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Jun 2020

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In vivo Optical Access to Olfactory Sensory Neurons in the Mouse Olfactory Epithelium
小鼠嗅上皮嗅觉感觉神经元的体内光学通路研究   

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Abstract

In neuroscience, it is fundamental to understand how sensory stimuli are translated into neural activity at the entry point of sensory systems. In the olfactory system, odorants inhaled into the nasal cavity are detected by ~1,000 types of odorant receptors (ORs) that are expressed by olfactory sensory neurons (OSNs). Since each OSN expresses only one type of odorant receptor, the odor-evoked responses reflect the interaction between odorants and the expressed OR. The responses of OSN somata are often measured by calcium imaging and electrophysiological techniques; however, previous techniques require tissue dissection or cell dissociation, rendering it difficult to investigate physiological responses. Here, we describe a protocol that allows us to observe odor-evoked responses of individual OSN somata in the mouse olfactory epithelium in vivo. Two-photon excitation through the thinned skull enables highly-sensitive calcium imaging using a genetically encoded calcium indicator, GCaMP. Recording of odor-evoked responses in OSN somata in freely breathing mice will be fundamental to understanding how odor information is processed at the periphery and higher circuits in the brain.

Keywords: Olfactory system (嗅觉系统), Olfactory sensory neuron (嗅感觉神经元), Olfactory epithelium (嗅觉上皮), Two-photon calcium imaging (双光子钙成像), GCaMP (GCaMP)

Background

Animals recognize their environmental cues using sensory systems. The mammalian olfactory system is able to detect and discriminate a large repertoire of odorants. Odorants inhaled into the nasal cavity are detected by ~1,000 types of odorant receptors (ORs) expressed by olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) of mice. Since each OSN expresses only one type of OR, the odor-evoked responses reflect the interaction between odorants and the expressed OR. To understand how odor information is translated into neural activity at the entry point of the olfactory system, it is important to study OSN responses in the olfactory epithelium in vivo.


The responses of OSN somata are often measured by calcium imaging and electrophysiological techniques (Maue and Dionne, 1987; Cygnar et al., 2010; Jarriault and Grosmaitre, 2015; Zhang, 2018); however, previous techniques require tissue dissection or cell dissociation, rendering it difficult to investigate physiological responses. Electroolfactograms can be used in vivo, but they cannot distinguish single-cell activities.


Here, we describe a protocol that allowed us to observe the odor-evoked responses of individual OSN somata in the OE in vivo (Iwata et al., 2017; Inagaki et al., 2020; Zak et al., 2020). Two-photon excitation through the thinned skull enables highly-sensitive calcium imaging using a genetically encoded calcium indicator, GCaMP (Yang et al., 2010). The preparation for in vivo imaging is simple and usually completed within 1 h. This method may apply not only to calcium imaging but also to other types of fluorescence imaging of OSNs.


Materials and Reagents

  1. 1.5 ml plastic tubes (Bio-bik, catalog number: CF-0150)

  2. 27 G needles for injection (Terumo, catalog number: NN-2719S)

  3. 1 ml syringe (Terumo, catalog number: 170215)

  4. 50 ml centrifuge tubes (Greiner, catalog number: 227261)

  5. Teflon tube (Chiyoda, catalog number: TF-4-10)

  6. KimWipes (Crecia, catalog number: S-200)

  7. Cotton buds (Suzuran, catalog number: 102046)

  8. Toothpicks (Yanagi, catalog number: J-613)

  9. Disposable balance tray (Bio-bik, catalog number: AS-DS)

  10. Cement solution (GC, Product name: Unifast II liquid 100 g)

  11. Cement powder (GC, Product name: Unifast II powder A3 35 g)

  12. Saline (Otsuka, catalog number: 3311401A7028)

  13. Ketamine (Daiichi-Sankyo, catalog number: S9-019780)

  14. Xylazine (Bayer, Product name: Rompun 2% w/v solution for injection 25 ml)

  15. Vaseline (Wako, catalog number: 227-01211)

  16. 70% ethanol (Shinwa, catalog number: WK2-75)

