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Aug 2016
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Measuring Behavioral Individuality in the Acoustic Startle Behavior in Zebrafish
测量斑马鱼声音惊恐行为中的行为个性   

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Abstract

The objective of this protocol is to provide a detailed description for the construction and use of a behavioral apparatus, the zBox, for high-throughput behavioral measurements in larval zebrafish (Danio rerio). The zBox is used to measure behavior in multiple individuals simultaneously. Individual fish are housed in wells of multi-well plates and receive acoustic/vibration stimuli with simultaneous recording of behavior. Automated analysis of behavioral movies is performed with MATLAB scripts. This protocol was adapted from two of our previously published papers (Levitz et al., 2013; Pantoja et al., 2016). The zBox provides an easy to setup flexible platform for behavioral experiments in zebrafish larvae.

Keywords: Zebrafish (斑马鱼), Behavior (行为), Individuality (个性), Neuromodulation (神经调节), High-throughput (高通量), Startle (惊恐), Larvae (幼虫)

Background

Behavioral differences between individuals in a population are ubiquitous and likely play a role in adaptation to varying selective pressures during evolution. However, variation in behavior among individuals is often ignored when the quantitation of the behavior of groups is presented as means and associated dispersions. In this protocol, we describe an approach to characterize individuality in the habituation of the acoustic startle behavior at the population level in zebrafish (Danio rerio) larvae. Our approach and set-up can be easily adapted to studies of individuality in other zebrafish larval behaviors.

Materials and Reagents

  1. Petri dishes
  2. Pipette tip  
  3. 48-well multi-well plate
  4. Zebrafish embryos
  5. Instant ocean sea salt mix (Spectrum Brands, catalog number: SS15-10 )
  6. DI H2O
  7. E3 medium (see Recipes)

Equipment

  1. P1000 PIPETMAN pipettor
  2. Stir plate
  3. zBox parts list (Table 1)

    Table 1. ZBox parts list
    Manufacturer/Item description
    Part number
    Quantity
    Newegg - Computer


    Minimum requirements: PC, 8 GB RAM, 2 GHz processor

    1
    PCIe Firewire 800 card
    9SIA24G28M5974
    1
    National Instruments (DAQ)


    NI PCI-6229
    779068-01
    1
    RC68-68 Cable (1 m)
    192061-01
    2
    CB-68LP - Unshielded
    777145-01
    2
    McMaster Carr (Structural external)


    80/20 1”x1” framing 96”
    47065T123
    4
    80/20 corners
    47065T177
    20
    80/20 nuts 80x
    47065T147
    12
    Black PVC, 48”x48”x.
    84775K942
    1
    236” (back)Black PVC 36”x48”x.236
    84775K742
    3
    sides and top Super Adhesive Hook
    94985K614
    10ft
    Loop
    9489K874
    10ft
    Loctite Black Silicone sealant
    74945A81
    1
    Black gaffer’s tape
    7612A94
    1
    Right angle 80/20
    47065T223
    2
    80/20 fastener
    47065T139
    2
    Acrylic cement
    7528A13
    1
    PTFE tape
    4591K11
    1
    General Purpose Epoxy
    Permatex 84101
    1
    Nylon 6/6 Thumb Screw
    96295A336
    1
    Thorlabs (Structural internal)


    Breadboard 24”x36”
    MB2436
    1
    XT66 Rail
    XT66-500
    1
    XT66 Dovetail
    XT66DP-500
    1
    XT66 Dovetail clamp 40 mm
    XT66C2
    2
    XT66 right angle cross piece
    XT66CB
    1
    XT66 base plate
    XT66P1
    1
    XT66 pivot platform
    XT66RC
    2
    Plate holder
    FP01
    2
    10” mounting post
    P10
    1
    Pedestal Base adapter
    PB4
    1
     Clamping fork
    PF175
    1
    Post mounting clamp
    C1501
    1
    Blackout fabric
    BK5
    1
    Right angle plate
    AP90
    1
    Edmund Optics


    101 x 127 mm, 45 Degree AOI, Hot Mirror
    43-958
    2
    Pike F-245 2/3” CCD camera
    59-221
    1
    Pike Tripod adapter
    59-227

    Plastic IR filter
    43-949
    1
    Navitar


    50MM F/2.8 TELECENTRIC LENS
    TC-5028
    1
    Unibrain (1394store.com)


