Protocol to Quantitatively Assess the Structural Integrity of Perineuronal Nets ex vivo
体外定量分析神经元周围基质网的结构完整性方法   

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Marta Miquel Marta Miquel
Joshua Brumberg Joshua Brumberg
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Nature Communications
Nov 2018

 

Abstract

Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system diseases including schizophrenia, Alzheimer disease and epilepsy present with qualitative alteration in PNNs, however prior studies failed to quantitatively assess such changes at single PNN level and correlate them with functional changes in disease. We describe a method to quantify the structural integrity of PNNs using high magnification image analysis of Wisteria Floribunda Agglutinin (WFA)-labeled PNNs in combination with cell-type-specific marker such as PV and NeuN. A polyline intensity profile of WFA along the entire perimeter of cell shows alternate segments with and without WFA labeling, indicating the intact chondroitin sulfate proteoglycan (CSPG) and holes of PNN respectively. This line intensity profile defines CSPG peaks, where intact PNN is present, and CSPG valleys (holes) where the PNN is missing. The average number of peaks reflect the integrity of the lattice assembly of PNN. The average size of PNN holes can be readily computed using image analysis software. Furthermore, degradation of PNNs using a bacterial-derived enzyme, Chondroitinase ABC (ChABC), allows to experimentally manipulate PNNs in situ brain slices during which biophysical properties can be assessed by patch-clamp recordings. We describe optimized experimental parameters to degrade PNNs in brain slices before as well as during recordings to study the possible change in function in real time. Our protocols provide effective and appropriate methods to modulate and quantify the PNN’s experimental manipulations.

Keywords: Perineuronal nets (神经元周围基质网), Brain slices (脑切片), Chondroitinase ABC (硫酸软骨素酶ABC), Wisteria floribunda agglutinin (紫藤凝集素), PV (PV), Extracellular matrix (细胞外基质)

Background

The extracellular spaces of the central nervous system are filled by an amorphous interstitial matrix that is composed of dense network of proteoglycans, hyaluronic acid (HA), tenascins and link proteins, and is contiguous with well-organized lattice-like structures called PNNs that enclose the somata, axons and dendrites of PV expressing interneurons in the cerebral cortex (Härtig et al., 1999). The lattice of PNNs is composed of chondroitin sulfate proteoglycans (CSPGs), HA, tenascins, various link proteins (Deepa et al., 2006), etc. Owing to the high density of sulfated proteoglycans, PNNs are highly negatively charged (Morawski et al., 2015). The development of PNN coincides with the closure of ocular dominance critical period and studies have shown reinstatement of ocular dominance plasticity upon removal of the PNNs thereby suggesting inhibitory role of PNNs on neuronal plasticity (Pizzorusso et al., 2002). PNNs are also suggested to modulate the biophysical properties PV interneurons (Balmer, 2016, Favuzzi et al., 2017, Tewari et al., 2018), and are prone to degradation by ECM remodeling enzymes. Most notably, under pathological conditions including epilepsy (McRae et al., 2012, Rankin-Gee et al., 2015, Dubey et al., 2017, Tewari et al., 2018, Patel et al., 2019), schizophrenia and Alzheimer disease (Testa et al., 2018) ECM remodeling is common.

One of the major challenges in reporting on the status of PNNs is the quantification of the structural integrity of their lattice-like assembly. Essentially all published studies use the CSPG binding lectin, WFA, as non-specific PNN marker to visualize PNNs, and use WFA intensity to quantify the overall CSPG levels without detailed analysis of individual PNN’s architecture (Slaker et al., 2015 and 2016, Lensjø et al., 2017, Ueno et al., 2018). We developed a method to quantify the structural integrity of individual PNNs using high-magnification fluorescence imaging. This method also minimizes the errors due to procedure-related variations in the WFA intensity. After acquiring multichannel high magnification images (40x objective lens with 5 digital zoom) of individual PNNs, a polyline is drawn around the periphery of the cell. The fluorescence intensity of WFA along this line shows a discrete pattern of peaks separated by low-intensity valleys. Each peak represents the continuous CSPG and the gap between the two consecutive peaks represent a hole in the PNN (Tewari et al., 2018). The average number of peaks and average width of holes can be derived from the WFA intensity data points and are reflective of the PNN’s structural integrity. The peaks in the line profile can be counted manually or by using clampfit program (Molecular Devices).

A vast majority of studies, which explore the function of PNNs and their influence on the neuronal physiology, use ChABC, which is a bacterial enzyme that cleaves the chondroitin sulfate side chains thereby dismantling the PNN assembly (Dityatev et al., 2007, Balmer, 2016, Testa et al., 2018). The most common approach in such studies involves degradation of PNN in-vivo/in-situ brain slices/primary cultures and studying the function afterward by comparing two different populations of samples. However, it would be advantageous to record from PNN bearing neurons during the real-time degradation of PNNs to evaluate the physiological role of PNN on neuronal function. Such studies are largely absent in the literature. Here, we report two variants of experimental PNN degradation in the brain slices, which reliably degrade PNNs to allow studying their function. The first variant includes degradation of PNNs by incubating brain slices with ChABC in an incubation chamber followed by performing experiments to compare their properties with non-treated controls. The other variant is to study the baseline physiology in the presence of intact PNNs followed by superfusing ChABC solution and recording the functional activity during and after PNN depletion in the experimental set-up. Both the variants involve controlled degradation of PNNs and identifiable traces of degraded PNNs around the cells can be observed to confirm the presence/absence of PNNs on the experimental cell/tissue after post-experimental WFA staining of the samples. These methods provide unparalleled advantages to study the physiological functions associate with the PNNs.

