CELL BIOLOGICAL STUDIES OF DEVELOPING CNS CELLS

发育中的中枢神经系统细胞的细胞生物学研究

基本信息

批准号：
6290621
负责人：
JEFFERY BARKER
金额：
--
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Summary of Work: The embryonic and early postnatal mammalian central nervous system (CNS) dynamically changes in cell number and anatomical structure as cells proliferate, die, migrate and differentiate into neurons and glia. This project line complements research conducted in project Z01 NS 02019-27 (Physiological properties developing on CNS cells), most of which involves direct recording of cell physiology in real time (electrophysiology), or near real-time (membrane potential and calcium imaging). Our long-term goal involves explanation of specific steps inCNS morphogenesis. This includes: 1) characterization of the physiological correlates of cell lineage progression emerging in vivo using surface labeling, physiological indicator dyes and flow cytometry and 2) the roles of transmitters and their corresponding receptors in mitogenic, motogenic and morphogenic activities. Previously, we used immunocytochemistry of surface epitopes (gangliosides) in conjunction with flow cytometry to characterize the cellular distribution of physiological properties emerging in vivo among precursors and neuronal and glial progenitors at the end of neurogenesis in the embryonic rat cortex. These results demonstrated contrasting patterns of transmitter receptor and ion channel expression emerging in vivo among proliferating precursors and cells progressing along neuronal and glial lineages. During FY99, we developed a novel FACS strategy to isolate for the first time in a prospective manner proliferating precursor (stem) cells at the beginning of neurogenesis in the cortex. We found that cells, which did not express surface epitopes characteristic of either neurons or glia or apoptosis (naturally occurring cell death), accounted for about 20% of the total. After sorting, the unlabeled cells were cultured in a serum-free, defined medium where they rapidly proliferated and then began to differentiate into neurons, astrocytes or oligodendrocytes. The cells could be frozen, thawed and cultured again with the same outcome as occurred with the primary sort. These self-renewing and multipotential characteristics identify the unlabeled population as neural stem cells. We used Ca2+ imaging to survey the cellular distribution of physiological properties expressed by stem cells and their progeny in vitro. Stem cells initially did not respond to transmitters from all the known classes. Over 24 hours, cytosolic Ca2+ (Ca2+c) responses to acetylcholine (ACh) and adenosine triphosphate (ATP) appeared, which were blocked by atropine and suramin, respectively. As cells progressed along neuronal and glial lineages they expressed contrasting and changing patterns of physiological properties similar to those identified in vivo at the end of neurogenesis. Thus, in vitro stem cells can differentiate in a defined medium into the three major cell types, recapitulating the developmental appearance of their physiological properties emerging in vivo. The early appearance and widespread cellular distribution of atropine-sensitive ACh receptors, identifying them as muscarinic in type, led us to investigate their role in neurogenesis. We treated explants of the early embryonic cortex with atropine, then used immunocytochemistry and flow cytometry to quantify the development of precursors and progenitors. Blockade of muscarinic receptors expressed by cells in cortical explants decreased the relative number of proliferating (BrdU+) precursors and neuronal and glial progenitors. These effects were closely mimicked by exposing the explants to BAPTA-AM, which blocks muscarinic receptor-mediated Ca2+c responses. Thus, endogenous cholinergic signaling at muscarinic receptors regulates proliferation during early neurogenesis. We used immunocytochemistry and flow cytometry to identify subpopulations of cells with choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, in dissociates of the early embryonic cortex. ChAT+ cells were primarily restricted to cells progressing along a neuronal lineage. Together, these results demonstrate a major mitogenic role for ACh during the earliest phases of neurogenesis in the cortex. Previously, we used flow cytometry and immunocytochemistry to quantify the developmental appearance and cellular distribution of GABAergic system components among neurons in the embryonic rat cortex. During neurogenesis, neuronal progenitors migrate from the ventricular zone to the cortical plate. We found that GABA was a primary chemoattractant in the rat, while glutamate was a primary motogen in the mouse. In FY99, we found that GABAergic neurons dissociated from the cortical plate region of the rat directed the migration of neuronal progenitors dissociated from the ventricular zone via GABA(B) receptor-coupled pathways involving pertussis toxin-sensitive G proteins, Ca2+ fluctuations, and mitogen-activated phosphorylating enzyme activity. Since cortical plate neurons also secrete other amino acids like taurine and beta-alamine, we tested their chemoattractant activities and found both to be effective. Although both amino acids have demonstrated efficacies at GABA(A) receptor/chloride channels we found that their chemoattractant effects occurred via GABA(B) receptors. These results thus implicate an array of amino acids in cortical cell migration. We studied GABA?s morphogenic roles in neurite outgrowth of cortical plate neurons, which are transiently GABAergic. In vitro, neurite outgrowth was eliminated by suppressing GABA synthesis or blocking GABA(A) receptor/chloride channels. It could largely be restored in neurons no longer synthesizing GABA by addition of agonists at GABA(A) receptors, by astrocyte-conditioned medium, which contains GABA, taurine and beta-alanine and by modest elevation in K+, which, like agonists at GABA(A) receptor/chloride channels, depolarizes neurons, thereby activating Ca2+ entry via L-type channels. Hence, the transient and widespread appearance of GABA in neurons throughout the embryonic CNS coincides with, and likely promotes neurite outgrowth.

