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Apr 2019
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Vestibular Organ Dissection and Whole-Mount Immunolabeling in Mouse
小鼠前庭器官解剖和整体免疫标记   

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Abstract

The vestibular sensory apparatus contained in the inner ear is a marvelous evolutionary adaptation for sensing movement in 3 dimensions and is essential for an animal’s sense of orientation in space, head movement, and balance. Damage to these systems through injury or disease can lead to vertigo, Meniere’s disease, and other disorders that are profoundly debilitating. One challenge in studying vestibular organs is their location within the boney inner ear and their small size, especially in mice, which have become an advantageous mammalian model. This protocol describes the dissection procedure of the five vestibular organs from the inner ear of adult mice, followed by immunohistochemical labeling of a whole mount preparation using antibodies to label endogenous proteins such as calretinin to label Type I hair cells or to amplify genetically expressed fluorescent proteins for confocal microscopic imaging. Using typical lab equipment and reagents, a patient technician, student, or postdoc can learn to dissect and immunolabel mouse vestibular organs to investigate their structure in health and disease.

Keywords: Inner ear (内耳), Vestibular (前庭), Hair cells (毛发细胞), Dissection (解剖), Immunohistochemistry (免疫组化), Whole mount (整装)

Background

The vestibular system senses head movements by decomposing motion into different angular and linear directions. The three semicircular canals are oriented along horizontal and vertical planes with respect to the head and sense angular movements along their axes. The two otolith organs, the utricle and sacculus, are oriented roughly horizontally and vertically and sense linear movements in those directions. Each of the five sensory structures contain a neuroepithelium. In the base of each semicircular canal, a swelling called the crista ampularis houses the neuroepithelium. The neuroepithelium of the otolith organs are called maculae. A more general term that describes vestibular neuroepithelia is ‘end organ’. Each end organ contains hair cells whose stereocilia are embedded in a structure that is deflected by the movement of endolymph. Sensory transduction occurs at hair cells that are similar to those in the auditory system. Hair cells are mechanosensitive and release graded amounts of glutamate onto the peripheral afferents of the vestibular ganglion cells depending on the deflection of their stereocilia. The graded signal is converted to a spike rate signal by the peripheral afferents that carry it to the brain via their central-projecting axons that make up the vestibular division of the vestibulocochlear nerve. A comprehensive resource for the structure and function of the vestibular system can be found in Goldberg et al. (2012).


Immunolabeling can be used to identify specific hair cell types, such as calretinin-expressing Type I hair cells, or to localize proteins involved in mechanotransduction, cell structure, or vestibular disorders. Many publications present exemplary micrographs of whole-mounted vestibular end organs from adult mice (Li et al., 2008; Lysakowski et al., 2011; Golub et al., 2012; Spitzmaul et al., 2013; Krey et al., 2015; Bucks et al., 2017; Hoffman et al., 2018; Yoshimura et al., 2018). The purpose of this protocol is to describe the procedure in sufficient detail that others may be encouraged to study the anatomy of the vestibular system. The procedure described here was used to dissect, label, and image vestibular organs reported in Balmer and Trussell (2019).

Materials and Reagents

  1. SecureSeal imaging spacer (Grace Bio-Labs, catalog number: SS8X9)

  2. Superfrost Plus microscope slides (Fisher Scientific, catalog number: 12-550-15)

  3. Coverslips #1.5 (Fisher Scientific, catalog number: 12-544-DP)

  4. CFM-3 mounting media (Citiflour, catalog number: CFM-3)

  5. 35 mm culture dishes (Thermo Scientific, catalog number: 171099)

  6. Paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15700)

  7. Heparin (Sigma, catalog number: H5515)

  8. Na2HPO4 (Sigma, catalog number: S0876)

  9. NaH2PO4 (Sigma, catalog number: S5011)

  10. NaCl (Sigma, catalog number: S9888)

  11. Phosphate buffered saline (PBS) (Growcells, catalog number: MRGF-6235)

  12. Bovine serum albumin (BSA) used to coat typical 200 µL pipette tips (Sigma, catalog number: A7906)

  13. Triton X-100 (Sigma, catalog number: X100)

  14. Normal Donkey Serum (NDS) (Jackson ImmunoResearch, catalog number: 017-000-121)

  15. Sylgard 184 (Fisher Scientific, catalog number: 50-366-794)

  16. Heparinized saline (see Recipes)

  17. 4% PFA in 0.1 M PB (see Recipes)

Equipment

  1. Dissection microscope (Zeiss, model: Stemi 305)

  2. Fiber light (World Precision Instruments, catalog number: Z-LITE-186)

  3. Water bath (Fisher Scientific, catalog number: S28124)

  4. Fine forceps (Fine Science Tools, catalog number: 11254-20)

  5. Fine curved forceps (Fine Science Tools, catalog number: 11272-30)

  6. Laminectomy forceps (Fine Science Tools, catalog number: 11223-20)

  7. Spring scissors (Fine Science Tools, catalog number: 15000-08)

  8. Large scissors (Fine Science Tools, catalog number: 14101-14)

  9. Large forceps (Fine Science Tools, catalog number: 11000-12)

Procedure

  1. Transcardiac perfusion

    1. Perform transcardiac perfusion with heparinized saline warmed to 37°C (see Recipe 1) until the fluid coming out of the heart runs almost clear, ~6 min.

