搜索

A Fluorescent Dye Method Suitable for Visualization of One or More Rat Whiskers
适合观察一根或多根大鼠触须的荧光染料方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Jun 2017

Abstract

Visualization and tracking of the facial whiskers is critical to many studies of rodent behavior. High-speed videography is the most robust methodology for characterizing whisker kinematics, but whisker visualization is challenging due to the low contrast of the whisker against its background. Recently, we showed that fluorescent dye(s) can be applied to enhance visualization and tracking of whisker(s) (Rigosa et al., 2017), and this protocol provides additional details on the technique.

Keywords: Whisker (触须), Tracking (跟踪), Fluorescence (荧光), Dye (染料), Tactile perception (触觉感知), Barrel cortex (体觉皮层)

Background

Over the last 10 years, neuroscientists have begun to focus on sensorimotor processing in behaving rodents. A critical step is to be able to quantify the signals that enter the nervous system from the outside world. For whisker-mediated touch, visualization and tracking of the facial whiskers are required in order to characterize the sensory input. Though many approaches have been employed, only high-speed videography has proven adequate for measuring whisker motion and deformation during interaction with an object. However, whisker visualization and tracking is challenging for multiple reasons, primary among them the low contrast of the whisker against its background. This protocol details a technique for increasing contrast by rendering whiskers fluorescent.

Materials and Reagents

  1. Latex gloves (DOCzero)
  2. Face mask (Benefis)
  3. Eppendorf PCR tube (2 ml)
  4. Cotton pads or gauzes (Megapharma)
  5. Cotton swabs (DaklaPack)
  6. Laboratory film (Parafilm)
  7. Borosilicate glass capillary inside Ø 0.58 mm (Hilgenberg) of 5 cm length
  8. Syringe (Nipro Europe N.V.)
  9. Standard aluminum foil
  10. Animals: Wistar rats (450-550 g) from Envigo-Harlan
    Notes:
    1. Wistar rats (450-550 g) individually housed or with one cage mate. 14/10 light/dark cycle.
    2. In principle, the method can be applied to any whiskered animal, but bleaching darker whiskers could require longer time and could produce less satisfactory results, affecting the quality of the fluorescence.
  11. Epigel (Ceva, Italy)
  12. Hydrogen peroxide (diluted solution 3%)
  13. Blue bleaching powder (DECO+)
  14. Distilled water (Marco Viti Farmaceutici)
  15. Kitchen vinegar
  16. Domitor (Medetomidine hydrochloride)
  17. Antisedan (Atipamezole hydrochloride)

Equipment

  1. Cage for rat (Tecniplast, catalog number: 1291H )
    Note: Rat is isolated in cage.
  2. Animal scale
  3. Pulse-oximeter
  4. 1 x scissor (Swiss Scissors, 9 cm) (World Precision Instruments, catalog number: 504613 )
  5. 1 x tweezer (#7, World Precision Instruments, catalog number: 504504 )
  6. Tissue and heating pad for body thermoregulation (Panlab s.l.)
  7. Fluorescent dye
    Note: We used and tested hair semi-permanent fluo rinse (‘UV Red’ or ‘UV Green’, Star Gazer).
  8. Excitation light
    Note: We used a custom made lamp with 7 bulbs: ILH-OO01-DEBL-SC211-WIR200, Wavelength 455 nm, Flux @700 mA 1,400 mW, Radiance angle ± 60°; concentrator lens FA11205_Tina-D-OSL FWHM angle ± 6°. The light was custom made to yield uniform illumination from different directions: this improves the homogeneity of the light emitted by the fluorescent dye and then improves the image quality. Each bulb was assembled using a commercial LED coupled with a compound parabolic concentrator (CPC), also called a ‘Winston cone’, in order to collect and project into the arena all the available light.
  9. Black neoprene (RS Components, catalog number: 733-6753 )
    Note: We covered the experimental setup with black neoprene because a black, non-reflective, background helps in increasing the overall contrast of the image.
  10. Safety goggles (Univet, catalog number: 5X7.03.00.04 )
    Note: Absorbance > 4 for Wavelength < 510 nm. Certification 0068/ETI-DPI/070-2009 Rev.2.
  11. High-speed camera and lens
    Note: Camera and lens depend on the experimenter’s choice. We used the camera CamRecord 450 (Optronis) combined with Computar TV lens–50 mm 1:1.3 lens. Each investigator must choose the video device according to frame-rate required by the experiment: for high resolution rat whisker kinematics 1,000 fps is an acceptable frame rate (Fassihi et al., 2014 and 2017). Attention has to be paid to the camera frame rate: the higher the rate, the stronger the light should be. For a frame rate of 1,000 fps, the duration of a single frame is 1 msec, giving the maximum exposure time. We used the entire frame duration, 1 msec, as exposure time. Lower exposure times typically generate better still images but require additional light: a trade-off has to be optimized according to the specific requirements of each individual experimental setup. In case the experimenter wants to reduce the intensity of the stimulus light, or in case a different (e.g., less bright) camera lens is used, some cameras allow setting of the internal gain of the sensor: this improves the sensitivity of the camera sensor at the cost of getting more salt-pepper noise in the resulting images.
  12. Long-pass filter
    Note: Long-pass filter is needed to visualize one or multiple whiskers stained with different dyes. We tested FEL0500 Long-pass filter cut-on wavelength 500 nm (Thorlabs) to visualize multiple fluorescent dyes and red/orange plexiglass to visualize only the red fluorescent dye. The filter choice should fit the lens dimension, while plexiglass, which is sold in sheets, can be cut to suit custom design, then it can be adapted to the lens dimension. As for camera and lens, the filter has to be chosen to match the dye color and suppress the color of the illumination source. This prevents the camera sensor from being bloomed by the source, while receiving the lower-intensity light emitted by the fluorescent dye.