  17. Superglue (Sankyo, Product name: aron alpha A 0.5 g × 5)

  18. Kwil-sil (WPI, catalog number: KWIK-SIL)

  19. Valeraldehyde (Tokyo Chemical Industry, catalog number: V0001)

  20. Mineral oil (Sigma, catalog number: M5310-500 ML)

  21. Phosphate-buffered saline (PBS)


Mouse:

The OSN-specific GCaMP transgenic mouse line, OSN-GCaMP3 (OMP-tTA; TRE-GCaMP3 compound heterozygous bacterial artificial chromosome transgenic mice, 8-16 weeks of age) was used (Iwata et al., 2017; Inagaki et al., 2020). OMP-tTA (Accession# CDB0506T) and TRE-GCaMP3 (Accession# CDB0505T) are available from RIKEN (http://www2.clst.riken.jp/arg/index.html).

Note: Transgenic mouse lines expressing any indicators could be used, but sparsely and brightly labeled lines are preferred so that you can easily distinguish OSN responses. In OSN-GCaMP3 mice, GCaMP3 is expressed in 57.9% of the total OSNs (Iwata et al., 2017). A mouse line based on the Tet-system may be suitable for OE imaging in terms of labeling density and fluorescence intensity.

Equipment

  1. Micropipette (Gilson, model: P200 and P1000)

  2. Heating pad (Natsume, catalog number: KN-475-3-40)

  3. Forceps (KFI, catalog number: 1-9749-32)

  4. Fine forceps (Ideal-tek, catalog number: 91-2427)

  5. Fine scissors (Mizuho, catalog number: 04-001-13)

  6. Fluorescent stereomicroscope (Leica, model: M205 C)

    Note: An epifluorescence microscope is useful for assessing the thickness of the skull above the OE (detailed below).

  7. External light source for fluorescence excitation (Leica, model: EL6000)

  8. Filter cube (Leica, model: GFP)

  9. Head holder for surgery (Narishige, model: SG-4N)

  10. Custom-made aluminum nose bar (Figure 1)

    Note: We installed a custom-made nose bar to Narishige SG-4N (Figure 1A), as the original nose bar was too long and prevented surgical access to the OE. The size should be adjusted to your head holder and to make the skull over the OE accessible during surgery (Figure 1C). Any head holders can be used as long as the OE is accessible for surgery.



    Figure 1. Installation of a custom-made nose bar to a head holder. A. An original nose bar of a head holder. B. The design of a custom-made nose bar. C. The custom-made nose bar is installed into SG-4N.


  11. Custom-made aluminum head bar (4 × 22 mm)

    Note: The head bar was designed for a custom-built head holder as described in a previous study (Guo et al., 2014).

  12. Custom-built head holder for imaging

    Note: The head holder was built as described previously (Guo et al., 2014). Any head-holding system can be used for this protocol if the OE is accessible for in vivo imaging.

  13. Φ1 mm drill tip (Meisinger, catalog number: ST1 HP010)

  14. Dental drill (Leutor, model: LP-120)

  15. Dust blower (UN, catalog number: UN-1321)

  16. Two-photon microscope (Olympus, model: FV1000MPE)

  17. Fluoview software (Olympus, model: FV10-ASW)

  18. 25× objective lens (Olympus, model: XLPLN25XWMP)

  19. Custom-built olfactometer

    Note: The design of the olfactometers has been described elsewhere (Slotnick and Restrepo, 2005; Burton et al., 2019). Briefly, the olfactometer consists of an air pump (AS ONE, catalog number: 1-7482-11), activated charcoal filter (Advantec, model: TCC-A1-S0C0 and 1TS-B), and flowmeters (Kofloc, model: RK-1250)]

Procedure

  1. Prepare a head holder for surgery. We used a combination of a commercial head holder and a custom-made nose bar to make the OE region accessible for surgery (Figure 1).