Materials and Reagents

Materials

  1. PNN degradation in brain slices outside experimental setup
    1. Carbogen inlet and outlet needles (BD PrecisionGlide needle, catalog number: 305198)
    2. Perfusion Tubes (Becton Dickinson, PE-160, catalog number: 427431)
    3. Carbogen bubbling tubes (Fisher Scientific, Tygon, catalog number: S3 E-3603)
    4. Multichannel splitter (Luner valve assortment, catalog number: WPI-14055)
    5. Transfer pipettes (Fisher brand, catalog number: 13-711-7M)
    6. Glass Petri dish (Cole-Parmer Instrument, catalog number: EW-34551-06)
    7. Polystyrene foam (from any commercial source)

  2. PNN degradation in brain slices in experimental setup
    1. 15 ml tube (Falcon, catalog number: 352097)
    2. Thin perfusion lines (PVC pump tubes-1.52 mm ID, catalog number: 116-0549-19)
    3. Bubbling tubes (Fisher Scientific, Tygon, catalog number: S3 E-3603)
    4. Perfusion Tubes (Becton Dickinson, PE-160, catalog number: 427431)

  3. PNN’s structural integrity analysis
    1. High magnification (40 x 5 or higher) images of WFA with PV or NeuN

  4. WFA staining of brain slices
    1. 24-well plate (Falcon, catalog number: 35-3226)
    2. Paint Brush (Any fine tipped clean brush)
    3. Coverslips (Fisher Finest, catalog number: 12-548-5M)
    4. Glass slide (Micro slides, VWR, catalog number: 48311-703)
    5. Mounting medium (Thermo Fisher, Invitrogen, catalog number: S36936)
    6. Marker (Lab marker, VWR, catalog number: 52877-310)
    7. Transfer pipettes (Fisher brand, catalog number: 13711-7M)

Reagents

  1. ChABC (Sigma-Aldrich, catalog number: C3667-10U)
  2. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A8806-1G)
  3. 4% Paraformaldehyde (PFA) (Electron Microscopy Science, catalog number: 15714-S)
  4. PBS (Fisher Bioreagent, catalog number: BP661-10)
  5. Biotinylated Wisteria Floribunda Agglutinin/Lectin (WFA/WFL) (Vector Laboratories, catalog number: B-1355)
  6. Streptavidin, Alexa FluorTM 555 Conjugate (Thermo Fisher, Invitrogen, catalog number: S32355)
  7. 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher, Life Tech, catalog number: D1306)
  8. Mounting medium (Thermo Fisher, Invitrogen, catalog number: S36936)
  9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418)
  10. ChABC (see Recipes)
  11. PBS (see Recipes)
  12. 4% PFA (see Recipes)
  13. ACSF (standard ringer’s ACSF for acute brain slices) (Papouin and Haydon, 2018, Tewari et al., 2018) (see Recipes)
  14. DAPI stock (see Recipes)

Equipment

  1. Measurement pipettes (Gilson, catalog number: F123602)
  2. Timer (from any commercial source)
  3. Sterile empty glass vials, 30 ml (such as Hospire, catalog number: 5816-31)
  4. Slice incubation assembly: assemble as shown in Figures 2B-2D
  5. Bath heater (Fisher Scientific, model: Isotemp 205)
  6. Peristaltic Perfusion pump (Gilson, model: mini-plus 3)
  7. In-line heater (Warner instrument, catalog number: TC324B)
  8. High resolution imaging microscope, such as Confocal microscope (Nikon Elements)
  9. 95% O2/5% CO2 tank (such as AirGas)
  10. -20 °C freezer (such as Norlake)

Software

  1. Nikon NIS-Elements
  2. Nikon NIS-Elements AR analysis
  3. (Optional) Clampfit (Molecular Devices)
  4. (Optional) Microsoft Excel/Origin (OriginLab)

Procedure

  1. Structural integrity of PNNs in high-magnification images
    1. Acquire high-magnification (40x objective lens with 5 digital zoom) multichannel (with PV or NeuN) single plane fluorescence images of individual PNNs in different experimental groups. The images should be taken from the optical plane that covers the largest perimeter of the cell.
    2. Draw a manual/automated (depending on the used software) polyline along the periphery of the cell (stained with NeuN/PV) with PNN (Figures 1A and 1B, large images).
      1. If using Nikon elements program, then use the auto ROI function to determine the perimeter of the cell followed by drawing a polyline along with this perimeter.
      2. If using Fiji/ImageJ, use the freehand line tool to draw a line along the cell perimeter followed by clicking on “Analyze” then “Plot profile” buttons.
      3. WFA peaks can be counted from the peak profile graph itself or numerical data for the plot profile can be exported to plot a graph in different software and count the peaks.
    3. After establishing the line profile, the software generates a line profile graph that shows the fluoresce intensity of WFA along the line (Figures 1A and 1B, bottom panels). The line profile graph and its numerical data can be exported in a Microsoft Excel file that has length (in µm) and fluorescence intensity as x and y-axis respectively. Peaks can be counted either from this graph generated by the acquisition program (as shown in Figures 1A and 1B, lower panels) or data can be plotted in Excel/analysis software such as Sigma plot or Origin for peak counting and representation.
    4. Mark and count the number of peaks in the line profile by setting a threshold of half the maximum intensity (Figures 1A-1B, lower panels). To determine the size of the biggest hole in the PNN, measure the distance between two consecutive peaks that appear to be farthest from each other (white double-headed arrows in lower panels in Figures 1A-1B). (Alternately, plot the data in Clampfit result window and save it as an .atf file. Open this file later and use the peak detection function to count the number of peaks. The parameters of peak detection function may detect few false peaks, which can be removed from the analysis. In addition, placing two cursers covering the gap and remarking the distance between them can be used to determine the size of the largest hole.)


