Craniotomy for Cortical Voltage-sensitive Dye Imaging in Mice

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Jun 2015



Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image sensor with a fast sampling rate (500 Hz) detects fluorescent changes in corrective activity. The procedure includes fixing a mouse brain to a stereotaxic apparatus, craniotomy on a large cortical area, VSD staining, and wide-field imaging of cortical activity. The entire procedure can be completed in 5 h (from the administration of anesthesia to the start of cortical VSD imaging).

Keywords: Voltage-sensitive dye imaging (电压敏感染料成像), Craniotomy (开颅手术), Cortical activity (皮层活动), Wide-field imaging (宽视场成像)

Materials and Reagents

  1. Fine needle (Bonn Micro Probes) (Fine Science Tools, catalog number: 10030-13 )
  2. Cover glass (Matsunami Glass Ind, catalog number: C024241 )
  3. Wild-type mice (Japan SLC, model: C57BL / 6JJmsSlc )
  4. Isoflurane (e.g., Wako Pure Chemical Industries, catalog number: 099-06571 )
  5. Lidocaine solution (80 mg/ml) (e.g., Xylocaine Pump Spray 8%) (AstraZeneca) for local anesthesia
  6. Dental cements (Super-Bond C&B) (Sun medical) (GC's Global, UNIFAST II)
  7. Voltage-sensitive dye (OPTICAL IMAGING LTD, catalog number: RH1691 )
  8. NaCl
  9. KCl
  10. MgCl2.6H2O
  11. CaCl2.2H2O
  12. HEPES
  13. Distilled water
  14. RH1691
  15. Ringer’s solution (see Recipes)
  16. Voltage-sensitive dye (VSD) solution (see Recipes)


  1. Anesthesia system for isoflurane (Shinano, catalog number: SN-487-0T )
  2. Electric clipper (e.g., Panasonic Corporation, catalog number: ER803P ) for cutting mouse hair
  3. Feedback-controlled heating pad (Bio Research Center, catalog number: BWT-100A )
    Note: The pad monitors the mouse body temperature and maintains it at the desired temperature.
  4. Head holder (stereotaxic apparatus) (NARISHIGE Group, model: SG-4N )
  5. Fine scissors (Fine Science Tools, catalog number: 91460-11 )
  6. Student Dumont #7 Forceps (Fine Science Tools, catalog number: 91197-00 )
  7. Dumont #5SF Forceps (Fine Science Tools, catalog number: 11252-00 )
  8. Cotton swab for absorbing the ringer’s solution
  9. Vacuum pump (e.g., AGC TECHNO GLASS CO., model: VPUMP-140 ) for removing the ringer's solution
  10. Surgical blade (e.g., Kai industries, catalog number: 310-A )
  11. Head-fixation plate (handmade, Figure 1A)
  12. Plate holder (custom-made, Figure 1B)
  13. Stereo Microscope (OLYMPUS, model: SZX7 )
  14. Green LED light (REVOX Inc., model: SLG-50S-G )
  15. Dental drill (SHOFU Inc., model: Tas-35LX )
  16. Dental round bur (SHOFU Inc., model: ELA Steel Bur HP-1 )
  17. Lens blower (e.g., HAKUBA, model: KMC-45 )
  18. Ultra-Fast CMOS Imaging System (Brainvision, model: MiCAM ULtima )
    Note: For reference imaging (see procedure step 13), the cortex is illuminated with a blue LED light (center wavelength: 465 nm) through a 506-nm dichroic mirror; green fluorescence is corrected through a 536/40-nm filter.
    For voltage-sensitive dye imaging, VSD fluorescence is excited with a red LED light (center wavelength: 625 nm). The excitation light is filtered with a 632/22-nm band-pass filter, reflected using a 655-nm dichroic mirror, and focused 375 μm below the cortical surface. Fluorescence is filtered with a 665-nm long pass filter.
  19. Blue LED light (Brainvision, model: LEX2-B )
  20. Red LED light (REVOX Inc., model: SLG-50S-R )
  21. Stimulator (e.g., NIHON KOHDEN CORPORATION, model: SEN-5201 )
  22. Metal electrodes for hindpaw stimulation (handmade, Figure 1C)
  23. Vortex mixer (e.g., Scientific Industries Inc., model: VORTEX-GENIE 2 )

    Figure 1. Head-fixation equipment. A. Handmade head-fixation plate; B. Custom-made plate holder; C. Handmade metal electrodes for hindpaw stimulation.