  2. Anesthetize a mouse using a ketamine/xylazine cocktail in saline (80 mg/kg and 16 mg/kg for ketamine and xylazine, respectively). Inject the ketamine/xylazine cocktail intraperitoneally using a 1-ml syringe and 27 G needle. During surgery, the depth of anesthesia needs to be assessed by the toe-pinch reflex, and supplemental doses should be administered when necessary.

  3. Hold the head under a fluorescent stereomicroscope using the head holder.

  4. Cover the eyes with Vaseline using a cotton bud to prevent drying.

  5. Apply 70% ethanol on the head.

    Note: This step is required to sterilize the surgical site and to remove hairs in the next step.

  6. Remove the scalp together with hairs using scissors and forceps (Figure 2A).

    Note: The scalp needs to be extensively removed to the back of the head to attach a custom-made head bar at the later step.

    Note: The hair can be shaved with a razor beforehand.

  7. Carefully remove the periosteum from the skull with forceps.

  8. Apply superglue to the periphery of the surgical site to prevent the scalp from being caught up by the rotation of the drill.

    Note: Superglue can harden quickly when PBS is overlaid. A used drill tip is useful for application of superglue and PBS.

  9. Carefully thin the skull over the OE using a dental drill (Φ 1 mm drill tip, 5,000-10,000 rpm) (Figure 2A-2D, Video 1).

    Note: The dorsal and rostral parts of the D zone (zone 1) and the dorsolateral part of the V zone (zone 4) can be imaged (Figure 2B). Other parts are difficult to drill due to the presence of a lot of blood vessels. To avoid overheating, do not continuously thin the same area of the skull. See Yang et al. (2010) for additional tips on thinned skull preparations.


    Video 1. Thinned-skull preparation for OE imaging. A drill tip was lightly touched to the skull and moved horizontally.



  10. Blow away the skull shavings with a dust blower (Video 1).

  11. Apply a small amount of PBS on the thinned skull and check if the blood vessels and fluorescence of OSN somata can be clearly observed (Figure 2D-2F).

  12. Continue thinning until the fluorescence of OSN somata is observed (Figure 2F, arrows).

    Note: You can also estimate the thickness of the skull based on the stiffness. If it is thin enough, the skull sinks a little when touched lightly with forceps.



    Figure 2. Thinned-skull preparation for in vivo imaging. A. The dorsal scalp was removed from the head. B. The dorsal and rostral parts of the D zone (zone 1) and the dorsolateral part of the V zone (zone 4) in the OE can be imaged. C. A close-up picture of the imaging area over zone 1 in the OE. The skull was thinned in the boxed area (A-C, 3-6 mm from the anterior edge of the olfactory bulb in the 12-week-old male mice). D. Brightfield image of zone 1 in the right OE taken through the thinned skull. The blood vessels should be clearly observed if the skull is sufficiently thinned (white arrows). E. A fluorescence image of zone 1 in the right OE taken by a fluorescent stereomicroscope. F. A close-up image of the square region indicated in (E). Arrows indicate fluorescence from OSN somata.


  13. Adjust the head angle to make sure that the dorsal surface of the OE is perpendicular to the light path.

  14. Apply superglue to the surface of the skull outside the imaging area to make a scaffold for the attachment of a custom-made head bar.

  15. Place ~0.3 g cement powder into a disposable balance tray. Pour ~0.3 ml cement solution onto the powder using a micropipette. Mix the powder and solution with a toothpick immediately to make the dental cement (Figure 3A).

    Note: Larger amounts of solution may dissolve the plastic tray. In that case, you can use a small silicone bowl instead.

  16. Attach a custom-made head bar perpendicular to the light path using dental cement (Figure 3B).

  17. Apply Kwik-sil at the periphery of the imaging area. PBS can be kept here to image using a water-immersion objective lens (Figure 3C).

  18. Fix the head under a two-photon microscope using the head bar and a custom-built head holder (Figure 3D) (Guo et al., 2014).



    Figure 3. Head-fixation under a two-photon microscope. A. Dental cement before (left) and after (right) mixing with a toothpick. B. A custom-made head bar was attached with dental cement perpendicular to the optical axis of the subsequent in vivo imaging. C. Kwik-sil was applied to the periphery of the imaging area to retain PBS during imaging. D. The imaging area was placed under an objective lens using a custom-made head holder.