      Figure 1. Analysis of WFA intensity peaks of PNN in two experimental conditions. A and B. High magnification (40x objective lens with 5 digital zoom) immunohistochemical staining images of WFA (green), PV (red) and overlay (large images on the right) showing single PNN in the cerebral cortex of a mouse model of glioma-associated epilepsy (A), and corresponding sham control (B). The polyline (right) along the perimeter of the PV, encompasses WFA staining and shows discrete WFA intensity peaks (marked by red arrows) separated by low WFA intensity baseline. White dotted line indicates the threshold (Maximum WFA intensity/2) to determine the WFA intensity peak. The two-headed white arrows show the highest apparent distance between two consecutive WFA peaks and indicate the size of the largest holes in the PNN. The total number of peaks are divided by the perimeter to account for the different size of cells and their PNNs. Scale bars: 5 µm.

  2. Pre-experimental degradation of PNNs in brain slices
    1. Prepare brain slices according to the standard brain slicing protocols and after completion of the recovery use for the experiments (Papouin and Haydon, 2018, Tewari et al., 2018).
    2. Prepare the slice incubation assembly using two glass vials; make two small holes on each cap to insert the inlet and outlet needles of carbogen gas supply. Insert an appropriate length piece of tube on the tip of needles to extend the gas supply close to the surface of the buffer. Do not dip these gas tubes into the solution (it can damage the slices).
    3. Make one needle inlet by connecting it to the carbogen supplying tube and leave the other needle open to function as outlet (Figure 2B).
    4. Next, fill 3 ml well-oxygenated ACSF in each vial and add ChABC stock to make a final concentration of 0.5 U/ml of ACSF in one of the vials labeled as treated.
    5. Transfer 2-3 brain slices in each vial using a modified transfer pipette (cut the tip of transfer pipette to broaden the tip for the ease of slice transfer as shown in Figure 2C).
    6. Set timer for 45 min from the time of ChABC addition and ensure that the carbogen gas supply through the inlet is working fine.
    7. Place both the vials into a custom-made floating polystyrene foam (Figure 2D) that allows vials to stay straight while floating in the water bath during incubation.
    8. Transfer both vials into the water bath, preset at 32 °C (Figure 2E).
    9. On completion of 45 min incubation, carefully take out vials, rinse slices with ACSF, and transfer slices from both the vials back to the recovery chamber (Figure 2G). The slices can be used for experimentation now.
    10. After completion of the experiment, carefully take out slices from the experimental setup and fix overnight with 4% PFA. Stain the fixed slices with WFA (and DAPI, if needed) to confirm the PNN degradation effect of ChABC as described in Procedure D.


      Figure 2. Degradation of PNNs in the acute brain slices using pre-experiment incubation method. A. Brain slice recovery chamber with cortical slices in the ACSF with continuous carbogen (95% O2, 5% CO2) bubbling. B. Fill the incubation vials (for control and ChABC-treated slices) with 3 ml ACSF (with/without ChABC) and assemble the carbogen gas supply tubes with an inlet and one outlet. C. Add 2-3 brain slices from (A) to each vial by modified transfer pipette (B on Petri dish) and subsequently insert the vials in the polystyrene foam to held them straight in the water bath (E) while incubating at 32 °C for 45 min (F). G. On completion of incubation, transfer slices back into the recovery chamber at room temperature for experimental purposes.

  3. PNN degradation during real time experimentation in brain slices
    1. Record the baseline activity of cells/slices for 5-10 min (depending on the specific experiment) in the recording setup.
    2. Superfuse 1 U/ml ChABC solution for 50 min while strictly maintaining the bath temperature at 32-33 °C (Figure 3A).
    3. After 50 min of recording, perfuse ACSF again and record the activity in the presence of ACSF for 5-10 min (Figure 3B). 
    4. Stop the experiment and transfer slices to a 24-well plate and add 500 µl, 4% PFA to fix the slice and keep at room temperature for overnight.
    5. Perform WFA staining next day as described in Procedure D.


      Figure 3. Degradation of PNNs in acute brain slices during real time data acquisition. A. ACSF containing ChABC with continuous carbogen bubbling is superfused and re-circulated at a speed of 2-3 ml/min to the experimental set-up using a peristaltic pump. B. Recording setup with an inline heater to keep the bath temperature constant at 32 °C.

