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Registration and Alignment Between in vivo Functional and Cytoarchitectonic Maps of Mouse Visual Cortex
小鼠视皮层的体内功能和细胞结构图谱的配准和比对   

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eLIFE
Jan 2017

Abstract

This protocol describes a method for registration of in vivo cortical retinotopic map with cytochrome c oxidase (CO) labeled architectonic maps of the same mouse brain through the alignment of vascular fiducials. By recording surface blood vessel pattern and sequential alignment at each step, this method overcomes the challenge imposed by tissue distortion during perfusion, mounting, sectioning and histology procedures. This method can also be generalized to register and align other types of in vivo functional maps like ocular dominance map and spatial/temporal frequency tuning map with various anatomical maps of mouse cortex.

Keywords: Architectonic map (构筑图谱), Retinotopic map (网膜代表图), Registration (配准), Cortex flattening (皮层展平), Tangential section (切向切面), Vasculature (脉管系统), Cytochrome c oxidase (细胞色素c氧化酶)

Background

The mouse visual cortex can be segregated into functionally distinct visual areas by in vivo retinotopic mapping (Marshel et al., 2011; Garrett et al., 2014; Zhuang et al., 2017) or by neuronal track-tracing techniques aided by architectonic structures (Olavarria and Montero, 1989; Wang and Burkhalter, 2007). These different visual areas have distinct response properties and corticocortical connectivity (Andermann et al., 2011; Marshel et al., 2011; Roth et al., 2012; Wang et al., 2011 and 2012). These results suggest that mouse visual areas form segregated visual streams processing different types of visual information (Murakami et al., 2017; Smith et al., 2017). Studying the mouse visual system in the context of visual area maps is essential to understanding the organization of visual cortex. However, although the functional maps and structure maps are broadly similar, the two maps have been shown not matching perfectly (Zhuang et al., 2017). For example, the primary visual cortex (V1) appears as an upward triangle in both maps, but the lateral edge of V1 in retinotopic map can be up to 300 micrometers more medial than that in anatomical map (Zhuang et al., 2017). Since the smallest visual areas in mouse cortex are only a few hundred micrometers wide, ignoring this mismatch will potentially bias our interpretation of visual area functions. Furthermore, both types of maps vary significantly across different individuals. Therefore, to study the functions of identified visual areas, it is important to be able to reliably generate and compare functional and anatomical maps in the same animal. However, the tissue distortion during perfusion, mounting, sectioning and histological procedure makes it difficult to directly compare functional maps recorded in vivo with anatomical maps recorded after histology. Here we describe a method to overcome these challenges, allowing direct comparison between these two types of maps.

Materials and Reagents

  1. Sponge (Patterson Veterinary Supply, catalog number: 07-847-3539 )
  2. Metal clips (Universal Small Binder Clips, Universal, catalog number: UNV10200 )
  3. Razor Blade (VWR, catalog number: 55411-050 )
  4. Spatula (Fine Science Tools, catalog number: 10090-13 )
  5. Gelatin subbed slides (SouthernBiotech, catalog number: SLD01-CS )
  6. Cover glass (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 12450S )
  7. Corning 500 ml filter system, 0.45 μm (Corning, catalog number: 430770 )
  8. Corning 24-well plate (Corning, Falcon®, catalog number: 351147 )
  9. Corning disposable Petri dish ( 100 x 15 mm, Corning, Falcon®, catalog number: 351029 )
  10. DyLight 649-labeled tomato lectin (Vector Laboratories, catalog number: DL-1178 )
  11. Dry ice
  12. Tissue-Tek OCT Compound (Sakura Finetek, VWR, catalog number: 4583 )
  13. 10x phosphate buffered saline (PBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9625 )
  14. Ethyl alcohol, 200 proof (Fisher Scientific, catalog number: 16-100-826)
    Manufacturer: Pharmco-Aaper, catalog number: 241ACS200CSGP .
  15. DPX mountants (Electron Microscopy Science, catalog number: 13512 )
  16. Paraformaldehyde (Sigma-Aldrich, catalog number: 441244 )
  17. Sodium hydroxide solution (NaOH) (1 N, Sigma-Aldrich, catalog number: S2770 )
  18. Hydrochloric acid (HCl) (36.5-38%, Sigma-Aldrich, catalog number: H1758 )
  19. Sodium phosphate monobasic (NaH2PO4) (anhydrous, Sigma-Aldrich, catalog number: S3139 )
  20. Sodium phosphate dibasic (Na2HPO4) (anhydrous, Sigma-Aldrich, catalog number: 255793 )
  21. Sucrose (Sigma-Aldrich, catalog number: S8501 )
  22. 3,3’-Diaminobenzidine (DAB, 1 mg/ml, Sigma-Aldrich, catalog number: D5637 )
  23. Trizma HCl (Sigma-Aldrich, catalog number: T5941 )
  24. Trizma base (Sigma-Aldrich, catalog number: T6066 )
  25. Cobalt(II) chloride (CoCl2) (Sigma-Aldrich, catalog number: 232696 )
  26. Cytochrome c (Sigma-Aldrich, catalog number: C2506 )
  27. Catalase (10,000-40,000 U/mg, 20-50 mg/ml, Sigma-Aldrich, catalog number: C30 )
  28. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  29. Xylene (Merck, catalog number: XX0060 )
  30. 4% (w/v) formaldehyde (4% PFA in PBS) (see Recipes)
  31. 1% (w/v) formaldehyde (1% PFA in PBS) (see Recipes)
  32. 0.2 M phosphate-buffer (PB) solution with 20% (w/v) sucrose stock (pH 7.4) (see Recipes)
  33. 5 mg/ml DAB stock in 0.05 M Tris-HCl buffer (pH 7.6) (see Recipes)
  34. Preincubation solution, 0.05 M Tris-HCl Buffer Stock Solution (see Recipes)
  35. Incubation solution (see Recipes)
  36. Rinse solution, 0.1 M PB with 10% sucrose (pH 7.4) (see Recipes)

