INTRACELLULAR CALCIUM IN DEVELOPING NEURONS

发育中神经元中的细胞内钙

基本信息

批准号：
3419035
负责人：
JAMES L KENYON
金额：
$ 12.85万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 1996-07-31
项目状态：
已结题

项目摘要

The experiments described in this proposal will investigate the function of ryanodine-sensitive intracellular Ca2+ stores in peripheral neurons, including their role in the differentiation and maturation of these cells from neural crest precursors. Our approach to this problem is fourfold. First, antibodies specific for isoforms of the avian ryanodine receptor and immunohistochemical techniques will be used to localize ryanodine receptors in neural crest cells and cells in developing peripheral ganglia. This work will identify cell types and times during development in which Ca2+ release from intracellular Ca2+ stores may be important. We will examine cells in sections of intact embryonic chicks to monitor the development of ryanodine receptors in situ. Similar observations made on cells differentiating and maturing in culture will characterize the development of ryanodine receptors under more defined conditions in vitro. Second, functional measurements of Ca2~+ using will be made using fluorescent Ca2+ indicator dyes to monitor bulk cytoplasmic Ca2+, and Ca2+ activated membrane conductances to monitor Ca2+ activity at the cell membrane. Functional Ca2+ stores will be identified by their caffeine- and ryanodine-sensitivity. These studies will be done using primary cultured cells from peripheral ganglia of E4 through post-hatch chicks in order to characterize the functional properties of Ca2+ stores in morphologically mature neurons. Third, cell survival assays will be used to investigate the role of intracellular Ca2+ stores, Ca2+-induced Ca2+ release, ryanodine receptors, and Ca2+ oscillations in the prevention of natural cell death of peripheral neurons by NGF or high K+. Ca2+ has been implicated in the cell saving action of both of these treatments and the existence of active intracellular Ca2+ stores raises the possibility that dynamic Ca2+ oscillations may contribute to this action or mimic it. Pharmacological agents, including ryanodine, thapsigargin, and caffeine will be used to manipulate intracellular Ca2+ stores in order to examine their effects on cell survival. Fourth, we will investigate the development of peripheral neurons in crooked neck dwarf mutant (cn/cn) chicks where the alpha isoform of the ryanodine receptor is not expressed normally in skeletal muscle. The goal of these experiments will be to establish whether or not this mutation affects peripheral neurons, to characterize the functional consequences of the mutation, and to investigate the suitability of cn/cn mutant birds as a system for future investigations of the role of neuronal ryanodine receptors in cell development and function.

本提案中描述的实验将研究该功能周围神经元中兰尼碱敏感的细胞内 Ca2+ 储存，包括它们在这些细胞的分化和成熟中的作用来自神经嵴前体。我们解决这个问题的方法有四个。首先，针对鸟类兰尼碱受体亚型的特异性抗体免疫组织化学技术将用于定位兰尼定神经嵴细胞和发育中的外周细胞中的受体神经节。这项工作将确定细胞类型和发育过程中的时间其中细胞内 Ca2+ 储存的 Ca2+ 释放可能很重要。我们将检查完整胚胎雏鸡切片中的细胞以进行监测兰尼碱受体的原位发育。做出了类似的观察细胞在培养物中的分化和成熟将表征在更明确的条件下开发兰尼碱受体体外。其次，Ca2~+使用的功能测量将使用荧光 Ca2+ 指示剂染料可监测大量细胞质 Ca2+，以及 Ca2+ 激活膜电导以监测细胞内的 Ca2+ 活性膜。功能性 Ca2+ 储存将通过其咖啡因来识别 - 和兰尼丁敏感性。这些研究将使用初级完成从 E4 外周神经节到孵化后雏鸡的培养细胞为了表征 Ca2+ 储存的功能特性形态上成熟的神经元。第三，将使用细胞存活测定研究细胞内 Ca2+ 储存、Ca2+ 诱导的 Ca2+ 的作用释放、兰尼碱受体和 Ca2+ 振荡以预防 NGF 或高 K+ 导致周围神经元自然细胞死亡。 Ca2+已与这两种治疗方法的细胞拯救作用有关活跃的细胞内 Ca2+ 储备的存在提出了以下可能性：动态 Ca2+ 振荡可能有助于或模仿这种作用。药理学制剂，包括兰尼定、毒胡萝卜素和咖啡因将用于操纵细胞内 Ca2+ 储存以检查它们对细胞存活的影响。第四，我们将调查歪颈侏儒突变体周围神经元的发育 (cn/cn) 不表达兰尼碱受体 α 亚型的雏鸡通常存在于骨骼肌中。这些实验的目标是确定这种突变是否影响周围神经元，以表征突变的功能后果，并研究 cn/cn 突变鸟类作为未来系统的适用性神经元兰尼碱受体在细胞中的作用研究发育和功能。