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Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice
立体定向外科手术治疗小鼠视交叉上核病变

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Abstract

Site-specific lesions are invaluable methods for investigating the function of brain regions within the central nervous system and can be used to study neural mechanisms of behaviors. Precise stereotaxic surgery is required to lesion small regions of the brain such as the suprachiasmatic nucleus (SCN), which harbors the master circadian clock. In this protocol, we describe stereotaxic surgery optimized for bilateral lesion of the mouse SCN by loading electric current. Success of the SCN lesion is verified histologically and behaviorally by monitoring arrhythmic locomotor activity. The SCN-lesioned mouse allows for the evaluation of behavioral, biochemical, and physiological consequences of ablation of the master circadian clock.

Keywords: Suprachiasmatic nucleus(视交叉上核), Stereotaxic surgery(立体定向手术), SCN-lesion(SCN病变), Circadian rhythm(昼夜节律), Hypothalamus(下丘脑)

Background

The suprachiasmatic nucleus (SCN) is a small region within the hypothalamus of the mammalian brain. It is positioned bilaterally above the optic chiasm and contains approximately 20,000 neurons. The SCN is known as the location of the master circadian oscillator (clock) and is required for synchronization with the light-dark cycle. Ablation of the SCN is a useful strategy for assessing the physiological influence of the master circadian clock.
   An electrolytic lesion of the SCN has advantages that enable fast and localized ablation of the master circadian clock in comparison to gene modification by virus injection or SCN-specific promoters. Location of the lesion by electrical impulse can be verified right after surgery by Nissl staining, and monitoring activity rhythm can be started one day after the surgery. In addition, lesion by administration of chemicals often results in non-specific damage and thus it is not as precise as lesion by electrical impulse, especially for small targets such as SCN. Therefore, this protocol provides a useful strategy to evaluate effects (outputs) of master circadian clock.

Materials and Reagents

  1. Adult mouse (C57BL/6J, usually 8-14 weeks old)
  2. Ketamine (Daiichi Sankyo Propharma, Ketalar for intramuscular injection 500 mg)
  3. Xylazine (Bayer, Serakutaru 2% injection)
  4. Bacteriostatic saline (Otsuka pharmaceutical, 20 ml ampoule)
  5. 70% ethanol
  6. For confirmation of SCN-lesioning
    1. Glacial acetic acid
    2. 0.2% cresyl violet acetate solution (see Recipes)
    3. Filter paper

Equipment

Note: Refer to Figures 1, 2, 3 for the Equipment used in this protocol.

  1. Stereotaxic frame (NARISHIGE, model: SR-6M-HT )
  2. Stereotaxic micromanipulator (NARISHIGE, model: SM-15R/L )
  3. Wide-field dissecting microscope (ZEISS, model: Stemi 2000 ) with boom stand
  4. Cold light source (ZEISS, model: CL 1500 ECO )


    Figure 1. Equipment list 1-4

  5. Auxiliary ear bar (NARISHIGE, model: EB-5N )
  6. Scissors (e.g., Fine Science Tools, catalog number: 14068-12 )
  7. Scalpel (FEATHER Safety Razor, catalog number: No.11 )
  8. Surgical needle with suture (e.g., NATSUME SEISAKUSHO, catalog number: C21-40 )
  9. Hemostats (e.g., Fine Science Tools, catalog number: 13008-12 )
  10. Forceps (e.g., Fine Science Tools, catalog number: 11009-13 or 11008-13 )
  11. Tuberculin syringe with needle (e.g., Terumo Medical, catalog numbers: SS-01T and NN-2719S )
  12. Razor (e.g., FEATHER Safety Razor, catalog number: FAS-10 )
  13. Cotton swabs (e.g., Q-tip)
  14. Heating pad (e.g. electric heating pad for pets)
  15. Hand drill with engraving cutter (DREMEL, model: 106 )


    Figure 2. Equipment list 5-15

  16. Lesion-making device (Ugo Basile, catalog number: 53500 )
  17. Electrode (100 μm, coated with epoxy except for 200 μm at the tip, Neuroscience)
  18. Plug connecter cables with crocodile clip
  19. Area sensor with an infrared detector for confirmation of SCN-lesioning (EK, catalog number: PS-3241 )


    Figure 3. Equipment list 16-18

Software

  1. ClockLab software

Procedure

This protocol is based on structural features on the skull surface (bregma and lambda) as reference points for determining the target position (Figure 5). Bregma and lambda are defined as the intersections of the sagittal suture with the coronal and lambdoid sutures, respectively. A mouse brain atlas (e.g., Paxinos and Franklin, 2001) allows the determination of the anterior-posterior, medial-lateral, and dorsal-ventral coordinates of the brain structures from measurement of these skull features.