  19. Perform two-photon imaging of odor-evoked responses in OSN somata with a custom-built olfactometer (Figure 4; Video 2). In this example, valeraldehyde was diluted at a concentration of 0.5% v/v in 1 ml mineral oil and soaked in a Kimwipe in a 50-ml centrifuge tube. Saturated odor vapor in the centrifuge tube was delivered to the nose via a Teflon tube at 1 L/min.

    Notes:

    1. The 50-ml centrifuge tube and Teflon tube should be replaced every time the odors are changed to avoid cross-contamination of the odors.

    2. We have never performed chronic imaging, but it may be possible (see also Zak et al., 2020).



    Figure 4. Two-photon calcium imaging of OSN somata. A. GCaMP3 fluorescence from OSN somata before (left) and during (right) odor stimulation. B. A pseudo-colored ∆F/F0 image of OSN somata responses to 0.5% valeraldehyde (see Video 2).


    Video 2. In vivo two-photon imaging of odor responses at the OSN somata in the OE. 0.5% valeraldehyde was delivered to the nose from 10 to 15 s. Gray-scale images show the fluorescence (pixel intensities).

Acknowledgments

This work was supported by grants from the PRESTO and CREST programs of the Japan Science and Technology Agency (JST), Japan (T.I.), the JSPS KAKENHI, Japan (grant numbers JP23680038, JP15H05572, JP15K14336, JP16K14568, JP16H06456, JP17H06261, and JP21H00205 to T.I., JP15K18353 to R.I., and JP21H02140 to S.I.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research, intramural grant from RIKEN Center for Developmental Biology (T.I.), and Grant-in-Aid for JSPS Research Fellow, Japan (JP15J08987 to R.I. and JP18J00899 to S.I.). We thank M.N. Leiwe for comments on the manuscript. This protocol has been used in our research (Iwata et al., 2017; Inagaki et al., 2020).

Competing interests

There are no conflicts of interests or competing financial interests.

Ethics

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Kyushu University (#A19-054, approved from 04/01/19 to 03/31/21).

References

  1. Burton, S. D., Wipfel, M., Guo, M., Eiting, T. P. and Wachowiak, M. (2019). A Novel Olfactometer for Efficient and Flexible Odorant Delivery. Chem Senses 44(3): 173-188.
  2. Cygnar, K.D., Stephan, A.B. and Zhao, H. (2010). Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording. J Vis Exp (37): 1850.
  3. Guo, Z. V., Hires, S. A., Li, N., O'Connor, D. H., Komiyama, T., Ophir, E., Huber, D., Bonardi, C., Morandell, K., Gutnisky, D., Peron, S., Xu, N. L., Cox, J. and Svoboda, K. (2014). Procedures for behavioral experiments in head-fixed mice. PLoS One 9(2): e88678.
  4. Inagaki, S., Iwata, R., Iwamoto, M. and Imai, T. (2020). Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo. Cell Rep 31(13): 107814.
  5. Iwata, R., Kiyonari, H. and Imai, T. (2017). Mechanosensory-based phase coding of odor identity in the olfactory bulb. Neuron 96(5): 1139-1152 e1137.
  6. Jarriault, D. and Grosmaitre, X. (2015). Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor. J Vis Exp 101: e52652.
  7. Maue, R. A. and Dionne, V.E. (1987). Patch-clamp studies of isolated mouse olfactory receptor neurons. J Gen Physiol 90(1): 95-125.
  8. Slotnick, B. and Restrepo, D. (2005). Olfactometry with mice. Curr Protoc Neurosci Chapter 8: Unit 820.
  9. Yang, G., Pan, F., Parkhurst, C.N., Grutzendler, J. and Gan, W.B. (2010). Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5(2): 201-208.
  10. Zak, J. D., Reddy, G., Vergassola, M. and Murthy, V. N. (2020). Antagonistic odor interactions in olfactory sensory neurons are widespread in freely breathing mice. Nat Commun 11(1):3350.
  11. Zhang, C. (2018). Calcium Imaging of Individual Olfactory Sensory Neurons from Intact Olfactory Turbinates. Methods Mol Biol 1820: 57-68.

