Final Report Summary - MAPPING FEAR MEMORY (Identification and selective targeting of neuronal networks underlying memory) Executive Summary:Advancing our understanding of the molecular and cellular mechanisms underlying learning and memory is among the most important questions in modern neuroscience, and highly relevant for translational efforts to improve the diagnosis, treatment, and prevention of the wide diversity of neurospsychiatric disorders in which cognitive impairments are a prominent feature. In this proposal, we use a combination of advanced methodologies in mouse genetics, molecular biology, and behavioural studies to advance our understanding of how neurons are recruited during learning for incorporation into memory networks, and precisely how memories are represented in neural circuits. Overall, we have identified novel cellular mechanisms by which neurons are selected during learning through differences in intrinsic excitability, while the encoding of memories appears to be accomplished by changes in the strength of synaptic connectivity. Moreover, we highlight important differences between distinct brain regions, regarding both the time-course and cellular mechanisms that are employed. The results of this project will impact both the fundamental neuroscience community interested in understanding the molecular and cellular biology of memory, as well as the translational research community exploring novel mechanisms of neuropsychiatric disorders for which cognitive impairments are a prominent feature.Summary description of the project context and objectives:The main aim of this proposal was to identify the cellular and molecular mechanisms that are necessary and sufficient for memory formation. Using novel genetic technologies, we asked fundamental questions not previously possible about how the brain encodes memories. For instance, how many neurons are required for learning? Are the network requirements similar in different brain regions? Will the number of neurons required to maintain a stable memory increase or decrease over time? And, probably most intriguingly, how is a neuron that is part of a memory trace different from its neighbouring neurons? In other words, what is the cellular and molecular basis of a memory?Previous studies documented how the sparse encoding of associative memories has limited the identification of neuronal ensembles responsible for storing a particular memory. Hence, these long-standing fundamental questions have remained unanswered. However, our recent results suggest a path forward, using novel genetic tools in mice that permit targeted manipulations of genes with both spatial and temporal-specificity. In particular, using genetic manipulations of proteins absolutely required for associative learning, we have effectively restricted the network space in which neuronal plasticity can occur, permitting the characterization of individual neurons responsible for encoding a specific memory. Importantly, the results of these studies not only expand our mechanistic understanding of the neurobiology of memory, but could also provide opportunities for clinical translation into cognitive neuropsychiatric disorders.Description of the main S&T results:Specific Aim 1: Time-dependent evolution of fear memory networksThe goal of Specific Aim 1 was to examine how neurons within a memory circuit are flexibly utilized during the acquisition, consolidation, and maintenance of a memory. In the these experiments, we have utilized HSV vectors to express either CREB, Kir2.1 or a dnKCNQ2 mutant, in order to modify the intrinsic excitability of lateral amygdala neurons to probe the importance for excitability in determining neuronal selection during memory formation versus influencing the consolidation or maintenance of a fear memory. Overall, we found that modulating the intrinsic excitability of a limited subset of lateral amygdala neurons was sufficient to bias the neuronal selection, for which increased excitability led to enhancements in fear learning. Interesting however, and consistent with the model of a time-limited neuronal selection, increasing excitability did not impact the maintenance of fear memories.The overall goal of Specific Aim 1 was to utilize novel transgenic methods to define the minimally-sufficient size of a neuronal ensemble required to encode a stable fear memory. Therefore, in collaboration with Ype Elgersma (Erasmus MC, dept. of Neuroscience) we have now completed the design and engineering of a powerful new transgenic mouse line in which endogenous CaMKII is inducibly activated through a cell-type-specific and temporally-regulated induction (LSL-CaMKII). Through this strategy, the endogenous CaMKII-alpha gene is initially disrupted by a loxP-STOP-loxP cassette inserted between exons 1 and 2. Then, upon tamoxifen-dependent cre recombination (using the CaMKII-CreERT2 transgenic line) or region-sepcific viral-mediated cre recombination (lentiviral Cre-EGFP), the intervening STOP cassette is deleted, allowing normal transcription of the endogenous CaMKII-alpha gene. Our preliminary data is highly encouraging, demonstrating a full expression of CaMKII after 4 daily injections of tamoxifen, while saline injected mice have a complete absence of CaMKII protein expression. Therefore, we are now proceeding temporally-graded reactivation to probe acquisition, consolidation, and long-term memory storage. Furthermore, using region-specific viral injections, we aim to define the minimally-sufficient neural network required for fear memory encoding.Specific Aim 2: Classical genetic rescue to identify the minimally-sufficient neural circuit to support fear conditioningFor Specific Aim 2, we sought utilize transgenic methodologies to readily identify individual neurons recruited into the fear memory network. Broadly, there are 2 major approaches being taken within the project workplan. The first was described above in Specific Aim 1, in which the novel LSL-CaMKII mouse line can now be used to define minimal criteria for neurons recruited into the fear memory network. The second utilizes the floxed CaMKII conditional deletion mouse line by which tamoxifen-inducible dose-dependent deletion is readily performed in adulthood. Using this approach, we have demonstrated that both amygdala and hippocampal-dependent forms of learning are highly sensitive to loss of CaMKII in adulthood. Furthermore, the requirement for CaMKII in both fear conditioning and spatial learning is dependent upon expression of CaMKII at the time of learning, rather than simply throughout development (manuscript in preparation). The sufficiency for expression of CaMKII at the time of learning will now be assessed using the LSL-CaMKII line. Furthermore, amygdala-dependent fear conditioning requires a more extensive deletion of CaMKII than hippocampal-dependent learning, highlighting important fundamental differences between these 2 widely studied brain regions underlying mnemonic function.Moreover, we aimed to characterize the molecular and cellular mechanisms by which fear memories are encoded, by examining neurons preferentially recruited into the fear memory network. Therefore, we have utilized a novel transgenic mouse line (Arc-dVenus), in which a mouse BAC including the endogenous Arc gene has been modified to express the dVenus cDNA, while disrupting the Arc gene. dVenus is a destabilized version of the EYFP variant, Venus fluorescent protein, to allow high signal-to-noise imaging with low background signal. This mouse line has the remarkable property of fluorescently labeling neurons that have been specifically activated during fear conditioning, allowing ex vivo confocal imaging, stereological quantification, and targeted whole-cell electrophysiological recordings. In particular, we find a significant increase in Arc+ neurons within the lateral amygdala, striatum, cortex, and hippocampus following fear conditioning. Specifically within the lateral amygdala, the percentage of activated neurons is approximately double, however the integrated quantitative increase in fluorescence intensity is an increase of nearly one order of magnitude. Electrophysiologically, we have identified robust changes in postsynaptically-mediated thalamo-amygdala EPSCs, spontaneous EPSCs, and intrinsic excitability, without detectable changes in passive membrane properties or single action potential characteristics.Ethics Review and Screening Requirements:All animal experiments were approved by the Dutch Ethical Committee. They have confirmed in their approval that the 3R’s have been properly applied, and that the importance for society justifies the animal usage.Potential impact:During the project, we have developed 2 novel experimental techniques that we likely useful to the broader neuroscience community. First, the development of the LSL-CaMKII line in collaboration with Ype Elgersma (EMC) has been a powerful tool for probing CaMKII function, and represents a unique approach to transgenic manipulation more generally. Second, we have extensively characterized the Arc-dVenus mouse line for conducting a broad array of activity-dependent gene expression studies. Regarding societal impact, the results of this project are of a pre-clinical nature. However, opportunities for clinical translation and confirmation in human subjects is actively ongoing.