Equipment

  1. Fume hood (Labcono)
  2. 4 °C fridge (Panasonic Healthcare, model: SR-L6111W-PA ; VWR, catalog number: 89031-974 )
  3. Scale (analytical balance, A&D Weighing, model: GH-252 )
  4. Hot plate stirrer (VWR, catalog number: 97042-646 )
  5. B10P Benchtop PH meter (VWR, catalog number: 89231-662 )
  6. Microtome (MICROM, model: Sliding Microtome HM 400R )
  7. Widefield microscope for both bright field and fluorescence imaging (ZEISS, model: Axio Imager 2 )
  8. Dissecting microscope (Leica Microsystems, model: Leica MZ10 F )
  9. Shaker (Corning, model: LSETM Low Speed )
  10. Incubator (Quincy Lab, model: Model 10 lab oven )
  11. Peristaltic pump (Harvard Apparatus, model: MA1-55-7766 )

Software

  1. Fiji (Schindelin et al., 2012) with TrakEM2 plugin (Cardona et al., 2012)

Procedure

  1. In vivo imaging
    Make two small fiducial marks indicating the anterior and medial directions of the cranial window respectively. Record vasculature structure image of the cranial window via a fluorescence or a brightfield image (using a green wavelength may give better image contrast of blood vessels). Name it image A. Generate in vivo retinotopic maps through the cranial window (i.e., intrinsic signal Juavinett et al., 2017 or fluorescence retinotopic map Zhuang et al., 2017). Name it image B. Image A and image B should be perfectly co-registered by nature given the imaging optical axis is perpendicular to the cranial window (Figure 2A/B).

  2. Perfusion and cortex flattening (modified from Wang and Burkhalter, 2007)
    1. Perform mouse cardiac perfusion under isoflurane anesthesia (5% isoflurane, Gage et al., 2012) with the following steps of perfusion fluids.
      1. Saline wash 10 ml/min for 100 ml.
      2. 5 µg/ml DyLight 649 lectin 5 ml/min for 25 ml to label blood vessel.
      3. Wait for 5 min for DyLight lectin to adsorb to tissue.
      4. 1% PFA (see Recipes) 5 ml/min for 90 ml.
    2. With brain within the skull, acquire fluorescence image of cranial window (filter setting: 655/670 nm). The fiducial marks made in Procedure A should be visible. Name it image C (Figure 2C).
    3. Collect brain tissue. Since the animal was perfused by 1% PFA, the brain tissue will be relatively soft for cortex flattening. Be careful not to make any damage.
    4. Optional: Acquire bright field and fluorescence images (filter setting: 655/670 nm) of surface vasculature of the whole brain using a dissecting scope. The major surface blood vessels should be visible in the bright field image (can be registered with image A) and the DyLight labeled blood vessels should be visible in the fluorescence image (can be registered with image D).
    5. Isolate the cortical sheet of windowed hemisphere (the procedure can be done in a Petri dish sitting on ice). Carefully keep track of the orientation of cortical sheet. For video guidance, please see this EJN video protocol (made by Hoey Sarah, Universität Zürich): http://www.ejnnews.org/video-protocol-isolation-adult-mouse-hippocampi/.
      1. Separate the two hemispheres of the brain with a razor blade. Keep the windowed hemisphere and discard the other hemisphere.
      2. Cut off olfactory bulb with a razor blade.
      3. Cut off brain tissue posterior to the neocortex (this include cerebellum, posterior midbrain and hind brain) with a razor blade.
      4. From the medial side, gently pull out the thalamus, septum and striatum by using a spatula. Cut off these subcortical tissue.
      5. Gently flip the hippocampal formation out and then separate it from cortex using a spatula.
    6. Flatten the isolated cortical sheet on a slide glass with the pia surface against the glass. Cover the other side of the cortical sheet with a piece of sponge. Cover the sponge with another piece of the slide glass. Space the two slides with two coins (we used United State dimes with thickness of 1.35 mm). Clip the both sides of slides (Figure 1).


      Figure 1. Sketch of the device used to flatten cortical sheet

    7. Immerse the ‘sandwich’ made in Step B6 in 1% PFA overnight (in a Petri dish in 4 °C fridge). Make sure the whole ‘sandwich’ is fully submerged.
    8. Remove 1% PFA and add 4% PFA (see Recipes) in the dish overnight.
    9. Remove 4% PFA and add 20% sucrose in the dish overnight.
    10. Remove the clips and remove the cortical sheet. Cut the outer edge of the sheet so that it is in an asymmetric shape and the orientation of the cortex (anterior, posterior, medial and lateral) is easy to identify.
    11. Take a fluorescence vasculature image (filter setting: 655/670 nm) of the flattened and cut cortex sheet before sectioning. Name it image D (Figure 2D).