    1m (15 ft) IEEE-1394b 9p to 9p screw lock cable

    1
    Digi-Key Electronics


    100Kohm resistor
    CMF100KHFCT-ND
    4
    2-pin recipient
    A99613-ND
    10
    2-pin socket
    A99620-ND
    20
    2-pin header
    A30770-ND
    10
    Yellow LEDs
    C503B-AAN-CY0B0251-ND
    48
    IR LEDs
    475-2919-ND
    48
    White LEDs
    C503C-WAS-CBADA151-ND
    48
     Blue LEDs
    C503B-BAN-CY0C0461-ND
    48
    1ohm resistor
    P1.0W-2BK-ND
    3
    270 ohm resistor
    PPC270W-1CT-ND
    16
    390 ohm resistor
    PPC390W-1CT-ND
    6
    Diode
    1N914B-ND
    2
    12 V power supply
    EPS377-ND
    1
    24 V power supply
    EPS357-ND
    1
    LEDsupply


    BuckPuck DC LED Drivers
    0302x-D-x-xxxx
    1
    Machine shop (plastics)


    Custom plastic fabrication

    1
    Whatman (GE) plates


    Multi-well plate of desired well number


    Visaton (speakers)
    8006
    2
    Pyle Audio (amplifier)
    PyleHome PCA1
    1
    60 Watt incandescent bulb

    1
    Simple designs home (lamp)
    LD1003-WHT
    1

    Notes: Assembly of zBox (see Figures 1-7):
    1. Use epoxy to adhere the two side walls into the grooves on the back panel (Figure 2).
    2. The screw holes in the LED platforms need to be drilled for 4-40 threads (Figure 3).
    3. Use the plastic thumbscrews to mount the 2 LED platforms (one for the imaging IR LEDs and one for LEDs for use in optogenetic experiments) to the slots in the side walls (Figure 4).
    4. LED arrays can be wired in 8 x 6 or 12 x 10 arrays depending on the desired amount of light. Due to perspective, the IR imaging LEDs are best arranged so that the arrays extend to the sides of the LED platform. Thus, given the distance from the plate, the IR LEDs can evenly light the extent of the multiwell plate.
    5. The BuckPack LED driver can be wired to power the LED arrays and be switched using TTL pulses from the DAQ
    6. The multiwell plate platform should be able to slide into the grooves on the side walls.
    7. Screw speakers to side of multiwell platform (Figures 4-6).
    8. Use diffuser paper to assure that IR light is evenly distributed throughout wells.
    9. Mount camera to P10 posts. XT66 rails should be constructed to hold IR mirrors (Figure 7).


      Figure 1. zBox plans


      Figure 2. Slide side walls into back panel grooves and epoxy in place


      Figure 3. Use thumb screws to mount LED platforms into side wall slots


      Figure 4. Insert multi-well plate platform into top grooves of side walls


      Figure 5. Detailed view of eaker mounting


      Figure 6. Completed zBox


      Figure 7. Camera mounting

Software

  1. Matlab2014a or later with Imaging Processing and Data Acquisition toolboxes installed

Procedure

  1. Zebrafish embryos are raised in Petri dishes at low densities (~2 fish per ml) at 28.5 °C in E3 medium.
  2. Larvae without inflated bladders and that do not respond to light taps on the dish on 4 dpf are removed.
  3. Larvae are individually pipetted in 300 µl of E3 medium with a P1000 PIPETMAN pipettor, with the pipette tip cut to prevent damage to the larvae, and individually transferred from Petri dishes to the wells of a 48-well multi-well plate.
  4. To prevent experimenter selection bias zebrafish larvae are randomly chosen for transfer to the 48-well plate.
  5. 48-well microplate is transferred to a plexiglass box (Figures 4-7) kept inside an isolated dark chamber. Interior of chamber illuminated with constant diffuse white light is supplied by a 60 W bulb.
  6. The behavioral apparatus is housed in a climate controlled environment at 22 °C.
  7. If exposed to different treatments, larvae from different groups are tested simultaneously in the same micro-well plate.
  8. Larvae used in single-day experiments are 6 dpf, whereas those used in the three-day long experiments are 5-7 dpf. For the three-day experiments, the larvae are kept in the microplates with 1,300 µl E3 overnight at 28.5 °C between measurements. The medium is refreshed the next day and then readjusted to 300 µl for the next habituation trial.
  9. Behavioral activity within the microplate is recorded with a CCD camera, which is backlit by an array of infrared LEDs.
  10. The behavioral assay consists of 15 min of acclimation to the high-throughput apparatus, followed by 10 min of spontaneous activity recording, and then the habituation protocol.
  11. Sound stimuli are administered by two speakers mounted to the same platform as the microplate. The speakers are powered by a 15 W amplifier and delivered 900 Hz square waves of ~3 msec duration. Stimuli are delivered via a Native Instruments PCI-6229 DAQ controlled by MATLAB.
  12. Habituation protocol consists of 10 stimuli at 90 sec ISI (0.4 V, ~95 dB) followed by 100 stimuli at 5 sec ISI (0.4 V, ~95 dB).
  13. For each behavior experiment, data is analyzed from randomly sampled larval zebrafish derived from multiple clutches obtained on different days. We empirically determined that measuring behavior in approximately 100 larvae is sufficient to quantify behavioral variation at the population level.