Procedure

  1. Dye application
    Notes:
    1. No sterile techniques are required for this protocol.
    2. Wear gloves and mask during the entire procedure.
    3. Prepare in advance a fresh hair discoloration preparation:
      1. Considering that it has an unpleasant and strong smell, you might consider using a hood.
      2. Fill at least one PCR tube with bleaching powder and the hydrogen peroxide, close the tube and mix it.
      3. A viscous preparation must be obtained to avoid it dripping off the whisker.
    4. Prepare in advance cotton pads (or gauzes) soaked in distilled water and vinegar.
    5. Wear protective goggles before switching the excitation source on.
    Before starting this protocol, the rat should be sedated (see Procedure B: ‘Animal sedation’: follow the ‘pre-anesthesia’ and ‘anesthesia’ procedures).
    1. Place the rat on the heating pad and set the body temperature to 37 °C.
    2. Put the Epigel on the rat’s eyes to protect them from light and dryness.
    3. Apply the discoloration preparation (Figure 1A) on all whiskers using cotton swabs. Since its smell is quite strong, avoid placing it close to the rat’s nose, because breathing it can reverse the effect of Domitor.
    4. Wait between 30 min and 1 h to bleach whiskers.
    5. Rinse the whiskers using distilled water with a cotton pad (Figure 1B).
    6. Dry the whiskers with air-flow or a cotton pad (Figure 1C).
    7. Place the rat’s head under illumination and tilt it a bit to reach the whisker(s) you are interested in.
    8. Isolate selected whiskers (Figure 1D)
      1. Cut a piece of Parafilm to cover the rat’s snout and cheeks (e.g., 6 cm L x 6 cm H).
      2. Make a small hole to let the whisker pass through. You can use a glass capillary or a tweezer to isolate the whisker, in both cases paying attention not to damage the whisker or the rat’s skin.
      3. The resulting working surface allows application of the dye(s) only on a selected subset of whiskers. This is very important in order to prevent the unwanted application of the dye.
    9. Flatten the working surface at the base of the selected whisker(s) to allow the application of the dye on the complete whisker(s) length.
    10. Apply the fluorescent dye on whisker(s) (Figure 1E). For each whisker:
      1. Cut a piece of Parafilm of circa 2 cm width and slightly longer than the whisker.
      2. Fill the syringe with the fluorescent dye and eject it abundantly onto the Parafilm in order to soak the whisker along its entire length. In order to avoid air bubbles, which would locally prevent staining, spread the dye with a cotton swab.
      3. The whisker now adheres weakly to the Parafilm because soaked in a gel. Any movement can make the whisker slip away. It will be helpful to hold the Paraffin film or even tighten it with aluminum foil.
    11. Wait between 30 min and 1 h from the last application to let the dye diffuse inside the cuticle.
    12. Remove the excessive dye and rinse the whisker(s) using a cotton pad or a gauze wet with vinegar, which tends to close the cuticle in complete safety for the animal.
      Notes:
      1. Clean one whisker at a time. Use a different cotton pad (or gauze) for each whisker in order to not mix the color).
      2. Since the vinegar smell is quite strong, avoid placing it close to the rat’s nose, because breathing it can reverse the effect of Domitor.
    13. Visualization test (Figure 1F): wear the protective glasses, turn off all the room lights and turn on the lamp.