  3. Tangential sectioning of flattened cortical sheet
    Note: This is the crucial step and do it with extra caution.
    1. Sufficiently cool the platform with dry ice before mounting (~10 min) the tissue and keep the platform frozen (dry ice always presented in the wells at the both end of the platform) for the whole sectioning process.
    2. Embed flattened cortex sheet in OCT with cortical surface facing up on microtome platform.
    3. Quickly put one glass slide on top of the cortex sheet before it freezes. Apply gentle pressure with fingers on the slides so that it flattens the tissue surface until it freezes.
    4. Raise and adjust the platform against the dissecting blade, until the blade is perfectly aligned with the top surface of the glass slide. Lock the platform.
    5. Lower the platform and warm the top glass slide with a finger to defrost the top surface. Remove top glass slide. Now the top surface of the cortical tissue should be perfectly parallel to the dissecting blade.
    6. Slowly raise the platform until the frozen tissue touches the blade.
    7. Check the alignment between frozen tissue surface and the blade carefully. Try very thin sections (~5 µm) to adjust alignment.
    8. Cut the first section with 150 µm thickness. This is to make sure the first section is across the whole cortical surface and contains sufficient surface vasculature for later alignment.
    9. Cut the remaining sections with 100 µm or 50 µm thickness through the whole cortex.
    10. Soak the sections in 1x PBS in sequence in a 24-well plate.

  4. CO staining (modified from Tootell et al., 1988)
    1. Wash the sections with excess PBS (pH 7.4), 3 x 5 min, ~40 ml per wash.
    2. Mount the sections on gelatin coated slides. Wait until completely dry.
    3. Preincubate sections with pre-incubation solution (see Recipes) at room temperature for 10 min.
    4. Rinse 4 x 5 min with rinse solution.
    5. Incubate sections with incubation solution (see Recipes) for 1-6 h at 37-40 °C in the dark (or foil covered).
    6. Check staining every 0.5-1 h until the reaction is sufficiently advanced and terminate the reaction by observing darkness of the tissue.
    7. Rinse sections with rinse solution (3 x 3 min, see Recipes)
    8. Rinse sections with dH2O (1 x 3 min)

  5. Dry mount (all procedure should be performed under a fume hood)
    1. Dry and defat through series of EtOH 50%, 70%, 90%, 3 min each.
    2. Wash with 100% EtOH: 2 x 3 min.
    3. Wash with xylene 1 x 5 min.
    4. Coverslip with DPX right after xylene without drying the xylene.
    5. Let the DPX solidify overnight.
    6. Take brightfield images of the sections (image series E, for example of a section across layer 4 in this series see Figure 2E, showing architectonic labeling of primary sensory cortices and retrosplenial cortex).

Data analysis

  1. Adjust the contrast and pixel resolution of images A, C, D, E so that the vasculature and cytoarchitectonic features are prominent and they all have roughly same pixel size.
  2. Image B should go through same transformations as image A, so that they remain co-registered (Figure 2A/B).
  3. Load all images into ImageJ TrakEM2 plugin.
  4. Use in vivo images (image A/B) as reference and align other images progressively. Align image C to image A/B → align image D to image A/B/C → align image series E to image A/B/C/D.
    1. Use non-linear transformation function (inside the TrakEM2 plugin) to align vasculature fiducials between adjacent image layers.
    2. Use surface vasculature to align images A, C, D (Figure 2F).
    3. Use the section outline and ascending/descending vessel cross sections to align image D and image series E (Figure 2G).
  5. Once all images are co-registered, hide all the intermediate image layers and superimpose image B and the image showing the most prominent cytoarchitectonic features in image series E. The overlay image allows a direct comparison between the in vivo functional map and the CO labeled architectonic map (Figure 2H).


    Figure 2. Images acquired at different steps and registration among them. A-E. Images from key steps in the processing of tissue from an Emx1-Ai96 mouse, each aligned to the CO image. A/B. Brightfield image of surface vasculature with overlaid visual area map. C. Fluorescence image of whole-mount brain, after perfusion, in which a subset of the surface vasculature is labeled with DyLight 649-lectin conjugate. D. Fluorescence image of the flattened cortex. E. Bright field image of a section through layer four after CO staining. F. Overlaid fluorescence images of surface vasculature in whole-mount (red, panel C) and after flattening (green, panel D). G. Overlaid images of the surface vasculature and CO staining in posterior barrel cortex and anterior V1. The contrast of the vasculature image is inverted for clarity. Arrowheads indicate small, circular regions that do not stain for CO and likely result from transverse cuts through ascending/descending vessels. Note the alignment of these putative vessels with likely locations of ascending/descending vessels in the fluorescence image of surface vasculature. H. Field sign map (panel A/B) aligned to chemoarchitectonic borders from the CO image (panel E). Borders of primary visual cortex, auditory cortex, and of barrels in primary somatosensory cortex) were drawn manually. Modified from Zhuang et al., 2017. Scale bar is 1 mm in panel H.

Notes

  1. The duration of Steps B2-B6 (after perfusion to flattening) should be as short as possible, longer delays may cause the brain to harden and affect the result of flattening.
  2. In images C (recorded in Step B2) and D (recorded in Step B11), only a subset of the cortical surface vasculature in image A (recorded in Procedure A) will be labeled.
  3. In image C recorded in Step B11, same cortical surface vasculature as that in image B should be visible.
  4. When rinsing the sections during CO staining, the rotation speed of the shaker should be less than 20 rpm to avoid displacing sections from the slide.
  5. DAB is GHS07, GSH08 hazardous material. Handle with caution.
  6. Paraformaldehyde is GHS02, GHS05, GHS07, GHS08 hazardous material. Handle with caution.
  7. The resolution of image D, image E and image series F should be high enough that the cross sections of ascending/descending vessels are visible (we used ~3 μm/pixel).
  8. Sometimes inverting the contrast of some images during image alignment may help visualize the fiducials across images.
  9. For image alignment, any image analysis software allowing the use of independent layers and nonlinear/warping transformations may be used; however, a suitable and widely available software is the TrakEM2 function (Cardona et al., 2012) in Fiji software (https://fiji.sc/, Schindelin et al., 2012).