    2. Perfuse with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) cooled to 4°C (on ice; see Recipe 2). Perfuse ~30 mL through heart.

      Note: In this protocol, this perfusion is the only fixation of the vestibular organs, so it must be done successfully. We have not found post-fixation to be necessary and were concerned that it may reduce the antigenicity of proteins during immunohistochemistry.


  2. Brain and temporal bone extraction

    1. Decapitate the mouse with large sharp scissors.

    2. Using small spring scissors and forceps, carefully remove the top of the skull to expose the brain (Figure 1A–B).

    3. Tip the brain forward and use small spring scissors to cut the vestibulocochlear nerves close to where they enter the brain (Figure 1C–D). The vestibular ganglion lies in the temporal bone at the base of the nerve, so take care not to damage it.

      Note: Put the brain in 4% PFA in PBS, if retaining for histology.

    4. The temporal bones (bulla) lie along the base of the skull on each side. Use scissors to cut them out of the base of the skull and place them PBS (Figure 1E–F).



      Figure 1. Brain and temporal bone extraction.

      A) Cuts are made along the skull sutures. B) Skull plates above the brain are removed. C) The vestibulocochlear nerve can be visualized when the skull below the brain is peeled downward. D) The vestibulocochlear nerve is cut where it enters the inner ear. CN- Cochlear Nucleus. PF- Paraflocculus. E) The temporal bones are revealed (outlined) when the brain is tipped forward and the trigeminal nerves are cut. F) Extracted temporal bones. * indicate the vestibulocochlear nerves.


  3. Vestibular organ dissection

    1. Place the bulla in a Sylgard 184-coated culture dish filled with PBS.

      Lighting the preparation with a fiber light at various oblique angles can improve the contrast of the tissue in the dish by creating shadows.

    2. Use large forceps to grip the entire bulla and hold it in place against the soft Sylgard on the bottom of the dish (Figure 2A).

      The Sylgard prevents the bulla from sliding. Optional: A permanent marker (sharpie) can be used to darken the outside of the dish to contrast it with the light-colored end organs and bone, or an inert dye, such as India ink, can be added to the Sylgard during preparation of the dishes.

    3. With your other hand, use fine forceps or a needle to remove the vestibular ganglion in one piece where it sits between the cochlea and labyrinths (Figure 1F). It may be difficult to tell where the vestibular ganglion is on the nerve, so it is advantageous to take out the whole nerve. Place the vestibular ganglion in a labeled 48-well plate, using a BSA-coated 200 µL pipette:

      1. The nerve may separate into two parts, presumably the auditory and vestibular divisions. Care must be taken when removing the nerve and vestibular ganglion to avoid damaging the utricle, which is attached to the base of the nerve. Do not insert the forceps too deep into where the nerve comes through the bone, as they might damage the utricle.

      2. Use of BSA-coated 200 µL pipette: Withdraw ~100 µL of PBS with the nerve into the pipette tip. It is optimal to keep the nerve (or end organ) suspended in solution in the pipette. It is more likely to stick to the inside of the pipette tip if it is floating at the top of the column of fluid in the tip.

      Prepare 10% BSA in PBS, cut the ends off the tips to make the opening large enough to accommodate the vestibular organs (~2 mm), pipette the 10% BSA into the pipette and out several times, and then eject and let dry. Store in a petri dish to prevent dust accumulation.

    4. Pigmented melanocytes that overlie the utricle can be visualized through the bone, which can help identify its position. Use laminectomy forceps or other stout forceps to break off small pieces of this bone, which will reveal the three vestibular end organs that are joined together- the utricle, horizontal canal crista and anterior canal crista. These may be removed together in one piece (Figure 2B–C).

    5. On the other side of the area from which the vestibular ganglion was removed, the vertical canal end organ can be removed in a similar manner, by carefully removing the overlying bone with laminectomy forceps and then sliding fine forceps underneath it to sever the nerve and detach it from the bone (Figure 2D).

    6. The sacculus is on the other side of the temporal bone near the cochlea and has no melanocytes, so it is difficult to see (Figure 2E). It needs to be scraped from the bone more than the other end organs, which makes the sacculus the most difficult to remove without damage.

    7. Put all the end organs in PBS as they are removed.

      1. It may be useful to have a picture of the vestibular system available to clarify the locations of the end organs. Keep in mind that the structures of the left and right ears are mirror images.

      2. Practice is required to reach the skill level required to dissect all five vestibular organs without damage or loss. After becoming skilled at this procedure, time and patience remain necessary, as a careful dissection of each ear may require 30–60 min.