  1. Anesthetize mouse
    1. Anesthetize the mouse with an intraperitoneal injection (20 µl/g of body weight) of a mixture of ketamine (7 mg/ml) and xylazine (0.44 mg/ml) dissolved in bacteriostatic saline. The animal should reach surgical anesthesia within 5-10 min. Shave the fur of the top of the skull after the mouse has reached full anesthesia.
      Note: Depth of anesthesia is assessed by the corneal blink and tail-pinch reflexes. Wait until the reflexes are disappeared, if the mouse is not well anesthetized. Anesthesia should persist for approximately 1 h.
  2. Mount mouse to a stereotaxic frame (Video 1)

    Video 1. Procedure of setting mouse to a stereotaxic frame. This video describes how to mount a mouse to a stereotaxic frame. The videoclip begins with insertion the auxiliary ear bar into the external auditory meatus.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    1. Before mounting the mouse to the stereotaxic frame, insert the auxiliary ear bar. Guide the tips of the auxiliary ear bar into the external auditory meatus and tighten the screw.
      Note: Small popping sounds are heard when the tips are properly inserted to the tympanic membranes. Make sure that the auxiliary ear bar and the midline of the mouse are vertically placed.
    2. Insert the ear bars of the stereotaxic frame into the auxiliary ear bar. Lock the ear bars tightly. When done correctly, the animal’s head should be centered by symmetrical adjustment of the ear bars.
    3. Slide the incisor bar into the mouth and place incisors into the hole. Apply downward pressure to the nose while sliding the incisor bar back until it stops. Tighten the nose clamp and incisor bar firmly (Figure 4).


      Figure 4. A mouse mounted in the stereotaxic frame

  3. Surgery (Video2)

    Video 2. Stereotaxic surgery. The video shows steps for the surgery.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    Note: To maintain body temperature, it is recommended to place the mouse on a heating pad (38 °C) during surgery.
    1. Disinfect the scalp with 70% ethanol and make an incision along the midline with a scalpel. Expose the skull and scrape away the pericranial tissues with a cotton swab.
    2. Fit the stereotaxic micromanipulator to the stereotaxic frame and attach the electrode.
    3. Perform dorsoventral measurements at bregma and lambda by lowering the tip of the electrode until it just touches the skull. Then, adjust the incisor bar to bring both bregma and lambda lines at the same horizontal level (Figure 5).
      Note: At this point, make sure that both of the bregma and the lambda are on the midline. If these are not on the midline, it is necessary to remount the mouse on the stereotaxic frame.


      Figure 5. Dorsal surface of the mouse skull. Bregma and lambda are defined as the points of intersections of the sagittal suture with the coronal and lambdoid sutures, respectively.

    4. Using a dissecting microscope, set zero at bregma. Set the electrode at the target coordinates: 0.2 mm caudal to bregma and 0.23 mm bilateral to the midline for an adult C57BL/6J mouse. Mark the targets with the pencil on the skull.
    5. Drill two small burr holes through the skull at the marked targets for the bilateral targets. Lower the electrode into the hole 5.9 mm below the skull surface for an adult C57BL/6J mouse. If bleeding is observed after drilling, stop bleeding by applying pressure to the whole by a cotton swab.
      Note: The correct target coordinates will vary depending on the age or strain of the mouse. Pilot surgeries may be necessary to refine the target coordinates.
  4. Setting lesion making device
    1. Connect the electrode to the binding post (+). Pinch the nose clamp with one of the clips of the plug connector cable and connect it to the binding post (-). Pinch the mouse tail with the other clip of the plug connector cables and connect to the binding post (G) (Figure 6).


      Figure 6. The connection of the lesion-making device to the mouse

  5. SCN-lesion
    1. Deliver an electric current of 0.8 mA for 3 sec using the lesion-making device. For the sham operation, use the same surgical procedure but do not deliver a current.
    2. Remove the electrode and suture the incision using surgical needle and thread (Video 3). Remove the mouse from the stereotaxic frame and auxiliary ear bars.
    3. Keep warm the mouse on a heating pad (38 °C) during recovery (approx. 30 min).
       
