Roger L. Clem - US grants

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.

Most psychiatric conditions have their highest incidence of onset in early life, and it is currently estimated that one in five adolescents will develop such a condition that persists into adulthood. A major risk factor for such disorders is the experience of childhood trauma. Among the brain regions most highly implicated in these conditions is the medial prefrontal cortex (mPFC), which forms a synaptic network that plays important roles in emotional regulation. During a developmental stage equivalent of human adolescence, rodents exhibit heightened susceptibility to stress-induced behavioral phenotypes that resemble human conditions like anxiety and depression, and that are accompanied by long-term changes in the structure and function of mPFC in adulthood. However, it remains unclear whether unique mechanisms confer susceptibility to stress in immature animals, or what processes immediately ensue upon exposure to stress in adults versus mice. The long-term goal of this project is to reveal how trauma impacts directly on mPFC neurons, delineate substrates and mechanisms in these effects, and understand the abnormal functional properties that result. In particular, our preliminary data suggest that early adolescence is a sensitive period for noradrenaline-dependent suppression of mPFC activity and excitability, and that engagement of this process during traumatic stress leads to a long- lasting increase in threat avoidance, a potential correlate of human anxiety. We will make unprecedented use of longitudinal calcium imaging in freely behaving mice to measure these physiological effects as well as changes in mPFC activity that signal abnormal avoidance behaviors. Cellular and synaptic mechanisms for hypoexcitability will be elucidated by electrophysiological recordings. Finally, we will use temporally specific optogenetic manipulations to test whether recovering mPFC activity within specific projection pathways is sufficient to reverse trauma-related phenotypes. We hope that be establishing this comprehensive paradigm of stress susceptibility, we can shed light on biological factors that potentially contribute to a high rate of childhood onset for psychiatric disorders.

Project summary Although single neurons occasionally project to a single downstream target, it is more often the case that their axons collateralize and project to multiple distinct anatomical areas. This feature of neuroanatomy has been appreciated for over 100 years and is theorized to be critical to coordinating brain-wide states. Despite this, collateral projections have largely been overlooked in contemporary neuroscience. This is because mapping collateral projections has practically been beyond the reach of empirical investigation, especially in non-human primates where single neurons can project over wide areas. To surmount these issues, our goal here is the comprehensive development of a sequencing-based approach that will allow us to reveal the patterns of connections of single neurons in non-human primates using Multiplexed Analysis of Projections by Sequencing (MAPseq). This approach allows the full collateral projections of potentially thousands of individual neurons to be mapped in a single animal. The first step towards our goal is to validate a sequencing-based connectomic approach in macaque monkeys that has previously been developed and validated in mice (Aim1). Then, once validated we will determine the local and long-range connections of individual neurons in one part of the limbic system in macaques, the amygdala. Our primary focus here is to determine the patterns of collateral projections from amygdala to the frontal cortex (FC) as these have been implicated in the pathophysiology of many psychiatric disorders. With the method for discerning the multiple projection targets of single neurons in the macaque brain in hand we will then compare the patterns that we see in amygdala to those in mice (Aim 2). We hypothesize that through the expansion and differentiation of FC in non-human primates, single amygdala neurons in non-human primates will be many more collateral projections compared to mice. In summary, when successful, our approach has the potential to fully discern the projection profiles of single neurons in non-human primates. This will enable novel insights into the neuroanatomical networks present in non-human primates, provide a powerful new tool for investigating comparative anatomy and aid interpretation of functional studies that target the amygdala in both non-human primates and mice.