Altering gene expression and function at single neuronal nicotinic synapses

描述(申请人提供)：尼古丁成瘾导致的烟草消费是美国可预防死亡的主要原因。在1995年至1999年期间，美国疾病控制与预防中心报告说，每年有44万人死于吸烟(癌症、呼吸系统和心血管疾病)引起的疾病，相关的经济损失为1570亿美元。神经元烟碱型乙酰胆碱受体(NAChRs)启动并加强尼古丁成瘾的过程。这些受体分子聚集在大脑和自主神经系统的突触前和突触后位置，一旦被胆碱能神经末梢释放的乙酰胆碱(ACh)激活，它们就可以调节和直接介导突触传递。最近的研究表明，长期暴露于吸烟者血液中的尼古丁水平，会导致突触前和突触后nAChRs的持续变化。人们普遍认为，这种变化会改变与尼古丁相关的成瘾行为的突触回路，可能还会改变其他常见的滥用药物。不幸的是，这些电路嵌入到大脑区域，在那里很难或不可能实现对突触前和突触后元素的实验控制，从而阻止了与尼古丁诱导的突触功能变化相关的精确机制的发现。为了解决这个问题，我们的实验室建议将最先进的单细胞基因导入和高分辨率突触刺激/记录方法结合在一个模型培养系统中，其中nAChRs和尼古丁突触丰富且可获得。一个目标是操纵突触前和突触后神经元的基因表达，随后可以在详细的电生理实验中进行检查(目标1)。实现这一目标将为突触形成、功能和调控的许多研究提供前沿进展。第二个目标是通过探测突触功能的变化来测试我们方法的实用性，这些变化预计会伴随尼古丁受体激活的持续变化(目标2)。预计后面的这些实验将提供新的答案，即尼古丁突触成分的变化如何改变电路功能，以启动和加强对尼古丁和其他滥用药物的上瘾。

DESCRIPTION (provided by applicant): Tobacco consumption, driven by addiction to nicotine, is the leading cause of preventable death in the United States. In the period from 1995-1999, the CDC reported 440,00 deaths/year from illnesses attributable to smoking (cancer, respiratory, and cardiovascular diseases) and $157 billion/year in related economic losses. Neuronal nicotinic acetylcholine receptors (nAChRs) initiate and reinforce the process of nicotine addiction. These receptor molecules aggregate at pre- and postsynaptic sites in the brain and autonomic nervous system, allowing them to both modulate and directly mediate synaptic transmission once activated by acetylcholine (ACh) released from cholinergic nerve terminals. Recent studies demonstrate that chronic exposure to levels of nicotine seen in smokers' blood, causes sustained changes in both pre- and postsynaptic nAChRs. It is widely presumed that such changes alter synaptic circuits underlying addictive behaviors associated with nicotine, and possibly other commonly abused drugs. Unfortunately, these circuits are embedded in brain regions where experimental control of pre- and postsynaptic elements is difficult or impossible to achieve, preventing discovery of precise mechanisms relevant to nicotine-induced changes in synaptic function. To address this problem, our laboratory proposes to combine state of the art single-cell gene transfection and high-resolution synaptic stimulation/recording methods in a model culture system where nAChRs and nicotinic synapses are abundant and accessible. One goal is to manipulate gene expression in pre- and postsynaptic neurons that can subsequently be examined in detailed electrophysiological experiments (Aim 1). Realizing this goal will provide a cutting-edge advance relevant to many studies of synapse formation, function and regulation. A second goal is to test the utility of our approach by probing for alterations in synaptic function expected to accompany sustained changes in nicotinic receptor activation (Aim 2). It is anticipated that these latter experiments will provide new answers concerning how alterations of nicotinic synaptic components modify circuit function to initiate and reinforce addiction to nicotine and other abused drugs.