简介

[摘要] 在神经科学中,了解感觉刺激如何在感觉系统的入口点转化为神经活动是至关重要的。在嗅觉系统中,吸入鼻腔的气味物质由约 1,000 种气味受体 (ORs) 检测,这些气味受体 (ORs) 由嗅觉感觉神经元 (OSNs) 表达。由于每个 OSN 只表达一种类型的气味受体,气味诱发的反应反映了气味和表达的 OR 之间的相互作用。OSN 胞体的反应通常通过钙成像和电生理技术来测量;^ h Ø wever,以前的技术需要组织剥离或细胞分离,使荷兰国际集团难以调查的生理反应。 在这里,我们描述了一个协议,使我们能够观察体内小鼠嗅觉上皮细胞中个体 OSN 胞体的气味诱发反应。通过变薄的头骨的双光子激发能够使用基因编码的钙指示剂 GCaMP进行高灵敏度的钙成像。在自由呼吸的小鼠在OSN胞体气味诱发反应的记录将是从根本上了解荷兰国际集团信息气味如何在大脑中的边缘和更高的电路进行处理。

[背景]动物使用感觉系统识别它们的环境线索。哺乳动物的嗅觉系统能够检测和区分大量的气味。吸入鼻腔的气味由小鼠嗅觉上皮 (OE)中的嗅觉感觉神经元 (OSN) 表达的约 1,000 种气味受体 (OR) 检测到。由于每个 OSN 只表达一种类型的 OR,气味诱发的反应反映了气味和表达的 OR 之间的相互作用。要了解气味信息如何在嗅觉系统的入口点转化为神经活动,研究体内嗅觉上皮中的OSN 反应很重要。

OSN 胞体的反应通常通过钙成像和电生理技术来测量(Maue 和 Dionne,1987;Cygnar等,2010;Jarriault和 Grosmaitre ,2015;Zhang ,2018 );^ h H但是,以前的技术需要组织剥离或细胞分离,使荷兰国际集团难以调查的生理反应。嗅觉图可以在体内使用,但不能区分单细胞活动。

在这里,我们描述了一个协议,允许我们观察体内OE中个体 OSN 胞体的气味诱发反应(Iwata等人,2017 年;Inagaki等人,2020 年;Zak等人,2020 年)。通过减薄头骨双光子激发使得能够高度-使用敏感钙成像遗传编码的钙指示剂,GCaMP (杨等人,2010。) 。体内成像的准备工作很简单,通常在 1 小时内完成。此方法毫安ý不仅适用于钙成像,而且其它类型的的OSN的荧光成像。

关键字:嗅觉系统, 嗅感觉神经元, 嗅觉上皮, 双光子钙成像, GCaMP



材料和[R eagents

1.5毫升塑料管(Bio-bik,目录号:CF-0150)
27 G注射针(Terumo,目录号:NN-2719S)
1毫升注射器(Terumo,目录号:170215)
50 ml离心管(Greiner,目录号:227261)
铁氟龙管(Chiyoda,目录号:TF-4-10)
KimWipes(Crecia,目录号:S-200 )
棉芽(Suzuran,目录号:102046 )
牙签(Yanagi,目录号:J-613)
一次性平衡托盘(Bio-bik,目录号:AS-DS )
水泥溶液(GC,产品名称:Unifast II 液体 100 g )
水泥粉(GC,产品名称:Unifast II Powder A3 35 g)
盐水(大冢,目录号:3311401A7028)
氯胺酮(Daiichi-Sankyo,目录号:S9-019780 )
甲苯噻嗪(拜耳,商品名:甲苯噻嗪2%w / v的小号olution用于我njection 25ml)中
凡士林(Wako,目录号:227-01211 )
70%乙醇(Shinwa,目录号:WK2-75 )
强力胶(Sankyo,产品名称:aron alpha A 0.5 g × 5)
Kwil-sil(WPI,目录号:KWIK-SIL)
戊醛(东京化学工业,目录号:V0001 )
矿物油(Sigma,目录号:M5310-500 ML )
磷酸盐缓冲盐水 (PBS )
 