      Figure 1. Dye application procedure. A. Whisker bleaching; B. Removal of bleaching factor; C. Whiskers drying in airflow; D. Isolation of a single whisker with paraffin film; E. Application of the dye; F. Visualization test. This figure was adapted using images from Supplementary Figure 1 of Rigosa et al., 2017.

      1. Set the shutter speed of the high speed camera according to experimenter’s needs.
        Note: The higher the shutter speed, the shorter the exposure time, the darker the grabbed image. Choose the highest exposure time possible for brightest images.
      2. Simulate experimental conditions placing the animal in a box covered with neoprene and installing the lamp at the proper distance upon experimenter’s needs.
        Note: Light conditions and reflections strongly depend on the neoprene and lamp installation.
      3. Visualize the whisker(s) and adjust the camera gain (Figures 2 and 3).
        Note: If the background is much darker than the visualized whisker, the gain of the camera sensor can be safely increased without introducing salt and pepper noise to the image. This feature depends on the specific camera model.
    14. Follow the guidelines for Post-anesthesia (see Procedure B: ‘Animal sedation’) to recover the rat.
    15. In case the visualization is not sufficient (e.g., the whisker did not absorb the dye along its entire length), plan another application of the dye. In case the time left before the current sedation ends is not sufficient for immediate re-staining, ask your veterinarian when another sedation for the same animal could be feasible.


      Figure 2. High-speed video setup. A cage equipped with a licking sensor is used to trigger and synchronize the high-speed video recording with the licking behavior. The field of view is illuminated using the custom made lamp and the optical path to the camera sensor was filtered using an orange transparent plexiglass.


      Figure 3. Grabbed frame from high-speed video. A behaving rat is illuminated using 4 LEDs of the lamp and an orange plexiglass plate as an optical filter. The video was shot at 1,000 frames per second. This figure was adapted using images from Supplementary Figure 3 of Rigosa et al., 2017.

  2. Animal sedation
    Notes:
    1. This protocol was performed without sterile techniques.
    2. Inform yourself about the features of Domitor: time of effect, site of injection, which reflexes are suppressed and time of recovery.
    3. Set ambient temperature to the room temperature (25 °C) and keep it constant.
    4. Wear gloves and mask during the entire procedure.
    5. The sedation should last between 2.5 and 3 h.
    6. Between two sedations the rat needs 24 h of recovery.
    7. Avoid more than two sedations a week.
    8. Do not obstruct the animal breathing with anything while it is sedated.
    9. Do not disturb the animal sedation with strong odors (e.g., by approaching the discoloration preparation or the vinegar).

    Pre-anesthesia procedure
    1. Make sure that the rat drank water in the last 5 h.
    2. Make sure that the rat is housed alone in the cage.
    3. Bring the rat to a silent and quiet place with constant room temperature.
    4. Weigh the rat and calculate the dose for the anesthetic; the dose of Domitor (Medetomidine hydrochloride) is 0.5 mg/kg [1 mg/ml] intraperitoneal (IP).
    5. Prepare the anesthetic in the syringe, ready to be injected.

    Anesthesia procedure
    1. From now on, the procedure should be as fast as possible to reduce the risk of stressing the animal.
    2. Make the IP injection:
      1. Restrain the animal, expose the abdomen and inject in the lower right quadrant to avoid inner organs.
        Note: In alternative, grasp the rat by the tail and raise its belly, with its posterior legs lifted, inner organs will move towards the head, reducing the risk of missing the target of the shot.
      2. Don’t keep the animal in the restrained position for a long time; it is unnatural and stressful.
      3. Inject the anesthetic IP in a single shot.
    3. Place the rat in the cage
      1. Use a heating pad or cover the rat body with a tissue or aluminum foil to diminish heat dissipation.
      2. Place the cage in a dark room.
    4. After 10 min test the reflexes (tail, hind paw, corneal).
    5. If the animal is sedated, apply the dye.
    6. During sedation, constantly monitor the heart beating and the breathing rate.