      Video 3. Suture of incision. This video provides a demonstration of suture of the incision in the end of surgery.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  6. Confirmation of SCN-lesion
    To confirm arrhythmicity induced by the SCN-lesion, monitor the rhythm of free-moving activity in constant darkness using an area sensor with an infrared detector (Figure 7). The activity rhythm can be analyzed using ClockLab software. The SCN lesion can also be confirmed by Nissl staining of the SCN slices using cresyl violet solution (Figure 8).


    Figure 7. The schematic chart of the infrared detector setup


    Figure 8. Confirmation of the SCN-lesion. Shown are the representative Nissl-stained coronal brain sections of a mouse 1 month after SCN-lesion (left panels) and the double-plotted actograms (right panels) of the SCN-lesioned mouse (upper panels) and sham-operated one (lower panels). In the Nissl-staining (left panels), the bilateral SCN is lost in the section of SCN-lesioned mouse (upper panel), while the SCN (arrows) in the section of sham-operated mouse is intact (lower panel). Electrolytic lesioning frequently results in formation of tissue debris (arrowhead). 3 V, third ventricle. OC, optic chiasm. Scale bar = 500 µm. When the activity is monitored under constant dark (DD) condition, the SCN-lesioned mouse shows arrhythmic locomotor behavior (upper panel), whilst the sham-operated mouse shows a normal circadian rhythm. Shaded areas on the actograms signify the dark period. (Data are cited from Shimizu et al., 2016).

Data analysis

Analysis for activity rhythm: ClockLab software collects and analyzes the data automatically. Chi-square periodgram analysis is used to assess rhythmicity. If the SCN lesion surgery is successful, no periodogram peaks (as defined with a statistical significance level P < 0.05) will be detected in a 24 h period.

Notes

  1. The entire surgical protocol takes approximately 30 min per animal.
  2. The most important step to ensure accuracy is to mount the head of the mouse correctly on the stereotaxic frame.

Recipes

  1. 0.2% cresyl violet acetate solution
    1. Dissolve 2.0 g cresyl violet in 990 ml distilled water
    2. Add 10 ml glacial acetic acid
    3. Make up to 1,000 ml with distilled water and agitate it for 1 h
    4. Filtrate the solution through a filter paper

Acknowledgments

This work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Paxinos, G. and Franklin, K. B. J. (2001). The Mouse Brain in Stereotaxic Coordinates, Deluxe Edition of the Atlas. Academic Press.
  2. Shimizu, K., Kobayashi, Y., Nakatsuji, E., Yamazaki, M., Shimba, S., Sakimura, K. and Fukada,Y. (2016). SCOP/PHLPP1β mediates circadian regulation of long-term recognition memory. Nat Commun 7: 12926.

简介

位点特异性病变是调查中枢神经系统中脑区功能的有价值的方法,可用于研究神经行为机制。 需要精确的立体定位手术来对大脑的小区域进行损伤,例如含有主昼夜节律钟的视交叉上核(SCN)。 在本协议中,我们介绍了通过加载电流优化小鼠SCN双侧病变的立体定位手术。 通过监测心律失常运动活动,组织学和行为学验证了SCN病变的成功。 SCN损伤的小鼠允许评估主昼夜节律钟的消融的行为,生化和生理后果。
【背景】视交叉上核(SCN)是哺乳动物脑下丘脑内的一个小区域。它位于视交叉的两侧,包含约20,000个神经元。 SCN被称为主昼夜节律振荡器(时钟)的位置,并且与亮 - 暗循环同步所需。 SCN的消融是评估主昼夜节律钟的生理影响的有用策略。
与通过病毒注射或SCN特异性启动子的基因修饰相比,SCN的电解损伤具有能够快速和局部消除主昼夜节律钟的优点。通过Nissl染色手术后可以通过电脉冲定位病变,手术后一天可以开始监测活动节律。此外,通过施用化学物质的病变通常导致非特异性损伤,因此其不像电脉冲的损伤那样精确,特别是对于诸如SCN的小靶标。因此,该协议提供了一个有用的策略来评估主昼夜节律钟的效果(输出)。

关键字:视交叉上核, 立体定向手术, SCN病变, 昼夜节律, 下丘脑

材料和试剂

  1. 成年老鼠(C57BL/6J,通常8-14周龄)
  2. 氯胺酮(Daiichi Sankyo Propharma,Ketalar for肌内注射500mg)
  3. 赛拉嗪(拜耳,Serakutaru 2%注射)
  4. 抑菌生理盐水(大冢制药,20ml安瓿)
  5. 70%乙醇
  6. 用于确认SCN损伤
    1. 冰醋酸
    2. 0.2%甲酚紫罗兰醋酸溶液(参见食谱)
    3. 滤纸

