Assessing Prepulse Inhibition of Startle in Mice
评估小鼠惊吓的前脉冲抑制   

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Molecular Psychiatry
Nov 2017

 

Abstract

Animal models are an important tool for studying neuropsychiatric disorders. However, a major challenge for researchers working with laboratory rodents is trying to reproduce ‘core’ symptoms of complex human disorders such as schizophrenia. Despite this challenge, however, it is still conceivable to use animal models designed to reproduce some of the disease’s ‘endo-phenotypes’. One example is the prepulse inhibition (PPI) of the startle reflex. PPI is a form of startle plasticity and is characterized by a normal reduction in startle magnitude that occurs when an intense startling stimulus (or pulse) is preceded by a weaker pre-stimulus (or prepulse). The PPI paradigm is commonly used to evaluate sensorimotor gating and it has been described in numerous species including humans and rodents. Deficits in PPI have been observed in subjects with schizophrenia and other neuropsychiatric diseases, as well as in established animal models of these disorders. The PPI paradigm is therefore largely used to explore genetic and neurobiological mechanisms underlying the sensorimotor gating phenotypes found in these disorders. Thus, it is necessary to set up reliable and reproducible protocols to study PPI in mice.

Keywords: Prepulse inhibition of startle (惊吓的前脉冲抑制), PPI (PPI), Animal models (动物模型), Schizophrenia (精神分裂症), Sensorimotor gating (感觉运动门控)

Background

Sensorimotor gating refers to the ability of a sensory event to suppress a motor response (Cryan and Reif, 2012). One form of sensorimotor gating that has been widely studied in humans and rodents is the prepulse inhibition (PPI) of startle. The startle reflex consists of involuntary contractions of whole-body musculature elicited by sufficiently sudden and intense stimuli. Specifically, the acoustic startle response is characterized by an exaggerated flinching response to an unexpected strong auditory stimulus. PPI is a form of startle plasticity and it is characterized by a normal reduction in startle magnitude that occurs when an intense startling stimulus (or pulse) is preceded by a brief, low intensity prestimulus (or prepulse) (Graham, 1975; Hoffman and Ison, 1980). The PPI paradigm is commonly used to evaluate sensorimotor gating and it has been described in numerous species, including humans (Schwarzkopf et al., 1993) and mice (Carter et al., 1999; Frankland et al., 2004). Impaired PPI is observed in schizophrenia (Braff et al., 2001; Swerdlow et al., 2008), as well as other neuropsychiatric disorders including obsessive-compulsive disorder (Ahmari et al., 2012), Tourette’s syndrome (Swerdlow et al., 2001), Huntington’s disease (Swerdlow et al., 1995) and bipolar disorder (Perry et al., 2001). In patients with psychotic disorders, deficits in sensorimotor gating are associated with cognitive fragmentation and psychotic symptoms (Kapur, 2003). As these deficits have been found both in psychotic patients as well as in animal models (Swerdlow and Light, 2016), the PPI paradigm is largely used in the study of neuropsychiatric diseases and has proven a useful tool for studying and characterizing the effects of several anti-psychotics (Xue et al., 2012), and for exploring the mechanisms underlying psychotic-like behaviors (Geyer, 1999; Ouagazzal et al., 2001).

Materials and Reagents

  1. Mice (C57BL6/N mice purchased from Janvier Labs, Le Genest-Saint-Isle, France)
    Note: If pharmacological treatments are applied before PPI performance, the reagents will depend on the control or drug solutions prepared. Depending on the treatments applied prior to the testing, the animals can be housed in either single or collective cages.
  2. 70% ethanol

Equipment

  1. SR-LAB startle apparatus with digitized electronic output (SR-Lab, San Diego Instruments, catalog number: 2325-0400 ) (Figure 1)
  2. Digital sound level meter (FLIR Systems, Extech, catalog number: 407730 )


    Figure 1. SR-LAB startle apparatus. A. Each experimental apparatus consists of an outer, lighted and ventilated, chamber that serves to prevent external noise or vibrations interfering with experiment. B. Inside the chamber a stabilimeter consisting of a Plexiglas cylinder is secured to a platform. C. A piezoelectric accelerometer-indicated by the red arrow-mounted under the cylinder transduces animal movements that are then digitized, rectified, and recorded by a computer and interface assembly. A loudspeaker-indicated by the blue arrow-generates the startling acoustic stimuli, according to the desired settings.