工作摘要：随着细胞的增殖，死亡，死亡，迁移并分化为神经元和神经胶质，胚胎和早期哺乳动物中枢神经系统（CNS）动态变化细胞数和解剖结构。该项目线补充了在项目Z01 NS 02019-27（在CNS细胞上开发的生理特性）中进行的研究，其中大多数涉及实时直接记录细胞生理学（电生理学）或接近实时的（膜电位和钙成像）。我们的长期目标涉及解释特定步骤的形态发生。这包括：1）使用表面标记，生理指示剂染料和流式细胞仪体内出现细胞谱系进展的生理相关性，以及2）2）发射器及其相应受体在有丝分裂，动作和形态发育活性中的作用。以前，我们使用了与流式细胞仪结合表面表面表现（神经节苷脂）的免疫细胞化学，以表征前体和神经元和神经胶质祖细胞中在胚胎鼠的神经发生结束时体内出现的生理特性的细胞分布。这些结果表明，在沿神经元和神经胶质谱系的增殖前体和细胞中，发射器受体和离子通道表达在体内出现的对比模式。在99财年期间，我们制定了一种新颖的FACS策略，以预期的方式第一次分离出在皮质中神经发生的开始时，以预期的方式增殖前体（STEM）细胞。我们发现，没有表达神经元，神经胶质或凋亡（天然发生的细胞死亡）的表面表演的细胞约占总数的20％。分类后，将未标记的细胞培养在无血清，定义的培养基中，它们快速增殖，然后开始分化为神经元，星形胶质细胞或少突胶质细胞。可以将细胞冷冻，融化和培养，并以与主要排序相同的结果进行培养。这些自我更新和多能特征将未标记的种群识别为神经干细胞。我们使用CA2+成像来调查干细胞表达的生理特性的细胞分布及其体外后代。干细胞最初没有对所有已知类别的发射机做出反应。在24小时内，出现了对乙酰胆碱（ACH）和三磷酸腺苷（ATP）的胞质Ca2+（Ca2+ c）的反应，分别被阿托品和苏拉明蛋白阻塞。随着细胞沿着神经元和神经胶质谱系的发展，它们表达了与神经发生结束时体内鉴定的生理特性的对比和变化的生理特性模式。因此，体外干细胞可以在定义的培养基中分化为三种主要细胞类型，从而概括了其体内出现的生理特性的发育外观。阿托品敏感的ACH受体的早期外观和广泛的细胞分布在类型上鉴定为毒蕈碱，使我们研究了它们在神经发生中的作用。我们用阿托品处理了早期胚胎皮层的外植体，然后使用免疫细胞化学和流式细胞仪来量化前体和祖细胞的发展。皮质外植体中细胞表达的毒蕈碱受体的阻断减少了增生（BRDU+）前体的相对数量，以及神经元和神经胶质祖细胞。通过将外植体暴露于Bapta-AM，从而密切模仿这些效果，后者阻塞了毒蕈碱受体介导的Ca2+C反应。因此，毒蕈碱受体的内源性胆碱能信号传导调节早期神经发生过程中的增殖。我们使用免疫细胞化学和流式细胞仪来鉴定用胆碱乙酰转移酶（CHAT）的细胞亚群，该酶是早期胚胎皮层解离物中合成ACH的酶。聊天+细胞主要局限于沿神经元谱系进展的细胞。总之，这些结果证明了在皮质中神经发生的最早阶段ACH的主要有丝分裂作用。以前，我们使用流式细胞术和免疫细胞化学来量化胚胎大鼠皮质中神经元之间GABA能系统成分的发育外观和细胞分布。在神经发生过程中，神经元祖细胞从心室区域迁移到皮质板。我们发现GABA是大鼠中的主要趋化剂，而谷氨酸是小鼠中的主要摩托原。在FY99中，我们发现，与大鼠皮质板区分离的GABA能神经元指示神经元祖细胞的迁移通过GABA（B）受体偶联途径从心室区分离出来，涉及涉及杂质G蛋白，CA2++ FLEPUCTUATION，CAC2+ EDZERATION ESTRADION和MITECENTAIN和MITECOGEN的途径和MITACTACTACTACTACTACTACEN和MITRACTIVERNODY cANTIVER和MITRACTIVER。由于皮质板神经元还分泌牛磺酸和β-丙氨酸等其他氨基酸，因此我们测试了它们的趋化性活性，并发现两者都有效。尽管两种氨基酸都表现出在GABA（A）受体/氯化物通道上的疗效，但我们发现它们的趋化剂作用通过GABA（B）受体发生。因此，这些结果暗示了一系列氨基酸在皮质细胞迁移中。我们研究了GABA的形态学作用在瞬时GABA能的皮质板神经元的神经突生长中。在体外，通过抑制GABA合成或阻断GABA（A）受体/氯化物通道来消除神经突生长。通过在GABA（A）受体上添加激动剂，通过星形胶质细胞条件培养基添加激动剂，它可以在很大程度上恢复在神经元中，该培养基中包含GABA，牛磺酸和β-丙氨酸，以及通过K+中的较小升高来恢复GABA（A）受体/氯化剂caba+ alimant+ after in+ neurons neuron-cains treemty limal-cab+ sevials，deppe tyly-neurons，deppe tyler-gaba（a）。频道。因此，在整个胚胎中枢神经系统中，神经元中GABA的瞬时和广泛外观与神经突生长一致，并可能促进神经突的生长。