Sleep-dependent engram reactivation during hippocampal memory consolidation associated with subregion-specific biosynthetic changes.

Post-learning sleep is essential for hippocampal memory processing, including contextual fear memory consolidation. We labeled context-encoding engram neurons in the hippocampal dentate gyrus (DG) and assessed reactivation of these neurons after fear learning. Post-learning sleep deprivation (SD) selectively disrupted reactivation of inferior blade DG engram neurons, linked to SD-induced suppression of neuronal activity in the inferior, but not superior DG blade. Subregion-specific spatial profiling of transcripts revealed that transcriptomic responses to SD differed greatly between hippocampal CA1, CA3, and DG inferior blade, superior blade, and hilus. Activity-driven transcripts, and those associated with cytoskeletal remodeling, were selectively suppressed in the inferior blade. Critically, learning-driven transcriptomic changes differed dramatically between the DG blades and were absent from all other regions. Together, these data suggest that the DG is critical for sleep-dependent memory consolidation, and that the effects of sleep loss on the hippocampus are highly subregion-specific.

Neuromodulation via muscarinic acetylcholine pathway can facilitate distinct, complementary, and sequential roles for NREM and REM states during sleep-dependent memory consolidation.

Across vertebrate species, sleep consists of repeating cycles of NREM followed by REM. However, the respective functions of NREM, REM, and their stereotypic cycling pattern are not well understood. Using a simplified biophysical network model, we show that NREM and REM sleep can play differential and critical roles in memory consolidation primarily regulated, based on state-specific changes in cholinergic signaling. Within this network, decreasing and increasing muscarinic acetylcholine (ACh) signaling during bouts of NREM and REM, respectively, differentially alters neuronal excitability and excitatory/inhibitory balance. During NREM, deactivation of inhibitory neurons leads to network-wide disinhibition and bursts of synchronized activity led by firing in engram neurons. These features strengthen connections from the original engram neurons to less-active network neurons. In contrast, during REM, an increase in network inhibition suppresses firing in all but the most-active excitatory neurons, leading to competitive strengthening/pruning of the memory trace. We tested the predictions of the model against in vivo recordings from mouse hippocampus during active sleep-dependent memory storage. Consistent with modeling results, we find that functional connectivity between CA1 neurons changes differentially at transition from NREM to REM sleep during learning. Returning to the model, we find that an iterative sequence of state-specific activations during NREM/REM cycling is essential for memory storage in the network, serving a critical role during simultaneous consolidation of multiple memories. Together these results provide a testable mechanistic hypothesis for the respective roles of NREM and REM sleep, and their universal relative timing, in memory consolidation. Significance statement: Using a simplified computational model and in vivo recordings from mouse hippocampus, we show that NREM and REM sleep can play differential roles in memory consolidation. The specific neurophysiological features of the two sleep states allow for expansion of memory traces (during NREM) and prevention of overlap between different memory traces (during REM). These features are likely essential in the context of storing more than one new memory simultaneously within a brain network.

Hypnotic treatment reverses NREM sleep disruption and EEG desynchronization in a mouse model of Fragile X syndrome to rescue memory consolidation deficits.

Fragile X syndrome (FXS) is a highly-prevalent genetic cause of intellectual disability, associated with disrupted cognition and sleep abnormalities. Sleep loss itself negatively impacts cognitive function, yet the contribution of sleep loss to impaired cognition in FXS is vastly understudied. One untested possibility is that disrupted cognition in FXS is exacerbated by abnormal sleep. We hypothesized that restoration of sleep-dependent mechanisms could improve functions such as memory consolidation in FXS. We examined whether administration of ML297, a hypnotic drug acting on G-protein-activated inward-rectifying potassium channels, could restore sleep phenotypes and improve disrupted memory consolidation in Fmr1 -/y mice. Using 24-h polysomnographic recordings, we found that Fmr1 -/y mice exhibit reduced non-rapid eye movement (NREM) sleep and fragmented NREM sleep architecture, alterations in NREM EEG spectral power (including reductions in sleep spindles), and reduced EEG coherence between cortical areas. These alterations were reversed in the hours following ML297 administration. Hypnotic treatment following contextual fear or spatial learning also ameliorated disrupted memory consolidation in Fmr1 -/y mice. Hippocampal activation patterns during memory recall was altered in Fmr1 -/y mice, reflecting an altered balance of activity among principal neurons vs. parvalbumin-expressing (PV+) interneurons. This phenotype was partially reversed by post-learning ML297 administration. These studies suggest that sleep disruption could have a major impact on neurophysiological and behavioral phenotypes in FXS, and that hypnotic therapy may significantly improve disrupted cognition in this disorder.

