Regulation of calcium-permeable AMPA receptors in associative memory

描述(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(GluR1-4)的各种多聚体组合中。缺乏GluR2亚单位的AMPAR因其独特的电导特性而被称为钙通透性AMPAR(CP-AMPAR)，在某些情况下会在体外和体内突触强化过程中被招募到突触中。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，研究还没有涉及CP-AMPAR在联想记忆形成中的作用，联想记忆的形成是通过突触特异性地加强谷氨酸能传递而发生的。初步数据表明，CP-AMPAR存在于小鼠杏仁外侧核(LA)的丘脑输入部位，杏仁外侧核是联想恐惧记忆中突触修改的关键部位。假设LA神经元中存在额外的CP-AMPAR池，或可能对行为激活做出反应而合成CP-AMPAR；突触结合和这些受体的移除可能分别在恐惧记忆的获得和消除中发挥作用。该项目将基于初步数据检验我们的预测，即通过与C-激酶-1(PICK1)相互作用的蛋白质移除或突触外保留含有GluR2的AMPAR有助于CP-AMPAR的突触表达，并且这一机制是恐惧记忆所必需的。由于初步实验表明，由于长期抑制(LTD)，CP-AMPAR选择性地从突触中移位，因此实验将检查调节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。为此，将利用LA蛋白匀浆的生化分级和脊髓兴奋性突触的双光子谷氨酸浸渍来研究CP-AMPAR电流的分子和解剖学基础，而常规的全细胞电生理学将在听觉恐惧条件反射后以更长的间隔检测突触CP-AMPAR。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。由于我们推断CP-AMPAR很可能包含GluR1，GluR1的去磷酸化将作为一种机制在GluR1磷酸化突变体和模拟磷酸盐小鼠的去增强和恐惧消退过程中被检测到。这些目标将促进我们对潜在的记忆形成和擦除过程的理解，进而促进我们有效治疗记忆损伤的能力。此外，这些实验可能会导致干预人类焦虑的衰弱障碍的新治疗策略，人类焦虑的特征是杏仁核活动异常高水平。

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.