      3. Best results are obtained when the dissection is followed by immunohistochemical processing on the same day.

      4. In addition, the cochlea can be similarly removed in a few sections and processed following these guidelines.



      Figure 2. Vestibular organ dissection.

      A) The temporal bone grasped with forceps. Acronyms for the five vestibular end-organs are positioned over their locations. UT- Utricle. SA- Sacculus. AC- Anterior Canal. HC- Horizontal Canal. VC- Vertical Canal. B) After removal of some of the overlying bone, four of the vestibular end organs are revealed. C) Utricle and attached anterior canal and horizontal canal cristae. D) Dissected vertical canal crista. E) Dissected sacculus. F) Applying a coverslip to the vestibular organs positioned within the spacer.


  4. Immunohistochemistry of vestibular end organs

    1. To transfer the end organs during rinses, etc., use a BSA-coated 200 µL pipette tip, as described above. Transfer the end organs to a different well in the 48-well plate containing the new solution (at least 200 µL in a 48-well plate).

      1. Moving all of the end organs down one row may help keep them organized.

      2. The previous solution that is transferred with the end organ in the pipette should be as small in volume as possible. A gentle flick may be necessary to cause the end organ to fall to the bottom of the column of solution that is suspended in the pipette tip and can then be ejected with minimal fluid.

    2. Permeabilize and block in 2% Triton X-100, 5% NDS 1 h room temperature on an orbital shaker.

      1. This high concentration of detergent may be necessary for optimal staining of this preparation.

    3. Remove the end organs from the permeabilization and blocking buffer and place in primary antibody diluted in 5% NDS. Incubate in primary antibody overnight on an orbital shaker at 4 °C (Table 1). Incubation for up to 3 days may enhance antibody penetration.

      1. We avoid rinsing between these steps as each transfer of the end organs represents a risk of damage or loss.


        Table 1. Primary antibodies

        Antibody Source Concentration
        Rabbit polyclonal anti-calretinin Swant, 7697 1:500
        Chicken polyclonal anti-GFP Aves Labs, GFP-1020 1:500
        Goat polyclonal anti-mCherry Sicgen, AB0040-200 1:500


    4. Rinse 3 × 10 min in PBS by removing end organs from primary and placing into a different well in the 48-well plate containing fresh PBS.

    5. Transfer end organs into secondary antibodies diluted in 5% NDS and incubate overnight 4°C on orbital shaker (Table 2).


      Table 2. Secondary antibodies

      Antibody Source Concentration
      Donkey polyclonal anti-rabbit Cy3 Jackson ImmunoResearch Labs, 711-165-152 1:500
      Donkey polyclonal anti-chicken Alexa Fluor 488 Jackson ImmunoResearch Labs, 703-545-155 1:500
      Donkey polyclonal anti-goat Cy3 Jackson ImmunoResearch Labs, 705-165-147 1:500


    6. Rinse 3 × 10 min in PBS by removing end organs from primary and placing into a different well in the 48-well plate containing fresh PBS.


  5. Mounting and coverslipping

    1. Place an 8-well 120 µm thick spacer onto a microscope slide (SecureSeal): First remove the plastic side and put the spacer on the slide. Flatten it down by running a flat object along it, taking care to not contaminate the glass of the microscope slide. Then, remove the paper side. It will be sticky from now until the coverslip is placed over it, so be extremely careful to avoid accidentally touching it. In our experience, this is preferred over trying to remove the paper and expose the adhesive after laying out all the end organs as their positioning is easily disrupted. Alternatively, each end organ can be placed on a separate slide with a single well spacer.

    2. Place one end organ on the slide within the spacer in a pool of PBS. Using fine forceps, remove the melanocytes or any membranes that would be between the coverslip and the face of the end organ. The canal cristae typically lie on their side, which is optimal for imaging. The utricle and sacculus can be laid such that the nerve is against the microscope slide with the hair cell side pointing upward toward the coverslip. This step is critical for good imaging. Apply more PBS so that it does not dry out while mounting the others, but not so much that it is free to float away from the microscope slide. Repeat for each end organ and the vestibular ganglion or nerve in separate wells of the spacer.

    3. Ensure that all the end organs are oriented properly, and then remove excess PBS with a normal 200 µL pipette tip (unmodified). Let it dry slightly so that they stick to the positively charged Superfrost Plus microscope slides.

    4. Apply a generous amount of CFM-3 mounting media to each well. Apply coverslip to the adhesive SecureSeal spacer (Figure 2F).

      Notes:

      1. It is far better for excess media to flow out when applying the coverslip than for air bubbles to be trapped in the wells; apply ample mountant.

      2. Note that repositioning of the coverslip is nearly impossible due to the adhesive face of the SecureSeal spacer, so apply it carefully.


  6. Confocal imaging

    1. This preparation can be imaged with typical confocal imaging described elsewhere. Long-working distance objectives may be necessary to image through the entire end organ and underlying nerve. We used a Zeiss 25× 0.8 NA oil immersion objective on a Zeiss LSM 880 for full end organ imaging (Figure 3). For imaging of smaller areas, we used a Zeiss 63× 1.4 NA oil immersion objective (Figure 4).