  1. Acquisition software (Brainvision, model: UL-Acq)
  2. Analysis software (Brainvision, model: BV_Ana)


All animal experiments were performed in accordance with institutional guidelines and were approved by the Animal Experiment Committee from the RIKEN BSI.

  1. Preparation for surgery (timing, 60-70 min)
    1. Anesthetize the mouse with isoflurane (1-2%, vol/vol in air).
    2. Cut the hair on the mouse’s head with an electric clipper.
    3. Place the mouse onto a heating pad (37 °C), temporarily stabilize the head with ear bars of a head holder, and insert the nostril into a tube for anesthetizing (Figure 2A). We did not use the nose clamp of the head holder.
    4. Using scissors, remove the skin (about 1 cm in diameter) covering the skull of both hemispheres (Figure 2B).
    5. Apply lidocaine solution to the wound.
      Note: Do not apply lidocaine to the brain because it blocks the voltage-gated sodium channels and will suppress the VSD response.
    6. Gently remove the periosteum with a surgical blade by scraping the skull (Figure 2B). This will help the glue adhere to the bone.
    7. Glue a head-fixation plate onto the exposed skull with dental cement (Super-Bond C&B) (Figure 2C). Do not allow any gaps between the skull and cement to prevent solution leakage.
    8. After drying out the dental cement (about 10 min), transfer the mouse to a plate holder, fix the head-fixation plate in the plate holder, and maintain body temperature at 37 °C with the heating pad (Figure 2D).
    9. Make a chamber by heavily coating and piling up dental cement (UNIFAST II) as a bank (about 1 or 2 mm in height) around the head-fixation plate hole (Figure 2E). This chamber will be used for staining by filling the chamber with voltage-sensitive dye (VSD) solution (step D15).

      Figure 2. Experimental preparation for the craniotomy. A. The hair on the head has been cut. B. The skin and periosteum have been removed. C. The head-fixation plate has been attached onto the exposed skull. D. The mouse has been transferred to a plate holder, and the head-fixation plate has been fixed. E. The chamber has been created with dental cement onto the head-fixation plate.

  2. Craniotomy (timing, 20-40 min)
    1. Perform a craniotomy over a large portion of the right hemisphere (details are shown below). During the craniotomy, a green LED light source (center wavelength: 525 nm) can help visualize blood vessels to avoid surgical damage. We did not control brain swelling.
      1. Using a dental drill (head diameter: 0.8 mm), make a groove by scraping the skull and an island in the right hemisphere (Figure 3A).
        Note: To prevent the heat damage generated by drilling, you should not drill the skull at the same spot continuously (5 sec maximum). It is helpful to cool down the drilled area with Ringer’s solution or by using an air blower.
        Note: The groove does not penetrate the skull at this step.
      2. Check the grooved skull thickness by gently pushing on the island with the fine forceps.
      3. If the island moves up and down when lightly pushing, carefully scratch the groove with a fine needle in order to cut the grooved skull.
      4. Filling the chamber with a ringer’s solution for wetting the skull, which allows us to easily remove the island from the skull.
      5. Remove the island from the rest of the skull using the fine forceps (Figure 3B and 3D).
        Note: Do not touch the brain, and leave the dura matter intact to avoid damage. When the cortical blood vessels are damaged, you can see the blood spread under the dura matter. In this case, you should prepare a new mouse and repeat the procedure.
      6. Gently wash the exposed cortex with the ringer’s solution until the superficial cortical vessels become clearly visible (Figure 3C).
      7. If the blood on the dura mater cannot be removed with the wash, gently dry the exposed dura with a lens blower and repeat steps B10f-g until clear (Figure 3C).
        Note: This procedure is helpful for stopping the bleeding.