Recipes

  1. 4% (w/v) formaldehyde (4% PFA in PBS, under fume hood)
    1. For 1 L of 4% formaldehyde, add 800 ml of PBS to a glass beaker on a stir plate in a fume hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil
    2. Add 40 g of paraformaldehyde powder to the heated PBS solution
    3. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears
    4. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered
    5. Adjust the volume of the solution to 1 L with PBS
    6. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9
    7. The solution can be aliquoted and frozen or stored at 2-8 °C for up to one month
  2. 1% (w/v) formaldehyde (1% PFA in PBS, under fume hood)
    1. For 1 L of 4% formaldehyde, add 800 ml of PBS to a glass beaker on a stir plate in a fume hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil
    2. Add 10 g of paraformaldehyde powder to the heated PBS solution
    3. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise with a pipette until the solution clears
    4. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered
    5. Adjust the volume of the solution to 1 L with PBS
    6. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9
    7. The solution can be aliquoted and frozen or stored at 2-8 °C for up to one month
  3. 0.2 M PB solution with 20% (w/v) sucrose stock (pH 7.4, 1,000 ml)
    NaH2PO4 (anhydrous) 0.04 M: 4.8 g
    Na2HPO4 (anhydrous) 0.16 M: 22.72 g
    Sucrose: 200 g
    Adjust pH to 7.4
    Add distilled water to 1,000 ml
  4. 5 mg/ml DAB stock in 0.05 M Tris-HCl buffer (pH 7.6, 100 ml, under fume hood)
    500 mg DAB
    Tris-HCl: 0.788 g
    Tris base: 0.606 g
    Adjust pH to 7.6
    Add distilled water to 100 ml
    Aliquot into 1 ml, frozen (-20 °C) for storage
  5. Pre-incubation solution, 0.05 M Tris-HCl Buffer Stock Solution (500 ml) with 275 mg/L CoCl2 and 10% sucrose (pH 7.4, 500 ml)
    Tris-HCl: 3.305 g
    Tris base: 0.485 g
    CoCl2: 137.5 mg (final concentration: 275 mg/L)
    Sucrose: 50 g (final concentration: 10% w/v)
    Adjust pH to 7.4
    Add distilled water to 500 ml
  6. Incubation solution (25 ml, under fume hood)
    1. 0.2 M PB with 20% sucrose (pH 7.4): 20 ml
    2. 4 ml DAB stock solution (5 mg/ml, final DAB concentration: 0.5 mg/ml)
    3. 3 mg cytochrome c (final concentration: 0.075 mg/ml)
    4. 0.008 ml catalase (final concentration: 64-640 units/ml)
    5. 0.1 ml DMSO (final concentration: 0.25%)
    6. Add distilled water to 25 ml (to reduce over reacting, this can be diluted to 40 ml)
  7. Rinse solution, 0.1 M PB with 10% sucrose (pH 7.4) (200 ml)
    0.2 M PB with 20% sucrose (pH 7.4): 100 ml
    Add distilled water to 200 ml

Acknowledgments

The project described here was supported by the Allen Institute for Brain Science and award number R01NS078067 from the National Institute of Mental Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of health and the National Institute of Neurological Disorders and Stroke. We thank the many staff members of the Allen Institute, especially the In Vivo Sciences team for surgeries and Marina Garrett for advice. We thank the Allen Institute founders, Paul G Allen and Jody Allen, for their vision, encouragement and support. The authors declare that there is no conflict of interest.

