Hippocampal neurons' cytosolic and membrane-bound ribosomal transcript profiles are differentially regulated by learning and subsequent sleep

Abstract

The hippocampus is essential for consolidating transient experiences into long-lasting memories. Memory consolidation is facilitated by postlearning sleep, although the underlying cellular mechanisms are largely unknown. We took an unbiased approach to this question by using a mouse model of hippocampally mediated, sleep-dependent memory consolidation (contextual fear memory). Because synaptic plasticity is associated with changes to both neuronal cell membranes (e.g., receptors) and cytosol (e.g., cytoskeletal elements), we characterized how these cell compartments are affected by learning and subsequent sleep or sleep deprivation (SD). Translating ribosome affinity purification was used to profile ribosome-associated RNAs in different subcellular compartments (cytosol and membrane) and in different cell populations (whole hippocampus, Camk2a+ neurons, or highly active neurons with phosphorylated ribosomal subunit S6 [pS6+]). We examined how transcript profiles change as a function of sleep versus SD and prior learning (contextual fear conditioning; CFC). While sleep loss altered many cytosolic ribosomal transcripts, CFC altered almost none, and CFC-driven changes were occluded by subsequent SD. In striking contrast, SD altered few transcripts on membrane-bound (MB) ribosomes, while learning altered many more (including long non-coding RNAs [lncRNAs]). The cellular pathways most affected by CFC were involved in structural remodeling. Comparisons of post-CFC MB transcript profiles between sleeping and SD mice implicated changes in cellular metabolism in Camk2a+ neurons and protein synthesis in highly active pS6+ (putative "engram") neurons as biological processes disrupted by SD. These findings provide insights into how learning affects hippocampal neurons and suggest that the effects of SD on memory consolidation are cell type and subcellular compartment specific.

Keywords: bioinformatics; memory consolidation; ribosomes; synaptic plasticity; translation.

Fig. 1.

TRAP-based profiling of hippocampal cell populations and isolation of subcellular fractions. (A, Left) Confocal images showing expression of HA (Camk2a), phosphorylated S6 (pS6), and parvalbumin in area CA1 of dorsal hippocampus. Highlighted neurons are parvalbumin, pS6, and HA. (Scale bar, 100 μm.) (Right) Schematic of protocol for isolating mRNAs from subcellular fractions and different cell populations using TRAP. (B) Camk2a+ (cyan) and pS6+ (orange) TRAP mRNA enrichment values were calculated (versus Input) for activity-dependent (Arc, Cfos, Homer1a), excitatory neuron (Glua1, Vglut1), inhibitory neuron (Parv, Sst), and glial (Mbp, Gfap) transcripts. *** indicates P < 0.001 for enrichment value differences between Camk2a+ and pS6+ neuronal populations (t test, n = 7/group). (C) Camk2a+ and pS6+ TRAP enrichment in supernatant (solid bars) and pellet (hatched bars) fractions (versus Input) for transcripts encoding secreted (Bdnf), transmembrane (Grin2a, Grin2b), ER (Hspa5), and cytosolic (Cfos, Homer1a) proteins. (t test, n = 9/group, *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively). (D) PCA plot (variance stabilizing transformation [VST], Deseq2) for RNA-seq data from the three cell populations (Input, Camk2a+ neurons, and pS6+ neurons) and two fractions (supernatant and pellet). Data are shown for n = 30 biological replicates (i.e., bilateral hippocampi) from 30 total mice across the four treatment groups.

Fig. 2.

Cytosolic ribosomal transcripts are altered primarily by SD, while MB ribosomal transcripts are altered primarily by learning. (A, Left) Experimental paradigm for RNA-seq experiments, showing the four treatment groups. n values indicate the number of mice per group; one mouse’s bilateral hippocampi constituted one biological replicate. At lights on, mice were either left in their HC or underwent single-trial CFC. All mice were then either permitted ad libitum sleep or were SD over the following 3 h. (Right) Transcript comparisons for quantifying effects of SD (yellow) included both HC and CFC animals. To quantify effects of CFC, CFC + Sleep (blue), and CFC + SD (red), mice were analyzed separately. Following behavioral manipulations, cytosolic and MB fractions for different cell populations were isolated as described in Fig. 1. (B) Proportional Venn diagrams reflect the number of significantly altered transcripts in each cell populations and subcellular fractions (i.e., Camk2a+/MB), based on comparisons shown in A. Complete transcript lists for each comparison are available in Datasets S4, S5, and S6. (C) Transcripts altered by SD and CFC in different subcellular fractions in Camk2a+, pS6+, and Input populations were used to construct Venn diagrams. Full transcript lists are included in Dataset S7.

Fig. 3.

mRNAs altered by SD on cytosolic ribosomes encode transcriptional regulators. (A) Proportional and overlapping Venn diagrams of transcripts significantly altered by SD, CFC + Sleep, and CFC + SD in cytosolic fractions from Camk2a+ neurons, pS6+ neurons, and Input. (B, Top) The seven most-enriched molecular and cellular function categories (ranked by P_adj value) for transcripts altered by SD alone in Camk2a+ neurons, pS6+ neurons, and Input. (Bottom) The 10 most-enriched canonical pathways of SD-affected transcripts are listed in order P_adj value (indicated by circle diameter), with z-scores reflecting direction of pathway regulation (indicated by hue). There were no significant canonical pathways present in the Input fraction. (C, Left) The 10 transcripts most significantly affected in CFC + Sleep (blue) and CFC + SD (red) conditions for Camk2a+ (Top) and pS6+ (Bottom) neurons, ranked by P_adj value. Transcripts that were also significantly altered as a function of SD alone are highlighted in yellow. (Right) Results of transcript-level analysis (56) show transcripts for transcript isoforms altered in Camk2a+ (Top) and pS6+ (Bottom) neurons following CFC. Transcript isoforms that were significantly altered as a function of SD are highlighted in yellow. Functional category analysis available in Dataset S8.

Fig. 4.

Transcripts altered by CFC on MB ribosomes encode regulators of neuronal morphology, intracellular trafficking, and lncRNAs. (A) Proportional and overlapping Venn diagrams of transcripts significantly altered by SD, CFC + Sleep, and CFC + SD in MB fractions from Camk2a+ neurons, pS6+ neurons, and Input. (B) The seven most-significant molecular functions (ranked by P_adj value) for transcripts altered by CFC + Sleep (Top) and CFC + SD (Bottom) in Camk2a+ neurons, pS6+ neurons, and Input. (C) The 10 transcripts most significantly affected in CFC + Sleep (blue) and CFC + SD (red) conditions for Camk2a+ and pS6+ neurons, ranked by P_adj value. Transcripts that were also significantly altered as a function of SD alone are highlighted in yellow. Functional category analysis available in Dataset S10.

Fig. 5.

MB ribosomal transcript networks affected by CFC vary as a function of subsequent sleep or SD. Canonical pathway network analysis of transcripts altered on MB ribosomes from Camk2a+ (A and B) or pS6+ (C and D) neurons following CFC + Sleep (A, C) or CFC + SD (B, D). Hub size and color denote Padj value and z-score, respectively, in each condition, while connecting lines indicate commonly expressed genes between hubs. Canonical pathways are available in Dataset S10.