        Figure 3. Craniotomy for wide-field cortical VSD imaging. A. A grooved skull (right hemisphere). B. The superficial blood vessels immediately after the craniotomy. C. The blood vessels after washing. D. The removed skull.

    2. Transfer the mouse to a recording setup (an epi-fluorescent microscope) with the heating pad, fix the head-fixation plate, and reduce isoflurane concentration to 1.0% (vol/vol in air) for anesthetized experiments. For experiments with awake animals, the following steps are performed without anesthesia.
      Note: Heart rate should be monitored and the anesthesia level should be controlled to prevent unforeseen death and to get a good response.

  3. Finding a region of interest (ROI) for imaging (timing, 10 min)
    1. Fill the chamber with the ringer’s solution, and place a thin cover glass over the solution.
    2. Search an ROI with the fluorescent microscope and capture the ROI image before VSD imaging. This is because VSD staining during the next step darkens the cortex (Figure 4), and researchers are unable to precisely create an activity map on the cortex.
      Note: Positions of superficial blood vessels on the cortex can be a reference for the response area (Figure 5).

  4. VSD staining (timing, 2.5 h-3 h)
    1. Remove the cover glass, and suck the ringer’s solution out of the chamber.
    2. Fill the chamber with VSD solution, and place a thin cover glass over the chamber to avoid drying (Figure 4A).
    3. Overlay an aluminum sheath onto the chamber to keep the chamber out of the light (Figure 4B).
      Note: Be careful not to allow air bubbles to enter the VSD solution to prevent irregular staining.
    4. Apply the VSD solution for 90 min so that the dye solution reaches the cortex through the dura matter.
    5. Remove the VSD solution, and rinse the chamber every 15 min with a fresh ringer’s solution to remove unbound dye until the solution becomes clear (60-90 min).
    6. After washing, fill the chamber with the ringer’s solution, and place a thin cover glass over the solution (Figure 4C).

      Figure 4. VSD staining. A. The chamber filled with VSD solution was covered with a thin cover glass. B. The chamber was covered by an aluminum sheath. C. After washing the cortex, the craniotomy is filled with the ringer’s solution and covered with a thin cover glass.

  5. Cortical VSD imaging (timing, depending on your experiment)
    1. Record cortical activity (Figure 5A). Electrically stimulate the contralateral hindpaw 3 sec after starting the imaging, and monitor the VSD responses for 4 sec (3 sec before stimulation and 1 sec after stimulation) at every imaging (Figure 5B). By referencing the positions of blood vessels captured beforehand (step C13; Figure 5C), overlay the cortical activity map and vessel positions in order to identify a functional cortical map (Figure 5D).

      Figure 5. Example of VSD imaging under anesthesia. A. Experimental configuration during the VSD imaging under anesthesia. B. Spatiotemporal dynamics of cortical VSD responses evoked by contralateral hindpaw stimulation (single pulse, 0.1 ms duration, 100 V) under anesthesia. The time post-stimulation is indicated. Images were collected with 2-ms temporal and 80-μm spatial resolution. The letters A and P denote anterior and posterior directions, respectively. A dot indicates the bregma location. C. The superficial blood vessels in the imaging area. D. Overlaid image.


To prevent phototoxicity of the dye to neurons, the intensity of the excitation light and exposure time should be minimized. We could obtain stable imaging for 2 h when we use 4 sec exposure time every 40 sec. Proper washing of the unbinding dye is helpful in improving the single-to-noise ratio. However, excessive washing decreases stable imaging time.


  1. Ringer’s solution (pH 7.2 with NaOH)
    Dissolve 7.89 g NaCl (135 mM), 0.40 g KCl (5.4 mM), 0.20 g MgCl2.6H2O (1 mM), 0.26 g CaCl2.2H2O (1.8 mM), and 1.19 g HEPES (5 mM) in distilled water, for a total volume of 1 L
    Stored at 4 °C
    Warm to body temperature before use
  2. Voltage-sensitive dye (VSD) solution (2 mg/ml)
    Dissolve 10 mg RH1691 in the 5-ml ringer’s solution, and mix with a vortex mixer
    Stored at -20 °C, and keep in the dark
    Warm to body temperature, and mix with a vortex mixer before use


This protocol was adapted from Manita et al. (2015). The authors acknowledge support from a Grant-in-Aid for Young Scientists (A) from the JSPS (Japan Society for the Promotion of Science), the Uehara Memorial Foundation, and the Japan Prize Foundation to M. M.; from a Grant-in-Aid for Challenging Exploratory Research from the JSPS to T. S.