      Figure 3. Vestibular end organ whole mounts imaged with 25× objective.

      A) The utricular macula, anterior canal crista, and horizontal canal crista can be dissected out of the temporal bone in one piece. Calretinin (magenta) is expressed in Type I hair cells. Single nerve fibers are labeled with green fluorescent protein (GFP) that has been amplified with Alexa Fluor 488. B-C) The vertical canal (B) and sacculus (C) are separated during the dissection and are therefore processed and mounted separately.



      Figure 4. Vestibular hair cells and peripheral vestibular afferents imaged with 63× objective.

      A) Single optical plane showing Myo7A (cyan), present in all hair cells; calretinin (magenta), present in Type I hair cells; GFP (green), expressed by an retrograde-AAV injected into the cerebellum and amplified with an anti-GFP antibody; and merged image showing that the retrolabeled peripheral afferent is a flask shape and surrounds a calretinin expressing hair cell. B) Maximum intensity projection of 24 µm thick image stack showing calretinin (magenta) and GFP (green).

Recipes

  1. Heparinized saline

    Add 3.6 g NaCl, 0.022 g heparin (calculated to be 10 U/mL final concentration), and 400 mL of dH2O

  2. 4% PFA in 0.1 M PB

    Add 10 mL of 16% PFA, 6.2 mL of 0.5 M Na2HPO4, 0.9 mL of 1 M NaH2PO4, and raise to 40 mL with dH2O

Acknowledgments

We thank Drs. Jocelyn Krey and Peter Barr-Gillespie for their expertise in this procedure and inner ear anatomy. Funding was provided by NIH F32 DC014878, NIH K99 DC016905, and Hearing Health Foundation Emerging Research Grants to TSB, NIH R01 NS028901, NIH RO1 DC004450 to LOT, and NIH P30 NS0618000 to S Aicher. This protocol was derived from Balmer and Trussell (2019).

Competing interests

The authors declare no competing interests.

Ethics

All procedures were approved by the Oregon Health and Science University’s Institutional Animal Care and Use Committee and met the recommendations of the Society for Neuroscience.

References

  1. Balmer, T. S. and Trussell, L. O. (2019). Selective targeting of unipolar brush cell subtypes by cerebellar mossy fibers. Elife 8: e44964.
  2. Bucks, S. A., Cox, B. C., Vlosich, B. A., Manning, J. P., Nguyen, T. B. and Stone, J. S. (2017). Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice. Elife 6: e18128.
  3. Goldberg, J. M., Wilson, V. J., Cullen, K. E., Angelaki, D. E., Broussard, D. M., Buttner-Ennever, J., Fukushima, K. and Minor, L. B. (2012). The Vestibular System: A Sixth Sense. New York: Oxford University Press. Available at: https://oxford.universitypressscholarship.com/10.1093/ acprof:oso/9780195167085.001.0001/acprof-9780195167085 [Accessed November 24, 2021].
  4. Golub, J. S., Tong, L., Ngyuen, T. B., Hume, C. R., Palmiter, R. D., Rubel, E. W. and Stone, J. S. (2012). Hair cell replacement in adult mouse utricles after targeted ablation of hair cells with diphtheria toxin. J Neurosci 32(43): 15093-15105.
  5. Hoffman, L. F., Choy, K. R., Sultemeier, D. R. and Simmons, D. D. (2018). Oncomodulin Expression Reveals New Insights into the Cellular Organization of the Murine Utricle Striola. J Assoc Res Otolaryngol 19(1): 33-51.
  6. Krey, J. F., Sherman, N. E., Jeffery, E. D., Choi, D. and Barr-Gillespie, P. G. (2015). The proteome of mouse vestibular hair bundles over development. Scientific Data 2(1): 150047.
  7. Li, A., Xue, J. and Peterson, E. H. (2008). Architecture of the mouse utricle: macular organization and hair bundle heights. J Neurophysiol 99(2): 718-733.
  8. Lysakowski, A., Gaboyard-Niay, S., Calin-Jageman, I., Chatlani, S., Price, S. D. and Eatock, R. A. (2011). Molecular microdomains in a sensory terminal, the vestibular calyx ending. J Neurosci 31(27): 10101-10114.
  9. Spitzmaul, G., Tolosa, L., Winkelman, B. H., Heidenreich, M., Frens, M. A., Chabbert, C., de Zeeuw, C. I. and Jentsch, T. J. (2013). Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models. J Biol Chem 288(13): 9334-9344.
  10. Yoshimura, H., Shibata, S. B., Ranum, P. T. and Smith, R. J. H. (2018). Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation. Sci Rep 8(1): 2980.