Software

  1. SR-Lab Analysis software (SR-Lab San Diego Instruments, catalog number: 2325-0400)

Procedure

  1. Designing the protocol
    Here, we describe the experimental design used in our lab to study PPI response in mice (Busquets-Garcia et al., 2017), but the protocol can be modified by adjusting the pulse and prepulse intensities, the number of trials, inter-trial intervals etc., appropriate for exploring different experimental questions.
    1. Begin the session with a 5-min acclimation period. During the acclimation period, the constant background noise of 70-dB white noise is presented for the animal to adapt to the animal holder, startle box and background noise.
    2. The session then proceeds through the presentation of 90 different trials (Figure 2): 


      Figure 2. Representative session using the described experimental design. The first five trials consist of five pulse-alone trials (A), the intermediate 80 trials are divided into 10 blocks of randomized pulse-alone trials, prepulse-alone trials, combinations of prepulse-pulse trials and no-stimulus trials (B) and the session is concluded with a final block of five consecutive pulse-alone trials (C). Prepulse intensities (73 dB, 76 dB and 82 dB) are above the 70 dB background.

      1. The first five trials consist of five pulse-alone trials where 120 dB of white noise is presented in isolation for a duration of 20 msec (i.e., with no prepulse). These trials serve to habituate and stabilize the animals to the startle response.
      2. Subsequently, ten blocks of trials are presented. Each block consists of one pulse-alone trial, three prepulse-alone trials (+3, +6, or +12 units above the background of 70 dB), three combinations of prepulse-pulse trials, and one no-stimulus trial (i.e., background only) (Table 1). The 8 trials are presented in a randomized order within each block, with the inter-trial interval (ITI) varying randomly between 10 and 30 sec, intended to minimize habituation to startle across trials.
        Notes:
        1. The advantage of randomized ITIs is in the fact that the animal cannot predict the time when the next stimulus presentation will occur. For example, attention to the prepulse can increase the animal’s efficacy in suppressing startle responses. ITIs below 10 sec should be avoided in order to exclude effects caused by muscle fatigue and refractory periods of muscle responses.
        2. The intensities of the prepulse should be kept at levels above the background noise but also low enough that they do not elicit a significant startle response on their own, the margin being approximately 2-20 dB above background levels (e.g., +3, +6 or +12 dB above a background of 70 dB). It is important to note that sensitivity to the prepulse may also vary between strain, gender or age of the animals.

          Table 1. Representation of the different types of trial used in the behavioral protocol


      3. The session is concluded with a final block of five consecutive pulse-alone trials, as in the first block.
        Note: Stimulus rise-time, duration, and intensity are variables that affect startle reflex magnitude (Graham, 1975; Hoffman and Searle, 1968). All the parameters should be carefully decided after taking into account the strain, age, sex and genetic background of the animals, since different lines can exhibit different responses to the startle stimuli (Willott et al., 1995 and 2003). 

  2. Running the experiment
    1. Calibrate the loudspeakers and the sensitivity of the transducer platform of the startle chambers (Figure 3). Follow manufacturer’s guidelines for effective sound and movement calibration.
      Note: Calibration of the sound and the movement sensors is very important for obtaining valid test results. Consequently, these must be routinely calibrated before each experiment. 


      Figure 3. Representative images of loudspeakers (A) and movement sensitivity detector (B) used for the calibration of the startle chambers

    2. Having created your experimental protocol (A1, A2), you can create a study database using the SR-LAB startle apparatus software, defining both the experimental sessions and the subjects that will be tested.
    3. Transport the mice to the testing room. You can simultaneously test as many mice as the number of chambers you have available (software compatible with up to a maximum of 16 chambers). For housing conditions, see Note 1. Take care not to stress the mice before starting the experiment and, to that end, make no changes to the home cage (e.g., bedding) for at least 24 h before the experiment. Illumination and noise levels in the testing room should be comparable to those in the housing rooms in order to minimize environmental effects on the behavioral outcome. Tubular animal enclosure minimizes stress from being restrained while animal remains centered over the sensor for consistently reliable results.
    4. In each experimental session, place the mouse in the cylinder inside the testing chamber and secure the door shut.
    5. Run the experimental session according to the experimental design described above. The session will stop automatically at the end of the protocol (after approximately 35-40 min).
    6. Remove each mouse from the chamber at the end of the experimental session and return it to its home cage. Wipe clean the animal holders and chambers with water and allow to dry before introducing the next animal.
    7. Select the next session on the screen and repeat the procedure for all the animals.
    8. At the end of all the sessions, clean the cylinders and chambers with 70% ethanol and leave them to dry. Save the data obtained for a subsequent detailed analysis of acoustic startle and acoustic prepulse inhibition responses.

