NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO

体外神经元发育和突触形成

• 批准号: 2702949
• 负责人: Darwin K BERG
• 金额: $ 21.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702949
• 关键词: animal genetic material tag; chick embryo; cholinergic receptors; ciliary ganglion; developmental neurobiology; embryogenesis; gene induction /repression; nonmammalian vertebrate embryology; protein structure function; receptor expression; synaptogenesis; tissue /cell culture; transfection

The functions of nicotinic acetylcholine receptors (AChRs) in the nervous system are almost completely unknown. One of the most abundant AChRs in both the CNS and PNS is a species that binds alpha-bungarotoxin (alphaBgt), contains alpha7 subunits, and has a high relative permeability to calcium. This proposal examines the function of such receptors (alphaBgt-AChRs) in a simple vertebrate neural circuit. The experiments use whole-cell patch clamp on neurons in situ to test the hypothesis that alphaBgt-AChRs generate most of the synaptic current despite having a perisynaptic location. Patch clamp recording from presynaptic calyces will be used to test the hypothesis that presynaptic alphaBgt-AChRs also are present and modulate transmitter release. Subunit-specific monoclonal antibodies (mAbs) will be used to identify presynaptic AChR species, determine their subunit composition, and visualize their locations via confocal immunofluorescence. Sharp electrode intracellular recording-will be used to determine whether alphaBgt-AChRs increase synaptic efficacy and support high frequency transmission through the ganglion. Application of alphaBgt in vivo, together with surgical manipulations, will be used to determine whether the receptors alter neuronal survival, synaptic development, or receptor distribution during embryogenesis. A major goal will be the development and use of strategies for manipulating the levels of neuronal AChR gene products in vivo and in cell culture. The approaches will include transient transfection of postmitotic neurons in culture and retroviral infection of embryos in vivo as two vehicles for introducing epitope- tagged mutant constructs, dominant negatives, and targeted ribozymes. The strategies will be employed to alter patterns of AChR expression and discover receptor roles in the development and function of identified neural circuits. The biomedical relevance of neuronal AChRs is clear: they must participate in the powerful addictive quality of nicotine responsible for vast numbers of chronic smokers world-wide. Neuronal AChRs have also been implicated in a range of brain functions including state of arousal, level of attention, and short-term memory formation. AChR changes have been reported in patients suffering from Alzheimer's disease, Parkinson's disease, and other cognitive and behavioral disorders. The studies outlined here represent basic research intended to illuminate the role of nicotinic receptors in the nervous system. More broadly, they may provide new insight into fundamental aspects of synaptic function and lay groundwork for considering therapeutic strategies involving nicotinic receptors.

烟碱型乙酰胆碱受体（AChRs）在脑内的功能 神经系统几乎完全未知。一个最 CNS和PNS中丰富的AChR是一种结合 α-银环蛇毒素（alphaBgt），含有α 7亚基， 对钙相对渗透性高。本提案审查了 在简单脊椎动物中这种受体（α Bgt-AChR）的功能 神经回路实验采用全细胞膜片钳技术， 神经元原位测试的假设，α Bgt-AChRs产生 大部分突触电流，尽管具有突触周围位置。 将使用突触前肾盏的膜片钳记录来测试 突触前α Bgt-AChR也存在的假设， 调制发射器释放。亚单位特异性单克隆抗体 （单克隆抗体）将用于识别突触前AChR种类， 它们的亚基组成，并通过共聚焦显微镜观察它们的位置。 免疫荧光锐电极细胞内记录-将 用于确定α Bgt-AChRs是否增加突触 功效和支持高频率传输通过 神经节alphaBgt在体内的应用，连同外科手术 操作，将用于确定受体是否改变 神经元存活、突触发育或受体分布 在胚胎发育过程中。一个主要目标将是开发和使用 操纵神经元AChR基因水平的策略 产品在体内和细胞培养。这些方法将包括 有丝分裂后神经元的瞬时转染和逆转录病毒 体内感染胚胎作为引入表位的两种载体- 标记的突变体构建体，显性阴性，和靶向的 核酶这些策略将被用来改变 AChR的表达和发现受体在发育和 识别神经回路的功能。的生物医学意义 神经元AChRs是明确的：他们必须参与强大的 尼古丁的成瘾性导致了大量的慢性 全世界的吸烟者神经元乙酰胆碱受体也与 一系列的大脑功能，包括唤醒状态， 注意力和短期记忆的形成。AChR的变化 据报道，在患有阿尔茨海默病的患者中， 帕金森氏症，以及其他认知和行为障碍。 这里概述的研究代表了基础研究， 阐明烟碱受体在神经系统中的作用。更 从广义上讲，它们可能会提供新的见解， 突触功能，并为考虑治疗奠定基础 涉及烟碱受体的策略。