Gene Expression Analysis of Aplysia Neural Network

描述（由申请人提供）：加州海蛾是主要的无脊椎模式生物之一，用于研究行为、学习和长期记忆的神经生物学。最近研究的一个重要发现是，记忆存储的功能和结构变化都是突触特异性的，需要局部翻译。为了理解长期记忆储存的分子机制，包括其结构基础，确定记忆形成过程中突触中mrna群体如何被翻译将是至关重要的。为了研究这个问题，我们将在神经递质5 -羟色胺（参与学习相关的长期促进）和FMRFamide（参与长期抑制）治疗后，从神经节和培养的个体感觉和运动神经元的运动蛋白转运复合物中提取mrna。在治疗后的几个时间点，我们还将描述在自由和膜结合多体上被积极翻译的mrna。后者应该包括靶向突触囊泡和膜的蛋白质。从所有3个群体（运动蛋白复合物，自由多聚体，结合多聚体）分离的mrna将被杂交到含有大多数中枢神经系统（CNS）基因特异性特征的applysia cDNA微阵列中。这个微阵列是我们现有阵列的扩展，将从我们已经从单个神经元、踏板胸膜神经节和整个澳大利亚中枢神经系统中产生的EST文库中的独特克隆构建。虽然我们估计这些克隆覆盖了近80%的海兔神经元中表达的所有基因，但由于编码信息不足，只有一小部分文库被注释。通过对来自相同中枢神经系统来源的全长cdna进行测序，几乎可以实现文库的完整注释。全长cDNA文库和微阵列将为研究这种重要模式生物的学习和其他行为范式提供重要资源。

DESCRIPTION (provided by applicant): Aplysia californica has been one of the dominant invertebrate model organisms utilized for research on the neurobiology of behavior, learning, and long-term memory. A key finding in recent studies is that both the functional and structural changes for memory storage are synapse-specific and require local translation. To understand the molecular mechanisms underlying long-term memory storage including its structural underpinning, it will be essential to determine how the population of mRNAs is destined for translation in synapses during memory formation. To examine this issue, we will extract mRNAs from kinesin transport complexes from ganglia and cultured individual sensory and motor neurons following treatment with the neurotransmitter serotonin (involved in learning-related long-term facilitation) and with FMRFamide (involved in long-term inhibition). At several time points after treatment, we will also characterize the mRNAs being actively translated on free and membrane-bound polysomes. The latter should include proteins targeted to the synaptic vesicles and membranes. Isolated mRNAs from all 3 populations (kinesin complexes, free polysomes, bound polysomes) will be hybridized to an Aplysia cDNA microarray containing features specific for the majority of central nervous system (CNS) genes. This microarray, an expansion of our current array, will be constructed from the unique clones in the EST libraries we have already generated from individual neurons, pedal-pleural ganglia, and the whole CNS of Aplysia. While we estimate these clones cover nearly 80% of all genes expressed in Aplysia neurons, only a fraction of the library has been annotated due to insufficient coding information. Nearly full annotation of the libraries will be achieved by sequencing full-length cDNAs from the same CNS sources. The full-length cDNA libraries and the Aplysia microarray will provide important resources for investigators to study learning and other behavior paradigms in this important model organism.