Data analysis

Reactivity scores obtained on the first and last blocks of five consecutive pulse-alone trials can be analyzed separately to evaluate startle habituation. The data obtained from the remaining 80 trials are categorized into three different subsets according to their relevance to distinct behavioral constructs.
First, startle reactivity (S) is assessed from the reactivity scores obtained in the pulse-alone trials (excluding the first and last blocks of five consecutive pulse-alone trials). The 100 millisecond response window after the presentation of the 120 dB pulse is analyzed by the software and the maximal response peak amplitude is used to determine the acoustic startle response as a control index for the animal’s reaction to the startle pulse (Figure 4A).
Second, reactivity on prepulse-pulse trials (PPiS) relative to the pulse-alone/startle trials (S) is used to evaluate prepulse inhibition (PPI) (Figure 4B). The amount of prepulse inhibition is calculated as a percentage score for each acoustic prepulse trial type using the following formula: 
% PPI = 100 x (S - PPiS)/S (Figure 4B)
Third, to measure prepulse-elicited reactivity (PP), data from prepulse-alone trials are included.
The output of a typical experiment should show increasing PPI levels with increasing prepulse intensities, with relatively low variability (Figure 4C). Well-established effects of particular drugs on PPI should be reproducible when using the same strain, sex, and drug dosages. Startle response data typically show more variability and less reliability than PPI response data.


Figure 4. Expected results of a typical PPI experiment. A. Software output of a 100 millisecond response window after the presentation of a pulse-alone and a prepulse-pulse trial. The arrows indicate the maximal response peak amplitude, which is used to determine the acoustic startle response and the maximal response time as an index of the animal’s reactivity to the stimuli. B. Reactivity on prepulse-pulse trials (PPiS) relative to pulse-alone/startle trials (S) is utilized to evaluate prepulse inhibition (PPI). C. Percent prepulse inhibition (%PPI) is shown for naïve wild-type mice with a BL6N background produced in our institute. As expected, PPI levels increase with increasing prepulse intensity (above background), and variability is low.

Notes

  1. In all PPI experiments performed in our institute, animals were housed in collective cages of 4-5 animals per cage. While social isolation can alter startle variables in rats, the effects of isolation on startle measures in mice have not been well characterized. Therefore, the use of mice that have been separated and single-housed in startle experiments is not ideal.
  2. All sessions of the experimental protocol were performed in the morning (from 9 AM to 2 PM).
  3. Strain: Strain differences in startle reflex and PPI have been described in mice (Bullock et al., 1997; Tarantino et al., 2000; Willott et al., 2003). This protocol was tested in several mouse lines. We found similar results between the C57/BL6N mice and the wild-type animals with a BL6N background produced in our institute, although each batch of mice can vary slightly in their responses.
  4. Sex: Both male and female mice have been used in the protocol of startle reactivity and PPI. If used on the same experimental day, the cleaning process of the testing chambers must be performed meticulously in order to avoid effects on the behavioral outcome.
  5. Age: Age is an important variable in measures of acoustic startle and PPI. Age-related hearing loss, which can alter startle reactivity and PPI levels, has been reported for many inbred strains including the C57BL/6 (Willott et al., 1995). Moreover, studies have shown that early adolescent mice can display altered startle reactivity and PPI compared to adult animals (Pietropaolo and Crusio, 2009).
  6. Different psychotogenic drugs (e.g., cannabinoids, amphetamine etc.) can be used as positive controls (i.e., PPI alteration) for the test.

Recipes

The current protocol does not contain any recipes. In case any psychotogenic or other drug is used as a positive control for the test, the preparation should be done according to relevant protocols and manufacturer’s guidelines.