References

  1. Andermann, M. L., Kerlin, A. M., Roumis, D. K., Glickfeld, L. L. and Reid, R. C. (2011). Functional specialization of mouse higher visual cortical areas. Neuron 72(6): 1025-1039.
  2. Cardona, A., Saalfeld, S., Schindelin, J., Arganda-Carreras, I., Preibisch, S., Longair, M., Tomancak, P., Hartenstein, V. and Douglas, R. J. (2012). TrakEM2 software for neural circuit reconstruction. PLoS One 7(6): e38011.
  3. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. J Vis Exp (65).
  4. Garrett, M. E., Nauhaus, I., Marshel, J. H. and Callaway, E. M. (2014). Topography and areal organization of mouse visual cortex. J Neurosci 34(37): 12587-12600.
  5. Juavinett, A. L., Nauhaus, I., Garrett, M. E., Zhuang, J. and Callaway, E. M. (2017). Automated identification of mouse visual areas with intrinsic signal imaging. Nat Protoc 12(1): 32-43.
  6. Marshel, J. H., Garrett, M. E., Nauhaus, I. and Callaway, E. M. (2011). Functional specialization of seven mouse visual cortical areas. Neuron 72(6): 1040-1054.
  7. Murakami, T., Matsui, T. and Ohki, K. (2017). Functional segregation and development of mouse higher visual areas. J Neurosci 37(39): 9424-9437.
  8. Olavarria, J. and Montero, V. M. (1989). Organization of visual cortex in the mouse revealed by correlating callosal and striate-extrastriate connections. Vis Neurosci 3(1): 59-69.
  9. Roth, M. M., Helmchen, F. and Kampa, B. M. (2012). Distinct functional properties of primary and posteromedial visual area of mouse neocortex. J Neurosci 32(28): 9716-9726.
  10. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  11. Smith, I. T., Townsend, L. B., Huh, R., Zhu, H. and Smith, S. L. (2017). Stream-dependent development of higher visual cortical areas. Nat Neurosci 20(2): 200-208.
  12. Tootell, R. B., Hamilton, S. L., Silverman, M. S. and Switkes, E. (1988). Functional anatomy of macaque striate cortex. I. Ocular dominance, binocular interactions, and baseline conditions. J Neurosci 8(5): 1500-1530.
  13. Wang, Q. and Burkhalter, A. (2007). Area map of mouse visual cortex. J Comp Neurol 502(3): 339-357.
  14. Wang, Q., Gao, E. and Burkhalter, A. (2011). Gateways of ventral and dorsal streams in mouse visual cortex. J Neurosci 31(5): 1905-1918.
  15. Wang, Q., Sporns, O. and Burkhalter, A. (2012). Network analysis of corticocortical connections reveals ventral and dorsal processing streams in mouse visual cortex. J Neurosci 32(13): 4386-4399.
  16. Zhuang, J., Ng, L., Williams, D., Valley, M., Li, Y., Garrett, M. and Waters, J. (2017). An extended retinotopic map of mouse cortex. Elife 6: e18372.

简介

该协议描述了通过血管基准点的对齐使用细胞色素c氧化酶(CO)标记的相同小鼠脑的构建图来注册体内皮质视网膜地图的方法。 通过记录每个步骤的表面血管图案和顺序对准,该方法克服了在灌注,贴壁,切片和组织学程序期间由组织变形所施加的挑战。 这种方法也可以推广到注册和对齐其他类型的体内功能地图,如眼优势地图和空间/时间频率调整地图与小鼠皮层的各种解剖图。

【背景】通过体内视网膜映射(Marshel等人,2011; Garrett等人,),可以将小鼠视觉皮层分隔成功能上不同的视觉区域。 (Olavarria and Montero,1989; Wang and Burkhalter,2007),或者通过建筑结构辅助的神经元追踪技术,这些不同的视觉区域具有不同的响应特性和皮质皮质连接(Andermann等人,2011; Marshel等人,2011; Roth等人, >,2012; Wang等人,2011和2012)。这些结果表明,鼠标视觉区域形成处理不同类型的视觉信息的分离的视觉流(Murakami等人,2017; Smith等人,2017)。在视觉区域地图的背景下研究鼠标视觉系统对于理解视觉皮层的组织是至关重要的。然而,虽然功能图和结构图大致相似,但是这两幅图显示的并不完美(Zhuang et al。,2017)。例如,初级视觉皮层(V1)在两幅地图中都呈现为向上的三角形,但是在视网膜地图上V1的侧边缘可能比解剖图中的内侧多300μm以上(Zhuang et al。 / ,2017)。由于小鼠皮层中最小的视觉区域只有几百微米宽,忽略这种不匹配将潜在地偏向我们对视觉区域功能的解释。而且,这两种地图在不同的个体之间差异很大。因此,为了研究识别的视觉区域的功能,能够可靠地生成和比较同一动物中的功能和解剖图是很重要的。然而,灌注,贴壁,切片和组织学过程中的组织变形使得难以直接比较体内记录的功能图与组织学记录后的解剖图。在这里我们介绍一种方法来克服这些挑战,允许直接比较这两种类型的地图。

关键字:构筑图谱, 网膜代表图, 配准, 皮层展平, 切向切面, 脉管系统, 细胞色素c氧化酶

材料和试剂

  1. 海绵(Patterson Veterinary Supply,目录号:07-847-3539)
  2. 金属夹(Universal Small Binder Clips,Universal,目录号:UNV10200)
  3. 剃刀刀片(VWR,目录号:55411-050)
  4. 刮刀(精细科学工具,目录号:10090-13)
  5. 明胶底层幻灯片(SouthernBiotech,目录号:SLD01-CS)
  6. 盖玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:12450S)
  7. 康宁500毫升过滤系统,0.45微米(Corning,目录号:430770)
  8. 康宁24孔板(Corning,Falcon ,目录号:351147)
  9. 康宁一次性培养皿(100×15mm,Corning,Falcon ,目录号:351029)
  10. DyLight 649标记的番茄凝集素(Vector Laboratories,目录号:DL-1178)
  11. 干冰
  12. 组织-Tek OCT化合物(Sakura Finetek,VWR,目录号:4583)
  13. 10x磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9625)
  14. 乙醇,200标准(Fisher Scientific,目录号:16-100-826)
    制造商:Pharmco-Aaper,目录号:241ACS200CSGP。
  15. DPX封装剂(电子显微镜科学,目录号:13512)
  16. 多聚甲醛(Sigma-Aldrich,目录号:441244)
  17. 氢氧化钠溶液(NaOH)(1N,Sigma-Aldrich,目录号:S2770)
  18. 盐酸(HCl)(36.5-38%,Sigma-Aldrich,目录号:H1758)
  19. 磷酸二氢钠(NaH 2 PO 4)(无水,Sigma-Aldrich,目录号:S3139)
  20. 磷酸二氢钠(Na 2 HPO 4)(无水,Sigma-Aldrich,目录号:255793)
  21. 蔗糖(Sigma-Aldrich,目录号:S8501)
  22. 3,3'-二氨基联苯胺(DAB,1mg / ml,Sigma-Aldrich,目录号:D5637)
  23. Trizma HCl(Sigma-Aldrich,目录号:T5941)
  24. Trizma碱(Sigma-Aldrich,目录号:T6066)
  25. 氯化钴(II)(CoCl 2)(Sigma-Aldrich,目录号:232696)
  26. 细胞色素c(Sigma-Aldrich,目录号:C2506)
  27. 过氧化氢酶(10,000-40,000U / mg,20-50mg / ml,Sigma-Aldrich,目录号:C30)
  28. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  29. 二甲苯(Merck,目录号:XX0060)
  30. 4%(w / v)甲醛(PBS中4%PFA)(见食谱)
  31. 1%(w / v)甲醛(PBS中1%PFA)(见食谱)
  32. 含20%(w / v)蔗糖原液(pH7.4)的0.2M磷酸盐缓冲液(PB)溶液(见食谱)
  33. 在0.05M Tris-HCl缓冲液(pH7.6)中的5mg / ml DAB储备液(见配方)
  34. 预温育溶液,0.05M Tris-HCl缓冲液储备液(见食谱)
  35. 孵化解决方案(见食谱)
  36. 冲洗溶液,含10%蔗糖的0.1M PB(pH 7.4)(见食谱)