鼠标:


的OSN-特异性G CAMP转基因小鼠品系,OSN-GCaMP3(OMP-tTA的; TRE-GCaMP3复合杂细菌人工染色体的转基因小鼠,8 - 16周龄)瓦特为使用(岩田等人,2017 ;稻垣等人. , 2020) 。OMP-tTA(登录号 CDB0506T)和 TRE-GCaMP3(登录号 CDB0505T)可从 RIKEN ( http://www2.clst.riken.jp/arg/index.html ) 获得。


注意:可以使用表达任何指标的转基因小鼠系,但首选稀疏和明亮标记的系,以便您可以轻松区分 OSN 响应。在OSN-GCaMP3小鼠,GCaMP3在57.9%表示的总的OSN (岩田等人,2017) 。基于所述的Tet-系统米鼠标线AY适合用于标记密度和荧光强度方面OE成像。

设备

微量移液器(Gilson,型号:P200 和 P1000)
加热垫(Natsume,目录号:KN-475-3-40)
镊子(KFI,目录号:1-9749-32)
细镊子(Ideal-tek,目录号:91-2427 )
细剪刀(Mizuho,目录号:04-001-13)
荧光立体显微镜(徕卡,型号:M205 C)
注意:一个落射荧光显微镜是有用用于评估荷兰国际集团的OE(下面详细说明)上面的头骨的厚度。


用于荧光激发的外部光源(Leica,型号:EL6000)
滤光片(徕卡,型号:GFP)
手术用头部支架(Narishige,型号:SG-4N)
定制铝制鼻梁(图1 )
注意:我们为 Narishige SG-4N (图 1A)安装了一个定制的鼻梁,因为原始鼻梁太长并阻止了手术进入 OE。尺寸应根据您的头部支架进行调整,并使手术过程中可以接触到 OE 上的头骨(图 1C )。只要 OE 可用于手术,就可以使用任何头部固定器。

 


图 1.将定制鼻梁安装到头架上。A. 头部固定器的原始鼻梁。B.该ð ESIGN一个特制的鼻子吧。C. 将定制的鼻梁安装到 SG-4N 中。

定制铝头条(4 × 22 mm)
注意:头杆是为定制的头架设计的,如先前的研究(Guo 等人,2014 年)所述。


用于成像的定制头架
注意:头部固定器的构建如前所述(Guo 等人,2014)。如果 OE 可用于体内成像,则任何头部固定系统均可用于该协议。


Φ 1毫米d细沟尖端(Meisinger,目录号:ST1 HP010)
牙钻(Leutor,型号:LP-120)
除尘器(UN,目录号:UN-1321)
双光子显微镜(奥林巴斯,型号:FV1000MPE)
Fluoview 软件(奥林巴斯,型号:FV10-ASW)
25 ×物镜(奥林巴斯,型号:XLPLN25XWMP)
定制的嗅觉计
注意:所述的设计的olfactometers已经别处描述(Slotnick和雷斯特雷波,2005 ; Burton等人,2019 )。简而言之,嗅觉计由一个气泵(AS ONE,目录号:1-7482-11)、活性炭过滤器(Advantec,型号:TCC-A1-S0C0 和 1TS-B)和流量计(Kofloc,型号:RK -1250)]

程序

准备一个头部支架进行手术。我们结合使用商业头架和定制的鼻梁,使 OE 区域可用于手术(图 1)。
使用盐水中的氯胺酮/赛拉嗪鸡尾酒麻醉小鼠(氯胺酮和赛拉嗪分别为 80 毫克/千克和 16 毫克/千克)。注射氯胺酮/赛拉嗪腹膜内鸡尾酒使用一个1 -毫升注射器和27号针。手术过程中,需要通过脚趾捏反射来评估麻醉深度,必要时应给予补充剂量。
使用头部支架将头部固定在荧光立体显微镜下。
用棉签用凡士林盖住眼睛以防止干燥。
在头部涂抹 70% 乙醇。
注意:此步骤需要对手术部位进行消毒并在下一步中去除毛发。