Enriched binocular experience followed by sleep optimally restores binocular visual cortical responses in a mouse model of amblyopia.

Studies of primary visual cortex have furthered our understanding of amblyopia, long-lasting visual impairment caused by imbalanced input from the two eyes during childhood, which is commonly treated by patching the dominant eye. However, the relative impacts of monocular vs. binocular visual experiences on recovery from amblyopia are unclear. Moreover, while sleep promotes visual cortex plasticity following loss of input from one eye, its role in recovering binocular visual function is unknown. Using monocular deprivation in juvenile male mice to model amblyopia, we compared recovery of cortical neurons' visual responses after identical-duration, identical-quality binocular or monocular visual experiences. We demonstrate that binocular experience is quantitatively superior in restoring binocular responses in visual cortex neurons. However, this recovery was seen only in freely-sleeping mice; post-experience sleep deprivation prevented functional recovery. Thus, both binocular visual experience and subsequent sleep help to optimally renormalize bV1 responses in a mouse model of amblyopia.

Atypical hypnotic compound ML297 restores sleep architecture immediately following emotionally valenced learning, to promote memory consolidation and hippocampal network activation during recall.

Sleep plays a critical role in consolidating many forms of hippocampus-dependent memory. While various classes of hypnotic drugs have been developed in recent years, it remains unknown whether, or how, some of them affect sleep-dependent memory consolidation mechanisms. We find that ML297, a recently developed candidate hypnotic agent targeting a new mechanism (activating GIRK1/2-subunit containing G-protein coupled inwardly rectifying potassium [GIRK] channels), alters sleep architecture in mice over the first 6 hr following a single-trial learning event. Following contextual fear conditioning (CFC), ML297 reversed post-CFC reductions in NREM sleep spindle power and REM sleep amounts and architecture, renormalizing sleep features to what was observed at baseline, prior to CFC. Renormalization of post-CFC REM sleep latency, REM sleep amounts, and NREM spindle power were all associated with improved contextual fear memory (CFM) consolidation. We find that improvements in CFM consolidation due to ML297 are sleep-dependent, and are associated with increased numbers of highly activated dentate gyrus (DG), CA1, and CA3 neurons during CFM recall. Together our findings suggest that GIRK1/2 channel activation restores normal sleep architecture- including REM sleep, which is normally suppressed following CFC-and increases the number of hippocampal neurons incorporated into the CFM engram during memory consolidation.

Perspective - ultrastructural analyses reflect the effects of sleep and sleep loss on neuronal cell biology.

Recent electron microscopic analyses of neurons in the Drosophila and rodent brain demonstrate that acute or chronic sleep loss can alter the structures of various organelles, including mitochondria, nucleus, and Golgi apparatus. Here, we discuss these findings in the context of biochemical findings from the sleep deprived brain, to clarify how these morphological changes may related to altered organelle function. We discuss how, taken together, the available data suggest that sleep loss (particularly chronic sleep loss) disrupts such fundamental cellular processes as transcription, translation, intracellular transport, and metabolism. A better understanding of these effects will have broad implications for understanding the biological importance of sleep, and the relationship of sleep loss to neuropathology.

Heterogeneous mechanisms for synchronization of networks of resonant neurons under different E/I balance regimes.

Rhythmic synchronization of neuronal firing patterns is a widely present phenomenon in the brain-one that seems to be essential for many cognitive processes. A variety of mechanisms contribute to generation and synchronization of network oscillations, ranging from intrinsic cellular excitability to network mediated effects. However, it is unclear how these mechanisms interact together. Here, using computational modeling of excitatory-inhibitory neural networks, we show that different synchronization mechanisms dominate network dynamics at different levels of excitation and inhibition (i.e. E/I levels) as synaptic strength is systematically varied. Our results show that with low synaptic strength networks are sensitive to external oscillatory drive as a synchronizing mechanism-a hallmark of resonance. In contrast, in a strongly-connected regime, synchronization is driven by network effects via the direct interaction between excitation and inhibition, and spontaneous oscillations and cross-frequency coupling emerge. Unexpectedly, we find that while excitation dominates network synchrony at low excitatory coupling strengths, inhibition dominates at high excitatory coupling strengths. Together, our results provide novel insights into the oscillatory modulation of firing patterns in different excitation/inhibition regimes.

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.

The Engram's Dark Horse: How Interneurons Regulate State-Dependent Memory Processing and Plasticity.