  4. WFA and DAPI staining in fixed acute brain slices
    1. Rinse slices 4 times with PBS with 5 min interval between each wash.
    2. Add 0.5 ml PBS that contains Biotinylated-WFA solution (1:300 in PBS), in each slice and incubate for 1 h at room temperature followed by 4 rinses with PBS at every 5 min interval.
    3. Add 0.5 ml PBS that contains Streptavidin-conjugated Alexa fluor-555 solution (1:300 in PBS), in each slice and incubate for 1 h at room temperature followed by 4 rinses with PBS at every 5 min interval.
    4. Incubate slices with PBS that contains DAPI (1:1,000 from 10 mg/ml stock in DMSO) for 4 min and rinse 3 times with PBS at every 5 min.
    5. Mount slices in between 2 coverslips that allows user to access both the surfaces of the slice for imaging. Use mounting medium and do not press hard the coverslips to prevent tissue distortion.
    6. Keep mounted tissue on a glass slide and take images of recorded cells/or regions of brain to confirm the degradation of PNNs.

Data analysis

  1. PNN’s structural integrity
    Average the number of peaks/unit perimeter length of the cell. Use > 5 PNNs per mouse and pool data from all mice of the same experimental group. Compare the number of peaks in different experimental groups, apply appropriate statistical tests and plot the graph. Average the lengths of biggest gaps in the individual PNNs and pool data from all mice of the same experimental group. Compare the average size of holes (gap length) in different experimental groups, apply appropriate statistical tests, and plot the graph.
  2. Confirmation of experimental PNN degradation
    Take images from both sides of the slices using a fluorescence microscope. In case of pre-experimental degradation (Procedure B), both the surfaces of slice do not show any intact PNNs. However, magnified images show faint outlines of WFA around the PNN expressing cells (Inset image in Figure 4B). Only ACSF treated slices show intact PNNs (Figure 4A). In case of PNN degradation in experimental setup (Procedure C), only the top or exposed surface of slice shows the degradation effect of ChABC (Figure 4D). The high magnification images (20x) show faint outline of PNNs around the PNN expressing neurons (Inset image in Figure 4D). In case of whole cell patch clamp experiments, a traces dye can also be added to the pipette buffer to label the recorded cell, and presence of faint WFA outline around the recorded cells suggests that cells expressed PNN before the ChABC-treatment (Tewari et al., 2018).


    Figure 4. Confirmation of PNN degradation after ChABC-treatment of acute slices in incubation chamber and in recording setup. A and B. Confocal images of DAPI (top) and WFA (middle) fluorescence of acute cortical slices after post-experimental fixation from control (A), and ChABC-treated (B) groups. Inset images in overlay show the traces of controlled degradation of PNNs. C and D. Confocal images of DAPI (top) and WFA (middle) fluorescence from the ChABC-unexposed bottom surface (C), and ChABC-exposed top surface (D) of an acute brain slice in which PNNs were degraded in the experimental setup simultaneously with the experimental recording. The unexposed surface (C) shows relatively intact PNNs compared to the exposed surface (D), which shows degraded PNNs due to direct exposure of ChABC. Inset images in overlay are from the marked rectangular areas in the slices and show the traces of controlled PNN degradation. Scale bars: 100 µm in main images, 10 µm in inset images.

Notes

  1. In Procedures B and C, bath temperature is crucial to maintain at 32-33 °C for reliable PNN degradation.
  2. In Procedure B, the incubation time should not exceed 45 min otherwise; PNNs will be completely degraded making it difficult to find the traces of the degraded PNNs.
  3. In Procedure C, recirculating the ChABC solutions into the recording setup is recommended to minimize the quantity of the enzyme.
  4. In Procedure C, the total length of perfusion tubing for recirculation of ChABC should be kept minimum to reduce the time lag and the volume of total ChABC solution.
  5. For more precise comparison of the change in function upon PNN degradation, brain slices can be cut into 2 halves separating the cerebral hemispheres and one half can be treated with ChABC and other can be used as a control.
  6. For PNN’s structural integrity analysis in immunohistochemistry (IHC), freely accessible software including ImageJ/Fiji can also be utilized, provided all the experimental groups are analyzed with the same software.
  7. The high magnification images for PNNs structural integrity analysis should have the whole range of fluorescence signal with minimal saturation to minimize the variations in WFA intensity due to IHC procedure.

Recipes

  1. ChABC
    1. Reconstitute ChABC from Proteus vulgaris in a 0.01% BSA aqueous solution according to the manufacturer’s instruction to make 1 U/40 µl stock solution
    2. Prepare aliquots of 2 U and store at -20 °C until used
    3. Just before experiment, add appropriate amount directly into the bubbling ACSF to make the working concentration
  2. PBS
    Dissolve PBS salts mixture in deionized water (DI) to make PBS solution
  3. 4% PFA
    Dilute the supplied 32% PFA to 1(PFA):7(PBS) to make 4% working solution
  4. ACSF
    Standard ringer’s buffer (125 NaCl, 3 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1.3 MgSO4, 25 glucose (all in mM); pH 7.3; 310 ± 5 mOsm) used for acute slice recordings (Tewari, Chaunsali et al., 2018)
  5. DAPI stock
    Dissolve DAPI in DMSO to make 10 mg/ml stock and store at -20 °C until use

Acknowledgments

This work was supported by NIHRO1-NS036692, NIH-RO1-NS082851, and NIH-RO1-NS052634.

Competing interests

The authors declare no competing interests

Ethics

All animal procedures were approved and performed in accordance with the ethical guidelines set by Virginia Tech Institutional Animal Care and Use Committee (IACUC) under protocols 15-241 (02/11/2016-02/09/2019), 15-106 (07/31/2015-07/30/2018), and 18-126 (07/18/2018-07/15/2021).