简介

内耳中包含的前庭感觉器官是一种奇妙的进化适应,用于感知 3 维运动,对于动物在空间、头部运动和平衡方面的方向感至关重要。受伤或疾病对这些系统的损害会导致眩晕、梅尼埃病和其他严重削弱身体的疾病。研究前庭器官的一个挑战是它们在骨内耳内的位置和它们的小尺寸,特别是在小鼠中,它们已成为一种有利的哺乳动物模型。该协议描述了成年小鼠内耳的五个前庭器官的解剖过程,然后使用抗体对整个安装制剂进行免疫组织化学标记,以标记内源性蛋白质,如钙调蛋白,以标记 I 型毛细胞或放大基因表达的荧光蛋白用于共聚焦显微成像。使用典型的实验室设备和试剂,患者技术人员、学生或博士后可以学习解剖和免疫标记小鼠前庭器官,以研究它们在健康和疾病中的结构。

背景

前庭系统通过将运动分解为不同的角度和线性方向来感知头部运动。三个半规管相对于头部沿水平和垂直平面定向,并沿其轴线感知角运动。两个耳石器官,椭圆囊和球囊,大致水平和垂直定向,并感知这些方向的线性运动。五个感觉结构中的每一个都包含一个神经上皮。在每个半规管的底部,称为壶腹的肿胀容纳神经上皮。耳石器官的神经上皮称为黄斑。描述前庭神经上皮的更通用术语是“末端器官”。每个末端器官都包含毛细胞,其静纤毛嵌入一个结构中,该结构会因内淋巴的运动而偏转。感觉转导发生在与听觉系统中的毛细胞相似的毛细胞上。毛细胞是机械敏感的,根据其静纤毛的偏转,将分级量的谷氨酸释放到前庭神经节细胞的外周传入神经上。分级信号被外周传入神经转换为尖峰率信号,这些传入神经通过构成前庭耳蜗神经前庭分裂的中央突出轴突将其传送到大脑。关于前庭系统结构和功能的综合资源可以在 Goldberg等人中找到。 (2012)。
免疫标记可用于识别特定的毛细胞类型,例如表达钙调蛋白的 I 型毛细胞,或定位参与机械转导、细胞结构或前庭疾病的蛋白质。许多出版物展示了成年小鼠前庭终末器官的示例性显微照片(Li等人,2008; Lysakowski 等。 , 2011;戈卢布等人。 , 2012;斯皮茨莫尔 等。 , 2013;克里 等。 , 2015;雄鹿等人。 , 2017;霍夫曼等人。 , 2018;吉村等人。 , 2018) 。该协议的目的是足够详细地描述该程序,以鼓励其他人研究前庭系统的解剖结构。此处描述的程序用于解剖、标记和成像 Balmer 和 Trussell (2019) 中报告的前庭器官。

关键字:内耳, 前庭, 毛发细胞, 解剖, 免疫组化, 整装

材料和试剂
1. SecureSeal成像垫片(Grace Bio-Labs,目录号: SS8X9)
2. Superfrost Plus 显微镜载玻片(Fisher Scientific,目录号: 12-550-15)
3. 盖玻片#1.5(Fisher Scientific,目录号: 12-544-DP)
4. CFM-3 封固剂( Citiflour ,目录号: CFM-3)
5. 35毫米培养皿(Thermo Scientific,目录号: 171099)
6. 多聚甲醛(PFA)(电子显微镜科学,目录号:15700)
7. 肝素(Sigma,目录号:H5515)
8. Na 2 HPO 4 (Sigma,目录号:S0876)
9. NaH 2 PO 4 (Sigma,目录号:S5011)
10. NaCl(Sigma,目录号:S9888)
11. 磷酸盐缓冲盐水(PBS)( Growcells ,目录号:MRGF-6235)
12. 牛血清白蛋白(BSA)用于涂覆典型的 200 µ L 移液器吸头(Sigma,目录号: A7906)
13. Triton X-100(Sigma,目录号: X100)
14. 正常驴血清(NDS)(Jackson ImmunoResearch ,目录号: 017-000-121)
15. Sylgard 184(Fisher Scientific,目录号: 50-366-794)
16. 肝素盐水(见食谱)
17. 0.1 M PB 中的 4% PFA(见食谱)




设备


1. 解剖显微镜(蔡司,型号: Stemi 305 )
2. 光纤(World Precision Instruments,目录号: Z-LITE-186)
3. 水浴(Fisher Scientific,目录号: S28124)
4. 精细镊子(Fine Science Tools,目录号: 11254-20)
5. 精细弯曲镊子(Fine Science Tools,目录号: 11272-3 0)
6. 椎板切除钳(Fine Science Tools,目录号: 11223-20)
7. 弹簧剪刀(Fine Science Tools,目录号: 15000-08)
8. 大剪刀(Fine Science Tools,目录号: 14101-14)
9. 大镊子(Fine Science Tools,目录号: 11000-12 )




程序


A. 经心灌注


1. 用加热至 37 ° C 的肝素盐水进行经心脏灌注(见配方 1),直到流出心脏的液体几乎清澈,约 6 分钟。
2. ° C的 0.1 M 磷酸盐缓冲液 (PB) 中注入 4% 多聚甲醛 (PFA) (在冰上;参见配方 2)。通过心脏灌注约 30 mL。
注意:在本协议中,这种灌注是前庭器官的唯一固定,因此必须成功完成。我们没有发现后固定是必要的,并且担心它可能会降低免疫组织化学过程中蛋白质的抗原性。


