耦合星形胶质细胞网络的大小、形状和方向性分析
Summary
在这里, 我们提出了一个评估胶质网络组织的协议。所述方法最大限度地减少偏倚, 以提供这些网络的描述性度量, 如细胞计数、大小、面积和原子核内的位置。各向异性的评估与矢量分析。
Abstract
越来越清楚的是, 星形胶质细胞调节神经元功能不仅在突触和单细胞水平, 而且在网络水平。星形胶质细胞通过缝隙连接紧密相连, 通过这些结点的耦合是动态和高度调节的。一个新的概念是, 胶质功能是专门的和适应的功能, 神经元电路与他们的联系。因此, 需要测量胶质网络各种参数的方法, 以便更好地描述其通信和耦合的规则, 进一步了解它们的功能。
在这里, 利用图像分析软件 (ImageJFIJI), 描述了一种分析染料耦合胶质网络共焦图像的方法。这些方法允许 1) 自动和无偏检标记的细胞, 2) 计算网络的大小, 3) 计算染料在网络内传播的优先方向, 4) 在感兴趣的区域内重新定位网络.
该分析可用于描述某一特定区域的胶质网络, 比较与不同功能相关的不同区域的网络, 或比较在不同条件下获得的网络对耦合有不同的影响。这些观察可能会导致重要的功能考虑。例如, 我们分析了三叉核的胶质网络, 我们以前曾表明, 胶质耦合对于神经元将其射击模式从补品转变为有节奏的1的能力至关重要。通过测量这个原子核中胶质网络的大小、约束和优先方向, 我们可以建立它们所限制的功能域的假说。一些研究表明, 一些其他的大脑区域, 包括桶皮质, 侧高橄榄, 嗅觉肾小球, 以及丘脑和视觉皮层的感觉细胞核, 可以从类似的分析中获益。
Introduction
许多研究已经描述了神经元-星形胶质细胞在子蜂窝或突触水平上的对话如何对神经元功能和突触传递产生影响。研究表明, 星形胶质细胞对周围神经元活动敏感;事实上, 他们有许多神经递质的受体, 包括谷氨酸, GABA, 乙酰胆碱和 ATP (见以前发表的评论2,3,4)。作为回报, 胶质处理 ensheath 突触元素, 并通过调节细胞外离子动态平衡和释放几个因素或发射机, 如谷氨酸、d-丝氨酸和 ATP, 影响到 extrasynaptic 的神经活动。5,6,7。
星形胶质细胞也可以在网络水平上调节神经元功能的想法已经出现, 证据表明胶质耦合是空间调控的, 对应于具有清晰解剖特征的区域的神经元分割。划分 (如与感官表示的区域), 表明星形胶质细胞将对其他功能相同的星形胶质细胞, 而不仅仅是那些附近的。例如, 在外侧高级橄榄, 大多数胶质网络是定向正交到 tonotopic 轴8, 而在桶皮层或 olfactoty 肾小球, 星形胶质细胞之间的通信更强在桶或肾小球并且较弱在相邻部分之间9,10。在这两种情况下, 胶质网络都面向团伞花序或桶9,10的中心。
我们最近表明, 胶质活动通过减少细胞外钙的浓度2 + ([Ca2 +]e) 来调节神经元的发射, 大概是通过释放 S100β, 一个 Ca2 +结合蛋白11。这一效应在三叉神经主要感觉细胞核 (NVsnpr) 背部的三叉神经 rhythmogenic 神经元中表现出来, 认为在咀嚼运动的产生中起着重要作用, 这是由于这些神经元的节律性射击依赖于持续的 Na+电流, 这是由 [Ca2 +]e11,12的减少所促进的。这些神经元的节律性射击可以通过刺激他们的输入或人工减少 [Ca2 +]e来诱发 “生理上的”。我们进一步表明, 胶质耦合是需要的神经元节律性射击1。这就提高了胶质网络可能形成可同步和协调神经元活动的界限功能域的可能性。为了评估这个假说, 我们首先需要开发一种方法来严格记录这些网络在 NVsnpr 中的组织。
以前对胶质网络的研究大多描述了细胞数和密度和覆盖范围的耦合程度。试图评估胶质网络的形状和染料耦合的方向主要是通过比较两个轴 (x 和 y) 在桶皮层9, 海马13,14的网络大小,15, barreloid 的丘脑16, 外侧高级橄榄8, 嗅肾小球10, 皮质14。此处描述的方法使网络中标记的单元格具有无偏数的计数, 并对所覆盖的区域进行估计。我们还开发了工具来定义网络中耦合的首选方向, 并评估首选方向是朝向原子核的中心还是朝着不同的方向。与以前使用的方法相比, 本协议提供了一种方法来描述胶质网络在结构上的组织和定位, 如背三叉神经主要感觉细胞核没有已知清楚的解剖划分。在上面的研究中, 网络定向被描述为与结构本身的形状的关系, 这已经被记录下来 (例如, 丘脑的 barreloid, 皮层中的桶, 海马和皮质层, 肾小球在嗅球等)。此外, 矢量分析允许比较在不同条件下揭示的耦合方向。为了分析这些参数是否根据网络在原子核中的位置而变化, 我们还开发了一个方法来代替每个网络, 并参照原子核的边界。这些工具可以很容易地适应其他领域, 以调查耦合细胞网络。
Protocol
Representative Results
Discussion
有许多电生理方法来评估星形胶质细胞23,24之间的功能耦合。然而, 这些方法并没有提供有关胶质网络解剖安排的信息。一些研究已经表明, “染料-或示踪-耦合”, 在这里做, 只发生在一小部分的耦合细胞检测的电生理方法25,26,27, 表明,使用此方法检测到的单元格数被低估。然而, 这仍然?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作由加拿大卫生研究所资助, 赠款/奖励编号: 14392。
Materials
| NaCl | Fisher Chemicals | S671-3 | |
| KCl | Fisher Chemicals | P217-500 | |
| KH2PO4 | Fisher Chemicals | P285-500 | |
| MgSO4 | Fisher Chemicals | M65-500 | |
| NaHCO3 | Fisher Chemicals | S233-500 | |