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简介

摘要:perineuronal网(PNNs)是高负电荷蛋白多糖的细胞外基质组装,包裹在大脑皮质中表达中间神经元的快速尖峰小白蛋白(PV)。 PNN在神经元可塑性中起重要作用,并调节封闭的中间神经元的生物物理特性。包括精神分裂症,阿尔茨海默病和癫痫在内的各种中枢神经系统疾病都存在PNNs的定性改变,但是之前的研究未能在单一PNN水平上定量评估这些变化并将其与疾病的功能变化相关联。我们描述了使用Wisteria Floribunda凝集素(WFA)标记的PNN与细胞类型特异性标记物如PV和NeuN的组合的高放大倍数图像分析来量化PNN的结构完整性的方法。沿着细胞的整个周边的WFA的折线强度分布显示具有和不具有WFA标记的交替区段,分别指示完整的硫酸软骨素蛋白多糖(CSPG)和PNN的孔。该线强度分布定义了存在完整PNN的CSPG峰,以及缺少PNN的CSPG谷(空穴)。峰的平均数量反映了PNN的晶格组件的完整性。使用图像分析软件可以容易地计算PNN孔的平均尺寸。此外,使用细菌衍生的酶,软骨素酶ABC(ChABC)降解PNN,允许通过实验操作PNNs 原位脑切片,在此期间可通过膜片钳记录评估生物物理特性。我们描述了优化的实验参数,以在记录之前和记录期间降低脑切片中的PNN,以实时研究功能的可能变化。我们的协议提供了有效和适当的方法来调整和量化PNN的实验操作。

背景:中枢神经系统的细胞外空间由无定形间质基质填充,该基质由蛋白聚糖,透明质酸(HA),肌腱蛋白和连接蛋白的密集网络组成,并且与称为PNN的组织良好的晶格状结构邻接。 PV表达大脑皮层中间神经元的胞体,轴突和树突(Härtig et al。,1999)。 PNNs的晶格由硫酸软骨素蛋白聚糖(CSPGs),HA,tenascins,各种连接蛋白(Deepa et al。,2006),等组成。由于硫酸化蛋白多糖的高密度,PNN具有高度负电荷(Morawski 等人,,2015)。 PNN的发展与眼优势关键期的闭合相吻合,并且研究表明,在去除PNN后恢复眼优势可塑性,从而表明PNNs对神经元可塑性的抑制作用(Pizzorusso et al。,2002 )。 PNN也被建议用于调节PV中间神经元的生物物理特性(Balmer,2016,Favuzzi et al。,2017,Tewari et al。,2018),并且易于降解通过ECM重塑酶。最值得注意的是,在包括癫痫在内的病理条件下(McRae et al。,2012,Rankin-Gee et al。,2015,Dubey et al。 ,2017,Tewari et al。,2018,Patel et al。,2019),精神分裂症和阿尔茨海默病(Testa et al。,2018 )ECM重塑很常见。

报告PNN状态的主要挑战之一是量化其网格状组件的结构完整性。基本上所有已发表的研究都使用CSPG结合凝集素WFA作为非特异性PNN标记来显示PNN,并使用WFA强度来量化整体CSPG水平,而无需详细分析单个PNN的结构(Slaker 等。,2015年和2016年,Lensjø等人,2017,Ueno et al。,2018)。我们开发了一种使用高倍率荧光成像来量化各个PNN的结构完整性的方法。该方法还最小化了由于WFA强度的程序相关变化引起的误差。在获取单个PNN的多通道高放大率图像(具有5个数字变焦的40倍物镜)之后,在单元的周边绘制折线。沿着该线的WFA的荧光强度显示由低强度谷分开的离散峰图案。每个峰代表连续的CSPG,两个连续峰之间的间隙代表PNN中的一个洞(Tewari et al。,2018)。孔的平均峰数和平均宽度可以从WFA强度数据点导出,并且反映了PNN的结构完整性。线轮廓中的峰可以手动计数或使用clampfit程序(Molecular Devices)计数。

绝大多数探索PNN功能及其对神经元生理学影响的研究使用ChABC,这是一种切割硫酸软骨素侧链的细菌酶,从而拆解PNN组件(Dityatev et al。,2007,Balmer,2016,Testa et al。,2018)。这类研究中最常见的方法是降解PNN 体内 / 原位脑切片/原代培养物,然后通过比较两种不同的样本群体来研究其功能。然而,在PNN的实时降解期间记录来自携带PNN的神经元以评估PNN对神经元功能的生理作用将是有利的。这些研究在文献中基本上不存在。在这里,我们报告脑切片中实验性PNN降解的两种变体,其可靠地降解PNN以允许研究它们的功能。第一种变体包括通过在孵育室中将脑切片与ChABC一起孵育来降解PNN,然后进行实验以比较它们的性质与未处理的对照。另一种变体是在完整PNN存在下研究基线生理学,然后灌注ChABC溶液并在实验装置中记录PNN耗尽期间和之后的功能活性。两种变体都涉及PNN的受控降解,并且可以观察到细胞周围的可识别痕迹的降解的PNN,以证实在样品的实验后WFA染色后实验细胞/组织上存在/不存在PNN。这些方法为研究与PNN相关的生理功能提供了无可比拟的优势。