Data analysis

  1. Behavioral analysis is performed with MATLAB scripts (supplemental materials and Figure 8). README.txt file contains detailed information on how to run scripts. Basic skills in MATLAB programming are recommended.


    Figure 8. Screenshot of MATLAB user interface

  2. Run habituationProtocol.m script. Escapes are detected using changes in pixel values between frames before and after sound stimulus presentation. An escape is counted if the difference of the integrated pixel values from the two frames immediately following the stimulus is statistically higher (P < 0.01, 2-sample t-test) than the distribution of pixel-change values taken from the non-escape portion spontaneous activity taken across all 120 movies (720 frames). The accuracy of this method was verified by visual inspection of the movies (Figure 9).


    Figure 9. Representative image of 48-well plate during sound-induced escape

  3. For analysis of behavioral movies run the analyzeEscapes.m script. We utilize the habituation index (HI) as a metric to describe acoustic startle habituation in individual larvae. The HI is defined as the difference in escape probability to the last 40 high-level stimuli under habituating conditions (10 sec inter-stimulus interval [ISI]) with the first 10 high-level stimuli under non-habituating conditions (90 sec ISI), divided by the probability of escape to the last 40 stimuli (HI =[Plast40 - Pfirst10]/Plast40). Only highly-responsive fish, probability of response 90% or more to the initial 10 high-level stimuli, are used in calculating the HI (Figure 10).


    Figure 10. Graphic example of acoustic startle repose quantification. A. Frequency distribution of probability of escape values during the first 10 stimuli (ISI = 90 sec, n = 230) of habituation series. B. Frequency distribution of HIs for highly responsive fish in (A) (ISI = 10 sec, n = 196). 

  4. To determine if differences in HI between experimental groups are significant, we use the Mann-Whitney U-Test, which is included in the script zBoxStat.m. The HIscores.mat file in the supplemental materials provides an example of data to analyze.

Recipes

  1. E3 embryo medium
    Note: It is not necessary to use filtered or sterile solutions.
    1. Making 50x stock solution:
      15 g instant ocean sea salt mix
      1 L DI H2O
      Mix on stir plate for ~5 min
    2. Working solution:
      200 ml 50x E3
      10 L DI H2O
      2 drops (~0.1 ml) of methylene blue

Acknowledgments

This work supported by the National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (PN2EY018241) and the Human Frontier Science Program (RGP0013/2010).
The zBox was initially developed by Dr. David Schoppik in Dr. Alex Schier’s laboratory. The work and experimental approach described here have been adapted from previous research published in Nature Neuroscience (Levitz et al., 2013) and Neuron (Pantoja et al., 2016). Further information about uses for the zBox are reported in a study that investigated the role of neuropeptides in the partition of arousal behaviors in zebrafish (Woods et al., 2014). In addition, work by Zhou et al. can be used as a complementary set of methods for the utilization of this apparatus (Zhou et al., 2014).

References

  1. Levitz, J., Pantoja, C., Gaub, B., Janovjak, H., Reiner, A., Hoagland, A., Schoppik, D., Kane, B., Stawski, P., Schier, A. F., Trauner, D. and Isacoff, E. Y. (2013). Optical control of metabotropic glutamate receptors. Nat Neurosci 16(4): 507-516.
  2. Pantoja, C., Hoagland, A., Carroll, E. C., Karalis, V., Conner, A. and Isacoff, E. Y. (2016). Neuromodulatory regulation of behavioral individuality in zebrafish. Neuron 91(3): 587-601.
  3. Woods, I. G., Schoppik, D., Shi, V. J., Zimmerman, S., Coleman, H. A., Greenwood, J., Soucy, E. R. and Schier, A. F. (2014). Neuropeptidergic signaling partitions arousal behaviors in zebrafish. J Neurosci 34(9): 3142-3160.
  4. Zhou, Y., Cattley, R. T., Cario, C. L., Bai, Q. and Burton, E. A. (2014). Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications. Nat Protoc 9(7): 1533-1548.