    Post-anesthesia procedure
    1. Put the rat back in the cage.
    2. Prepare the dose of the Antisedan (Atipamezole hydrochloride), standard dose for IP injection is 0.2 ml.
    3. Make the IP injection.
    4. Cover the rat with a tissue to keep its body temperature at 37 °C until it wakes up.

Notes

The whisker staining (protocol ‘Dye application’) is executed on the sedated animal (protocol ‘Animal sedation’). The sedation should last between 2.5/3 h and between two sedation there should be a resting period of at least 24 h, and in any case no more than twice a week.
The dye is subject to photo-bleaching. Though in Rigosa et al. (2017) a time constant of about 14 h was reported (i.e., ca. 25,000 trials of 2 sec each), this result depends on the light density and the amount of dye that was absorbed by the whisker sample. If necessary, the investigator can re-stain the same whisker, as this would not alter its mechanical properties. If staining multiple whiskers, the investigator can obtain the same result by staining one whisker at a time across different sessions.

Acknowledgments

The authors declare no competing financial interests. We acknowledge the financial support of the Human Frontier Science Program (http://www.Hfsp.org; project RG0015/2013), the European Research Council Advanced grants CONCEPT (http://erc.europa.eu; project 294498) and MicroMotility (project 340685), and Italian MIUR grant HANDBOT (http://hubmiur.pubblica.istruzione.it/web/ricerca/home; project GA 280778). GN gratefully acknowledges support by SISSA through the excellence program NOFYSAS 2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Fassihi, A., Akrami, A., Esmaeili, V. and Diamond, M. E. (2014). Tactile perception and working memory in rats and humans. PNAS 111: 2331-2336.
  2. Fassihi, A., Akrami, A., Pulecchi, F., Schönfelder, V. and Diamond, M. E. (2017). Transformation of perception from sensory to motor cortex. Curr Biol 27: 1585-1596.
  3. Rigosa, J., Lucantonio, A., Noselli, G., Fassihi, A., Zorzin, E., Manzino, F., Pulecchi, F. and Diamond, M. E. (2017). Dye-enhanced visualization of rat whiskers for behavioral studies. Elife 6.

简介

可视化和跟踪面部胡须对许多啮齿动物行为研究至关重要。 高速摄影是用于表征晶须运动学的最稳健的方法,但由于晶须与其背景的低对比度,晶须可视化具有挑战性。 最近,我们发现可以应用荧光染料来增强晶须的可视化和追踪(Rigosa等人,2017),并且该协议提供了关于该技术的其他细节。


【背景】在过去的10年中,神经科学家已经开始关注感觉运动处理来处理啮齿类动物。 关键的一步是能够量化来自外界的进入神经系统的信号。 对于晶须介导的触觉,为了表征感觉输入,需要对面部晶须进行可视化和追踪。 尽管已经采用了许多方法,但只有高速摄像证明足以测量与物体相互作用期间的胡须运动和变形。 然而,晶须可视化和跟踪由于多种原因而具有挑战性,其中主要是晶须与其背景的低对比度。 该协议详细描述了一种通过使胡须发荧光来增加对比度的技术。

关键字:触须, 跟踪, 荧光, 染料, 触觉感知, 体觉皮层

材料和试剂

  1. 乳胶手套(DOCzero)
  2. 面膜(Benefis)
  3. Eppendorf PCR管(2 ml)
  4. 棉垫或纱布(Megapharma)
  5. 棉签(DaklaPack)
  6. 实验室电影(Parafilm)
  7. 硼硅酸盐玻璃毛细管内Ø0.58毫米(Hilgenberg)5厘米长度
  8. 注射器(Nipro Europe N.V.)
  9. 标准铝箔
  10. 动物:来自Envigo-Harlan的Wistar大鼠(450-550g)
    注意:
    1. Wistar大鼠(450-550克)单独饲养或与一个笼子配对。 14/10明/暗周期。
    2. 原则上,该方法可以应用于任何有胡须的动物,但漂白较暗的胡须可能需要较长的时间并且可能产生较不令人满意的结果,影响荧光的质量。
  11. Epigel(Ceva,意大利)
  12. 过氧化氢(稀释溶液3%)
  13. 蓝色漂白粉(DECO +)
  14. 蒸馏水(Marco Viti Farmaceutici)
  15. 厨房醋
  16. Domitor(盐酸美托咪啶)
  17. Antisedan(阿替美唑盐酸盐)