B. 脑和颞骨提取


1. 用锋利的大剪刀将鼠标斩首。
2. 使用小弹簧剪刀和镊子,小心地取出头骨顶部以暴露大脑(图 1A-B)。
3. 将大脑向前倾斜并使用小弹簧剪刀将前庭耳蜗神经切开靠近它们进入大脑的位置(图 1C-D)。前庭神经节位于神经基部的颞骨中,因此请注意不要损坏它。
注意:如果保留用于组织学,请将大脑放入 PBS 中的 4% PFA 中。
4. 颞骨(大泡)位于颅底两侧。用剪刀将它们从头骨底部剪下来,并将它们放置在 PBS(图 1E-F)中。


 


图 1. 脑和颞骨提取。
A) 沿着颅骨缝合线进行切割。 B) 大脑上方的颅骨板被移除。 C) 当大脑下方的头骨向下剥离时,可以看到前庭耳蜗神经。 D) 前庭耳蜗神经在进入内耳的位置被切断。 CN- 耳蜗核。 PF-副絮状体。 E) 当大脑向前倾斜并切断三叉神经时,颞骨显露(勾勒)。 F) 提取的颞骨。 *表示前庭蜗神经。


C. 前庭器官解剖


1. 将大泡放入装有 PBS 的Sylgard 184 涂层培养皿中。
以不同的倾斜角度用光纤照明制剂可以通过产生阴影来提高培养皿中组织的对比度。
2. 使用大镊子夹住整个大泡,并将其固定在盘子底部的软Sylgard上(图 2A)。
Sylgard可防止大泡滑动。可选:可以使用永久性记号笔(记号笔)使盘子的外部变暗,以使其与浅色的末端器官和骨骼形成对比,或者可以在制备过程中将惰性染料(例如印度墨水)添加到Sylgard盘子。
3. 用另一只手,使用细镊子或针头将位于耳蜗和迷路之间的前庭神经节取出(图 1F)。前庭神经节在神经上的位置可能很难分辨,因此取出整个神经是有利的。使用涂有 BSA 的 200 μL 移液器将前庭神经节置于标记的 48 孔板中:
a. 神经可能分为两部分,大概是听觉和前庭部分。移除神经和前庭神经节时必须小心,以免损坏连接到神经基部的椭圆囊。不要将镊子插入太深的神经穿过骨骼的位置,因为它们可能会损坏椭圆囊。
b. 使用涂有 BSA 的 200 μL 移液器:将约 100 μL 的 PBS 与神经一起抽入移液器尖端。最好将神经(或末端器官)悬浮在移液管中的溶液中。如果移液器吸头漂浮在吸头液柱的顶部,则它更有可能粘在吸头内部。
在 PBS 中制备 10% BSA,切掉尖端的末端,使开口足够大以容纳前庭器官(~2 毫米),将 10% BSA 移入移液器并移出数次,然后弹出并晾干。存放在培养皿中以防止灰尘积聚。
4. 可以通过骨骼看到覆盖在椭圆囊上的色素沉着的黑色素细胞,这可以帮助识别其位置。使用椎板切除钳或其他粗壮的钳子折断这块骨头的小块,这将露出三个连接在一起的前庭末端器官——椭圆囊、水平管嵴和前管嵴。这些可以一起移除(图 2B-C)。
5. 在切除前庭神经节的区域的另一侧,可以以类似的方式切除垂直管末端器官,方法是用椎板切除钳小心地切除覆盖的骨头,然后在其下方滑动细镊子以切断神经并分离它来自骨骼(图 2D)。
6. 球囊位于颞骨的另一侧,靠近耳蜗,没有黑色素细胞,因此很难看到(图 2E)。它比其他末端器官更需要从骨头上刮下来,这使得球囊最难在没有损伤的情况下去除。
7. 将所有末端器官放入 PBS 中,因为它们被移除。
a. 前庭系统的图片可用于阐明终末器官的位置,这可能是有用的。请记住,左右耳的结构是镜像。
b. 需要练习才能达到解剖所有五个前庭器官而不损坏或丢失所需的技能水平。在熟练掌握此过程后,仍然需要时间和耐心,因为仔细解剖每只耳朵可能需要30-60分钟。
c. 当解剖后在同一天进行免疫组织化学处理时,可获得最佳结果。
d. 此外,耳蜗可以类似地在几个部分中移除,并按照这些指南进行处理。




 


图 2.前庭器官解剖。 
A) 用镊子夹住颞骨。五个前庭末端器官的首字母缩写词位于它们的位置上。 UT-Utricle。 SA-球囊。 AC-前根管。 HC-水平运河。 VC-垂直运河。 B) 去除一些覆盖的骨头后,四个前庭末端器官显露出来。 C) Utricle 和附着的前管和水平管嵴。 D) 解剖的垂直管嵴。 E) 解剖球囊。 F) 将盖玻片应用于位于垫片内的前庭器官。