  1. Manita, S., Suzuki, T., Homma, C., Matsumoto, T., Odagawa, M., Yamada, K., Ota, K., Matsubara, C., Inutsuka, A., Sato, M., Ohkura, M., Yamanaka, A., Yanagawa, Y., Nakai, J., Hayashi, Y., Larkum, M. E. and Murayama, M. (2015). A top-down cortical circuit for accurate sensory perception. Neuron 86(5): 1304-1316.


皮层皮层相互作用在各种大脑功能中起关键作用。 在这里,我们提供皮质电压敏感染料(VSD)成像允许监测活体小鼠皮质活动的时空动力学的详细的外科手术。 上层(层1-3)中的皮层神经元用VSD染色,并且具有快速采样率(500Hz)的图像传感器检测校正活性中的荧光变化。 该程序包括将小鼠脑固定到立体定位装置,在大皮质区域上的颅骨切开术,VSD染色和皮层活动的宽视野成像。 整个过程可以在5小时内完成(从麻醉的施用到皮质VSD成像的开始)。

关键字:电压敏感染料成像, 开颅手术, 皮层活动, 宽视场成像


  1. 细针(Bonn Micro Probes)(Fine Science Tools,目录号:10030-13)
  2. 盖玻璃(Matsunami Glass Ind,目录号:C024241)
  3. 野生型小鼠(日本SLC,型号:C57BL/6JJmsSlc)
  4. 异氟烷(例如,Wako Pure Chemical Industries,目录号:099-06571)
  5. 用于局部麻醉的利多卡因溶液(80mg/ml)(例如Xylocaine Pump Spray 8%)(AstraZeneca)
  6. 牙科用水泥(Super-Bond C& B)(Sun medical)(GC's Global,UNIFAST II)
  7. 电压敏感染料(OPTICAL IMAGING LTD,目录号:RH1691)
  8. NaCl
  9. KCl
  10. MgCl 2 6H <2> O
  11. CaCl 2 2H O
  12. HEPES
  13. 蒸馏水
  14. RH1691
  15. 林格的解决方案(参见配方)
  16. 电压敏感染料(VSD)溶液(见配方)


  1. 异葎草的麻醉系统(Shinano,目录号:SN-487-0T)
  2. 用于切割鼠标毛的电动剪刀( Panasonic Corporation,目录号:ER803P)
  3. 反馈控制加热垫(Bio Research Center,目录号:BWT-100A)
  4. 头支架(立体定位装置)(NARISHIGE Group,型号:SG-4N)
  5. 精剪刀(Fine Science Tools,目录号:91460-11)
  6. 学生Dumont#7镊子(Fine Science Tools,目录号:91197-00)
  7. Dumont#5SF镊子(Fine Science Tools,目录号:11252-00)
  8. 棉签用于吸收戒指的溶液
  9. 用于去除振铃溶液的真空泵(例如AGC TECHNO GLASS CO。,型号:VPUMP-140)
  10. 手术刀(例如:Kai industries,目录号:310-A)
  11. 头固定板(手工制作,图1A)
  12. 板架(定制,图1B)
  13. 立体显微镜(OLYMPUS,型号:SZX7)
  14. 绿色LED灯(REVOX Inc.,型号:SLG-50S-G)
  15. 牙钻(SHOFU Inc.,型号:Tas-35LX)
  16. 牙轮钻(SHOFU Inc.,型号:ELA Steel Bur HP-1)
  17. 镜头鼓风机(例如HAKUBA,型号:KMC-45)
  18. 超快CMOS成像系统(Brainvision,型号:MiCAM ULtima)
  19. 蓝色LED灯(Brainvision,型号:LEX2-B)
  20. 红色LED灯(REVOX Inc.,型号:SLG-50S-R)
  21. 刺激器(例如,NIHON KOHDEN CORPORATION,型号:SEN-5201)
  22. 用于后爪刺激的金属电极(手工制作,图1C)
  23. 涡旋混合器(例如Scientific Industries Inc.,型号:VORTEX-GENIE 2)

