突触量化：基于一个免疫细胞化学检测，以量化突触数量
Summary
该协议的细节，如何量化，在分离神经元的文化和突触数量在脑切片，用免疫细胞化学。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。
Abstract
在神经科学中最重要的目标之一是要了解分子线索指示突触形成的早期阶段。因此，它已成为当务之急，制定客观的方法来量化突触连接的变化。从样品固定开始，这个协议的细节如何量化无论是在分离的神经元文化和在脑切片，用免疫细胞化学突触的数量。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。这些colocalizations量化Puncta分析器（巴里Wark，可根据要求，c.eroglu @ cellbio.duke.edu书面）使用ImageJ分析软件平台下的一个插件。在本议定书中所描述的突触检测可应用于任何神经组织或文化的准备，你有选择性的前和突触后标记。这突触法是一种宝贵的的工具，可以被广泛利用在突触发展的研究。
Protocol
准备的解决方案： 抗体缓冲液： 150 mM氯化钠 50 mM的三基（费舍尔，猫编号：BP152 – 5，50毫米） – 1.21克 1％BSA（Sigma公司，没有猫：A2153，1％） – 2.0克 100毫米L -赖氨酸（西格玛，猫编号：L – 1137，100毫米） – 3.65克调整pH值至7.4 0.04％叠氮音量调整到200毫升，用蒸馏水H 2 O 通过0.22μm的过滤器的过滤器（Millipore公司，卡?…
Discussion
上文所述突触检测是基于在我们的实验目标，其中主要集中于视网膜神经节细胞兴奋的预测中，无论是在纯化的文化，或在大脑部分。我们提供了一个参考表中列出的标签（见表1）兴奋性突触的抗体，工作。
这种突触的检测，可适应量化任何神经元的人口或任何其他的突触亚型，这是有选择性的前和突触后标记的突触数量。例如，突触实验描述在这里可以应用到研究的GABA – …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Puncta分析仪插入图像J的巴里Wark（地址：Physion咨询）写在本A.巴雷斯实验室（斯坦福大学）。
资金;
- Alfred P. Sloan基金会
- 以斯帖A.和约瑟夫Klingenstein基金公司
- 广泛的生物医学研究基金会
- 治疗亨廷顿氏病倡议
Materials
| Antigen | Species | Monoclonal/Polyclonal | Vendor | Catalog Number | Dilution | Works in Culture | Works in Sections | |
| Presynaptic | Synapsin | Rabbit | Polyclonal | Synaptic Systems | 106004 | 1:750 | Y | N.D. |
| Synapsin | Mouse | Monoclonal | Synaptic Systems | 106001 | 1:500 | Y | Y | |
| Bassoon | Mouse | Monoclonal | Assay Designs | VAM-PS003F | 1:500 | Y | Y | |
| Bassoon | Guinea Pig | Polyclonal | Synaptic Systems | 141004 | 1:1000 | Y | N.D. | |
| Synaptotagmin 1 | Rabbit | Polyclonal | Synaptic Systems | 105002 | 1:750 | Y | N | |
| Synaptobrevin 2 (C1.69.1) |
Mouse | Monoclonal | Synaptic Systems | 104211 | 1:500 | Y | Y | |
| Synaptophysin (C1.7.2) |
Mouse | Monoclonal | Synaptic Systems | 101011 | 1:500 | Y | Y | |
| VGlut1 | Mouse | Monoclonal | Millipore | MAB5502 | 1:2500 | N | Y | |
| VGlut1 | Guinea pig | Polyclonal | Millipore | AB5905 | 1:2500 | N | Y | |
| VGlut2 | Guinea pig | Polyclonal | Millipore | AB2251 | 1:2500 | N | Y | |
| Postsynaptic | PSD-95 (6G6-1C9 clone) |
Mouse | Monoclonal | Affinity Bio Reagents | MA1-045 | 1:750 | Y | N |
| PSD-95 | Rabbit | Polyclonal | Zymed | 51-6900 | 1:500 | N | Y | |
| Homer | Mouse | Monoclonal | Synaptic Systems | 160011 | 1:500 | Y | N.D. | |
| Homer | Rat | Polyclonal | Millipore | AB5875 | 1:500 | Y | Y | |
| Gephyrin | Rabbit | Polyclonal | Synaptic Systems | 147003 | 1:500 | Y | Y | |
| Gephyrin | Mouse | Monoclonal | Synaptic Systems | 147 | 1:200 | Y | Y |
Table 1: Lists examples of good pre- and postsynaptic markers that we have successfully utilized in our synapse assay. Be aware that this is not an exhaustive list of all available markers. Y = Yes, N = No, N.D. = Not Determined.
| Reagent | Company | Cat. No. |
| PBS | Invitrogen | 20012-027 |
| poly-d-lysine | Sigma | P6407 |
| Laminin | Cultrex | 3400-010-01 |
| Triton X-100 | Roche Diagnostics Gmbh | 9002-93-1 |
| Normal Goat Serum | Gibco | 16210 |
| VectaShield with DAPI | Vector Laboratories | H-1200 |
| OCT | Tissue-Tek | 4583 |
| Tris-Base (50 mM) | Fisher | BP152-5 |
| Bovine Serum Albumin | Sigma | A2153 |
| l-lysine | Sigma | L-1137 |
| 16% PFA solution | Electron Microscopy Sciences | 15711 |
| Granular PFA | Electron Microscopy Sciences | 19210 |
| 24-well culture plate | Falcon | 35-3047 |
| Goat anti-mouse Alexa conjugated antibodies | Invitrogen | — |
| Supply | Company | Cat. No. |
| 12mm, No. 0 glass coverslips | Karl Hecht Gmbh | 1105209 |
| No. 1.5 glass coverslip (for slices) | VWR Scientific | 48393241 |
| Glass slides | VWR Scientific | 48311-703 |
References
- Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron. 1, 791-803 (1988).
- Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron. 15, 805-819 (1995).
- Eroglu, C. Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell. 139, 380-392 (2009).
- Pfrieger, F. W., Barres, B. A. Synaptic efficacy enhanced by glial cells in vitro. Science. 277, 1684-1687 .
- Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. Control of synapse number by glia. Science. 291, 657-661 (2001).
- Christopherson, K. S. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell. 120, 421-433 (2005).
- Godement, P., Salaun, J., Imbert, M. Prenatal and postnatal development of retinocollicular projections in the mouse. J. Comp. Neurol. 230, 552-575 (1985).
- Schmidt, J. T. Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism. Cell. Mol. Neurobiol. 5, 65-84 (1985).
- Sachs, G. M., Jacobson, M., Caviness, V. S. Postnatal changes in arborization patterns of murine retinocollicular axons. J. Comp. Neurol. 246, 395-408 (1986).
- Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors. J. Neurosci. 25, 3638-3650 (2005).
- Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis. Moll. Cell. Neurosci. 43, 136-145 (2010).
Tags
QuantifySynapsesImmunocytochemistry-based AssaySynapse NumberNeuroscienceMolecular CuesObjective ApproachesSynaptic ConnectivitySample FixationDissociated Neuronal CultureBrain SectionsImmunocytochemistryCompartment-specific AntibodiesPresynaptic TerminalsPostsynaptic SpecializationColocalizationSignalsMarkersPuncta AnalyzerImageJ Analysis Software PlatformSynapse AssayCulture Preparation
Cite This Article
Ippolito, D. M., Eroglu, C. Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number. J. Vis. Exp. (45), e2270, doi:10.3791/2270 (2010).