设备

  1. 大鼠笼(Tecniplast,目录号:1291H)
    注意:鼠被隔离在笼子里。
  2. 动物秤
  3. 脉搏血氧仪
  4. 1把剪刀(瑞士剪刀,9厘米)(世界精密仪器公司,目录号:504613)
  5. 1 x镊子(#7,世界精密仪器,产品目录号:504504)

  6. 用于身体体温调节的组织和加热垫(Panlab s.l.)
  7. 荧光染料
    注意:我们使用并测试了头发半永久性氟化物冲洗剂('UV Red'或'UV Green',Star Gazer)。
  8. 激发灯
    注:我们使用一个定制的灯泡,使用7个灯泡:ILH-OO01-DEBL-SC211-WIR200,波长455nm,通量@ 700mA 1,400mW,发光角度±60°;聚光透镜FA11205_Tina-D-OSL FWHM角度±6°。该灯是为了从不同的方向产生均匀的照明而定制的:这改善了荧光染料发出的光的均匀性,然后改善了图像质量。每个灯泡使用商用LED与复合抛物面聚光器(CPC)(也称为“Winston锥体”)组装,以便收集和投射到舞台上所有可用的光。
  9. 黑色氯丁橡胶(RS Components,产品目录号:733-6753)
    注:我们用黑色氯丁橡胶覆盖了实验装置,因为黑色的非反射性背景有助于增加图像的整体对比度。

  10. 安全护目镜(Univet,目录号:5X7.03.00.04) 注:吸光度&gt; 4用于波长&lt; 510纳米。认证0068 / ETI-DPI / 070-2009 Rev.2。
  11. 高速相机和镜头
    注意:相机和镜头取决于实验者的选择。我们使用相机CamRecord 450(Optronis)与Computar TV镜头-50 mm 1:1.3镜头相结合。每个研究者必须根据实验所需的帧率选择视频设备:对于高分辨率大鼠晶须运动学,1,000 fps是可接受的帧频(Fassihi等,2014和2017)。必须注意相机的帧速率:速率越高,光线应该越强。对于1,000 fps的帧速率,单帧的持续时间为1毫秒,从而提供最大的曝光时间。我们使用整个帧持续时间1毫秒作为曝光时间。较低的曝光时间通常会产生更好的静止图像,但需要额外的光线:必须根据每个单独实验装置的特定要求对折衷进行优化。在实验者想要降低刺激光的强度的情况下,或者在使用不同的(例如,较不明亮的)相机镜头的情况下,一些相机允许设置传感器的内部增益:这提高了相机传感器的灵敏度这样做的代价是在所得图像中获得更多的椒盐噪音。
  12. 长通过滤器
    注意:需要长通过滤器来观察用不同染料染色的一个或多个晶须。我们测试了FEL0500长波滤光片截止波长500 nm(Thorlabs),以显现多种荧光染料和红色/橙色有机玻璃,仅观察红色荧光染料。过滤器的选择应该适合透镜的尺寸,而有机玻璃可以切成适合定制的设计,然后它可以适应透镜的尺寸。至于相机和镜头,必须选择过滤器来匹配染料颜色并抑制照明光源的颜色。这样可以防止相机传感器在接收到由荧光染料发出的较低强度的光线时被光源打开。

程序

  1. 染料应用
    注意:
    1. 此协议不需要无菌技术。
    2. 在整个过程中戴上手套和口罩。
    3. 预先准备一种新的头发变色制剂:
      1. 考虑到它有令人不愉快和强烈的气味,你可以考虑使用罩子。
      2. 用漂白粉和过氧化氢填充至少一个PCR管,关闭管并混合。
      3. 必须获得粘性制剂以避免其从晶须上滴落。
    4. 预先准备浸泡在蒸馏水和醋中的棉垫(或纱布)。
    5. 在打开激发源之前,戴上防护眼镜。
    在开始本方案之前,应对大鼠进行镇静(参见程序B:'动物镇静':遵循'麻醉前'和'麻醉'程序)。
    1. 将老鼠放在加热垫上,并将体温设置为37°C。
    2. 将Epigel放在老鼠的眼睛上,以保护它们免受光照和干燥。
    3. 使用棉签在所有晶须上涂上变色制剂(图1A)。由于它的气味非常强烈,请避免将其放在老鼠鼻子附近,因为呼吸可以扭转Domitor的效果。
    4. 等待30分钟到1小时,以漂白胡须。
    5. 用化妆棉用蒸馏水冲洗晶须(图1B)。

