PROJECT SUMMARY Molecular, cellular and circuit mechanisms for age-related deficits in memory-linking. Age is the strongest risk factor for Alzheimer’s disease (AD), and therefore, targeting factors that contribute to the onset of this disorder, such as age-related changes in microglia(1, 2) and Locus Coeruleus (LC) (3, 4), may be key for early prevention and treatment(5-7). Here, we propose that early age-dependent changes in these two factors contribute to the onset of memory deficits with aging and AD, including source and relational memory problems(8-12). Our memories depend not only on the ability to recall individual elements/items, but also on processes that link these elements/items (source/relational memory) (13, 14). Importantly, source/relational memory is more sensitive to age-related decline than item memory(8-12). We propose to study middle-aged mice because we found that although contextual memory is still preserved in these mice (14-18 months), they show robust impairments in their ability to link the memories for two contexts (15, 16) acquired close in time, a possible early sign of memory deficits relevant to AD. Two memories are linked when the recall of one triggers the recall of the other(15). In the previous period of this project, we discovered that the temporal window for the allocation of contextual memories to overlapping ensembles in hippocampal dorsal CA1 (dCA1) is critical for modulating the temporal window for contextual memory linking (16-18), and that this mechanism is disrupted in middle- aged mice before any deficits in contextual conditioning itself(16). Importantly, impairments in either LC activation or dopamine from LC in dCA1, dCA1 inhibition of microglia P2Y12 function, as well as increases in the expression of the chemokine receptor CCR5 seen with aging, resulted in both deficits in contextual memory ensemble overlap in dCA1, and in the loss of contextual memory linking (but not in contextual memory itself); In contrast, the depletion/repopulation of microglia or decreases in CCR5 function, resulted in dramatic extensions of the temporal windows for both dCA1 ensemble overlap and contextual memory linking (16-18). Here, we propose to use state-of-the-art imaging, chemogenetic and optogenetic tools to test the novel hypothesis that age-related changes in LC to dCA1 dopaminergic signaling, and associated changes in microglia function, including CCR5 receptor signaling, result in the premature closure of the temporal window for memory ensemble overlap, and therefore in age-related deficits in memory linking. To test this hypothesis, we propose to determine whether age-related changes in LC dopaminergic and microglia function, including CCR5 signaling, contribute to the loss of age-related dCA1 contextual memory ensemble overlap and contextual memory linking in middle-aged mice. Importantly, our preliminary evidence showed that dopaminergic activation in dCA1, decreases in CCR5 function, and microglia depletio...