使用剪刀和镊子去除头皮和头发(图 2A )。
注意:头皮需要大量去除到头后部,以便在后面的步骤中连接定制的头条。


注意:头发可以事先用剃须刀剃掉。


用镊子小心地从头骨上取出骨膜。
强力胶应用到手术部位的周边,以防止头皮被卷入由钻头的旋转。
注意:当覆盖 PBS 时,强力胶会迅速硬化。使用过的钻头可用于涂抹强力胶和 PBS。


使用牙钻(Φ 1 毫米钻尖,5,000 - 10,000 rpm)小心地将 OE 上的头骨变薄(图 2A - 2 D,视频 1 )。
注:背侧和喙部小号的d区(区域1)和V区域(区域4)的背外侧部分的可成像(图2B )。由于存在大量血管,其他部分难以钻孔。为避免过热,不要连续变薄头骨的同一区域。见杨等人。( 2010 年)有关薄化颅骨制备的其他提示。

 


视频 1.用于 OE 成像的薄化颅骨准备。钻尖轻轻接触头骨并水平移动。

用鼓风机吹掉头骨屑(视频 1 )。
在变薄的头骨上涂抹少量 PBS,检查是否可以清楚地观察到 OSN 胞体的血管和荧光(图 2D -2 F )。
继续稀释,直到观察到 OSN 胞体的荧光(图 2F ,箭头)。
注意:您还可以根据刚度估计头骨的厚度。如果它足够薄,用镊子轻轻接触时,头骨会下沉一点。

 


图 2. 用于体内成像的薄化头骨制备。A. 从头部去除背部头皮。B.该背侧和喙部小号在OE的d区(区域1)和V区的背外侧部分(4区)可以是我马吉德阿卜。C. OE 区域 1 上成像区域的特写照片。(A颅骨中方框区域减薄- C,3 -从嗅球的在12的前边缘6毫米-周龄雄性小鼠)。D. 通过变薄的头骨拍摄的右侧 OE 区域 1 的明场图像。如果颅骨足够薄 (白色箭头), 应清楚地观察血管。乙。右侧 OE 区域 1 的荧光图像由荧光立体显微镜拍摄。F. (E) 中所示方形区域的特写图像。箭头表示来自 OSN 胞体的荧光。

调整头部角度,以确保其在OE的背表面垂直于光路。
在成像区域外的头骨表面涂上强力胶,为定制头杆的附件制作支架。
将约 0.3 克水泥粉放入一次性平衡托盘中。使用微量吸管将 ~0.3 ml水泥溶液倒入粉末中。立即用牙签将粉末和溶液混合,制成牙科水泥(图 3A )。
注意:较大的溶液量可以溶解的塑料托盘。在这种情况下,您可以使用一个小的硅胶碗代替。


使用牙科水泥(图 3B )将定制的头条与光路垂直。
在成像区域的外围应用 Kwik-sil。PBS 可以保留在这里使用浸水物镜进行成像(图 3C ) 。
使用头杆和定制的头部支架(图 3D )(Guo等人,2014 年)将头部固定在双光子显微镜下。
 

图 3.双光子显微镜下的头部固定。A.用牙签混合之前(左)和之后(右)的牙科水泥。B. 一个定制的头杆与牙科水泥垂直于随后的体内成像的光轴。C. Kwik-sil 应用于成像区域的外围以在成像过程中保留 PBS。D.成像区域WA S置于使用定制的打印头保持器的物镜下。

使用定制的嗅觉计对 OSN 胞体中的气味诱发反应进行双光子成像(图 4;视频 2 )。在这个例子中,戊醛在稀释一浓度的0.5%体积/体积在1ml的矿物油,并在50浸泡在的Kimwipe -毫升离心管中。离心管中的饱和气味蒸汽通过特氟龙管以 1 L/min 的速度输送到鼻子。
注意小号:


