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

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.

Sleep loss drives acetylcholine- and somatostatin interneuron-mediated gating of hippocampal activity to inhibit memory consolidation.

Sleep loss disrupts consolidation of hippocampus-dependent memory. To characterize effects of learning and sleep loss, we quantified activity-dependent phosphorylation of ribosomal protein S6 (pS6) across the dorsal hippocampus of mice. We find that pS6 is enhanced in dentate gyrus (DG) following single-trial contextual fear conditioning (CFC) but is reduced throughout the hippocampus after brief sleep deprivation (SD; which disrupts contextual fear memory [CFM] consolidation). To characterize neuronal populations affected by SD, we used translating ribosome affinity purification sequencing to identify cell type-specific transcripts on pS6 ribosomes (pS6-TRAP). Cell type-specific enrichment analysis revealed that SD selectively activated hippocampal somatostatin-expressing (Sst+) interneurons and cholinergic and orexinergic hippocampal inputs. To understand the functional consequences of SD-elevated Sst+ interneuron activity, we used pharmacogenetics to activate or inhibit hippocampal Sst+ interneurons or cholinergic input from the medial septum. The activation of either cell population was sufficient to disrupt sleep-dependent CFM consolidation by gating activity in granule cells. The inhibition of either cell population during sleep promoted CFM consolidation and increased S6 phosphorylation among DG granule cells, suggesting their disinhibition by these manipulations. The inhibition of either population across post-CFC SD was insufficient to fully rescue CFM deficits, suggesting that additional features of sleeping brain activity are required for consolidation. Together, our data suggest that state-dependent gating of DG activity may be mediated by cholinergic input and local Sst+ interneurons. This mechanism could act as a sleep loss-driven inhibitory gate on hippocampal information processing.

Sleep Loss Drives Brain Region-Specific and Cell Type-Specific Alterations in Ribosome-Associated Transcripts Involved in Synaptic Plasticity and Cellular Timekeeping.

Sleep and sleep loss are thought to impact synaptic plasticity, and recent studies have shown that sleep and sleep deprivation (SD) differentially affect gene transcription and protein translation in the mammalian forebrain. However, much less is known regarding how sleep and SD affect these processes in different microcircuit elements within the hippocampus and neocortex, for example, in inhibitory versus excitatory neurons. Here, we use translating ribosome affinity purification (TRAP) and in situ hybridization to characterize the effects of sleep versus SD on abundance of ribosome-associated transcripts in Camk2a-expressing (Camk2a+) pyramidal neurons and parvalbumin-expressing (PV+) interneurons in the hippocampus and neocortex of male mice. We find that while both Camk2a+ neurons and PV+ interneurons in neocortex show concurrent SD-driven increases in ribosome-associated transcripts for activity-regulated effectors of plasticity and transcriptional regulation, these transcripts are minimally affected by SD in hippocampus. Similarly, we find that while SD alters several ribosome-associated transcripts involved in cellular timekeeping in neocortical Camk2a+ and PV+ neurons, effects on circadian clock transcripts in hippocampus are minimal, and restricted to Camk2a+ neurons. Taken together, our results indicate that SD effects on transcripts associated with translating ribosomes are both cell type-specific and brain region-specific, and that these effects are substantially more pronounced in the neocortex than the hippocampus. We conclude that SD-driven alterations in the strength of synapses, excitatory-inhibitory (E-I) balance, and cellular timekeeping are likely more heterogeneous than previously appreciated.SIGNIFICANCE STATEMENT Sleep loss-driven changes in transcript and protein abundance have been used as a means to better understand the function of sleep for the brain. Here, we use translating ribosome affinity purification (TRAP) to characterize changes in abundance of ribosome-associated transcripts in excitatory and inhibitory neurons in mouse hippocampus and neocortex after a brief period of sleep or sleep loss. We show that these changes are not uniform, but are generally more pronounced in excitatory neurons than inhibitory neurons, and more pronounced in neocortex than in hippocampus.