    图1.头部固定设备。 A.手工头部固定板; B.定制板架; C.用于后爪刺激的手工金属电极


  1. 采集软件(Brainvision,型号:UL-Acq)
  2. 分析软件(Brainvision,型号:BV_Ana)


所有动物实验根据机构指导进行,并由来自RIKEN BSI的动物实验委员会批准。

  1. 手术准备(时间,60-70分钟)
    1. 麻醉鼠标与异氟烷(1-2%,vol/vol在空气中)
    2. 用电动剪刀剪头发上的头发。
    3. 将鼠标放在加热垫(37°C),暂时稳定 头部带有头部支架的耳杆,并将鼻孔插入 麻醉管(图2A)。我们没有使用鼻夹 ?头架。
    4. 使用剪刀,去除覆盖两个半球头骨的皮肤(直径约1厘米)(图2B)。
    5. 将利多卡因溶液应用于伤口。
      注意:不要将利多卡因应用于大脑,因为它阻断了 电压门控钠通道并抑制VSD反应。
    6. 用刮刀轻轻取出骨膜 颅骨(图2B)。这将有助于胶水粘在骨头上。
    7. 用牙科骨水泥将头部固定板粘在暴露的颅骨上 (Super-Bond C& B)(图2C)。不要让任何间隙 颅骨和水泥,以防止溶液泄漏
    8. 干燥后 牙齿粘固剂(约10分钟),将小鼠转移到板夹, 将头部固定板固定在板支架上,并保持身体 用加热垫在37℃的温度下(图2D)
    9. 做一个 通过大量涂布和堆积牙科水泥(UNIFAST II)作为一个 ?围绕头部固定板孔排列(高度约1或2mm) (图2E)。该室将通过填充用于染色 腔室与电压敏感染料(VSD)溶液(步骤D15)

      图 ?2.开颅手术的实验准备。 A.头发在 头已被切。 B.除去皮肤和骨膜。 C.的 头部固定板已经附接到暴露的头骨上。 D.的 小鼠已转移到板支架,并进行头部固定 板已被固定。 E.腔室已经用牙科水泥制成 ?到头部固定板上。

  2. 切开术(时间,20-40分钟)
    1. 在右半球的大部分上进行开颅手术 (详情如下所示)。在开颅手术期间,绿色LED灯 源(中心波长:525nm)可以帮助使血管可视化 避免手术损伤。我们没有控制脑肿胀。
      1. 使用a ?牙钻(头直径:0.8mm),通过刮削形成凹槽 头骨和一个岛在右半球(图3A)。
        注意: 防止钻孔产生的热损伤,你不应该钻孔 颅骨连续在同一地点(最大5秒)。这是有帮助的 使用林格氏溶液或通过使用空气冷却钻孔区域 鼓风机。
      2. 通过用细镊子轻轻地推动岛上来检查沟槽的头骨厚度
      3. 如果岛上轻轻推动时小心上下移动 用细针头划伤凹槽,以便切割带槽的头骨。
      4. 填充室用一个ringer的解决方案润湿 头骨,这使我们能够很容易地从头骨去除岛
      5. 使用细镊子从颅骨的其余部分去除岛(图3B和3D)。
        注意:不要触摸大脑,并保持硬脑膜完整,以避免 ?损伤。当皮质血管损伤时,您可以看到 血液在硬膜物质下传播。在这种情况下,你应该准备一份 新鼠标并重复此过程。
      6. 轻轻洗净暴露 皮质与振荡器的解决方案,直到表面皮层血管 ?变得清晰可见(图3C)
      7. 如果硬脑膜上的血液 不能用洗涤去除,轻轻干燥暴露的硬脑膜 ?镜头鼓风机,并重复步骤B10f-g,直到清除(图3C) 注意:此程序有助于止血。