| C6H12O6 Dextrose anhydrous | Fisher Chemicals | D16-500 | |
| CaCl2 dihydrated | Sigma | C70-500 | |
| Sucrose | Sigma | S9378 | |
| D-gluconic acid potassium salt | Sigma | G45001 | |
| MgCl2 anhydrous | Sigma | M8266 | |
| HEPES | Sigma | H3375 | |
| EGTA | Sigma | E4378 | |
| ATPTris Salt | Sigma | A9062 | |
| GTPTris Salt | Sigma | G9002 | |
| Biocytin | Sigma | B4261 | |
| Carbenoxolone disodium salt | Sigma | C4790 | |
| avidin-biotin complex : ABC kit | Vestor laboratories | PK-4000 | |
| Streptavidine-alexa 594 | Molecular Probes | S11227 | |
| Triton | Fisher Chemicals | BP151-500 | |
| Xylene | Fisher Chemicals | X5-1 | |
| Aqueous mounting medium 1 : Fluoromount-G | SouthernBiotech | 0100-01 | |
| Toluen-based synthetic resin mounting medium : Permount | Fisher Chemicals | SP15-100 | |
| Slide Drying Bench | Fisherbrand | 11-474-470 | |
| Vibratome | Leica | VT 1000S | |
| Microscope cover glass | Fisherbrand | 12-544A | |
| Microscope slide ColorFrost | Fisherbrand | 12-550-413 | |
| PFA | Fisherchemicals | 04042-500 | |
| Olympus FluoView FV 1000 Confocal microscope | Olympus | ||
| 40X water-immersion lens | Olympus | LUMPLFLN40XW | |
| 20X water-immersion lens | Olympus | XLUMPLFL20XW | |
| 4X water-immersion lens | Olympus | XLFLUOR4X/340 | |
| Micropipette puller | Sutter Instrument | P97 | |
| Micromanipulator | Sutter Instrument | MP 225 | |
| Camera CCD | Sony | CX-ST50 | |
| Black and white monitor | Sony | SSM-125 | |
| Digidata | Molecular devices | 1322A | |
| Patch Clamp amplifier | Axon instrument | Mulitclamp 700A | |
| Electrophysiology acquisition software | Molecular devices | pClamp 8 | |
| Electrophysiology analysis software | Molecular devices | Clampfit 8 | |
| Imaging analysis software | ImageJFIJI | Open source software. FIJI version including plug in package. | |
| Vector image editor | Adobe | Illustrator CS4 | |
| Spreadsheet application | Microsoft Office | Excel 2010 |
References
- Condamine, S., Lavoie, R., Verdier, D., Kolta, A. Functional rhythmogenic domains defined by astrocytic networks in the trigeminal main sensory nucleus. Glia. 66 (2), 311-326 (2018).
- Verkhratsky, A., Orkand, R. K., Kettenmann, H. Glial calcium: homeostasis and signaling function. Physiological Review. 78 (1), 99-141 (1998).
- Christensen, R. K., Petersen, A. V., Perrier, J. F. How do glial cells contribute to motor control?. Current Pharmaceutical Design. 19 (24), 4385-4399 (2013).
- Verkhratsky, A., Steinhauser, C. Ion channels in glial cells. Brain Research Review. 32 (2-3), 380-412 (2000).