Acknowledgments

We thank Delphine Gonzales, Nathalie Aubailly, and all the personnel of the Animal Facility of the Neurocentre Magendie for mouse care. We also thank Christopher Stevens for the critical reading of the manuscript and Dr. Susanna Pietropaolo for the advice when the PPI was set up in our lab. This behavioral protocol was originally used in Busquets-Garcia et al., 2017. This work was supported by INSERM (to G.M.), EU–FP7 (PAINCAGE, HEALTH-603191 to G.M., FP7-PEOPLE-2013-IEF-623638 to A.B.-G., European Research Council (Endofood, ERC–2010–StG–260515; CannaPreg, ERC-2014-PoC-640923, to G.M.), Fondation pour la Recherche Medicale (DRM20101220445 and DPP20151033974, to G.M.), Human Frontiers Science Program (to G.M.), Region Aquitaine (to G.M.), French State/Agence Nationale de la Recherche (BRAIN ANR-10-LABX-0043 to G.M., ANR-10-IDEX-03-02 to A.B.-G, NeuroNutriSens ANR-13-BSV4-0006-02 to G.M. and ORUPS ANR-16-CE37-0010 to G.M.). The authors declare that they have no conflict of interest.

References

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  2. Braff, D. L., Geyer, M. A., Light, G. A., Sprock, J., Perry, W., Cadenhead, K. S. and Swerdlow, N. R. (2001). Impact of prepulse characteristics on the detection of sensorimotor gating deficits in schizophrenia. Schizophr Res 49(1-2): 171-178.
  3. Bullock, A. E., Slobe, B. S., Vazquez, V. and Collins, A. C. (1997). Inbred mouse strains differ in the regulation of startle and prepulse inhibition of the startle response. Behav Neurosci 111(6): 1353-1360.
  4. Busquets-Garcia, A., Soria-Gomez, E., Redon, B., Mackenbach, Y., Vallee, M., Chaouloff, F., Varilh, M., Ferreira, G., Piazza, P. V. and Marsicano, G. (2017). Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice. Mol Psychiatry 22(11): 1594-1603.
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  6. Cryan, F. J. and Reif, A. (2012). Behavioral neurogenetics. Springer.
  7. Frankland, P. W., Wang, Y., Rosner, B., Shimizu, T., Balleine, B. W., Dykens, E. M., Ornitz, E. M. and Silva, A. J. (2004). Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice. Mol Psychiatry 9(4): 417-425.
  8. Geyer, M. A. (1999). Assessing prepulse inhibition of startle in wild-type and knockout mice. Psychopharmacology (Berl) 147(1): 11-13.
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  10. Hoffman, H. S. and Ison, J. R. (1980). Reflex modification in the domain of startle: I. Some empirical findings and their implications for how the nervous system processes sensory input. Psychol Rev 87(2): 175-189.
  11. Hoffman, H. S. and Searle, J. L. (1968). Acoustic and temporal factors in the evocation of startle. J Acoust Soc Am 43(2): 269-82.
  12. Kapur, S. (2003). Psychosis as a state of aberrant salience: a framework linking biology, phenomenology, and pharmacology in schizophrenia. Am J Psychiatry 160(1): 13-23.
  13. Ouagazzal, A. M., Jenck, F. and Moreau, J. L. (2001). Drug-induced potentiation of prepulse inhibition of acoustic startle reflex in mice: a model for detecting antipsychotic activity? Psychopharmacology (Berl) 156(2-3): 273-283.
  14. Perry, W., Minassian, A., Feifel, D. and Braff, D. L. (2001). Sensorimotor gating deficits in bipolar disorder patients with acute psychotic mania. Biol Psychiatry 50(6): 418-424.
  15. Pietropaolo, S. and Crusio W. E. (2009). Strain-dependent changes in acoustic startle response and its plasticity across adolescence in mice. Behav Genet 39(6): 623-31.
  16. Schwarzkopf, S. B., Lamberti, J. S. and Smith, D. A. (1993). Concurrent assessment of acoustic startle and auditory P50 evoked potential measures of sensory inhibition. Biol Psychiatry 33(11-12): 815-828.
  17. Swerdlow, N. R., Karban, B., Ploum, Y., Sharp, R., Geyer, M. A. and Eastvold, A. (2001). Tactile prepuff inhibition of startle in children with Tourette's syndrome: in search of an "fMRI-friendly" startle paradigm. Biol Psychiatry 50(8): 578-585.
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简介