关键字:神经元周围基质网, 脑切片, 硫酸软骨素酶ABC, 紫藤凝集素, PV, 细胞外基质

材料和试剂


  1. 在实验装置外的脑切片中PNN降解
    1. Carbogen进针和出针(BD PrecisionGlide针,目录号:305198)
    2. 灌注管(Becton Dickinson,PE-160,目录号:427431)
    3. Carbogen鼓泡管(Fisher Scientific,Tygon,目录号:S3 E-3603)
    4. 多通道分流器(Luner阀门分类,目录号:WPI-14055)
    5. 转移移液器(Fisher品牌,目录号:13-711-7M)
    6. 玻璃培养皿(Cole-Parmer仪器,目录号:EW-34551-06)
    7. 聚苯乙烯泡沫(来自任何商业来源)

  2. 实验装置中脑切片中的PNN降解
    1. 15毫升管(猎鹰,目录号:352097)
    2. 薄灌注管(PVC泵管 - 1.52 mm内径,目录号:116-0549-19)
    3. 鼓泡管(Fisher Scientific,Tygon,目录号:S3 E-3603)
    4. 灌注管(Becton Dickinson,PE-160,目录号:427431)

  3. PNN的结构完整性分析
    1. 具有PV或NeuN的WFA的高放大倍率(40 x 5或更高)图像

  4. 脑切片的WFA染色
    1. 24孔板(Falcon,目录号:35-3226)
    2. 油漆刷(任何细尖的清洁刷)
    3. 盖弗(Fisher Finest,目录号:12-548-5M)
    4. 载玻片(微型载玻片,VWR,目录号:48311-703)
    5. 安装介质(Thermo Fisher,Invitrogen,目录号:S36936)
    6. 标记(实验室标记,VWR,目录号:52877-310)
    7. 转移移液器(Fisher品牌,目录号:13711-7M)

的试剂

  1. ChABC(Sigma-Aldrich,目录号:C3667-10U)
  2. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A8806-1G)
  3. 4%多聚甲醛(PFA)(电子显微镜科学,目录号:15714-S)
  4. PBS(Fisher Bioreagent,目录号:BP661-10)
  5. 生物素化的紫藤Floribunda凝集素/凝集素(WFA / WFL)(Vector Laboratories,目录号:B-1355)
  6. 链霉抗生物素蛋白,Alexa Fluor TM 555 Conjugate(Thermo Fisher,Invitrogen,目录号:S32355)
  7. 4',6-二脒基-2-苯基吲哚,二盐酸盐(DAPI)(Thermo Fisher,Life Tech,目录号:D1306)
  8. 安装介质(Thermo Fisher,Invitrogen,目录号:S36936)
  9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  10. ChABC(见食谱)
  11. PBS(见食谱)
  12. 4%PFA(见食谱)
  13. ACSF(标准振铃器用于急性脑切片的ACSF)(Papouin和Haydon,2018,Tewari et al。,2018)(见食谱)
  14. DAPI股票(见食谱)

设备

  1. 测量移液器(Gilson,目录号:F123602)
  2. 定时器(来自任何商业来源)
  3. 无菌空玻璃瓶,30毫升(如Hospire,目录号:5816-31)
  4. 切片孵育组件:如图2B-2D所示组装
  5. 浴缸加热器(Fisher Scientific,型号:Isotemp 205)
  6. 蠕动灌注泵(Gilson,型号:mini-plus 3)
  7. 直列式加热器(华纳仪器,目录号:TC324B)
  8. 高分辨率成像显微镜,如共聚焦显微镜(尼康元素)
  9. 95%O 2 / 5%CO 2 罐(如AirGas)
  10. -20°C冰柜(如Norlake)

软件

  1. 尼康NIS-Elements
  2. 尼康NIS-Elements AR分析
  3. (可选)Clampfit(Molecular Devices)
  4. (可选)Microsoft Excel / Origin(OriginLab)

程序

  1. 高放大率图像中PNN的结构完整性
    1. 获得不同实验组中各个PNN的高倍率(40倍物镜,5倍数码变焦)多通道(带PV或NeuN)单平面荧光图像。图像应取自覆盖电池最大周长的光学平面。
    2. 使用PNN(图1A和1B,大图像)绘制沿着细胞周边(用NeuN / PV染色)的手动/自动(取决于使用的软件)折线。
      1. 如果使用尼康元素程序,则使用自动ROI功能确定单元格的周长,然后绘制折线以及此周长。
      2. 如果使用斐济/ ImageJ,请使用手绘线工具沿单元格边界绘制一条线,然后单击“分析”,然后单击“绘图轮廓”按钮。
      3. 可以从峰轮廓图本身计算WFA峰值,也可以导出绘图轮廓的数值数据以绘制不同软件中的图形并计算峰值。
    3. 在建立线轮廓后,软件生成线轮廓图,显示沿线的WFA荧光强度(图1A和1B,底部面板)。线轮廓图及其数值数据可以导出为Microsoft Excel文件,其长度(以μm为单位),荧光强度分别为x和y轴。可以从采集程序生成的图表中计算峰值(如图1A和1B所示,下图),或者可以在Excel /分析软件(如Sigma图或Origin)中绘制数据以进行峰值计数和表示。
    4. 通过设置最大强度的一半的阈值来标记和计算线轮廓中的峰值数量(图1A-1B,下面板)。要确定PNN中最大孔的大小,请测量两个似乎彼此相距最远的连续峰之间的距离(图1A-1B中下方面板中的白色双头箭头)。 (或者,在Clampfit结果窗口中绘制数据并将其保存为.atf文件。稍后打开此文件并使用峰值检测功能计算峰值数量。峰值检测功能的参数可以检测到很少的假峰值,这可以从分析中移除。此外,放置两个光阑覆盖间隙并记录它们之间的距离可用于确定最大孔的大小。)