在50 -毫升离心管中并特氟隆管应该每次更换第ë气味被改变,以避免气味的交叉污染。
我们从未进行慢性成像,但它中号AY是可能的(小号EE也扎克等人,2020)。
 

图 4. OSN 胞体的双光子钙成像。A. 在气味刺激之前(左)和期间(右)来自 OSN 胞体的 GCaMP3 荧光。乙。OSN 胞体响应 0.5% 戊醛的伪彩色 F/F 0图像(参见视频 2)。

 

视频 2. OE 中 OSN 胞体气味反应的体内双光子成像。0.5% 戊醛在 10 到 15 秒内被输送到鼻子。灰度图像显示荧光(像素强度)。

致谢

这项工作是由从PRESTO的资金支持和CREST程序小号的日本科学技术振兴机构(JST),日本(TI)的JSPS KAKENHI,日本(授权号的小号JP23680038,JP15H05572,JP15K14336,JP16K14568,JP16H06456,JP17H06261 ,和JP21H00205到TI ,JP15K18353到RI ,并JP21H02140到SI ),牛逼他持田纪念基金会的医学和药物研究,从理化学研究所发育生物学中心(TI)校内津贴和格兰特在急救的JSPS研究员,日本(JP15J08987 到 RI 和 JP18J00899 到 SI)。我们感谢 MN Leiwe 对手稿的评论。该协议已用于我们的研究(Iwata等人,2017 年;Inagaki等人,2020 年)。

利益争夺

这里是没有冲突小号的利益或竞争经济利益小号。

伦理

所有的动物实验是由九州大学的机构动物护理和使用委员会(IACUC)(#A19-054,Appro公司批准v版从19年4月1日至21年3月31日)。

参考

Burton, SD、Wipfel, M.、Guo, M.、Eiting, TP 和 Wachowiak, M.(2019 年)。一种用于有效和灵活传递气味的新型嗅觉计。化学感官44(3):173-188。
Cygnar, KD, Stephan, AB 和 Zhao, H. (2010)。使用气相嗅觉电图记录分析小鼠嗅觉感觉神经元的反应。J Vis Exp (37):1850 年。
Guo, ZV, Hires, SA, Li, N., O'Connor, DH, Komiyama, T., Ophir, E., Huber, D., Bonardi, C., Morandell, K., Gutnisky, D., Peron , S., Xu, NL, Cox, J. 和 Svoboda, K. (2014)。头部固定小鼠的行为实验程序。PLoS 一号9(2):e88678。
Inagaki, S.、Iwata, R.、Iwamoto, M. 和 Imai, T. (2020)。体内小鼠嗅觉感觉神经元的广泛抑制、拮抗和协同作用。细胞代表31(13):107814。
Iwata, R.、Kiyonari, H. 和 Imai, T.(2017 年)。嗅球中气味特性的基于机械感觉的相位编码。神经元96(5):1139-1152 e1137。
Jarriault, D. 和 Grosmaitre, X. (2015)。完整神经上皮中小鼠嗅觉感觉神经元的穿孔膜片钳记录:表达已识别气味受体的神经元的功能分析。J Vis Exp 101:e52652。
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Slotnick, B. 和 Restrepo, D. (2005)。用小鼠进行嗅觉测量。Curr Protoc Neurosci第 8 章:820 单元。              
Yang, G., Pan, F., Parkhurst, CN, Grutzendler, J. 和 Gan, WB (2010) 。用于活小鼠皮层长期成像的薄颅骨颅窗技术。纳特Protoc 5(2):201- 20 8。
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张 C. (2018)。来自完整嗅觉鼻甲的个体嗅觉感觉神经元的钙成像。分子生物学方法1820:57-68。
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引用:Inagaki, S., Iwata, R. and Imai, T. (2021). In vivo Optical Access to Olfactory Sensory Neurons in the Mouse Olfactory Epithelium. Bio-protocol 11(12): e4055. DOI: 10.21769/BioProtoc.4055.
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