Normandie Univ, UNICAEN, INSERM, COMETE, 14000, Caen, France.
Normandie Univ, UNICAEN, INSERM, COMETE, 14000, Caen, France; University Normandie, IFR ICORE, Caen, 14032, France.
J Neurosci Methods. 2018 Jul 1;304:11-23. doi: 10.1016/j.jneumeth.2018.04.005. Epub 2018 Apr 13.
Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity.
We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL).
mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M mAChRs in both the hippocampus and the striatum.
Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue.
流式细胞术允许对外周生物样本进行单细胞分析，在许多研究和临床应用领域都非常有用，主要在血液学、免疫学和肿瘤学领域。在神经科学中，流式细胞术分离方法首先被应用于从健康或脑肿瘤组织中提取干细胞，最近也被用于对脑细胞、海马神经发生进行表型分析，并检测朊病毒蛋白。然而，它在定量与突触可塑性相关的膜受体方面的应用仍然很少。
我们旨在优化一种用于神经元和非神经元受体定量的流式细胞术程序。优化了神经分离过程、髓鞘分离、固定和细胞膜通透性程序，以最大限度地提高成年啮齿动物海马组织中细胞的存活率和分析效果。然后，我们旨在定量分析双侧前庭损失（BVL）大鼠的细胞膜毒蕈碱型乙酰胆碱受体（mAChR）。
在 BVL 后，对海马体和纹状体中的神经元和非神经元细胞进行了 mAChR 的定量分析。在 BVL 后第 30 天而非第 7 天，海马体和纹状体中表达 M mAChR 的神经元比例显著增加（P≤0.05）。
在这里，我们表明流式细胞术似乎是一种可靠的离体脑组织中膜受体定量方法。