      图1.在两个实验条件下分析PNN的WFA强度峰。 A和B.高倍率(40倍物镜,5倍数字变焦)WFA(绿色),PV(红色)的免疫组织化学染色图像并且覆盖(右侧的大图像)显示在神经胶质瘤相关性癫痫(A)的小鼠模型的大脑皮质中的单个PNN,以及相应的假对照(B)。沿着PV周边的折线(右)包括WFA染色并且显示由低WFA强度基线分开的离散WFA强度峰(由红色箭头标记)。白色虚线表示阈值(最大WFA强度/ 2)以确定WFA强度峰值。双头白色箭头显示两个连续WFA峰之间的最高视在距离,并指示PNN中最大孔的大小。峰的总数除以周长,以考虑不同大小的小区及其PNN。比例尺:5μm。

  2. 脑切片中PNNs的实验前降解
    1. 根据标准脑切片方案准备脑切片,并在完成实验的恢复使用后(Papouin和Haydon,2018,Tewari et al。,2018)。
    2. 使用两个玻璃瓶准备切片孵育组件;在每个盖子上做两个小孔,插入碳氢化合物气体供应的入口和出口针头。在针尖上插入适当长度的管子,使气体供应延伸到靠近缓冲液表面的位置。不要将这些气体管浸入溶液中(它可能会损坏切片)。
    3. 通过将一个针入口连接到碳氢化合物供应管并使另一个针打开以作为出口(图2B)。
    4. 接下来,在每个小瓶中填充3ml充分氧化的ACSF,并添加ChABC原液以在标记为处理的一个小瓶中制备0.5U / ml的ACSF的最终浓度。
    5. 使用改进的移液管移动每个小瓶中的2-3个脑切片(切割移液管的尖端以扩大尖端以便于切片转移,如图2C所示)。
    6. 从添加ChABC开始,将定时器设置为45分钟,并确保通过进样口的碳氢化合物气体供应正常。
    7. 将两个小瓶放入定制的浮动聚苯乙烯泡沫(图2D)中,使小瓶在孵化期间漂浮在水浴中时保持笔直。
    8. 将两个小瓶转移到水浴中,预设温度为32°C(图2E)。
    9. 孵育45分钟后,小心取出小瓶,用ACSF冲洗切片,然后将切片从两个小瓶转移回回收室(图2G)。切片现在可以用于实验。
    10. 完成实验后,小心地从实验装置中取出切片并用4%PFA固定过夜。如上所述,用WFA(和DAPI,如果需要)染色固定切片以确认ChABC的PNN降解效果。


      图2.使用实验前孵育方法对急性脑切片中的PNNs进行降解。 A.脑切片恢复室与ACSF中的皮质切片连续碳水化合物(95%O 2 ,5%CO 2 )鼓泡。 B.用3ml ACSF(有/无ChABC)填充孵育小瓶(用于对照和ChABC处理的切片)并组装具有入口和一个出口的碳氢化合物气体供应管。 C.通过改良的移液管(培养皿上的B)从(A)向每个小瓶中加入2-3个脑切片,随后将小瓶插入聚苯乙烯泡沫中,使其在水浴(E)中保持平直,同时在32°温育C持续45分钟(F)。 G.孵育完成后,将切片在室温下转移回回收室进行实验。

  3. 在脑切片中的实时实验中PNN降解
    1. 在记录设置中记录细胞/切片的基线活动5-10分钟(取决于具体实验)。
    2. 将1U / ml ChABC溶液超融合50分钟,同时严格保持浴温在32-33℃(图3A)。
    3. 记录50分钟后,再次灌注ACSF并在ACSF存在下记录活动5-10分钟(图3B)。 
    4. 停止实验并将切片转移至24孔板并加入500μl,4%PFA以固定切片并在室温下保持过夜。
    5. 如程序D所述,在第二天进行WFA染色。


      图3.实时数据采集过程中急性脑切片中PNNs的降解 A.含有连续碳氢化合物鼓泡的ChABC的ACSF被灌注并以2-3 ml / min的速度再循环至使用蠕动泵进行实验装置。 B.使用在线加热器进行记录设置,以保持浴温恒定在32°C。

  4. 固定急性脑切片中的WFA和DAPI染色
    1. 用PBS冲洗切片4次,每次洗涤间隔5分钟。
    2. 在每个切片中加入0.5ml含有生物素化-WFA溶液(PBS中1:300)的PBS,在室温下孵育1小时,然后每隔5分钟用PBS冲洗4次。
    3. 在每个切片中加入含有链霉抗生物素蛋白缀合的Alexa fluor-555溶液(PBS中1:300)的0.5ml PBS,并在室温下孵育1小时,然后每隔5分钟用PBS漂洗4次。
    4. 用含有DAPI的PBS(DMSO中10mg / ml原液1:1,000)孵育切片4分钟,每5分钟用PBS冲洗3次。
    5. 将切片安装在2个盖玻片之间,允许用户访问切片的两个表面以进行成像。使用安装介质,不要用力按压盖玻片以防止组织变形。
    6. 将安装的组织保持在载玻片上并拍摄记录的细胞/或脑区域的图像以确认PNN的降解。