Synergetic Effect of Water-Soluble PEG-Based Macromonomers and Cellulose Nanocrystals for the Stabilization of PMMA Latexes by Surfactant-Free Emulsion Polymerization.

The combination of cellulose nanocrystals (CNCs) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) was evaluated to synthesize stable latexes by surfactant-free emulsion polymerization of methyl methacrylate (MMA). Cellulose-particle interaction was provided due to the dual role of PEGMA, acting as water-soluble comonomer with MMA under emulsion polymerization conditions and able to interact with CNCs, recovered from sulfuric acid hydrolysis (H2SO4-CNCs). After preliminary experiments designed to validate the affinity between CNCs and PEG-stabilized PMMA particles obtained by MMA/PEGMA emulsion copolymerization, the effect of the PEGMA content and molar mass and also of the content of CNCs on the kinetics of the polymerization and the stability of the latexes were investigated. The use of PEGMA300 (Mn = 300 g mol-1, 2-10 wt %) allowed the formation of a stable latex, however, with a broad particle size distribution and the presence of both small (ca. 25-50 nm) and large (ca. 425-650 nm) particles (at 10 wt %, Dn = 278 nm and Dw/Dn = 1.34). Increasing the molar mass of PEGMA (PEGMA950 or PEGMA2080) significantly increased the fraction of small particles. This was explained by the nucleation and growth of small polymer particles adsorbed at the CNCs' surface, resulting in a particular organization where the CNCs were covered by several polymer particles. The influence of the initial amount of CNCs in these systems was finally evidenced, the polymerization being faster as the content of CNCs increased, but only the latexes prepared with 2 and 5 wt % of CNCs were stable.

Early Life Trauma Has Lifelong Consequences for Sleep And Behavior.

Sleep quality varies widely across individuals, especially during normal aging, with impaired sleep contributing to deficits in cognition and emotional regulation. Sleep can also be impacted by a variety of adverse events, including childhood adversity. Here we examined how early life adverse events impacted later life sleep structure and physiology using an animal model to test the relationship between early life adversity and sleep quality across the life span. Rat pups were exposed to an Adversity-Scarcity model from postnatal day 8-12, where insufficient bedding for nest building induces maternal maltreatment of pups. Polysomnography and sleep physiology were assessed in weaning, early adult and older adults. Early life adversity induced age-dependent disruptions in sleep and behavior, including lifelong spindle decreases and later life NREM sleep fragmentation. Given the importance of sleep in cognitive and emotional functions, these results highlight an important factor driving variation in sleep, cognition and emotion throughout the lifespan that suggest age-appropriate and trauma informed treatment of sleep problems.

Sleep loss disrupts Arc expression in dentate gyrus neurons.

Sleep loss affects many aspects of cognition, and memory consolidation processes occurring in the hippocampus seem particularly vulnerable to sleep loss. The immediate-early gene Arc plays an essential role in both synaptic plasticity and memory formation, and its expression is altered by sleep. Here, using a variety of techniques, we have characterized the effects of brief (3-h) periods of sleep vs. sleep deprivation (SD) on the expression of Arc mRNA and Arc protein in the mouse hippocampus and cortex. By comparing the relative abundance of mature Arc mRNA with unspliced pre-mRNA, we see evidence that during SD, increases in Arc across the cortex, but not hippocampus, reflect de novo transcription. Arc increases in the hippocampus during SD are not accompanied by changes in pre-mRNA levels, suggesting that increases in mRNA stability, not transcription, drives this change. Using in situ hybridization (together with behavioral observation to quantify sleep amounts), we find that in the dorsal hippocampus, SD minimally affects Arc mRNA expression, and decreases the number of dentate gyrus (DG) granule cells expressing Arc. This is in contrast to neighboring cortical areas, which show large increases in neuronal Arc expression after SD. Using immunohistochemistry, we find that Arc protein expression is also differentially affected in the cortex and DG with SD - while larger numbers of cortical neurons are Arc+, fewer DG granule cells are Arc+, relative to the same regions in sleeping mice. These data suggest that with regard to expression of plasticity-regulating genes, sleep (and SD) can have differential effects in hippocampal and cortical areas. This may provide a clue regarding the susceptibility of performance on hippocampus-dependent tasks to deficits following even brief periods of sleep loss.