设备

  1. 通风橱(Labcono)
  2. 4°C的冰箱(松下医疗,型号:SR-L6111W-PA; VWR,目录号:89031-974)
  3. 秤(分析天平,A& D称量,型号:GH-252)
  4. 热板搅拌器(VWR,目录号:97042-646)
  5. B10P台式PH计(VWR,目录号:89231-662)
  6. 切片机(MICROM,型号:Sliding Microtome HM 400R)
  7. 用于明场和荧光成像的宽场显微镜(ZEISS,型号:Axio Imager 2)
  8. 解剖显微镜(徕卡显微系统,型号:徕卡MZ10 F)
  9. 振荡器(康宁,型号:LSE 低速)
  10. 孵化器(昆西实验室,模型:10型实验室烤箱)
  11. 蠕动泵(哈佛仪器,型号:MA1-55-7766)

软件

  1. 使用TrakEM2插件(Cardona等人,2012)的斐济(Schindelin等人,2012)

程序

  1. 体内成像
    做两个小的基准点分别指示颅窗的前方和内侧方向。通过荧光或明场图像(使用绿色波长可以获得更好的血管图像对比度)记录颅窗的脉管系统结构图像。将其命名为A.体内生成视网膜透过颅窗( ie ,内在信号Juavinett 等,2017年)或荧光视网膜视图Zhuang 等。,2017)。将其命名为图像B.图像A和图像B应该完美地共同登记,因为成像光轴垂直于颅窗(图2A / B)。

  2. 灌注和皮层展平(从Wang和Burkhalter,2007年修改)
    1. 在异氟烷麻醉下(5%异氟烷,Gage等人,2012)进行小鼠心脏灌注,灌注流体如下步骤。
      1. 盐水洗10毫升/分钟为100毫升。
      2. 5微克/毫升DyLight 649凝集素5毫升/分钟,25毫升标签血管。
      3. 等待5分钟,DyLight凝集素吸附到组织。
      4. 1%PFA(见食谱)5毫升/分钟,90毫升。
    2. 用头颅内的脑,获取颅窗的荧光图像(滤波器设置:655/670 nm)。程序A中的基准标记应该是可见的。将其命名为C(图2C)。
    3. 收集脑组织。由于动物灌注1%PFA,脑组织将比较软皮层扁平化。小心不要造成任何损坏。
    4. 可选:使用解剖镜,获取全脑表面脉管系统的明场和荧光图像(滤波器设置:655/670 nm)。主要的表面血管应该在明视野图像中可见(可以用图像A注册),DyLight标记的血管应该在荧光图像中可见(可以用图像D注册)。
    5. 隔离开窗半球的皮层(程序可以在冰上的培养皿中完成)。仔细跟踪皮层的方向。有关视频指导,请参阅此EJN视频协议(由苏黎世大学Hoey Sarah制作): http://www.ejnnews.org/video-protocol-isolation-adult-mouse-hippocampi/
      1. 用剃须刀片分开大脑的两个半球。保持窗口半球,并放弃另一半球。

      2. 用剃刀切断嗅球

      3. 切断新皮层(包括小脑,后脑和后脑)后面的脑组织。
      4. 从内侧,用刮刀轻轻拔出丘脑,隔膜和纹状体。切除这些皮质下组织。
      5. 轻轻翻转海马的形成,然后用刮刀从皮层分开。
    6. 将pia表面贴靠在玻璃上,将幻灯片上的隔离皮质层压平。用一块海绵覆盖皮层的另一面。用另一片幻灯片覆盖海绵。用两个硬币(我们用美国硬币1.35毫米的厚度)两个幻灯片空间。剪下幻灯片的两边(图1)。


      图1.用于扁平化皮层的设备草图

    7. 将步骤B6中制成的“三明治”浸入1%PFA中过夜(在4℃冰箱中的培养皿中)。确保整个“三明治”完全被淹没。

    8. 除去1%PFA,并在培养皿中加入4%PFA(见食谱)