Brain states such as arousal and sleep play critical roles in memory encoding, storage, and recall. Recent studies have highlighted the role of engram neurons-populations of neurons activated during learning-in subsequent memory consolidation and recall. These engram populations are generally assumed to be glutamatergic, and the vast majority of data regarding the function of engram neurons have focused on glutamatergic pyramidal or granule cell populations in either the hippocampus, amygdala, or neocortex. Recent data suggest that sleep and wake states differentially regulate the activity and temporal dynamics of engram neurons. Two potential mechanisms for this regulation are either via direct regulation of glutamatergic engram neuron excitability and firing, or via state-dependent effects on interneuron populations-which in turn modulate the activity of glutamatergic engram neurons. Here, we will discuss recent findings related to the roles of interneurons in state-regulated memory processes and synaptic plasticity, and the potential therapeutic implications of understanding these mechanisms.

Acetylcholine-gated current translates wake neuronal firing rate information into a spike timing-based code in Non-REM sleep, stabilizing neural network dynamics during memory consolidation.

Sleep is critical for memory consolidation, although the exact mechanisms mediating this process are unknown. Combining reduced network models and analysis of in vivo recordings, we tested the hypothesis that neuromodulatory changes in acetylcholine (ACh) levels during non-rapid eye movement (NREM) sleep mediate stabilization of network-wide firing patterns, with temporal order of neurons' firing dependent on their mean firing rate during wake. In both reduced models and in vivo recordings from mouse hippocampus, we find that the relative order of firing among neurons during NREM sleep reflects their relative firing rates during prior wake. Our modeling results show that this remapping of wake-associated, firing frequency-based representations is based on NREM-associated changes in neuronal excitability mediated by ACh-gated potassium current. We also show that learning-dependent reordering of sequential firing during NREM sleep, together with spike timing-dependent plasticity (STDP), reconfigures neuronal firing rates across the network. This rescaling of firing rates has been reported in multiple brain circuits across periods of sleep. Our model and experimental data both suggest that this effect is amplified in neural circuits following learning. Together our data suggest that sleep may bias neural networks from firing rate-based towards phase-based information encoding to consolidate memories.

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.

Theta-gamma coupling emerges from spatially heterogeneous cholinergic neuromodulation.

Theta and gamma rhythms and their cross-frequency coupling play critical roles in perception, attention, learning, and memory. Available data suggest that forebrain acetylcholine (ACh) signaling promotes theta-gamma coupling, although the mechanism has not been identified. Recent evidence suggests that cholinergic signaling is both temporally and spatially constrained, in contrast to the traditional notion of slow, spatially homogeneous, and diffuse neuromodulation. Here, we find that spatially constrained cholinergic stimulation can generate theta-modulated gamma rhythms. Using biophysically-based excitatory-inhibitory (E-I) neural network models, we simulate the effects of ACh on neural excitability by varying the conductance of a muscarinic receptor-regulated K+ current. In E-I networks with local excitatory connectivity and global inhibitory connectivity, we demonstrate that theta-gamma-coupled firing patterns emerge in ACh modulated network regions. Stable gamma-modulated firing arises within regions with high ACh signaling, while theta or mixed theta-gamma activity occurs at the peripheries of these regions. High gamma activity also alternates between different high-ACh regions, at theta frequency. Our results are the first to indicate a causal role for spatially heterogenous ACh signaling in the emergence of localized theta-gamma rhythmicity. Our findings also provide novel insights into mechanisms by which ACh signaling supports the brain region-specific attentional processing of sensory information.

Aligning one's sights: The pulvinar provides context for visual information processing.

The pulvinar (lateral posterior [LP]), like other higher-order thalamic nuclei, receives input from-and sends output to-multiple neocortical structures. In this issue of Neuron, Blot et al. (2021) demonstrate that LP integrates multimodal inputs to put visual information in context.

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.

The role of clock genes in sleep, stress and memory.

Circadian clock genes serve as the molecular basis for animals' ~24-h internal timekeeping. Clock gene expression inside and outside of the mammalian brain's circadian pacemaker (i.e. the SCN) integrates temporal information into a wealth of physiological processes. Ample data suggests that in addition to canonical cellular timekeeping functions, clock proteins also interact with proteins involved in cellular processes not related to timekeeping, including protein regulation and the interaction with other signaling mechanisms not directly linked to the regulation of circadian rhythms. Indeed, recent data suggests that clock genes outside the SCN are involved in fundamental brain processes such as sleep/wakefulness, stress and memory. The role of clock genes in these brain processes are complex and divers, influencing many molecular pathways and phenotypes. In this review, we will discuss recent work on the involvement of clock genes in sleep, stress, and memory. Moreover, we raise the controversial possibility that these functions may be under certain circumstances independent of their circadian timekeeping function.