动物模型是研究神经精神疾病的重要工具。然而,研究实验室啮齿动物的研究人员面临的一个主要挑战是试图复制诸如精神分裂症等复杂人类疾病的“核心”症状。然而,尽管存在这样的挑战,仍然可以使用旨在再现疾病的一些“内表型”的动物模型。一个例子是惊恐反射的前脉冲抑制(PPI)。 PPI是惊恐可塑性的一种形式,其特征在于当强烈的惊吓刺激(或脉搏)之前有较弱的预刺激(或预脉冲)时,惊吓幅度正常减少。 PPI范例通常用于评估感觉运动门控,并已在包括人类和啮齿动物在内的许多物种中进行了描述。在精神分裂症和其他神经精神疾病患者以及已建立的这些疾病的动物模型中观察到PPI缺陷。因此,PPI范式主要用于探索在这些疾病中发现的感觉运动门控表型的基础和神经生物学机制。因此,有必要建立可靠和可重复的方案来研究小鼠的PPI。

【背景】感觉运动门控是指感觉事件抑制运动反应的能力(Cryan和Reif,2012)。已经在人类和啮齿动物中广泛研究的感觉运动门控的一种形式是惊吓的前脉冲抑制(PPI)。惊恐反射包括全身肌肉系统的不自主收缩,其由充分突然和强烈的刺激引起。具体而言,听觉惊吓反应的特征在于对意外的强烈听觉刺激的夸大的退缩反应。 PPI是惊恐可塑性的一种形式,其特征在于当强烈的惊人刺激(或脉搏)出现在短暂的低强度前刺激(或前脉冲)之前时惊吓幅度正常减少(Graham,1975; Hoffman and Ison ,1980)。 PPI范例通常用于评估感觉运动门控,并且已经在包括人类(Schwarzkopf等人,1993)和小鼠(Carter等人, ,1999; Frankland等人,2004)。在精神分裂症中观察到受损的PPI(Braff等人,2001; Swerdlow等人,2008),以及其他神经精神障碍,包括强迫症(Ahmari ,2012),图雷特综合征(Swerdlow等人,2001),亨廷顿舞蹈病(Swerdlow等人,1995)和双极障碍(Perry等人,2001)。在精神障碍患者中,感觉运动门控缺陷与认知碎裂和精神病症状相关(Kapur,2003)。由于这些缺陷在精神病患者和动物模型中均有发现(Swerdlow and Light,2016),PPI范式主要用于神经精神疾病的研究,并且已被证明是一种有用的工具,用于研究和表征几种(2012),以及探索精神病样行为的机制(Geyer,1999; Ouagazzal等人,2001)。

关键字:惊吓的前脉冲抑制, PPI, 动物模型, 精神分裂症, 感觉运动门控

材料和试剂

  1. 小鼠(从法国Le Genest-Saint-Isle的Janvier Labs购买的C57BL6 / N小鼠)
    注:如果在PPI性能之前应用药理学处理,则试剂将取决于所制备的对照或药物溶液。根据测试前应用的处理方式,动物可以安置在单笼或笼笼中。
  2. 70%乙醇

设备

  1. 具有数字化电子输出的SR-LAB惊奇设备(SR-Lab,San Diego Instruments,目录号:2325-0400)(图1)
  2. 数字声级计(FLIR Systems,Extech,目录号:407730)


    图1. SR-LAB惊吓装置。 :一种。每个实验装置由一个外部照明和通风室组成,用于防止外部噪音或振动干扰实验。 B.在腔室内部,由有机玻璃圆柱体组成的稳定仪固定在平台上。 C.压电式加速度计 - 由红色箭头指示 - 安装在圆柱体下方,可转换动物运动,然后将其数字化,校正并通过计算机和接口组件进行记录。根据所需的设置,扬声器 - 由蓝色箭头指示 - 生成令人吃惊的声音刺激。

软件

  1. SR-Lab分析软件(SR-Lab San Diego Instruments,目录号:2325-0400)

程序

  1. 设计协议
    在这里,我们描述了在实验室中用于研究小鼠PPI反应的实验设计(Busquets-Garcia等,2017),但协议可以通过调整脉冲和前脉冲强度来修改,试验次数,试用间隔时间等,适合探索不同的实验问题。
    1. 以5分钟的适应期开始训练。在适应期间,为了适应动物持有者,惊吓盒和背景噪声,呈现70-dB白噪声的恒定背景噪声。
    2. 然后,本次会议通过90个不同的试验(图2)进行演示: 


