Linking Intracellular Events to Network Reorganization in Sleep-Dependent Memory Consolidation
Delorme, James
2020
Abstract
Research in animal and human subjects has found that sleep loss profoundly disrupts the consolidation of hippocampus-dependent memories. One leading hypothesis regarding how sleep consolidates memories is by increasing protein synthesis, thereby translating any synaptic plasticity transcripts expressed during wakefulness. Still it remains unclear how translational processes or differences in hippocampal activity are modulated during sleep or which transcripts are necessary for memory consolidation. The studies outlined in this dissertation aim to address this question in the context of a well-studied form of sleep-dependent memory consolidation in mice, contextual fear memory (CFM). CFM is disrupted by sleep loss in the first few hours following CFM. To describe the contributions of sleep and learning on hippocampal protein translation, we modified existing translating ribosome affinity purification (TRAP) techniques to immunoprecipitate ribosome-bound mRNAs from multiple hippocampal cell populations in the same tissue and isolate transcripts from different subcellular fractions. Our protocol allowed us to quantify differences in ribosome-bound mRNAs from excitatory and ‘activated’ neurons, as well mRNA from whole hippocampal homogenate from the same mice. To further characterize differences in translation based on ribosomes intracellular localization, we separated free-floating from membrane-bound (MB) ribosomes and analyzed them separately. Our results identified divergent effects of SD and learning on cytosolic and MB ribosomes, respectively. At cytosolic ribosomes, SD increased the expression of synaptic plasticity genes and occluded the sparse expression of CFC-related genes in excitatory hippocampal neurons. In MB ribosomes, CFC induced overlapping cellular pathways in both sleep permitted and SD mice. However we also detected ribosome-associated enrichments of transcripts for components of bioenergetic pathways not observed in sleeping mice. These results reveal how SD differentially impairs CFC-related protein translation in two distinct subcellular compartments. Since sleep deprivation impairs protein synthesis and hippocampus-dependent memory tasks, we analyzed the phosphorylation of the ribosomal S6 protein (pS6) in mice following CFC and subsequent sleep or SD. S6 is phosphorylated in response to increased neuronal activity, and pS6 is correlated with elevated translational regulation. We characterized expression of pS6 the major subregions of the hippocampus (DG, CA1, & CA3) using immunohistochemistry. We found that 3 h of SD alone reduced S6 phosphorylation across all subregions whereas prior learning (CFC) increased pS6 expression in the DG. Furthermore, depriving mice of sleep following CFC selectively impaired pS6 expression in the DG and CA1, suggesting that memory processing during sleep invokes cellular circuits distinct from experimentally naïve mice. To characterize cell populations affected by SD, we used translating ribosome affinity purification (TRAP) to isolate cell type-specific transcripts associated with pS6-ribosomes in active neurons. Our results identified DG hilar somatostatin (SST+) interneurons to be highly enriched with pS6 during SD and express intracellular markers of plasticity and activity. Increasing SST+ interneuron activity during post-CFC sleep using pharmacogenetics reduced the number of activated neurons in the DG and impaired memory consolidation, suggesting that this interneuron population dampens network activity and potentially intracellular signaling events. To determine the aspect of sleep influencing SST activity, we tested reducing cholinergic input to the hippocampus following CFC and observed increased DG cFos+ neurons as well as improved memory retention 24 h later. Our research has identified a hippocampal SST microcircuit that serves to dampen hippocampal activity during SD and may contribute to disruptions in memory consolidation extended wakefulness.Subjects
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