NICOTINE REGULATION OF DEVELOPING BRAIN CATECHOLAMINES

描述：（申请人摘要） 最近的数据表明， 年轻女性，母亲吸烟对胎儿结局的潜在后果 是一个临床意义日益增加的问题。 体内数据表明， 产前尼古丁暴露对发育有显着影响， 脑儿茶酚胺（CA）系统，这可能是一些观察到的基础 行为缺陷 然而，尼古丁影响的确切机制 中央空调的发展还不清楚。 本申请的目的 是为了确定大脑CA神经元是否直接受烟碱 受体（nAChRS）在大脑发育早期。 与大多数 以前的研究，我们建议使用体外方法， 实验变量可以更好地控制和间接影响 最小化 定量解剖技术将被用于检查 nAChR mRNA和高亲和力结合位点的发育表达 在该位点的发育中的去甲肾上腺素（NE）和多巴胺（DA）细胞内 蓝斑（LC）和黑质/腹侧被盖区（SN/VTA）， 分别 联合同位素和非同位素原位杂交将 用于定量nAChR亚单位mRNA的发育表达 在同样表达CA合成酶酪氨酸的细胞内， 羟化酶 在这些发育中的细胞群中的高亲和力nAChR结合 也将使用[3H]野靛碱和[3H]地棘蛙素作为 放射性配体。 将按年龄和时间分析用于研究的动物。 性别，并将涵盖从胚胎日起CA发育的全方位 15成人 nAChRs在NE和DA调控中的功能作用 将使用递质释放来评估发育中的脑中的释放 测定。 使用脑切片，将检查尼古丁对NE的影响 以及从LC和SN/VTA的所选终端字段释放DA。 岁 将检查nAChR调节开始的时间，以及药理学 未成熟大脑中受体的特征。 特别是要 评估nAChRs调节LC功能的特性 随着年龄的变化而变化。 还将使用原代神经元细胞培养模型， 分析nAChRs在发育中的LC神经元上的特性，并检查 慢性nAChR激活的发育后果。 是 预计拟议的体外分析将提供大量 解释慢性围产期尼古丁影响数据的框架 体内给药。

DESCRIPTION: (Applicant's Abstract) With recent data indicating an increased incidence of cigarette use among young women, the potential consequences of maternal smoking on fetal outcome is an issue of increasing clinical significance. In vivo data indicate that prenatal nicotine exposure has significant effects on the development of brain catecholamine (CA) systems, and that this may underlie some observed behavioral deficits. However, the exact mechanism by which nicotine affects central CA development is unclear. The purpose of the present application is to determine whether brain CA neurons are directly regulated by nicotinic receptors (nAChRS) during early brain development. Unlike the majority of previous studies, we propose to use in vitro methodologies so that experimental variables can be better controlled and indirect effects minimized. Quantitative anatomical techniques will be used to examine the developmental expression of nAChR mRNA and high affinity binding sites within developing norepinephrine (NE) and dopamine (DA) cells of the locus coeruleus (LC) and substantia nigra/ventral tegmental area (SN/VTA), respectively. Combined isotopic and non-isotopic in situ hybridization will be used to quantitate the developmental expression of nAChR subunit mRNAs within cells which also express the CA synthetic enzyme, tyrosine hydroxylase. High affinity nAChR binding in these developing cell groups will also be examined using [3H]cytisine and [3H]epibatidine as radioligands. Animals to be used for the study will be analyzed by age and sex, and will encompass the full range of CA development from embryonic day 15 through adult. The functional role of nAChRs in controlling NE and DA release in developing brain will be evaluated using transmitter release assays. Using brain slices, the effects of nicotine will be examined on NE and DA release from selected terminal fields of the LC and SN/VTA. The age of onset of nAChR regulation will be examined, as will the pharmacological characteristics of the receptor in immature brain. In particular, we will evaluate the hypothesis that the properties of nAChRs regulating LC function change with age. A primary neuronal cell culture model will also be used to analyze the properties of nAChRs on developing LC neurons, and to examine the developmental consequences of chronic nAChR activation. It is anticipated that the proposed in vitro analysis will provide a substantial framework for interpreting data on the effects of chronic perinatal nicotine administration in vivo.