software to control the HPL.
    For optogenetic modulation of neuronal activity:
    1. Set the parameters in OTPG4 software to control the LPL (example: frequency = 20 Hz; Time ON duration = 10 msec, Number of Pulses = 100, Channel 3 in Figure 4).

      Figure 4. OTPG4 software interface

    2. Turn On the ANY-maze software on the computer
      1. Start by opening a New Experiment and name it.
      2. Create a New Protocol (Figure 5).

        Figure 5. ANY-maze software. How to create a new protocol.

      3. Select the video source corresponding to your camera and set up your image. Name a new apparatus (example CPA).
      4. Create two zones on the screen (Figure 6), name them and select the settings you want to record during the experiment (time in the zone).

        Figure 6. ANY-maze software. How to delimitate 2 zones.

      5. On the stages tab, select the duration of your experiment (600 sec = 10 min in our case) Select the type of data you want to collect in Results, Report and Data.
      6. Set up the parameters of your Experiment (number of animals, different phases. Figure 7).

        Figure 7. ANY-maze software. Set up for a new experiment.

      7. To start recording, go to the Test section → click on video recording → ‘Play’ button (double click until the chronometer starts).
        Note: For any additional parameters, please refer to ANY-maze software Guideline.

  3. Experiments
    1. For optogenetic manipulation of neuronal activity, prior to conducting the behavioral experiments, perform transcranial injections of optogenetic constructs. Anesthetize animals with isoflurane (1.5 to 2%). In all experiments, the virus is delivered to the anterior cingulate cortex (ACC) only. Rats are bilaterally injected with 0.5 μl of viral vectors at a rate of 0.1 μl/10 sec with a 26-G 5 μl Hamilton syringe at anteroposterior (AP) +2.6 mm, mediolateral (ML) ±1.6 mm, and dorsoventral (DV) -2.25 mm, with tips angled 28° toward the midline. The microinjection needles are left in place for 10 min, raised 1 mm and left for another minute to allow for diffusion of virus particles away from injection site, while minimizing spread of viral particles along the injection tract.
      Rats are then implanted with 200 μm optic fibers held in 2.5 mm ferrules in the ACC: AP +2.6 mm, ML ±1.6 mm, DV -1.25 mm. Fibers with ferrules are held in place by dental acrylic. Allow the virus to be properly expressed in the neurons (2 to 4 weeks depending of the viral vector serotype).
    2. For 1 week prior to beginning experiments, rats are habituated to the experimenter and the environment. Rats must be comfortable with the experimenter to give the best possible results. Rats are handled by the experimenter. If they seemed stressed or anxious, they are put back in their cages and resumed habituation the following day. During all the behavioral phases, animals are allowed to move unrestricted to either side of the box. The movements of rats in each chamber are automatically recorded by a camera and analyzed with the ANY-maze software (Stoelting).
    3. During the preconditioning phase (Video 1), place rats randomly on either the left or the right side of the box to start. An equal number of animals in each group starts on either the left or the right side of the box. All animals are allowed to explore the two chamber apparatus without restraint for 10 min. The movement of each rat is recorded and analyzed to verify the absence of any preconditioning chamber preference (Figure 9). Animals spending more than 500 sec or less than 100 sec of the total time in any chamber should be eliminated from further testing or analysis (< 20% of total animals).

      Video 1. Preconditioning phase. All animals are allowed to explore the two chamber apparatus for 10 min. The two chambers are delimited by an orange rectangle and the position of the animal is monitored by a red dot tracking the center of the animal’s body. Time spent in each chamber is recorded by the camera linked to the ANY-maze software.

    4. Following the preconditioning phase, the rats undergo conditioning for 10 min (Video 2): each chamber is paired with a stimulus (noxious or not, different noxious intensity). As an example, chamber A is paired with a thermal painful stimulus (laser-High Stimulus HS) and the other chamber (B) is paired with a thermal non-painful stimulus (laser-Non Noxious Stimulus NS). Stimuli are given to the hind-paw every 10 sec when the animal is immobile. For each stimulus applied to the rat’s hind paw, bring the laser tip closely to the animal (0.3-0.5 mm without touching the rodent’s skin), between the holes in the grid of the mesh table. Even if the animal is freely moving, it has some immobile periods during which it’s possible for the experimenter to apply the stimulus on a precise location, its hind paw. Paw withdrawal removes the stimulus as the experimenter would stop applying the stimulus to the rodent’s hind paw.