    6. 用气流或化妆棉擦干晶须(图1C)。
    7. 将老鼠的头放在照明下,倾斜一点,以便触及您感兴趣的晶须。
    8. 分离选定的晶须(图1D)
      1. 切一片Parafilm覆盖老鼠的鼻子和脸颊(,例如,6厘米长x 6厘米高)。
      2. 做一个小洞让晶须通过。您可以使用玻璃毛细管或镊子来隔离晶须,在这两种情况下注意不要损伤晶须或老鼠的皮肤。
      3. 所得到的工作表面仅允许将染料施加在选定的晶须子集上。这是非常重要的,以防止不必要的染料应用。
    9. 在选定的晶须底部铺平工作表面,以便在整个晶须长度上施加染料。
    10. 在晶须上涂上荧光染料(图1E)。对于每个晶须:

      1. 切一块大约2厘米宽的Parafilm,稍长于晶须。
      2. 用荧光染料填充注射器并将其大量喷射到石蜡膜上,以便将晶须浸泡在其整个长度上。为了避免气泡,这会局部防止染色,用棉签涂染染料。
      3. 由于浸泡在凝胶中,晶须现在粘附在Parafilm上。任何动作都会使晶须脱落。
        保持石蜡膜或用铝箔将其拉紧是有帮助的。

    11. 在最后一次使用后30分钟至1小时之间等待,让染料在角质层内扩散。
    12. 去除多余的染料,用化妆棉或用醋湿润的纱布冲洗晶须,这样可以使动物完全安全地关闭角质层。
      注意:
      1. 每次清洁一个胡须。
        为每种胡须使用不同的棉垫(或纱布)以便不混合颜色)。
      2. 由于醋的气味非常强烈,所以请避免将它放在老鼠鼻子附近,因为呼吸可以扭转Domitor的效果。
    13. 可视化测试(图1F):佩戴防护眼镜,关闭所有室内灯并打开灯。



      图1.染料施用程序 A.晶须漂白; B.去除漂白因子; C.气流在气流中干燥; D.用石蜡膜分离单个晶须; E.染料的应用; F.可视化测试。这个数字是根据Rigosa et al。的2017年补充图1中的图片进行调整的。


      1. 根据实验者的需要设置高速摄像机的快门速度。
        注意:快门速度越高,曝光时间越短,抓图像越暗。选择可能的最高曝光时间以获得最亮的图像。
      2. 模拟实验条件,将动物置于覆盖有氯丁橡胶的盒子中,并根据实验者的需要将灯泡安装在适当的距离。
        注:光照条件和反射强烈依赖于氯丁橡胶和灯泡的安装。
      3. 可视化晶须并调整相机增益(图2和3)。
        注:如果背景比可视化晶须暗得多,则可以安全地增加相机传感器的增益,而不会在图像中引入盐和胡椒噪声。此功能取决于特定的相机型号。
    14. 按照麻醉后的指导方针(见程序B:'动物镇静')来恢复大鼠。
    15. 如果可视化不充分(例如,晶须没有沿着其整个长度吸收染料),则计划染料的另一种应用。如果在当前镇静结束之前剩下的时间不足以立即重新染色,请询问兽医何时可以使用同一动物的另一种镇静剂。


      图2.高速视频设置。 配备舔传感器的笼子用于触发和同步高速视频录制与舔动行为。使用定制的灯照亮视野,使用橙色透明有机玻璃过滤相机传感器的光路。


      图3.高速视频抓取帧。 使用灯的4个LED和橙色有机玻璃板作为光学滤光片照亮一只表现不佳的老鼠。该视频以每秒1000帧的速度拍摄。这个数字是根据Rigosa等人2017年补充图3中的图片进行调整的。

  2. 动物镇静
    注意:
    1. 该协议是在无菌技术下进行的。
    2. 告诉你Domitor的特点:效果时间,注射部位,哪些反射被抑制,恢复的时间。
    3. 将环境温度设置为室温(25°C)并保持恒定。
    4. 在整个过程中戴上手套和口罩。
    5. 镇静应持续2.5至3小时。
    6. 大鼠需要24小时才能恢复。
    7. 每周避免超过两次sedations。
    8. 在镇静时不要用任何东西阻塞动物呼吸。
    9. 不要以强烈的气味来扰乱动物镇静(例如通过接近变色制剂或醋)。