        图3.宽视场皮质VSD成像的切开术。A.有凹槽的 头骨(右半球)。 B.表层血管立即 开颅后。 C.洗涤后的血管。 D.删除 ?头骨。

    2. 将鼠标传输到录音设置( epi荧光显微镜)与加热垫,固定头固定 ,并将异氟烷浓度降至1.0%(在空气中的体积/体积) 麻醉实验。对于清醒动物的实验 以下步骤不进行麻醉 注意:心率 应进行监测,麻醉水平应控制在 防止意外死亡和获得良好的反应。

  3. 找到感兴趣区域(ROI)进行成像(时间,10分钟)
    1. 给振动室注入振铃溶液,并在溶液上放置一层薄的玻璃盖
    2. 用荧光显微镜搜索ROI并捕获ROI VSD成像前的图像。这是因为VSD在下一次染色 步骤使皮质变暗(图4),研究人员不能 精确地在皮质上创建活动图。

  4. VSD染色(定时,2.5小时-3小时)
    1. 取下盖玻片,并将振铃器溶液从腔室中吸出
    2. 用VSD溶液填充室,并在室上放置薄的盖玻片以避免干燥(图4A)
    3. 将铝护套覆盖在腔室上以保持腔室不受光照(图4B)。
    4. 应用VSD溶液90分钟,以便染料溶液通过硬脑膜物质到达皮质。
    5. 取出VSD溶液,并每15分钟用一次冲洗室 新鲜的ringer溶液除去未结合的染料,直到溶液变成 ?透明(60-90分钟)。
    6. 洗涤后,用振荡器溶液填充室,并在溶液上放置薄的盖玻片(图4C)。

      图4.VSD染色。A.填充有VSD溶液的室 盖用一个薄的盖玻璃。 B.房间被一个 铝护套。 C.清洗皮质后,开颅手术 与振动器的解决方案和覆盖着薄盖玻片。

  5. 皮层VSD成像(时间,取决于您的实验)
    1. 记录皮层活动(图5A)。电刺激 对侧后爪3秒后开始成像,并监测 VSD响应4秒(刺激前3秒和刺激后1秒 刺激)(图5B)。通过引用位置 的血管(步骤C13;图5C),重叠 皮层活动图和血管位置,以便识别 功能皮质图(图5D)。

      图5. VSD的示例 麻醉下的成像 A.在VSD期间的实验配置 麻醉下成像。 B.皮层VSD的时空动力学 通过对侧后爪刺激诱发的反应(单脉冲,0.1 ?ms持续时间,100V)。刺激后的时间是 表示。以2-ms时间和80-μm空间收集图像 解析度。字母A和P表示前和后 方向。点表示前囟位置。 C.的 表面血管。 D.重叠图像。




  1. 林格氏溶液(pH7.2,NaOH)
    将7.89g NaCl(135mM),0.40g KCl(5.4mM),0.20g MgCl 2 .6 6H 2 O(1mM)溶解, ,0.26g CaCl 2·2H 2·2H 2 O(1.8mM)和1.19g HEPES(5mM)的蒸馏水溶液,总共音量1 L
  2. 电压敏感染料(VSD)溶液(2mg/ml)
    将10毫克RH1691溶于5毫升振荡溶液中,并用涡流混合器混合 储存于-20°C,保持阴暗


该协议改编自Manita等人(2015)。作者确认来自JSPS(日本科学促进会),上原纪念基金会和日本奖基金会的来自青年科学家的助学金(A)的支持。从用于挑战探索研究的Grant-in-Aid从JSPS到T. S.


  1. Manita,S.,Suzuki,T.,Homma,C.,Matsumoto,T.,Odagawa,M.,Yamada,K.,Ota,K.,Matsubara,C.,Inutsuka,A.,Sato, Ohkura,M.,Yamanaka,A.,Yanagawa,Y.,Nakai,J.,Hayashi,Y.,Larkum,MEand Murayama,M.(2015)。 从自上而下的皮层回路获得准确的感官知觉。 Neuron 86(5):1304-1316。
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引用:Suzuki, T. and Murayama, M. (2016). Craniotomy for Cortical Voltage-sensitive Dye Imaging in Mice. Bio-protocol 6(3): e1722. DOI: 10.21769/BioProtoc.1722.