Regulation of calcium-permeable AMPA receptors in associative memory

描述（由申请人提供）：AMPA型谷氨酸受体（AMPAR）存在于谷氨酸受体蛋白1-4（GluR 1 -4）的各种多聚体组合中。缺乏GluR 2亚基的AMPAR由于其独特的电导性质而通常被称为钙渗透性AMPAR（CP-AMPAR），并且在某些情况下在体外和体内突触强化期间被募集到突触。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，研究还没有解决CP-AMPAR在联想记忆形成中的作用，这是通过突触特异性加强突触能传递而发生的。初步数据表明，CP-AMPAR存在于小鼠丘脑向外侧杏仁核（LA）的输入，这是关联恐惧记忆中突触修饰的关键部位。据推测，额外的池CP-AMPAR存在于LA神经元或可能是合成的行为激活的反应，和突触的纳入和删除这些受体可能发挥作用，在收购和擦除的恐惧记忆，分别。该项目将测试我们的预测的基础上的初步数据，删除或突触外保留的GluR 2-含有AMPAR的蛋白质相互作用与C-激酶1（PICK 1）促进突触表达的CP-AMPAR，这一机制是必要的恐惧记忆。由于初步实验表明，CP-AMPAR被长期抑郁症（LTD）选择性地从突触中置换，因此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消退。为了实现这些目标，LA蛋白匀浆的生化分级分离和脊柱兴奋性突触处的2-光子谷氨酸释放将用于研究CP-AMPAR电流的分子和解剖基础，而常规的全细胞电生理学将用于在听觉恐惧条件反射后以增加的间隔检测突触CP-AMPAR。将使用小鼠突变体来测试PICK 1和GluR 2磷酸化在受体运输和记忆恢复中的作用。由于我们的理由，CP-AMPAR可能会被发现含有GluR 1，GluR 1的去磷酸化将被检查作为一种机制，CP-AMPAR去除在去增强和恐惧消退使用GluR 1磷酸突变体和磷酸模拟小鼠。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，并进一步提高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，用于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.