设备

注意:有关本协议中使用的设备,请参见图1,图2,图3

  1. 立体定位框架(NARISHIGE,型号:SR-6M-HT)
  2. 立体定向显微操纵器(NARISHIGE,型号:SM-15R/L)
  3. 宽幅解剖显微镜(ZEISS,型号:Stemi 2000),带有吊架
  4. 冷光源(ZEISS,型号:CL 1500 ECO)


    图1.设备列表1-4

  5. 辅助耳杆(NARISHIGE,型号:EB-5N)
  6. 剪刀(例如,,Fine Science Tools,目录号:14068-12)
  7. Scalpel(FEATHER安全剃须刀,目录号:No.11)
  8. 缝合线的手术针(例如,NATSUME SEISAKUSHO,目录号:C21-40)
  9. 止血剂(例如,,精细科学工具,目录号:13008-12)
  10. 镊子(例如,,Fine Science Tools,目录号:11009-13或11008-13)
  11. 带针的结核菌素注射器(例如,Terumo Medical,目录号:SS-01T和NN-2719S)
  12. 剃刀(例如,,FEATHER安全剃刀,目录号:FAS-10)
  13. 棉签(例如,,Q-tip)
  14. 加热垫(例如,,宠物电加热垫)
  15. 带雕刻刀的手钻(DREMEL,型号:106)


    图2.设备列表5-15

  16. 损伤装置(Ugo Basile,目录号:53500)
  17. 电极(100μm,涂有环氧树脂,除了尖端的200μm,Neuroscience)
  18. 用鳄鱼夹插入连接器电缆
  19. 带有用于确认SCN损伤的红外探测器的区域传感器(EK,目录号:PS-3241)


    图3设备列表16-18

软件

  1. ClockLab软件

程序

该协议基于颅骨表面(粗糙和λ)上的结构特征作为确定目标位置的参考点(图5)。 Bregma和lambda分别定义为矢状缝合与冠状和羔羊皮缝合线的交点。小鼠脑图谱(例如,Paxinos和Franklin,2001)允许通过测量这些头骨特征来确定脑结构的前后,内侧和外侧 - 腹侧坐标。

  1. 麻醉老鼠
    1. 用溶解在抑菌盐水中的氯胺酮(7mg/ml)和赛拉嗪(0.44mg/ml)的混合物腹膜内注射(20μl/g体重)麻醉小鼠。动物应在5-10分钟内达到手术麻醉。在鼠标达到完全麻醉后,刮去头骨顶部的毛皮。
      注意:通过角膜眨眼和尾部反射来评估麻醉深度。等到反射消失时,如果鼠标没有被麻醉。麻醉应持续约1小时。
  2. 将鼠标安装到立体定位框架(视频1)

    Video 1. Procedure of setting mouse to a stereotaxic frame. This video describes how to mount a mouse to a stereotaxic frame. The videoclip begins with insertion the auxiliary ear bar into the external auditory meatus.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    1. 将鼠标安装到立体定位框架之前,请插入辅助耳杆。将辅助耳杆的尖端引导到外耳道内并拧紧螺丝。
      注意:当尖端正确插入鼓膜时,会听到小声的声音。确保辅助耳杆和鼠标的中线垂直放置。
    2. 将立体定位框架的耳杆插入辅助耳杆。锁紧耳塞。正确完成后,动物的头部应以耳杆对称调整为中心。
    3. 将门牙滑入口中,将门牙放入孔中。向下施加向下的压力,同时将门牙杆向后滑动直至停止。牢固地拧紧鼻钳和门牙杆(图4)

      图4.安装在立体定位框架中的鼠标

  3. 手术(Video2)

    Video 2. Stereotaxic surgery. The video shows steps for the surgery.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    注意:为了维持体温,建议在手术过程中将鼠标放在加热垫(38°C)上。
    1. 用70%乙醇消毒头皮,并用手术刀沿中线切开。露出头骨,用棉签擦去颅骨组织。
    2. 将立体定位显微操纵器安装在立体定位框架上并附着电极。
    3. 通过降低电极尖端直到接触颅骨,进行前凸和鼻窦的背侧测量。然后,调整门牙栏,使两个粗线和lambda线都处于相同的水平位置(图5)。
      注意:在这一点上,确保bregma和lambda都在中线。如果这些不在中线,则需要在立体定位框架上重新安装鼠标。