D. 前庭终末器官的免疫组织化学


1. 要在冲洗等过程中转移末端器官,请使用涂有 BSA 的 200 µL 移液器吸头,如上所述。将末端器官转移到含有新溶液的 48 孔板中的不同孔中(48 孔板中至少 200 μL)。
a. 将所有末端器官向下移动一排可能有助于使它们保持井井有条。
b. 与移液器中的末端器官一起转移的先前溶液的体积应尽可能小。可能需要轻轻轻弹以使末端器官落到溶液柱的底部,该溶液柱悬浮在移液器吸头中,然后可以用最少的液体排出。
2. 在 2% Triton X-100、5% NDS 1 小时室温下,在轨道摇床上渗透和阻塞。
a. 这种高浓度的去污剂可能是该制剂最佳染色所必需的。
3. 从透化和封闭缓冲液中取出末端器官,并放入用 5% NDS 稀释的一抗中。在 4 ° C的轨道摇床上过夜孵育初级抗体(表 1)。长达 3 天的孵育可能会增强抗体的渗透性。
a. 我们避免在这些步骤之间进行冲洗,因为每次转移末端器官都存在损坏或丢失的风险。


表 1. 一抗


抗体 来源 浓度
兔多克隆抗钙调蛋白 斯旺特, 7697 1:500
鸡多克隆抗GFP Aves 实验室,GFP-1020 1:500
山羊多克隆抗mCherry , AB0040-200 1:500


4. 冲洗 3 × 10 分钟,方法是从初级去除末端器官并放入含有新鲜 PBS 的 48 孔板中的不同孔中。
5. 将末端器官转移到用 5% NDS 稀释的二级抗体中,并在轨道振荡器上孵育过夜 4 ° C(表 2)。


表 2. 二抗


抗体 来源 浓度
驴多克隆抗兔 Cy3 杰克逊免疫研究实验室, 711-165-152 1:500
驴多克隆抗鸡 Alexa Fluor 488 杰克逊免疫研究实验室, 703-545-155 1:500
驴多克隆抗山羊 Cy3 杰克逊免疫研究实验室, 705-165-147 1:500


6. 冲洗 3 × 10 分钟,方法是从初级去除末端器官并放入含有新鲜 PBS 的 48 孔板中的不同孔中。


E. 安装和盖玻片


1. 将 8 孔 120 µm 厚的垫片放在显微镜载玻片上 ( SecureSeal ):首先取下塑料侧并将垫片放在载玻片上。通过沿其运行扁平物体将其压平,注意不要污染显微镜载玻片的玻璃。然后,取出纸面。从现在开始直到盖玻片放在上面之前它都会很粘,所以要非常小心,以免意外触摸它。根据我们的经验,这比在布置完所有末端器官后尝试去除纸张并暴露粘合剂更可取,因为它们的定位很容易被破坏。或者,可以将每个末端器官放置在带有单个孔隔板的单独载玻片上。
2. 将一个末端器官放在 PBS 池中的垫片内的幻灯片上。使用细镊子,去除盖玻片和末端器官表面之间的黑色素细胞或任何膜。管嵴通常位于其一侧,这是成像的最佳选择。可以放置椭圆囊和球囊,使神经对着显微镜载玻片,毛细胞侧向上指向盖玻片。此步骤对于良好的成像至关重要。应用更多的 PBS,使其在安装其他 PBS 时不会变干,但不要太多以至于它可以自由地从显微镜载玻片上漂浮。在垫片的不同孔中对每个末端器官和前庭神经节或神经重复。
3. 确保所有末端器官的方向正确,然后用正常的 200 μL 移液器尖端(未修改)去除多余的 PBS。让它稍微干燥,以便它们粘在带正电的Superfrost Plus 显微镜载玻片上。
4. 在每口井上涂抹大量的 CFM-3 安装介质。将盖玻片涂在粘合剂SecureSeal垫片上(图 2F)。
笔记:
a. 应用盖玻片时,多余的介质流出要好于气泡被困在孔中;涂抹充足的封固剂。
b. 请注意,由于SecureSeal垫片的粘合面,几乎不可能重新定位盖玻片,因此请小心使用。


F. 共焦成像


1. 这种准备可以用其他地方描述的典型共焦成像进行成像。长工作距离物镜可能需要通过整个末端器官和底层神经进行成像。我们在蔡司 LSM 880 上使用蔡司 25 × 0.8 NA 油浸物镜进行全端器官成像(图 3)。对于较小区域的成像,我们使用了蔡司 63 × 1.4 NA 油浸物镜(图 4)。


 


