The molecular logic of persistent memory storage in the cerebellum

小脑持久记忆存储的分子逻辑

• 批准号: 9208822
• 负责人: DAVID J. LINDEN
• 金额: $ 35.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9208822
• 关键词: AMPA Receptors; Acute; Animal Model; Bacterial Artificial Chromosomes; Basic Science; Behavioral; Binding; Biological Models; Brain; Brain Diseases; Brain region; CREB1 gene; Calcineurin; Calcineurin inhibitor; Cell Nucleus; Cerebellum; Chelating Agents; Chemosensitization; DNA Binding; Dendrites; Dendritic Spines; Diagnostic; Disease; Dominant-Negative Mutation; Drug usage; Engineering; Epilepsy; Event; F-Actin; Fiber; Fragile X Syndrome; G Actin; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glutamates; Image; Individual; Knockout Mice; Logic; Measures; Mediating; Memory; Mental Depression; Mental Retardation; Molecular; Mus; Mutate; Mutation; Neurons; Nuclear; Nuclear Translocation; Optics; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Process; Promoter Regions; Protein Dephosphorylation; Purkinje Cells; Reporter; Role; Series; Signal Transduction; Site; Slice; Structure; Synapses; Synaptic plasticity; Tamoxifen; Testing; VP 16; Vertebral column; Work; addiction; cofactor; experimental study; gene induction; knock-down; memory consolidation; mutant; patch clamp; polymerization; promoter; public health relevance; receptor internalization; transcription factor; two-photon

 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.

 描述(由申请人提供)：由突触活动诱导的基因表达对于长期突触增强和抑制的后期阶段是必不可少的(LTP&LTD)。虽然神经元中的活性依赖的转录事件似乎是记忆巩固所必需的，但对特定的靶基因以及转录因子和这些靶基因的启动子区域之间的分子相互作用缺乏了解，这是突触可塑性的后期阶段所必需的。在这一努力中，该领域受到了一系列技术限制的阻碍。这些问题包括无法记录单个哺乳动物神经元中依赖基因表达的突触可塑性晚期，以及无法通过精确激活单个、已定义和成像的突触来研究晚期突触可塑性。此外，突变和设计候选可塑性基因的启动子区域，并对突触LTP/LTD过程中单个神经元中靶基因的表达进行成像并不是一件简单的事情。我们已经解决了这些技术限制，因此可以对培养和脑切片中单个浦肯野细胞中转录依赖的小脑LTD晚期进行长期膜片钳记录，该记录是由定义的树突棘处的谷氨酸去掉引起的，同时成像脊柱和核钙以及基因表达的荧光标记。我们在空白小鼠的神经元上进行了这些实验，并将工程细菌人工染色体(BAC)导入，以便轻松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。我们假设爬行纤维的激活驱动核Ca和CREB与Arc CRE6.9的结合，并且爬行纤维+平行纤维的共同激活是启动磷酸酶和MAL信号级联所必需的，这些信号级联起源于激活的平行纤维刺并传播到细胞核，导致MEF2D和SRF分别与Arc启动子中的MRE6.9和SRE6.9结合。这样，Arc启动子sare区的所有三个结合事件都是触发Arc转录和LTD晚期所必需的。如果我们的假设是正确的，这将是一个双重逻辑运算符编码在Arc启动子的结构中。关联性是许多记忆痕迹的标志，但在任何大脑区域或模式生物中，LTP/LTD的晚期几乎都没有被研究过。