      图2.使用描述的实验设计的代表性会话。前五项试验包括5项单独脉冲试验(A),中间80项试验分为10组随机脉冲单独试验,单独前脉冲试验,前脉冲试验组合和无刺激试验试验(B),本次会议结束时连续5次连续脉冲单独试验(C)。预冲强度(73 dB,76 dB和82 dB)高于70 dB背景。

      1. 前五个试验包括五个脉冲单独试验,其中120分贝的白噪声在20毫秒的持续时间内独立出现( ,没有预脉冲)。这些试验有助于习惯和稳定动物惊吓反应。
      2. 随后,提出了十块试验。每个模块包括一个单独的脉冲试验,三个单独的单脉冲试验(70分贝背景上+3,+6或+12单位),三个脉波试验组合和一个无刺激试验(<即时,仅限背景)(表1)。 8个试验在每个区块内以随机顺序呈现,试验间期(ITI)在10至30秒内随机变化,旨在最大限度地减少习惯,以便在试验中惊醒。
        备注:
        1. 随机ITIs的优势在于动物不能预测下一次刺激呈现将发生的时间。例如,注意前脉冲可以增加动物抑制惊吓反应的效力。应避免10秒以下的ITI,以排除肌肉疲劳和肌肉反应不应期造成的影响。
        2. 预脉冲的强度应保持在背景噪声以上的水平,但也足够低,以至于它们本身不会引起显着的惊吓反应,边界大约高于背景水平2-20分贝(例如+3 ,高于70dB背景的+6或+12dB)。重要的是要注意,对前脉冲的敏感度也可能因应变,性别或动物年龄而异。

          表1.在行为协议中使用的不同类型的试验的表示形式



      3. 在第一个区块中,会议以最后一个连续五个脉冲单独试验结束。
        注:刺激的上升时间,持续时间和强度是影响惊恐反射幅度的变量(Graham,1975; Hoffman和Searle,1968)。考虑到动物的品系,年龄,性别和遗传背景,所有参数都应仔细确定,因为不同的品系可能会对惊吓刺激产生不同的反应(Willott等,1995和2003)。&nbsp;

  2. 运行实验
    1. 校准惊吓腔的传感器平台的扬声器和灵敏度(图3)。按照制造商的指导进行有效的声音和移动校准。
      注:声音和运动传感器的校准对于获得有效的测试结果非常重要。因此,必须在每次实验前对这些进行常规校准。&nbsp;


      图3.用于校准惊吓腔的扬声器(A)和运动灵敏度检测器(B)的代表性图像

    2. 创建了您的实验方案(A1,A2)后,您可以使用SR-LAB惊吓仪器软件创建研究数据库,定义实验会议和将要测试的主题。
    3. 运送老鼠到测试室。您可以同时测试多少个小鼠,可以使用多少个小室(软件最多可容纳16个小室)。关于住房条件,请参阅注释1。在开始实验之前,请小心不要给小鼠施压,并且为此,至少24小时之前不要对家笼( eg ,铺垫)进行更改本实验。测试室内的照明和噪声水平应该与住房中的水平相当,以尽量减少环境对行为结果的影响。
      。管状动物外壳可以最大限度地减少压力,同时动物仍然集中在传感器上,以获得稳定可靠的结果。
    4. 在每个实验环节中,将鼠标放在测试室内的圆筒内,并关上门。
    5. 根据上述实验设计运行实验会话。会话将在协议结束时自动停止(约35-40分钟后)。
    6. 在实验结束时将每只小鼠从小室中取出并放回家中的笼子中。
      。在使用下一个动物之前,用水清洁动物支架和小室并让其干燥。

    7. 在屏幕上选择下一个会话并对所有动物重复该过程。
    8. 在所有会议结束时,用70%乙醇清洁圆柱体和腔室,并使其干燥。保存获得的数据,以便对声学惊恐和声学预脉冲抑制反应进行后续详细分析。