×物镜成像的前庭末端器官整体支架。
A) 椭圆囊黄斑、前管嵴和水平管嵴可以从颞骨中一块一块地解剖出来。 Calretinin(洋红色)在 I 型毛细胞中表达。单神经纤维用绿色荧光蛋白 (GFP) 标记,该蛋白已用 Alexa Fluor 488 放大。BC) 垂直管 (B) 和球囊 (C) 在解剖过程中分离,因此分别处理和安装。




 


×物镜成像的前庭毛细胞和外周前庭传入神经。
A) 单个光学平面显示 Myo7A(青色),存在于所有毛细胞中;钙调蛋白(洋红色),存在于 I 型毛细胞中; GFP(绿色),由注入小脑的逆行 AAV 表达,并用抗 GFP 抗体扩增;和合并的图像显示逆向标记的外周传入神经是一个烧瓶形状并围绕一个表达钙调蛋白的毛细胞。 B)显示 calretinin(洋红色)和 GFP(绿色)的 24 μm 厚图像堆栈的最大强度投影。




食谱


1. 肝素盐水
加入 3.6 g NaCl、0.022 g 肝素(计算为 10 U/mL 终浓度)和 400 mL dH 2 O


2. 0.1 M PB 中的 4% PFA
加入 10 mL 16% PFA、6.2 mL 0.5 M Na 2 HPO 4 、0.9 mL 1 M NaH 2 PO 4 ,用 dH 2 O升至 40 mL




致谢


我们感谢 Drs。 Jocelyn Krey和 Peter Barr-Gillespie 在此程序和内耳解剖方面的专业知识。资金由NIH F32 DC014878 、NIH K99 DC016905 和听力健康基金会新兴研究资助向 TSB、NIH R01 NS028901、NIH RO1 DC004450 向 LOT 和 NIH P30 NS0618000 向 S Aicher提供。该协议源自 Balmer 和 Trussell (2019) 。




利益争夺


作者声明没有竞争利益。




伦理


所有程序均经俄勒冈健康与科学大学机构动物护理和使用委员会批准,并符合神经科学学会的建议。




参考


Balmer, TS 和 Trussell, LO (2019)。小脑苔藓纤维选择性靶向单极刷细胞亚型。 生命8 : e44964 。
Bucks, SA, Cox, BC, Vlosich , BA, Manning, JP, Nguyen, TB 和 Stone, JS (2017)。支持细胞去除和替换成年小鼠平衡器官中的感觉受体毛细胞。 生命6 : e18128 。
Goldberg, JM, Wilson, VJ, Cullen, KE, Angelaki , DE, Broussard, DM, Buttner-Ennever , J., Fukushima, K. 和 Minor, LB (2012)。前庭系统:第六感。纽约:牛津大学出版社。网址为:https ://oxford.universitypressscholarship.com/10.1093/acprof :oso /9780195167085.001.0001/acprof-9780195167085 [2021 年 11 月 24 日访问]。
Golub, JS, Tong, L., Ngyuen , TB, Hume, CR, Palmiter , RD, Rubel, EW 和 Stone, JS (2012)。用白喉毒素靶向消融毛细胞后成年小鼠椭圆囊中的毛细胞替代物。 神经科学杂志 32(43):15093-15105 。
Hoffman, LF, Choy, KR, Sultemeier , DR 和 Simmons, DD (2018)。 Oncomodulin 表达揭示了对鼠 Utricle Striola 细胞组织的新见解。 J Assoc Res Otolaryngol 19(1):33-51。
Krey , JF, Sherman, NE, Jeffery, ED, Choi, D. 和 Barr-Gillespie, PG (2015)。小鼠前庭毛束过度发育的蛋白质组。 科学数据2(1):150047。
Li, A.、 Xue , J. 和 Peterson, EH (2008)。小鼠椭圆囊的结构:黄斑组织和毛束高度。 神经生理学杂志 99(2):718-733 。
Lysakowski , A., Gaboyard-Niay , S., Calin- Jageman , I., Chatlani , S., Price, SD 和Eatock , RA (2011)。分子微区位于感觉末端,前庭花萼末端。 神经科学杂志 31(27):10101-10114 。
Spitzmaul , G., Tolosa , L., Winkelman, BH, Heidenreich, M., Frens , MA, Chabbert , C., de Zeeuw , CI 和 Jentsch, TJ (2013)。小鼠模型揭示了 KCNQ4 和 KCNQ5 K+ 通道的前庭作用。 J Biol Chem 288(13):9334-9344。
Yoshimura, H.、Shibata, SB、 Ranum , PT 和 Smith, RJH (2018)。通过将耳道开窗与圆窗膜接种相结合,增强成年小鼠病毒介导的耳蜗基因传递。 科学代表8(1):2980。


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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 Balmer and Trussell. 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. Balmer, T. S. and Trussell, L. O. (2022). Vestibular Organ Dissection and Whole-Mount Immunolabeling in Mouse. Bio-protocol 12(10): e4416. DOI: 10.21769/BioProtoc.4416.
  2. Balmer, T. S. and Trussell, L. O. (2019). Selective targeting of unipolar brush cell subtypes by cerebellar mossy fibers. Elife 8: e44964.
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