Regulation of calcium-permeable AMPA receptors in associative memory

描述（由申请人提供）：ampa型谷氨酸受体（AMPARs）存在于谷氨酸受体蛋白1-4 （GluR1-4）的各种多聚体组合中。缺乏GluR2亚基的AMPARs由于其独特的电导特性通常被称为钙透性AMPARs (CP-AMPARs)，并且在某些情况下在体外和体内突触强化过程中被募集到突触中。然而，关于这种形式的可塑性与行为改变的相关性，仍然存在许多问题。特别是，研究尚未解决CP-AMPARs在联想记忆形成中的作用，联想记忆是通过突触特异性强化谷氨酸能传递而发生的。初步数据表明，CP- ampar存在于小鼠丘脑外侧杏仁核（LA）的输入区，而外侧杏仁核是联想恐惧记忆突触修饰的关键部位。假设LA神经元中存在额外的CP-AMPARs池，或者可能在行为激活的反应中合成；这些受体的突触结合和移除可能分别在恐惧记忆的获得和消除中发挥作用。该项目将验证我们基于初步数据的预测，即通过与c -激酶-1 （PICK1）的蛋白相互作用去除或保留含glur2的AMPARs促进CP-AMPARs的突触表达，并且这种机制是恐惧记忆所必需的。由于初步实验表明cp - ampar被长期抑郁（LTD）选择性地从突触中转移，实验将研究介导这一过程的分子事件，并确定它们是否有助于恐惧记忆的行为灭绝。为此，将利用LA蛋白匀浆的生化分离和脊椎兴奋性突触的2光子谷氨酸释放来研究CP- ampar电流的分子和解剖学基础，而传统的全细胞电生理学将用于检测听觉恐惧条件作用后突触CP- ampar的增加间隔。小鼠突变体将被用来测试PICK1和GluR2磷酸化在受体运输和记忆恢复中的作用。由于我们认为CP-AMPAR很可能含有GluR1，因此我们将使用GluR1磷化和拟磷小鼠来研究GluR1去磷酸化作为在去增强和恐惧消除过程中CP-AMPAR去除的机制。这些目标将进一步加深我们对记忆形成和消除过程的理解，并通过扩展我们有效治疗记忆障碍的能力。此外，这些实验可能会导致新的治疗策略，以干预人类焦虑的衰弱性疾病，其特征是异常高水平的杏仁核活动。

DESCRIPTION (provided by applicant): AMPA-type glutamate receptors (AMPARs) exist in various multimeric combinations of glutamate receptor proteins 1-4 (GluR1-4). AMPARs that lack the GluR2 subunit are commonly referred to as calcium- permeable AMPARs (CP-AMPARs) due to their unique conductance properties, and are recruited to synapses during in vitro and in vivo synaptic strengthening under some circumstances. However, many questions remain about the relevance of this form of plasticity to the modification of behavior. In particular, studies have not addressed the role of CP-AMPARs in the formation of associative memory, which occurs by synapse-specific strengthening of glutamatergic transmission. Preliminary data indicate that CP- AMPARs exist at thalamic inputs to the lateral amygdala (LA) of mice, a key site of synaptic modification in associative fear memory. It is hypothesized that additional pools of CP-AMPARs exist in LA neurons or may be synthesized in response to behavioral activation; and that synaptic incorporation and removal of these receptors may play a role in acquisition and erasure of fear memory, respectively. This project will test our predictions based on preliminary data that removal or extrasynaptic retention of GluR2-containing AMPARs by protein-interacting with C-kinase-1 (PICK1) facilitates the synaptic expression of CP-AMPARs, and that this mechanism is required for fear memory. Since preliminary experiments indicate that CP-AMPARs are selectively displaced from synapses by long-term depression (LTD), experiments will examine the molecular events that mediate this process and determine whether they contribute to behavioral extinction of fear memory. Towards these aims, biochemical fractionation of LA protein homogenates and 2-photon glutamate uncaging at spine excitatory synapses will be used to study the molecular and anatomical bases of CP-AMPAR currents, while conventional whole-cell electrophysiology will be used to detect synaptic CP- AMPARs at increasing intervals after auditory fear conditioning. Mouse mutants will be employed to test the role of PICK1 and GluR2 phosphorylation in receptor trafficking and memory retrieval. Since we reason that CP-AMPARs will likely be found to contain GluR1, dephosphorylation of GluR1 will be examined as a mechanism for CP-AMPAR removal during depotentiation and fear extinction using GluR1 phosphomutant and phosphomimetic mice. These aims will further our understanding of processes underlying memory formation and erasure, and by extension our ability to effectively treat memory impairments. In addition, these experiments may lead to new therapeutic strategies for intervention in debilitating disorders of human anxiety, which are characterized by abnormally high levels of amygdala activity.