数据分析

可以分别分析在五个连续脉搏单独试验的第一个和最后一个块上获得的反应性评分,以评估惊吓习惯。从其余80个试验中获得的数据根据其与不同行为构建的相关性分为三个不同的子集。
首先,从脉冲单独试验中获得的反应性评分(不包括五个连续脉冲单独试验的第一个和最后一个试验)评估惊恐反应性(S)。通过软件分析120毫秒脉冲呈现后的100毫秒响应窗口,并使用最大响应峰值幅度来确定声学惊跳反应作为动物对惊吓脉冲反应的对照指数(图4A)。
其次,脉冲前脉冲试验(PPiS)相对于脉冲单独/惊吓试验(S)的反应性用于评估前脉冲抑制(PPI)(图4B)。使用以下公式计算每种声学预脉冲试验类型的预脉冲抑制量:百分比分数:&nbsp;
%PPI = 100×(S-PPiS)/ S(图4B)
第三,为了测量前脉冲引发的反应性(PP),包括来自单独前脉冲试验的数据。
典型实验的结果应该显示随着前脉冲强度增加PPI水平增加,而变异性相对较低(图4C)。使用相同的菌株,性别和药物剂量时,特定药物对PPI的良好效果应该是可重复的。
惊愕反应数据通常表现出比PPI反应数据更多的可变性和可靠性。


图4.典型PPI实验的预期结果。 :一种。在出现单脉冲和脉冲前脉冲试验后,软件输出100毫秒的响应窗口。箭头指示最大响应峰值幅度,其用于确定声学惊跳反应和最大响应时间作为动物对刺激的反应性的指标。 B.预脉冲 - 脉冲试验(PPiS)相对于单脉冲/惊吓试验(S)的反应性被用来评估前脉冲抑制(PPI)。 C.本研究所生产的具有BL6N背景的幼稚野生型小鼠显示了前脉冲抑制百分比(%PPI)。正如预期的那样,PPI水平随前脉冲强度增加(高于背景)而增加,并且变异性较低。

笔记

  1. 在我们研究所进行的所有PPI实验中,将动物饲养在每笼4-5只动物的集合笼中。尽管社会隔离可以改变大鼠的惊吓变量,但隔离对小鼠惊吓措施的影响尚未得到很好的表征。因此,在惊吓实验中使用已分离和单独饲养的小鼠并不理想。

  2. 在早上(从上午9点到下午2点)进行实验方案的所有阶段。
  3. 在小鼠中已经描述了惊恐反射和PPI的应变差异(Bullock等人,1997; Tarantino等人,2000; Willott等人,2003)。该协议在几个鼠标线上进行了测试。我们发现C57 / BL6N小鼠和我们研究所生产的BL6N背景的野生型动物之间有类似的结果,尽管每批小鼠的反应都有轻微的变化。
  4. 性别:雄性和雌性小鼠都已经用于惊吓反应性和PPI的方案中。如果在同一个实验日使用,为了避免对行为结果的影响,必须仔细地执行检测室的清洁过程。
  5. 年龄:年龄是听觉惊吓和PPI测量的重要变量。已经报道了包括C57BL / 6在内的许多近交系的年龄相关的听力损失,其可以改变惊吓反应性和PPI水平(Willott等人,1995)。此外,研究表明,与成年动物相比,早期青春期小鼠可以显示改变的惊吓反应性和PPI(Pietropaolo and Crusio,2009)。
  6. 不同的致精神病药物( eg ,大麻素,苯丙胺等)可作为阳性对照( ie ,PPI改变) br />

食谱

目前的协议不包含任何配方。如果使用任何致精神病药物或其他药物作为阳性对照,则应按照相关规程和制造商的指导原则进行准备。

致谢

我们感谢Delphine Gonzales,Nathalie Aubailly以及Neurocentre Magendie动物设施的所有人员对鼠标的关怀。我们也感谢克里斯托弗·史蒂文斯对本手稿的批判性阅读以及Susanna Pietropaolo博士在我们实验室中设立PPI时的建议。该行为协议最初用于布斯克茨 - 加西亚等人2017年。这项工作得到了INSERM(对GM),EU-FP7(PAINCAGE,HEALTH-603191对GM,FP7-PEOPLE- 2013-IEF-623638至AB-G。,欧洲研究委员会(Endofood,ERC-2010-StG-260515; CannaPreg,ERC-2014-PoC-640923,GM),Fondation pour la Recherche Medicale(DRM20101220445和DPP20151033974) GM),人类前沿科学计划(通用汽车公司),阿基坦大区(通用汽车公司),法国国家通用汽车公司-G,NeuroNutriSens ANR-13-BSV4-0006-02转GM和ORUPS ANR-16-CE37-0010转GM)。作者宣称他们没有利益冲突。

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引用:Ioannidou, C., Marsicano, G. and Busquets-Garcia, A. (2018). Assessing Prepulse Inhibition of Startle in Mice. Bio-protocol 8(7): e2789. DOI: 10.21769/BioProtoc.2789.
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