      Video 2. Conditioning phase. Each chamber is paired with a stimulus. The left chamber is paired with a thermal painful stimulus (laser-High Stimulus HS) and the right chamber is paired with a thermal non-painful stimulus (laser-Non Noxious Stimulus NS). Stimuli are given to the hind-paw every 10 sec. Peripheral stimuli are applied to the hind paws through the mesh table.

      As you can observe on this picture (Figure 8), the tip of the laser (red arrow) is positioned between the holes of the mesh table, allowing to come as close as 0.3-0.5 mm to the rat’s hindpaw, standing above.
      Note: In some experiments, the thermal stimulus delivered by the HPL is paired with an optogenetic activation or inhibition of ACC neurons controlled by the LPL. If channel 1 controls HPL1 and channel 3 controls the LPL for the optogenetic stimulation you just have to click on ‘Start All’ on the OTPG4 software interface (Figure 4) and you will activate both lasers simultaneously.

      Figure 8. How to apply the stimulus

    5. Finally, the animals undergo a test phase (Video 3), where they are allowed to explore the two chamber apparatus for 10 min (Figure 9).

      Video 3. Test phase. All animals are allowed to explore the two chamber apparatus for 10 min. This phase is comparable to the preconditioning phase and aims to see the effect of the conditioning through a change of time spent in each chamber.

      Figure 9. Representative results of CPA behavior (original Figure 1.C [Zhang et al., 2017]: Rats recognize and seek to avoid the aversive value associated with HS). During conditioning, rats receive HS in one chamber and NS in the other chamber. After conditioning, rats spend less time in the chamber paired with HS during the post conditioning phase (blue bars) than during the preconditioning phase (white bars), and more time in the chamber paired with NS. n = 14; P < 0.0001; paired Student’s t-test.

    6. At the end of the test, place the animal back in his cage; clean the apparatus, and mesh table with a 70% ethanol solution.

Data analysis

The data analysis was conducted using GraphPad Prism version 7. The ANY-maze software provides us a value of time spent in each chamber and we analyze the videos offline to be sure that the tracking was efficient, and not erroneous because of tracking artifacts introduced by, for example, manual movement of the laser fiber.
A paired Student’s t-test was used to compare the time spent in each treatment chamber before and after conditioning (i.e., baseline vs. test phase for each chamber). Decreased time spent in a chamber during the test phase when compared with the baseline, indicates avoidance (aversion) for that chamber.


  1. All procedures in this study were approved by the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC) as consistent with the National Institute of Health Guide for the care and use of laboratory Animals to ensure minimal animal use and discomfort. Male Sprague-Dawley rats were purchased from Taconic Farms, Albany, NY and kept at Mispro Biotech Services Facility in the Alexandria Center for Life Science, with controlled humidity, temperature, and 12 h (6:30 AM to 6:30 PM) light-dark cycle. Food and water were available ad libitum. Animals arrived to the animal facility at 250 to 300 g and were given on average 10 days to adjust to the new environment prior to the onset of experiments.
  2. The apparatus was made of 6 black acrylic plexiglas panels (2 panels 16 x 13 and 2 panels 7 x 13 to build the rectangular base, 2 panels 1 x 13 to do the separation between the 2 chambers). The panels were glued together to make a two chamber apparatus, dimensions 16 x 7 x 13 cm (see Equipment).
  3. The box was divided into two chambers. To allow the animal to discriminate between the 2 chambers, each one is paired with different cues (any kind of smell, visual, or tactile cues). In our experiment, we applied different smell cues inside each lateral side of the 2 chambers (right chamber = cherry fruity smell/left chamber = mint smell).
  4. The habituation of the rats to the experimenter 1 week prior to the test is done to ensure that the rats are comfortable with the experimenter and the testing environment. This will reduce/mitigate the effect that the presence of the experimenter and novel environment will have on the rat’s movement during the experiments. The animals were handled by the experimenter daily for 5 min, until the animals showed signs of stress (start to urinate, defecate or vocalize), or escaped from the experimenter’s arms. No force or restraint was used and the time the animals are handled increases daily. The habituation was performed in the same room as the experiments will be conducted. In addition to the experimenter’s handling, the animals were also placed 5 min in the two-chamber apparatus for habituation to the environment. The experimenter is expected to stand in a neutral position when performing the test; a location where each chamber is equidistant from the experimenter. All the phases of the experiment (preconditioning, conditioning, and testing) should occur, one right after the other.