- Harada, K., Kamiya, T., Tsuboi, T. Gliotransmitter Release from Astrocytes: Functional, Developmental, and Pathological Implications in the Brain. Frontiers Neuroscience. 9, 499 (2015).
- Montero, T. D., Orellana, J. A. Hemichannels: new pathways for gliotransmitter release. Neurosciences. 286, 45-59 (2015).
- Araque, A., et al. Gliotransmitters travel in time and space. Neuron. 81 (4), 728-739 (2014).
- Augustin, V., et al. Functional anisotropic panglial networks in the lateral superior olive. Glia. 64 (11), 1892-1911 (2016).
- Houades, V., Koulakoff, A., Ezan, P., Seif, I., Giaume, C. Gap junction-mediated astrocytic networks in the mouse barrel cortex. Journal of Neuroscience. 28 (20), 5207-5217 (2008).
- Roux, L., Benchenane, K., Rothstein, J. D., Bonvento, G., Giaume, C. Plasticity of astroglial networks in olfactory glomeruli. Proceedings of the National Academy of Science of the United State of America. 108 (45), 18442-18446 (2011).
- Morquette, P., et al. An astrocyte-dependent mechanism for neuronal rhythmogenesis. Nature Neuroscience. 18 (6), 844-854 (2015).
- Brocard, F., Verdier, D., Arsenault, I., Lund, J. P., Kolta, A. Emergence of intrinsic bursting in trigeminal sensory neurons parallels the acquisition of mastication in weanling rats. Journal of Neurophysiology. 96 (5), 2410-2424 (2006).
- Anders, S., et al. Spatial properties of astrocyte gap junction coupling in the rat hippocampus. Philosophical Transactions of the Royal Society of London. Series B, Biological Science. 369 (1654), (2014).
- Houades, V., et al. Shapes of astrocyte networks in the juvenile brain. Neuron Glia Biology. 2 (1), 3-14 (2006).
- Rouach, N., Koulakoff, A., Abudara, V., Willecke, K., Giaume, C. Astroglial metabolic networks sustain hippocampal synaptic transmission. Science. 322 (5907), 1551-1555 (2008).
- Claus, L., et al. Barreloid Borders and Neuronal Activity Shape Panglial Gap Junction-Coupled Networks in the Mouse Thalamus. Cerebral Cortex. 28 (1), 213-222 (2018).
- Cameron, M. A., et al. Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging. Journal of Visualized Experiments. (120), (2017).
- Kafitz, K. W., Meier, S. D., Stephan, J., Rose, C. R. Developmental profile and properties of sulforhodamine 101–Labeled glial cells in acute brain slices of rat hippocampus. Journal of Neuroscience Methods. 169 (1), 84-92 (2008).
- Neher, E. Correction for liquid junction potentials in patch clamp experiments. Methods in Enzymology. , 123-131 (1992).
- Giaume, C., Leybaert, L., Naus, C. C., Saez, J. C. Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles. Frontiers in Pharmacology. 4, 88 (2013).
- Torres, A., et al. Extracellular Ca(2)(+) acts as a mediator of communication from neurons to glia. Science Signaling. 5 (208), ra8 (2012).
- Ye, Z. C., Wyeth, M. S., Baltan-Tekkok, S., Ransom, B. R. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. Journal of Neuroscience. 23 (9), 3588-3596 (2003).
- Ma, B., et al. Gap junction coupling confers isopotentiality on astrocyte syncytium. Glia. 64 (2), 214-226 (2016).
- Meme, W., Vandecasteele, M., Giaume, C., Venance, L. Electrical coupling between hippocampal astrocytes in rat brain slices. Neuroscience Research. 63 (4), 236-243 (2009).
- Ransom, B. R., Kettenmann, H. Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture. Glia. 3 (4), 258-266 (1990).
- Audesirk, G., Audesirk, T., Bowsher, P. Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis. Journal of Neurobiology. 13 (4), 369-375 (1982).
- Ewadinger, N., Syed, N., Lukowiak, K., Bulloch, A. Differential Tracer Coupling between Pairs of Identified Neurones of the Mollusc Lymnaea Stagnalis. Journal of Experimental Biology. 192 (1), 291-297 (1994).
- Griemsmann, S., et al. Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells. Cerebral Cortex. 25 (10), 3420-3433 (2015).
- Kuwajima, T., et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development. 140 (6), 1364-1368 (2013).
- Gourine, A. V., et al. Astrocytes control breathing through pH-dependent release of ATP. Science. 329 (5991), 571-575 (2010).
- Forsberg, D., Ringstedt, T., Herlenius, E. Astrocytes release prostaglandin E2 to modify respiratory network activity. eLife. 6, (2017).