      图5.小鼠颅骨的背表面。 Bregma和lambda分别定义为矢状缝合与冠状和羔羊皮缝合线的交叉点。

    4. 使用解剖显微镜,在正前方设置为零。将电极设置为目标坐标:对于成年C57BL/6J小鼠,尾部到尾标为0.2mm,中线为0.23mm。用骷髅上的铅笔标记目标。
    5. 在两个目标的标记目标上通过头骨钻两个小钻孔。将成年人C57BL/6J小鼠的电极放入颅骨表面5.9毫米以下的孔中。如果在钻孔后观察到出血,请用棉签向整体施加压力来停止出血。
      注意:正确的目标坐标将根据鼠标的年龄或应变而变化。可能需要引导手术来完善目标坐标。
  4. 设置病变制造装置
    1. 将电极连接到连接柱(+)上。用插头连接器电缆的一个夹子夹住鼻夹,并将其连接到装订柱( - )。用鼠标尾部夹住插头连接器电缆的另一个夹子,并连接到装订柱(G)(图6)。


      图6.病毒制作设备与鼠标的连接

  5. SCN病变
    1. 使用病变制造装置输送0.8 mA的电流3秒。对于假手术,请使用相同的外科手术,但不提供电流。
    2. 使用手术针和螺纹去除电极并缝合切口(视频3)。从立体定位框架和辅助耳杆上取下鼠标。
    3. 在恢复期间(约30分钟),将加热垫(38°C)上的鼠标温热。
       
      Video 3. Suture of incision. This video provides a demonstration of suture of the incision in the end of surgery.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  6. 确认SCN病变
    为了确认由SCN病变引起的心律失常,使用具有红外检测器的区域传感器在恒定黑暗中监测自由移动活动的节律(图7)。活动节奏可以使用ClockLab软件进行分析。 SCN病变也可以通过使用甲酚紫溶液的SCN切片的Nissl染色来确认(图8)。


    图7.红外探测器设置示意图


    图8. SCN病变的确认显示SCN损伤后1个月(左图)和双重绘制的活动图(右图)的小鼠的代表性Nissl染色的冠状脑切片的SCN损伤的小鼠(上图)和假手术的一个(下图)。在Nissl染色(左图)中,双侧SCN在SCN损伤的小鼠(上图)部分丢失,而假手术小鼠部分的SCN(箭头)完整(下图)。电解损伤常常导致组织碎片的形成(箭头)。 3 V,第三脑室。 OC,视交叉。刻度棒= 500μm。当在恒定暗(DD)条件下监测活动时,SCN损伤的小鼠显示心律失常运动行为(上图),而假手术小鼠显示正常的昼夜节律。活动图上的阴影区域表示黑暗时期。 (数据来自Shimizu等人,2016)。

数据分析

活动节奏分析:ClockLab软件自动收集和分析数据。卡方周期分析用于评估节律性。如果SCN损伤手术成功,则在24小时期间内不会检测到周期图峰值(定义为统计显着性水平 <0.05)。

笔记

  1. 整个手术方案每只动物需要约30分钟。
  2. 确保准确度最重要的一步是将鼠标的头部正确安装在立体定位框架上。

食谱

  1. 0.2%甲酚紫醋酸溶液
    1. 溶解2.0克甲酚紫990毫升蒸馏水
    2. 加入10毫升冰醋酸
    3. 用蒸馏水补充1000毫升,搅拌1小时
    4. 通过滤纸过滤溶液

致谢

这项工作得到了日本教育,文化,体育,科技部科学研究资助。

参考

  1. Paxinos,G.和Franklin,KBJ(2001)。< a class ="ke-insertfile"href ="http://braininfo.rprc.washington.edu/Source.aspx?ID=263&questID=1"target = "_blank">立体定位坐标中的小鼠脑,Atlas的豪华版。 学术出版社。
  2. Shimizu,K.,Kobayashi,Y.,Nakatsuji,E.,Yamazaki,M.,Shimba,S.,Sakimura,K。和Fukada,Y。 (2016)。 SCOP /PHLPP1β调节昼夜节律-term识别记忆。 7:12926.
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引用:Shimizu, K. and Fukada, Y. (2017). Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice. Bio-protocol 7(12): e2346. DOI: 10.21769/BioProtoc.2346.
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