The molecular logic of persistent memory storage in the cerebellum

 描述（由申请人提供）：通过突触活性诱导基因表达对于长时程突触增强和抑制（LTP & LTD）的后期阶段是必不可少的。虽然神经元中的活性依赖性转录事件似乎是记忆巩固所必需的，但对突触可塑性后期所需的特定靶基因以及转录因子与这些靶基因的启动子区域之间的分子相互作用的理解很少。在这一奋进中，该领域受到一系列技术限制的阻碍。这些包括无法记录单个哺乳动物神经元中突触可塑性的基因表达依赖性晚期，以及无法使用精确激活个体、定义和成像的突触来研究晚期突触可塑性。此外，突变和工程化候选可塑性基因的定义启动子区域并不简单，并且在经历突触LTP/LTD的单个神经元中成像靶基因的表达。我们已经解决了这些技术限制，因此可以在培养物和脑切片中的单个浦肯野细胞中对小脑LTD的转录依赖性晚期进行长期膜片钳记录，通过在确定的树突棘释放谷氨酸引起，同时成像棘和核Ca和基因表达的荧光标记物。我们在来自空小鼠的神经元中进行这些实验，并用工程化的细菌人工染色体（BAC）转染，以容易地操纵相关基因的启动子。这项工作揭示了LTD的晚期需要Arc基因的转录。我们假设，爬行纤维激活驱动核Ca和CREB结合到Arc CRE6.9，并且爬行纤维+平行纤维共激活是启动磷酸酶和MAL信号级联所必需的，所述磷酸酶和MAL信号级联起源于激活的平行纤维棘并传播到核，从而导致MEF 2D和SRF分别结合到Arc启动子中的MRE 6.9和SRE 6.9。以这种方式，所有三个结合事件的萨雷区域的Arc启动子是必要的触发Arc转录和后期的LTD。如果我们的假设是正确的，这将是一个双AND逻辑运算符编码的Arc启动子的结构。关联性是许多记忆痕迹的标志，但在任何大脑区域或模型生物中，LTP/LTD的晚期阶段几乎完全未被探索。

 DESCRIPTION (provided by applicant): The induction of gene expression by synaptic activity is essential for the later phases of long-term synaptic potentiation and depression (LTP & LTD). While activity-dependent transcriptional events in neurons appear to be necessary for memory consolidation, there is poor understanding of the specific target genes and the molecular interactions between transcription factors and the promoter regions of these target genes that are required for the later phases of synaptic plasticity. The field has been hampered by a series of technical limitations in this endeavor. These include an inability to record the gene expression-dependent late phase of synaptic plasticity in single mammalian neurons and to study late phase synaptic plasticity using precise activation of individual, defined and imaged synapses. Furthermore, it has not been straightforward to mutate and engineer defined promoter regions of candidate plasticity genes and to image the expression of target genes in individual neurons undergoing synaptic LTP/LTD. We have solved these technical limitations and so can perform long-term patch-clamp recording of the transcription-dependent late phase of cerebellar LTD in single Purkinje cells in cultures and brain slices, evoked by uncaging of glutamate at defined dendritic spines, while imaging spine and nuclear Ca and fluorescent markers of gene expression. We perform these experiments in neurons from null mice and transfected with engineered bacterial artificial chromosomes (BACs) to easily manipulate the promoters of relevant genes. This work has revealed that the late phase of LTD requires transcription of the Arc gene. We hypothesize that climbing fiber activation drives nuclear Ca and CREB binding to Arc CRE6.9 and that climbing fiber + parallel fiber co-activation is necessary to initiate phosphatase and MAL signaling cascades that originate in activated parallel fiber spines and propagate to the nucleus to result in MEF2D and SRF binding to MRE6.9 and SRE6.9 in the Arc promoter respectively. In this way, all three binding events in the SARE region of the Arc promoter are necessary to trigger Arc transcription and the late phase of LTD. If our hypothesis is correct, this would be a double-AND logical operator encoded in the structure of the Arc promoter. Associativity is the hallmark of many memory traces, yet remains almost entirely unexplored for late phases of LTP/LTD in any brain region or model organism.