This work was supported by the National Institute of General Medical Sciences (GM115384), National Institute of Neurological Disorders and Stroke (NS100065), (Bethesda, MD, USA) and the Anesthesia Research Fund of New York University Department of Anesthesiology (New York, NY, USA). The authors declare no competing financial interests. This protocol was adapted from LaBuda and Fuchs, 2000.


  1. Ding, H. K., Shum, F. W., Ko, S. W. and Zhuo, M. (2005). A new assay of thermal-based avoidance test in freely moving mice. J Pain 6(7): 411-416.
  2. He, Y., Tian, X., Hu, X., Porreca, F. and Wang, Z. J. (2012). Negative reinforcement reveals non-evoked ongoing pain in mice with tissue or nerve injury. J Pain 13(6): 598-607.
  3. Johansen, J. P., Fields, H. L. and Manning, B. H. (2001). The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci U S A 98(14): 8077-8082.
  4. LaBuda, C. J. and Fuchs, P. N. (2000). A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Exp Neurol 163(2): 490-494.
  5. LaGraize, S. C., Labuda, C. J., Rutledge, M. A., Jackson, R. L. and Fuchs, P. N. (2004). Differential effect of anterior cingulate cortex lesion on mechanical hypersensitivity and escape/avoidance behavior in an animal model of neuropathic pain. Exp Neurol 188(1): 139-148.
  6. McNabb, C. T., Uhelski, M. L. and Fuchs, P. N. (2012). A direct comparison of affective pain processing underlying two traditional pain modalities in rodents. Neurosci Lett 507(1): 57-61.
  7. Qu, C., King, T., Okun, A., Lai, J., Fields, H. L. and Porreca, F. (2011). Lesion of the rostral anterior cingulate cortex eliminates the aversiveness of spontaneous neuropathic pain following partial or complete axotomy. Pain 152(7): 1641-1648.
  8. van der Kam, E. L., De Vry, J., Schiene, K. and Tzschentke, T. M. (2008). Differential effects of morphine on the affective and the sensory component of carrageenan-induced nociception in the rat. Pain 136: 373-79.
  9. Zhang, Q., Manders, T., Tong, A. P., Yang, R., Garg, A., Martinez, E., Zhou, H., Dale, J., Goyal, A., Urien, L., Yang, G., Chen, Z. and Wang, J. (2017). Chronic pain induces generalized enhancement of aversion. Elife 6: e25302.


疼痛是一个复杂的经验。 已经通过对啮齿类动物有条件的地方厌恶来评估疼痛的厌恶成分。 但是,这种行为测试不允许评估对急性疼痛刺激的厌恶。 在Zhang等人(2017年)中,我们提供了一个更新版本的条件反应模式来应对这一挑战。 在这个协议中,描述了这个方法的详细版本。
【背景】疼痛是一个包含感官和情感成分的多维体验。因此,评估疼痛的感觉和情绪方面的行为测试对于理解整个疼痛体验是至关重要的。然而,大多数可用的行为测试取决于伤害性刺激的测量,并依赖于退缩阈值,这是自反应。伤害性撤退反射行为是在脊柱上控制下的,并且可以在没有脊柱上输入的情况下发生。另一方面,高阶疼痛行为,如条件性位置厌恶(CPA)评估疼痛的一些情绪成分。这些实验涉及让动物选择通过移动到盒子的另一个腔室来避免或逃避引起疼痛的行为和/或治疗(LaBuda和Fuchs 2000; Ding等人,2005; van der Kam等人,2008; McNabb等人,2012; He等人,2012)。前扣带皮层(ACC)的病变阻碍逃避 - 避免疼痛行为的发展(Johansen等人,2001; LaGraize等人,2004; Qu- et al。,2011)。然而,那些以前的行为分析总是暗示了一个回避的学习范式,并且调理持续了连续的几天。这些测定法还要求在多天调理阶段期间长时间应用疼痛刺激,并且因此大多数这些测定法采用持久性或慢性疼痛模型。结果,这些测定法不评估对急性疼痛刺激的厌恶响应。因此,迫切需要测试急性疼痛刺激的厌恶情况,以便准确和全面地评估急性疼痛和镇痛药物的筛选。