    9. 除去4%PFA,并在培养皿中加入20%蔗糖
    10. 删除剪辑和删除皮质表。切割片材的外缘,使其处于不对称的形状,并且皮层(前,后,内侧和外侧)的方向很容易识别。
    11. 在切片前取一张扁平皮质切片的荧光脉管系统图像(滤波器设置:655/670 nm)。将其命名为图像D(图2D)。

  3. 扁平皮质切片的切线切片
    注意:这是至关重要的一步,请谨慎操作。
    1. 在安装(〜10分钟)纸巾之前用干冰充分冷却平台,并保持平台冷冻(干冰总是在平台两端的井中出现),以便进行整个切片过程。

    2. 在OCT中将皮层表面镶嵌在皮肤表面朝上的切片机平台上。
    3. 在冻结之前,快速将一张载玻片放在皮质片上。
      。用手指轻轻按压载玻片,使组织表面变平直至冻结。
    4. 抬起并调整平台靠着解剖刀片,直到刀片完全与载玻片的上表面对齐。锁定平台。
    5. 降低平台,用手指温热顶部玻璃片,除去顶部表面。卸下顶部玻璃滑块。现在,皮质组织的顶部表面应该完美平行于解剖刀片。
    6. 慢慢升高平台,直到冰冻组织接触到刀片。
    7. 仔细检查冷冻组织表面和刀片之间的对齐情况。尝试非常薄的部分(〜5微米)来调整对齐。
    8. 切割150微米厚的第一部分。这是为了确保第一部分穿过整个皮质表面,并包含足够的表面脉管系统,以便以后对齐。

    9. 在整个皮层切下100μm或50μm厚的剩余部分

    10. 在1x PBS中将切片浸泡在24孔板中
  4. CO染色(由Tootell等人修改,1988)
    1. 用过量的PBS(pH7.4)洗涤切片,3×5分钟,每次洗涤〜40毫升。
    2. 将这些部分安装在涂有明胶的载玻片上。等到完全干燥。

    3. 室温预培养切片(见食谱)10分钟
    4. 用冲洗液冲洗4 x 5分钟。

    5. 在37-40°C的黑暗中(或覆盖铝箔)孵育孵育液(见食谱)1-6小时。
    6. 每0.5-1小时检查一次染色,直到反应充分进行,通过观察组织的黑暗终止反应。
    7. 用冲洗液冲洗部分(3×3分钟,见食谱)
    8. 用dH 2 O(1×3分钟)冲洗切片,

  5. 干式安装(所有程序应在通风橱下进行)
    1. 通过系列乙醇50%,70%,90%,每个3分钟干燥和脱脂。
    2. 用100%乙醇洗涤:2×3分钟。
    3. 用二甲苯洗1次5分钟。
    4. 在二甲苯之后立即盖上DPX,不用干燥二甲苯。
    5. 让DPX在一夜之间凝固。
    6. 以剖面的明视野图像为例(图像序列E,例如本系列第4层的剖面见图2E,显示初级感觉皮质和后皮层的建筑标记)。

数据分析

  1. 调整图像A,C,D,E的对比度和像素分辨率,使脉管系统和细胞构造特征显着,并且都具有大致相同的像素尺寸。
  2. 图片B应该经过与图片A相同的转换,以便它们保持共同注册(图2A / B)。
  3. 将所有图像加载到ImageJ TrakEM2插件中。
  4. 使用体内图像(图像A / B)作为参考,逐步对齐其他图像。将图像C对准图像A / B→将图像D对准图像A / B / C→将图像序列E对准图像A / B / C / D。
    1. 使用非线性变换函数(TrakEM2插件内)来调整相邻图像层之间的脉管系统基准点。
    2. 使用表面血管来对齐图像A,C,D(图2F)。

    3. 使用截面轮廓和上升/下降血管横截面对齐图像D和图像系列E(图2G)
  5. 一旦所有的图像被共同注册,隐藏所有的中间图像层,并叠加图像B和图像系列E中显示最突出的cytoarchitectonic特征的图像。叠加图像允许直接比较体内功能图和CO标记的架构图(图2H)。


    图2.不同步骤获取的图像以及其中的注册。 A-E。来自Emx1-Ai96小鼠组织处理关键步骤的图像,每个图像都与CO图像对齐。 A / B。与重叠的视觉区域地图的表面脉管系统的明场图像。 C.全脑组织的荧光图像,在灌注后,其中一部分表面脉管系统用DyLight 649-凝集素缀合物标记。 D.扁平皮质的荧光图像。 E. CO染色后穿过第四层的切片的明场图像。 F.整体安装(红色,图C)和扁平化(绿色,图D)后的表面脉管系统的荧光图像重叠。 G.覆盖在后面的桶状皮质和V1前面的表面脉管系统和CO染色的图像。为了清楚起见,脉管系统图像的对比反转。箭头表示小的圆形区域,不会因为CO而染色,而且可能是通过上升/下降血管的横向切割造成的。注意这些假定的血管与表面脉管系统的荧光图像中上升/下降血管的可能位置对齐。 H.现场标志图(图A / B)与CO图像(图E)对齐到chemoarchitectonic边界。初级视觉皮层,听觉皮层和初级躯体感觉皮层中的桶的边界)被手动绘制。根据Zhuang et al。修改,2017年。面板H中的比例尺为1 mm。