简介

该协议的目的是提供一个行为装置的构造和使用的详细描述,zBox,用于幼虫斑马鱼(Danio rerio)中的高通量行为测量。 zBox用于同时测量多个人的行为。 个体鱼被放置在多孔板的孔中并且接收声/振动刺激同时记录行为。 使用MATLAB脚本对行为电影进行自动分析。 这个协议是从我们之前发表的两篇文章(Levitz et al。,2013; Pantoja et al。,2016)改编而成。 zBox为斑马鱼幼虫的行为实验提供了一个易于设置的灵活平台。
【背景】人口中的个体之间的行为差异是普遍存在的,并且可能在适应进化过程中不同选择压力时发挥作用。 然而,当群体的行为的定量被表示为手段和相关的分散体时,往往忽略个体之间行为的变化。 在这个协议中,我们描述了一种在斑马鱼(Danio rerio)幼虫的群体级别的声学惊吓行为习惯中表征个性的方法。 我们的方法和设置可以轻松适应其他斑马鱼幼虫行为的个性研究。

关键字:斑马鱼, 行为, 个性, 神经调节, 高通量, 惊恐, 幼虫

材料和试剂

  1. 培养皿
  2. 移液器尖端
  3. 48孔多孔板
  4. 斑马鱼胚胎
  5. 即时海洋海盐混合(Spectrum Brands,目录号:SS15-10)
  6. DI H 2 O
  7. E3介质(见配方)

设备

  1. P1000 PIPETMAN移液器
  2. 搅拌板
  3. zBox零件清单(表1)

    表1. ZBox零件清单
    制造商/物品描述
    零件号
    数量
    Newegg - 电脑


    最低要求:PC,8 GB RAM,2 GHz处理器

    1
    PCIe Firewire 800卡
    9SIA24G28M5974
    1
    国家仪器(DAQ)


    NI PCI-6229
    779068-01
    1
    RC68-68 Cable (1 m)
    192061-01
    2
    CB-68LP - 非屏蔽
    777145-01
    2
    麦克马斯特·卡尔 (结构外部)


    80/20 1"x1" 框架 96"
    47065T123
    4
    80/20 角落
    47065T177
    20
    80/20 坚果 80x
    47065T147
    12
    黑色PVC, 48"x48"x.
    84775K942
    1
    236" (背)黑色PVC 36"x48"x.236
    84775K742
    3
    侧面和顶级超级粘合钩
    94985K614
    10ft
    循环
    9489K874
    10ft
    乐泰黑硅胶密封剂
    74945A81
    1
    黑色的黑色胶带
    7612A94
    1
    直角 80/20
    47065T223
    2
    80/20 紧固件
    47065T139
    2
    亚克力水泥
    7528A13
    1
    PTFE胶带
    4591K11
    1
    通用环氧树脂
    Permatex 84101
    1
    Nylon 6/6 Thumb Screw
    96295A336
    1
    Thorlabs (结构内部)


    面包板 24"x36"
    MB2436
    1
    XT66 轨
    XT66-500
    1
    XT66 燕尾
    XT66DP-500
    1
    XT66 燕尾钳 40 mm
    XT66C2
    2
    XT66 直角横梁
    XT66CB
    1
    XT66 底盘
    XT66P1
    1
    XT66 枢纽平台
    XT66RC
    2
    Plate holder
    FP01
    2
    10" 安装柱
    P10
    1
    基座适配器
    PB4
    1
    夹子叉
    PF175
    1
    后安装夹
    C1501
    1
    遮光布
    BK5
    1
    直角板
    AP90
    1
    Edmund Optics


    101 x 127毫米,45度AOI,热镜
    43-958
    2
    派克F-245 2/3"CCD相机
    59-221
    1
    派克三脚架适配器
    59-227

    塑料红外滤光片
    43-949
    1
    Na vitar


    50MM F/2.8 TELECENTRIC LENS
    TC-5028
    1
    Unibrain (1394store.com)