关键字:条件性位置厌恶, 急性疼痛, 激光刺激, 光遗传学刺激


  1. 雄性Sprague-Dawley大鼠,250-300g(Taconic Biosciences,目录号:SD-M)
  2. 70%乙醇溶液(Decon Labs,目录号:2716)
  3. 妮维雅唇部护理亲吻樱桃果味唇部护理0.17盎司。 (Walgreen,妮维雅,目录号:331781)
  4. 妮维雅唇部护理薄荷糖之吻矿物刷新的嘴唇关心0.17盎司。 (Walgreen,妮维雅,目录号:443307)
  5. 异氟醚(Piramal Critical care,NDC 66794-017-25)
  6. AAV1.CAMKII.ChR2-eYFP.WPRE.hGH(Penn载体核心,宾夕法尼亚大学)
  7. AAV1.CAMKII.NpHR-eYFP.WPRE.hGH(Penn载体核心,宾夕法尼亚大学)


  1. Hamilton注射器,75 ASN,26 G,5μl(汉密尔顿,目录号:87989)
  2. 两个高功率蓝色DPSS激光器HPL1和HPL2,1000毫瓦,473纳米(上海梦幻激光技术公司,产品目录号:SDL-473-1000T)
  3. 低功率蓝光DPSS激光LPL,200mW,473nm(上海梦幻激光技术公司,产品目录编号:SDL-473-10T)
  4. 低功率黄色DPSS激光LPL,50mW,589nm(Ultralasers,型号:MGL-III-589-50)
  5. 数字光功率和能量计(Thorlabs,型号:PM100D)
  6. 标准光电二极管功率传感器(Thorlabs,型号:S121C)
  7. 网表(IITC生命科学,目录号:410)
  8. USB 3.0单色工业相机(The Imaging Source,目录号:DMK 23U618)
  9. 变焦手动光圈镜头(Computar,目录号:T3Z3510CS)
  10. 1x2光纤旋转接头(Doric镜头,型号:FRJ_1x2i_FC-2FC)
  11. TTL脉冲发生器(Doric镜头,型号:OTPG_4)
  12. Dohm发声机(Marpac,目录号:Marpac Dohm DS)
  13. 标准台式电脑或笔记本电脑
  14. 金属支架(Humboldt,目录号:H-21330)
  15. (厚度为1/4“(5.6mm)(美国纽约州Canal Plastics Center)的黑色(颜色编号2025)不透明丙烯酸树脂制成的2室装置(在实验室中设计,16×7×13cm)
  16. 迷你胶枪,15瓦,1/4英寸(史丹利黑色和迪克,型号:GR10,目录号:3NZW9Mfr)
  17. 透明热熔胶棒,1/4“直径,4”长度,24 PK(史丹利黑&德克,型号:GS10DT,目录号:2FDB9制造商)
  18. 陶瓷LC MM箍1.25毫米(Thorlabs,目录号:CFLC230-10)
  19. 陶瓷分体式套筒(Thorlabs,产品目录号:ADAL1)
  20. 光缆(Thorlabs,目录号:M83L01)

  21. 牙科水泥Unifast Trad粉末(Pearson牙科供应,目录编号:G 05-12-24)和液体(Pearson Dental Supply,目录编号:G 05-12-26)


  1. 任意迷宫跟踪软件(Stoelting Co.,Illinois,USA,
  2. GraphPad Prism版本7(GraphPad Software公司,美国加利福尼亚州,
  3. OTPG4软件(Doric Lenses公司,魁北克,加拿大,




  1. 硬件设置(详细图2)

    图2.用于执行条件位置反转实验的完整设置的示意图。 CPA设备放置在网格桌上。通过连接到ANY-maze软件的相机记录动物。高功率激光器(HPL)提供由实验者施加的热刺激,而低功率激光器(LPL)提供光遗传光刺激。 LPL将2根光纤电缆连接到植入动物头部的2根光纤,用于ACC的双侧光遗传刺激。所有激光器都通过OPTG4软件中的TTL脉冲发生器进行控制,以同步传送光线刺激。

    1. 我们使用了先前公布的修改过的逃避避税范例(LaBuda and Fuchs,2000)。该装置由有机玻璃制成,尺寸为16×7×13cm,放置在网状桌面上(图3)。箱子被分成两个房间。为了让动物区分两个房间,每个房间都配有不同的线索(任何种类的气味,视觉或触觉线索)。在我们的实验中,我们在两个房间的每个侧面(右房=樱桃果味/左房=薄荷味)内应用不同的气味线索。

      图3. CPA设备中的老鼠。允许老鼠在实验的三个阶段期间在两个室之间自由移动。它的运动是通过连接到ANY-maze软件的相机来记录的,该软件可以计算每个腔室的时间。