    预麻醉程序
    1. 确保老鼠在过去5小时内喝了水。
    2. 确保老鼠独自安置在笼子里。
    3. 将老鼠带到室温恒定的安静而安静的地方。
    4. 称量大鼠并计算麻醉剂的剂量;
      Domitor(盐酸美托咪啶)的剂量是0.5 mg / kg [1 mg / ml]腹膜内(IP)。
    5. 在注射器中准备麻醉剂,准备注射。

    麻醉程序
    1. 从现在起,程序应尽可能快,以减少动物受到压力的风险。
    2. 进行IP注入:

      1. 。抑制动物,暴露腹部并注射到右下象限,以避免内脏。
        注意:或者,抓住老鼠的尾巴,抬起它的腹部,抬起其后腿,内部器官会向头部移动,从而减少错过拍摄目标的风险。 />
      2. 不要将动物长时间保持在限制位置;这是不自然和压力。

      3. 注射麻醉剂IP
    3. 将老鼠放入笼中

      1. 。使用加热垫或用纸巾或铝箔覆盖老鼠身体以减少散热。
      2. 将笼子放在黑暗的房间里。
    4. 10分钟后测试反射(尾巴,后爪,角膜)。
    5. 如果动物镇静,则使用染料。
    6. 在镇静期间,不断监测心脏跳动和呼吸频率。

    麻醉后手术
    1. 把老鼠放回笼子里。
    2. 准备Antisedan的剂量(阿替美唑盐酸盐),IP注射的标准剂量为0.2毫升。
    3. 进行IP注射。

    4. 。用纸巾覆盖大鼠,使其体温保持在37°C直到醒来。

笔记

晶须染色(方案'染料应用')在镇静动物上执行(方案'动物镇静')。镇静应该持续2.5 / 3小时之间,两次镇静之间应该有至少24小时的休息时间,并且无论如何每周不超过两次。
染料受到光漂白。尽管在Rigosa等人(2017年)报道了约14小时的时间常数( ie , ca。每次2秒的25,000次试验),这个结果取决于晶须样品吸收的光密度和染料的量。如有必要,研究人员可以重新染色同一个晶须,因为这不会改变其机械性能。如果染色多个胡须,研究人员可以通过在不同阶段一次对一个胡须染色获得相同的结果。

致谢

作者声明没有竞争的财务利益。我们承认人类前沿科学计划( http://www.Hfsp.org ;项目RG0015 / 2013),欧洲研究理事会高级会议授予CONCEPT( http://erc.europa.eu ;项目294498 )和MicroMotility(项目340685)和意大利MIUR授予HANDBOT( http://hubmiur.pubblica .istruzione.it / web / ricerca / home ;项目GA 280778)。 GN非常感谢SISSA通过2012年卓越计划NOFYSAS提供的支持。出资者在研究设计,数据收集和分析,决定发表或准备手稿方面没有任何作用。

参考

  1. Fassihi,A.,Akrami,A.,Esmaeili,V.和Diamond,M.E。(2014)。 老鼠和人类的触觉和工作记忆 PNAS 111:2331-2336。
  2. Fassihi,A.,Akrami,A.,Pulecchi,F.,Schönfelder,V.and Diamond,M.E。(2017)。 将感知从感觉转变为运动皮层 Curr Biol 27:1585-1596。
  3. Rigosa,J.,Lucantonio,A.,Noselli,G.,Fassihi,A.,Zorzin,E.,Manzino,F.,Pulecchi,F.和Diamond,M.E。(2017)。 用于行为研究的大鼠晶须的染料增强可视化 Elife 6.
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Rigosa et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rigosa, J., Lucantonio, A., Noselli, G., Fassihi, A., Zorzin, E., Manzino, F., Pulecchi, F. and Diamond, M. E. (2018). A Fluorescent Dye Method Suitable for Visualization of One or More Rat Whiskers. Bio-protocol 8(5): e2749. DOI: 10.21769/BioProtoc.2749.
  2. Rigosa, J., Lucantonio, A., Noselli, G., Fassihi, A., Zorzin, E., Manzino, F., Pulecchi, F. and Diamond, M. E. (2017). Dye-enhanced visualization of rat whiskers for behavioral studies. Elife 6.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。