    1米(15英尺)IEEE-1394b 9p至9p螺丝锁电缆

    1
    Digi-Key Electronics


    100KΩ电阻器
    CMF100KHFCT-ND
    4
    2针收件人
    A99613-ND
    10
    2针插座
    A99620-ND
    20
    2针头
    A30770-ND
    10
    黄色LED
    C503B-AAN-CY0B0251-ND
    48
    红外LED
    475-2919-ND
    48
    白色LED
    C503C-WAS-CBADA151-ND
    48
     蓝色LED
    C503B-BAN-CY0C0461-ND
    48
    1欧姆电阻
    P1.0W-2BK-ND
    3
    270欧姆电阻器
    PPC270W-1CT-ND
    16
    390欧姆电阻器
    PPC390W-1CT-ND
    6
    二极管
    1N914B-ND
    2
    12 V电源
    EPS377-ND
    1
    24 V电源
    EPS357-ND
    1
    LED供应


    BuckPuck DC LED驱动器
    0302x-D-x-xxxx
    1
    机械车间(塑料)


    定制塑料制造

    1
    Whatm(GE)板


    所需孔数多孔板


    Visaton (扬声器)
    8006
    2
    Pyle奥迪 o (放大器)
    PyleHome PCA1
    1
    60瓦白炽灯泡

    1
    简单设计家居(灯)
    LD1003-WHT
    1

    注意:组装zBox(见图1-7):
    1. 使用环氧树脂将两个侧壁粘合到后面板上的凹槽中(图2)。
    2. 在LED平台上的螺丝孔需要钻4-40线(图3)。
    3. 使用塑胶指旋螺丝将两个LED平台(一个用于成像红外LED,另一个用于光生代实验中的LED)安装到侧壁上的插槽(图4)。/em>
    4. 根据所需的光量,LED阵列可以连接在8 x 6或12 x 10阵列中。由于透视,最佳配置成像LED,使得阵列延伸到LED平台的侧面。因此,考虑到板的距离,IR LED可以均匀地点亮多孔板的厚度。
    5. 可以将BuckPack LED驱动器连接起来为LED阵列供电,并使用DAQ
      中的TTL脉冲进行切换
    6. 多孔板平台应能够滑入侧壁上的凹槽。
    7. 将扬声器拧到多井平台的侧面(图4-6)。
    8. 使用扩散纸确保红外光均匀分布在整个井中。
    9. 将相机安装到P10帖子。 XT66导轨应构造成夹持红外线镜(图7)。


      图1. zB ox 计划


    图2.在 位置
    中将侧壁滑入后面板凹槽和环氧树脂

    图3.使用拇指螺钉将LED平台安装到侧壁插槽中


    图4.将多孔板 pla tform 插入侧墙顶部


    F  igure 5. sp eaker 安装
    的详细视图

    F

    图7. Cam 时代 安装

软件

  1. Matlab2014a或更高版本安装了成像处理和数据采集工具箱

程序

  1. 斑马鱼在E3培养基中以28.5℃的低密度(〜2只鱼/毫升)在培养皿中培养。
  2. 没有充气气囊的幼虫和4 dpf上没有对盘上的小水龙头的反应被删除。
  3. 幼虫通过P1000 PIPETMAN移液器在300μlE3培养基中单独移液,移液管尖端切割以防止对幼虫的损伤,并单独从培养皿转移到48孔多孔板的孔中。
  4. 为了防止实验者选择偏倚斑马鱼幼虫随机选择转移到48孔板
  5. 将48孔微板转移到保持在孤立的暗室内的有机玻璃盒(图4-7)。用恒定漫反射白光照亮的室内部由60W灯泡提供。
  6. 行为装置安置在22°C的气候控制环境中。
  7. 如果暴露于不同的处理方法,则在同一微孔板中同时测试来自不同组的幼虫
  8. 在单天实验中使用的幼虫为6 dpf,而在三天长实验中使用的幼虫为5-7 dpf。对于为期三天的实验,将测量之间的幼虫在28.5℃下在1,300μlE3保持在微孔板中过夜。该培养基在第二天更新,然后重新调整至300μl进行下一次习惯性试验。
  9. 微量板中的行为活动用CCD相机记录,CCD相机由红外LED阵列背光
  10. 行为测定包括15分钟适应高通量仪器,随后10分钟的自发活动记录,然后是习惯方案。
  11. 声音刺激由安装在与微孔板相同的平台上的两个扬声器施用。扬声器由15 W放大器供电,并提供约3毫秒持续时间的900 Hz方波。刺激通过由MATLAB控制的Native Instruments PCI-6229 DAQ传递。
  12. 习惯协议由90秒ISI(0.4 V,〜95 dB)的10次刺激,5秒ISI(0.4 V,〜95 dB)的100次刺激而成。
  13. 对于每个行为实验,从随机取样的幼虫斑马鱼分析来自多个离合器在不同日期获得的数据。我们根据经验确定,约100只幼虫的测量行为足以量化群体层面的行为变化。