    2. 将相机安装在2室装置上方的金属架上。
    3. 将激光器连接到TTL脉冲发生器。
    4. 将TTL脉冲发生器连接到电脑。
    5. 打开Dohm音响机器。
    6. 打开HPL和LPL。让他们热身15分钟。

    7. 使用功率计将HPL设置为所需的功率 HPL1无毒刺激NS-50毫瓦,和HPL2高有害刺激HS-250毫瓦;

    8. 使用光遗传刺激功率计(功率= 10 mW)将LPL设置为所需功率。

  2. 软件设置
    确保通道编号对应于TTL脉冲发生器的正确端口。连接到端口入口#1的HPL1将在通道1中被控制,连接到端口入口#2的HPL2将在通道2中被控制,连接到端口入口#3的LPL将被控制在通道3中(图4) > 在OTPG4软件中设置参数来控制HPL。
    1. 设置OTPG4软件中的参数来控制LPL(例如:频率= 20 Hz;时间开启持续时间= 10毫秒,脉冲数= 100,图4中的通道3)。

      图4. OTPG4软件界面

    2. 打开电脑上的ANY-maze软件
      1. 首先打开一个新的实验并命名。
      2. 创建一个新的协议(图5)。

        图5. ANY-maze软件如何创建一个新的协议。

      3. 选择与您的相机对应的视频源并设置您的图像。命名新设备(例如CPA)。
      4. 在屏幕上创建两个区域(图6),命名它们并选择您想要在实验过程中记录的设置(区域中的时间)。

        图6. ANY-maze软件如何界定2个区域。

      5. 在阶段选项卡上,选择实验的持续时间(在我们的例子中为600秒= 10分钟)在结果,报告和数据中选择要收集的数据类型。
      6. 设置你的实验参数(动物数量,不同阶段,图7)。

        图7. ANY-maze软件设置一个新的实验。

      7. 要开始录制,进入测试部分→点击录像→“播放”按钮(双击直到天文台开始)。

  3. 实验
    1. 对于神经元活动的光遗传操作,在进行行为实验之前,进行经颅注射光遗传学构建体。用异氟醚麻醉动物(1.5-2%)。在所有实验中,病毒仅被递送到前扣带皮层(ACC)。在正位(AP)+ 2.6mm,内侧(ML)±1.6mm和背腹(DV)处,用26-G5μlHamilton注射器以0.1μl/ 10秒的速率向大鼠双侧注射0.5μl病毒载体。 -2.25毫米,尖端向中线倾斜28°。将显微注射针留置10分钟,升高1毫米,再放置一分钟以使病毒颗粒从注射部位扩散,同时最小化病毒颗粒沿注射道的扩散。
      然后在ACC:AP + 2.6mm,ML±1.6mm,DV -1.25mm的大鼠中植入200μm光纤保持在2.5mm套圈中的大鼠。带有金属环的纤维由牙科丙烯酸材料固定。允许病毒在神经元中正确表达(2到4周,取决于病毒载体血清型)。
    2. 开始实验前1周,使大鼠适应实验者和环境。大鼠必须对实验者感到满意,才能获得最好的结果。大鼠由实验者处理。如果他们看起来有些紧张或焦虑,就会被关在笼子里,并在第二天恢复习惯。在所有的行为阶段,允许动物不受限制地移动到盒子的任一侧。每个房间的老鼠的动作是由相机自动记录,并用ANY迷宫软件(Stoelting)进行分析。
    3. 在预处理阶段(视频1),将大鼠随机放在盒子的左侧或右侧开始。每个组的动物数量相等,从盒子的左侧或右侧开始。允许所有的动物不受限制地探测两个腔室装置10分钟。记录和分析每只大鼠的运动,以验证没有任何预处理室的偏好(图9)。


    4. 在预处理阶段之后,将大鼠进行调理10分钟(视频2):每个室与刺激物(有毒或无毒,不同的有害强度)配对。作为一个例子,房间A与热疼痛刺激(激光高刺激HS)配对,而另一个房间(B)与热无痛刺激(激光 - 无毒刺激NS)配对。当动物不动时,每10秒给予后爪刺激。对于应用于大鼠后爪的每一种刺激,将激光尖端紧贴动物(0.3-0.5毫米,不接触啮齿动物的皮肤),在网格台的网格孔之间。即使动物是自由移动的,它也有一些不动的时期,实验者有可能在精确的位置,即后爪上施加刺激。爪子撤回消除刺激,因为实验者将停止对啮齿动物的后爪施加刺激。