笔记

  1. 步骤B2-B6的时间(灌注到扁平后)应尽可能短,延迟时间可能会导致大脑硬化并影响扁平化结果。
  2. 在图像C(在步骤B2中记录)和D(在步骤B11中记录)中,图像A中的皮层表面血管系统的一个子集(记录在程序A中)将被标记。
  3. 在步骤B11记录的图像C中,与图像B中相同的皮层表面脉管系统应该是可见的。
  4. 在CO染色过程中冲洗切片时,摇床的旋转速度应低于20 rpm,以避免从切片上移动切片。
  5. DAB是GHS07,GSH08有害物质。谨慎处理。
  6. 多聚甲醛是GHS02,GHS05,GHS07,GHS08有害物质。谨慎处理。
  7. 图像D,图像E和图像序列F的分辨率应该足够高,使得上升/下降血管的横截面可见(我们使用〜3μm/像素)。
  8. 有时在图像对齐过程中反转一些图像的对比度可能有助于可视化图像间的基准点。
  9. 对于图像对齐,可以使用允许使用独立层和非线性/变形转换的任何图像分析软件;然而,斐济软件( https://fiji.sc/ ,Schindelin ,,2012)。

食谱

  1. 4%(w / v)甲醛(4%PFA的PBS溶液,通风橱)
    1. 对于1L的4%甲醛,在通风橱中搅拌板上的玻璃烧杯中加入800ml的PBS。在搅拌下加热至约60℃。注意解决方案不煮沸
    2. 将40克多聚甲醛粉末加入到加热的PBS溶液中
    3. 粉末不会立即溶解到溶液中。从移液管中滴加1N氢氧化钠缓慢提高pH值,直到溶液清除
    4. 一旦多聚甲醛溶解,溶液应冷却并过滤
    5. 用PBS
      将溶液的体积调整到1L
    6. 重新检查pH值,并用少量稀HCl调整至约6.9
    7. 解决方案可以分装和冷冻或2-8°C保存长达一个月
  2. 1%(w / v)甲醛(1%PFA的PBS溶液,通风橱)
    1. 对于1L的4%甲醛,在通风橱中搅拌板上的玻璃烧杯中加入800ml的PBS。在搅拌下加热至约60℃。注意解决方案不煮沸
    2. 加入10克多聚甲醛粉末加热的PBS溶液
    3. 粉末不会立即溶解到溶液中。用移液管滴加1N氢氧化钠缓慢升高pH值直至溶液清除
    4. 一旦多聚甲醛溶解,溶液应冷却并过滤
    5. 用PBS
      将溶液的体积调整到1L
    6. 重新检查pH值,并用少量稀HCl调整至约6.9
    7. 解决方案可以分装和冷冻或2-8°C保存长达一个月
  3. 含有20%(w / v)蔗糖原液(pH 7.4,1000毫升)的0.2M PB溶液
    NaH2PO4(无水)0.04M:4.8g
    Na 2 HPO 4(无水)0.16 M:22.72g。
    蔗糖:200克
    调整pH值到7.4
    加蒸馏水到1000毫升
  4. 在0.05M Tris-HCl缓冲液(pH7.6,100ml,通风橱)中的5mg / ml DAB储备液 500毫克DAB
    Tris-HCl:0.788克
    Tris碱:0.606克
    调整pH值到7.6
    加蒸馏水到100毫升
    分装成1毫升,冷冻(-20°C)存储
  5. 预孵育溶液,含275mg / L CoCl 2和10%蔗糖(pH 7.4,500ml)的0.05M Tris-HCl缓冲液储备溶液(500ml)。
    Tris-HCl:3.305克
    Tris碱:0.485克
    CoCl 2:137.5mg(最终浓度:275mg / L)
    蔗糖:50g(最终浓度:10%w / v)
    调整pH值到7.4
    加蒸馏水到500毫升
  6. 孵育溶液(25毫升,在通风橱下)
    1. 含20%蔗糖的0.2M PB(pH7.4):20ml
    2. 4毫升DAB储备液(5毫克/毫升,最终DAB浓度:0.5毫克/毫升)
    3. 3毫克细胞色素C(终浓度:0.075毫克/毫升)
    4. 0.008毫升过氧化氢酶(终浓度:64-640单位/毫升)
    5. 0.1ml DMSO(终浓度:0.25%)
    6. 加入蒸馏水至25毫升(减少过度反应,这可以稀释到40毫升)
  7. 漂洗溶液,含10%蔗糖的0.1M PB(pH7.4)(200ml)
    含20%蔗糖的0.2M PB(pH7.4):100ml
    加蒸馏水到200毫升

致谢

这里描述的项目得到了艾伦脑科学研究所的支持,并得到了国立精神卫生研究所的奖励号码R01NS078067的支持。其内容完全是作者的责任,不一定代表国立卫生研究院和国家神经疾病和中风研究所的官方观点。我们感谢艾伦研究所的许多工作人员,特别是体内科学团队的手术和滨海加勒特的建议。我们感谢艾伦研究所的创始人Paul G Allen和Jody Allen的远见,鼓励和支持。作者声明不存在利益冲突。

参考

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Copyright Zhuang 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. Zhuang, J., Wang, Q., Takeno, M. and Waters, J. (2018). Registration and Alignment Between in vivo Functional and Cytoarchitectonic Maps of Mouse Visual Cortex. Bio-protocol 8(4): e2731. DOI: 10.21769/BioProtoc.2731.
  2. Zhuang, J., Ng, L., Williams, D., Valley, M., Li, Y., Garrett, M. and Waters, J. (2017). An extended retinotopic map of mouse cortex. Elife 6: e18372.
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