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3-/-) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3-/- mice show normal visual, photic, and non-photic circadian function. In addition, HCN3-/- mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.

Liver-derived ketone bodies are necessary for food anticipation.

The circadian system has endowed animals with the ability to anticipate recurring food availability at particular times of day. As daily food anticipation (FA) is independent of the suprachiasmatic nuclei, the central pacemaker of the circadian system, questions arise of where FA signals originate and what role components of the circadian clock might play. Here we show that liver-specific deletion of Per2 in mice abolishes FA, an effect that is rescued by viral overexpression of Per2 in the liver. RNA sequencing indicates that Per2 regulates ß-hydroxybutyrate (ßOHB) production to induce FA leading to the conclusion that liver Per2 is important for this process. Unexpectedly, we show that FA originates in the liver and not in the brain. However, manifestation of FA involves processing of the liver-derived ßOHB signal in the brain, indicating that the food-entrainable oscillator is not located in a single tissue but is of systemic nature.

A specific role for the REV-ERBα-controlled L-Type Voltage-Gated Calcium Channel CaV1.2 in resetting the circadian clock in the late night.

Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting have been shown to be largely dependent on both membrane depolarization and intracellular second-messenger signaling. In both of these processes, voltage-gated calcium channels (VGCCs) mediate voltage-dependent calcium influx, which propagates neural impulses by stimulating vesicle fusion and instigates intracellular pathways resulting in clock gene expression. Through the cumulative actions of these processes, the phase of the internal clock is modified to match the light cycle of the external environment. To parse out the distinct roles of the L-type VGCCs, we analyzed mice deficient in Cav1.2 (Cacna1c) in brain tissue. We found that mice deficient in the Cav1.2 channel exhibited a significant reduction in their ability to phase-advance circadian behavior when subjected to a light pulse in the late night. Furthermore, the study revealed that the expression of Cav1.2 mRNA was rhythmic (peaking during the late night) and was regulated by the circadian clock component REV-ERBα. Finally, the induction of clock genes in both the early and late subjective night was affected by the loss of Cav1.2, with reductions in Per2 and Per1 in the early and late night, respectively. In sum, these results reveal a role of the L-type VGCC Cav1.2 in mediating both clock gene expression and phase advances in response to a light pulse in the late night.

Activity and social behavior in a complex environment in rats neonatally exposed to alcohol.

Environmental complexity (EC) is a powerful, stimulating paradigm that engages animals through a variety of sensory and motor pathways. Exposure to EC (30 days) following 12 days of wheel running preserves hippocampal neuroplasticity in male rats neonatally exposed to alcohol during the third-trimester equivalent (binge-like exposure on postnatal days [PD] 4-9). The current experiment investigates the importance of various components of EC (physical activity, exploration, social interaction, novelty) and examines whether neonatal alcohol exposure affects how male rats interact with their environment and other male rats. Male pups were assigned to 1 of 3 neonatal conditions from PD 4-9: suckle control (SC), sham-intubated (SI), or alcohol-exposed (AE, 5.25 g/kg/day). From PD 30-42 animals were housed with 24-h access to a voluntary running wheel. The animals were then placed in EC from PD 42-72 (9 animals/cage, counterbalanced by neonatal condition). During EC, the animals were filmed for five 30-min sessions (PD 42, 48, 56, 64, 68). For the first experiment, the videos were coded for distance traveled in the cage, overall locomotor activity, time spent near other animals, and interaction with toys. For the second experiment, the videos were analyzed for wrestling, mounting, boxing, grooming, sniffing, and crawling over/under. AE animals were found to be less active and exploratory and engaged in fewer mounting behaviors compared to control animals. Results suggest that after exposure to wheel running, AE animals still have deficits in activity and social behaviors while housed in EC compared to control animals with the same experience.