数据分析

  1. 使用MATLAB脚本执行行为分析(补充材料和图8)。 README.txt文件包含有关如何运行脚本的详细信息。推荐MATLAB编程的基本技能

    图8. MATLAB用户界面的屏幕截图

  2. 运行habituationProtocol.m脚本。使用声音刺激呈现之前和之后的帧之间的像素值的改变来检测逃逸。如果来自紧跟在刺激之后的两个帧的积分像素值的差异在统计学上较高(


    <0.01,2-sample -test),则计数逃逸比从所有120部电影(720帧)拍摄的非逃避部分自发活动取得的像素变化值的分布。这种方法的准确性通过电影的目视检查来验证(图9)

    图9.代表 im 年龄 48孔板在声音引发的逃生

  3. 为了分析行为电影,运行analyzeEscapes.m脚本。我们利用习惯指数(HI)作为衡量单个幼虫的声学惊奇习惯的度量标准。 HI被定义为在习惯条件(10秒刺激间隔[ISI])前的最后40个高级刺激与非习惯条件下的前10个高级刺激(90秒ISI)的逃避概率的差异, ,除以最后40次刺激的逸出概率(HI =最后40次/秒)。只有高度响应的鱼,在初始10次高水平刺激下90%以上的响应概率被用于计算HI(图10)。


    ure 10。声学惊吓定量的图示例 A.习惯系列的前10个刺激(ISI = 90秒,n = 230)中逃脱值概率的频率分布。 B.(A)(ISI = 10秒,n = 196)中高反应性鱼的HIs频率分布。 

  4. 为了确定实验组之间的差异是否显着,我们使用Mann-Whitney U-Test,它包含在脚本zBoxStat.m中。 补充材料中的HIscores.mat文件提供了要分析的数据的示例。

食谱

  1. E3胚胎培养基
    1. 制造50x库存解决方案:
      15克即时海洋盐水混合物
      1 L DI H 2 O
      混合搅拌板〜5分钟
    2. 工作解决方案:
      200毫升50x E3
      10 L DI H 2 O O
      2滴(约0.1ml)亚甲蓝

致谢

这项工作由国立卫生研究院生物功能光学控制纳米医学发展中心(PN2EY018241)和人类前沿科学计划(RGP0013/2010)支持。
zBox最初是由Dr. David Schoppik博士在Alex Schier博士的实验室开发的。这里描述的工作和实验方法已经从以前在Nature Neuroscience发表的研究中得到改编(Levitz >,2013)和Neuron(Pantoja ,2016)。关于zBox的用途的进一步信息在一项研究中被报道,该研究调查了神经肽在斑马鱼唤醒行为分配中的作用(Woods em> ,2014)。另外,周恩来 可以用作这种装置的使用方法的补充(Zhou 。 ,2014)。

参考文献

  1. Levitz,J.,Pantoja,C.,Gaub,B.,Janovjak,H.,Reiner,A.,Hoagland,A.,Schoppik,D.,Kane,B.,Stawski,P.,Schier,AF,Trauner ,D.和Isacoff,EY(2013)。  光学代谢型谷氨酸受体的控制。 16(4):507-516。
  2. Pantoja,C.,Hoagland,A.,Carroll,EC,Karalis,V.,Conner,A.and Isacoff,EY(2016)。  斑马鱼行为个性的神经调节调节。 91(3 ):587-601。
  3. Woods,IG,Schoppik,D.,Shi,VJ,Zimmerman,S.,Coleman,HA,Greenwood,J.,Soucy,ER and Schier,AF(2014)。  神经肽能信号分布在斑马鱼中的唤醒行为。 Neurosci 34(9):3142-3160。
  4. Zhou,Y.,Cattley,RT,Cario,CL,Bai,Q. and Burton,EA(2014)。  使用开源MATLAB应用程序在多孔板中定量幼虫斑马鱼运动功能。 em> Protoc 9(7):1533-1548。
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引用:Pantoja, C., Hoagland, A., Carroll, E., Schoppik, D. and Isacoff, E. Y. (2017). Measuring Behavioral Individuality in the Acoustic Startle Behavior in Zebrafish. Bio-protocol 7(7): e2200. DOI: 10.21769/BioProtoc.2200.
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