      如图8所示,激光的尖端(红色箭头)位于网格台的孔之间,与老鼠的后爪接近0.3-0.5mm,位于上方。 br /> 注意:在一些实验中,由HPL传递的热刺激与由LPL控制的ACC神经元的光遗传激活或抑制配对。如果通道1控制HPL1,通道3控制LPL进行光遗传刺激,则只需点击OTPG4软件界面上的“全部开始”(Start All),您将同时激活两个激光器。 br />


    5. 最后,动物进行测试阶段(视频3),在那里允许他们探索两个腔室装置10分钟(图9)。


      图9. CPA行为的代表性结果(原图1.C [Zhang等人,2017]:大鼠认识到并试图避免与HS有关的厌恶价值) 。在调理过程中,大鼠在一个房间内接受HS,而在另一个房间内接受NS。在调理后,大鼠在调理后阶段(蓝色条)与HS配对的时间少于预处理阶段(白色条),在NS与NS配对的时间更长。 n = 14; P &lt; 0.0001;配对学生的 t - 测试。

    6. 在测试结束时,将动物放回笼中;清洁设备,并用70%的乙醇溶液筛网。


数据分析使用GraphPad Prism版本7进行。ANY-maze软件为我们提供了在每个房间花费的时间,我们离线分析视频以确保跟踪是有效的,而不是错误的,因为跟踪文物例如,激光纤维的手动移动。
使用配对的Student's test实验来比较在调节之前和之后在每个处理室中花费的时间(即,每个室的基线对测试阶段)。在测试阶段与基线相比,在一个小室中花费的时间减少表明对该小室的回避(厌恶)。


  1. 本研究中的所有程序均得到了纽约大学医学院动物护理和使用委员会(IACUC)的批准,与国家卫生研究院指南一致,用于保护和使用实验动物,以确保动物的最少使用和不适。雄性Sprague-Dawley大鼠购自纽约州奥尔巴尼Taconic Farms,保存在亚历山大生命科学中心的Mispro Biotech Services Facility中,控制湿度,温度和12h(6:30 AM至6:30 PM)光 - 黑暗循环。食物和水随意获得。动物到达动物设施250至300克,并平均10天,以适应实验开始之前的新环境。
  2. 该设备由6个黑色丙烯酸有机玻璃面板(2个面板16×13和2个面板7×13来构建矩形底座,2个面板1×13来在两个室之间进行分离)制成。将面板粘在一起,制成一个尺寸为16×7×13厘米(见设备)的两室设备。
  3. 箱子被分成两个房间。为了让动物区分两个房间,每个房间都配有不同的线索(任何种类的气味,视觉或触觉线索)。在我们的实验中,我们在两个房间的每个侧面(右房=樱桃果味/左房=薄荷味)内应用不同的气味线索。
  4. 在测试前1周对大鼠进行习惯化处理以确保大鼠对实验者和测试环境舒适。这将减少/减轻实验者和新环境的存在对实验期间鼠的运动的影响。实验者每天处理动物5分钟,直到动物出现压力迹象(开始排尿,排便或发声),或从实验者的手臂逃跑。没有使用武力或约束,动物的处理时间每天增加。习惯在同一房间进行,因为将进行实验。除了实验者的处理之外,还将动物放置在双室设备中5分钟以适应环境。在实验中,实验者应该处于中立的地位。每个室与实验者等距的位置。实验的所有阶段(预处理,调理和测试)应该一个接一个地发生。


本研究由美国国立综合医学研究所(GM115384),美国国立神经疾病与卒中研究所(NS100065)(美国马里兰州贝塞斯达)和纽约大学麻醉科麻醉科麻醉研究基金(纽约, NY,USA)。作者宣称没有竞争的经济利益。本议定书改编自LaBuda和Fuchs,2000年。


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Urien, L., Zhang, Q., Martinez, E., Zhou, H., Desrosier, N., Dale, J. and Wang, J. (2017). Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion. Bio-protocol 7(21): e2595. DOI: 10.21769/BioProtoc.2595.
  2. Zhang, Q., Manders, T., Tong, A. P., Yang, R., Garg, A., Martinez, E., Zhou, H., Dale, J., Goyal, A., Urien, L., Yang, G., Chen, Z. and Wang, J. (2017). Chronic pain induces generalized enhancement of aversion. Elife 6.