数据分析

  1. PNN的结构完整性
    平均细胞的峰/单位周长数。使用>每只小鼠5个PNN和来自同一实验组的所有小鼠的池数据。比较不同实验组中的峰数,应用适当的统计检验并绘制图。平均来自同一实验组的所有小鼠的各个PNN和池数据中的最大间隙的长度。比较不同实验组的平均孔尺寸(间隙长度),应用适当的统计检验,并绘制图形。
  2. 确认实验PNN退化
    使用荧光显微镜从切片的两侧拍摄图像。在实验前降解的情况下(程序B),切片的两个表面都没有显示任何完整的PNN。然而,放大的图像显示PNN表达细胞周围的WFA的微弱轮廓(图4B中的插图)。只有ACSF处理的切片显示完整的PNN(图4A)。在实验装置(程序C)中PNN降解的情况下,仅切片的顶部或暴露表面显示ChABC的降解作用(图4D)。高放大率图像(20x)显示PNN表达神经元周围的PNN的微弱轮廓(图4D中的插入图像)。在全细胞膜片钳实验的情况下,还可以将痕量染料添加到移液管缓冲液中以标记记录的细胞,并且在记录的细胞周围存在微弱的WFA轮廓表明细胞在ChABC处理之前表达PNN(Tewari et al。,2018)。


    图4.孵育室和记录设置中ChABC处理急性切片后PNN降解的确认。 A和B.急性皮层切片的DAPI(上图)和WFA(中图)荧光的共聚焦图像在对照(A)和ChABC处理的(B)组的实验后固定后。叠加中的插入图像显示了PNN的受控降级的痕迹。 C和D.来自ChABC未暴露的底部表面(C)的DAPI(顶部)和WFA(中部)荧光的共聚焦图像,以及在实验中PNNs降解的急性脑切片的暴露于ChABC的顶部表面(D)与实验记录同时设置。与暴露表面(D)相比,未暴露表面(C)显示相对完整的PNN,其显示由于ChABC的直接暴露而降解的PNN。叠加中的插入图像来自切片中标记的矩形区域,并显示受控PNN降级的痕迹。比例尺:主图像为100μm,插入图像为10μm。

笔记

  1. 在程序B和C中,浴温对于维持在32-33°C以确保可靠的PNN降解至关重要。
  2. 在程序B中,孵育时间不应超过45分钟; PNN将完全降级,因此难以找到降级的PNN的痕迹。
  3. 在程序C中,建议将ChABC溶液再循环到记录装置中以最小化酶的量。
  4. 在程序C中,用于ChABC再循环的灌注管的总长度应保持最小,以减少时间滞后和总ChABC溶液的体积。
  5. 为了更精确地比较PNN降解时功能的变化,可以将脑切片切成分开大脑半球的两半,一半可以用ChABC处理,其他可以用作对照。
  6. 对于PNN在免疫组织化学(IHC)中的结构完整性分析,如果使用相同的软件分析所有实验组,也可以使用包括ImageJ / Fiji的可自由访问的软件。
  7. 用于PNNs结构完整性分析的高放大倍数图像应具有最小饱和度的荧光信号的整个范围,以最小化由于IHC程序引起的WFA强度的变化。

食谱

  1. ChABC
    1. 根据制造商的说明,在0.01%BSA水溶液中从普通变形虫中重建ChABC,制成1U /40μl储备液
    2. 准备2U的等分试样并储存在-20°C直至使用
    3. 在实验前,将适量添加到鼓泡ACSF中以达到工作浓度
  2. PBS
    将PBS盐混合物溶解在去离子水(DI)中以制备PBS溶液
  3. 4%PFA
    将提供的32%PFA稀释至1(PFA):7(PBS)以制备4%的工作溶液
  4. ACSF
    标准振铃器的缓冲液(125 NaCl,3 KCl,1.25 NaH 2 PO 4 ,25 NaHCO 3 ,2 CaCl 2 ,1.3 MgSO 4 ,25葡萄糖(均为mM); pH 7.3; 310±5 mOsm)用于急性切片记录(Tewari,Chaunsali et al。,2018 )
  5. DAPI股票
    将DAPI溶解在DMSO中制成10 mg / ml的原液并储存在-20°C直至使用

致谢

这项工作得到NIHRO1-NS036692,NIH-RO1-NS082851和NIH-RO1-NS052634的支持。

利益争夺

作者宣称没有竞争利益

伦理

所有动物程序均按照弗吉尼亚理工大学动物护理和使用委员会(IACUC)根据协议15-241(02/11 / 2016-02 / 09/2019),15-106(07)设定的伦理指南进行批准和执行。 / 31 / 2015-07 / 30/20188)和18-126(07/18 / 2018-07 / 15/2021)。

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引用:Tewari, B. P. and Sontheimer, H. (2019). Protocol to Quantitatively Assess the Structural Integrity of Perineuronal Nets ex vivo. Bio-protocol 9(10): e3234. DOI: 10.21769/BioProtoc.3234.
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