Causal role for sleep-dependent reactivation of learning-activated sensory ensembles for fear memory consolidation.

Learning-activated engram neurons play a critical role in memory recall. An untested hypothesis is that these same neurons play an instructive role in offline memory consolidation. Here we show that a visually-cued fear memory is consolidated during post-conditioning sleep in mice. We then use TRAP (targeted recombination in active populations) to genetically label or optogenetically manipulate primary visual cortex (V1) neurons responsive to the visual cue. Following fear conditioning, mice respond to activation of this visual engram population in a manner similar to visual presentation of fear cues. Cue-responsive neurons are selectively reactivated in V1 during post-conditioning sleep. Mimicking visual engram reactivation optogenetically leads to increased representation of the visual cue in V1. Optogenetic inhibition of the engram population during post-conditioning sleep disrupts consolidation of fear memory. We conclude that selective sleep-associated reactivation of learning-activated sensory populations serves as a necessary instructive mechanism for memory consolidation.

Dynamical Mechanism Underlying Scale-Free Network Reorganization in Low Acetylcholine States Corresponding to Slow Wave Sleep.

Sleep is indispensable for most animals' cognitive functions, and is hypothesized to be a major factor in memory consolidation. Although we do not fully understand the mechanisms of network reorganisation driving memory consolidation, available data suggests that sleep-associated neurochemical changes may be important for such processes. In particular, global acetylcholine levels change across the sleep/wake cycle, with high cholinergic tone during wake and REM sleep and low cholinergic tone during slow wave sleep. Furthermore, experimental perturbation of cholinergic tone has been shown to impact memory storage. Through in silico modeling of neuronal networks, we show how spiking dynamics change in highly heterogenous networks under varying levels of cholinergic tone, with neuronal networks under high cholinergic modulation firing asynchronously and at high frequencies, while those under low cholinergic modulation exhibit synchronous patterns of activity. We further examined the network's dynamics and its reorganization mediated via changing levels of acetylcholine within the context of different scale-free topologies, comparing network activity within the hub cells, a small group of neurons having high degree connectivity, and with the rest of the network. We show a dramatic, state-dependent change in information flow throughout the network, with highly active hub cells integrating information in a high-acetylcholine state, and transferring it to rest of the network in a low-acetylcholine state. This result is experimentally corroborated by frequency-dependent frequency changes observed in vivo experiments. Together, these findings provide insight into how new neurons are recruited into memory traces during sleep, a mechanism which may underlie system memory consolidation.

Impacts of Sleep Loss versus Waking Experience on Brain Plasticity: Parallel or Orthogonal?

Recent studies on the effects of sleep deprivation on synaptic plasticity have yielded discrepant results. Sleep deprivation studies using novelty exposure as a means to keep animals awake suggests that sleep (compared with wake) leads to widespread reductions in net synaptic strength. By contrast, sleep deprivation studies using approaches avoiding novelty-induced arousal (i.e., gentle handling) suggest that sleep can promote synaptic growth and strengthening. How can these discrepant findings be reconciled? Here, we discuss how varying methodologies for the experimental disruption of sleep (with differential introduction of novel experiences) could fundamentally alter the experimental outcome with regard to synaptic plasticity. Thus, data from experiments aimed at assessing the relative impact of sleep versus wake on the brain may instead reflect the quality of the waking experience itself. The highlighted work suggests that brain plasticity resulting from novel experiences versus wake per se has unique and distinct features.

Network and cellular mechanisms underlying heterogeneous excitatory/inhibitory balanced states.

Recent work has explored spatiotemporal relationships between excitatory (E) and inhibitory (I) signaling within neural networks, and the effect of these relationships on network activity patterns. Data from these studies have indicated that excitation and inhibition are maintained at a similar level across long time periods and that excitatory and inhibitory currents may be tightly synchronized. Disruption of this balance-leading to an aberrant E/I ratio-is implicated in various brain pathologies. However, a thorough characterization of the relationship between E and I currents in experimental settings is largely impossible, due to their tight regulation at multiple cellular and network levels. Here, we use biophysical neural network models to investigate the emergence and properties of balanced states by heterogeneous mechanisms. Our results show that a network can homeostatically regulate the E/I ratio through interactions among multiple cellular and network factors, including average firing rates, synaptic weights and average neural depolarization levels in excitatory/inhibitory populations. Complex and competing interactions between firing rates and depolarization levels allow these factors to alternately dominate network dynamics in different synaptic weight regimes. This leads to the emergence of distinct mechanisms responsible for determining a balanced state and its dynamical correlate. Our analysis provides a comprehensive picture of how E/I ratio changes when manipulating specific network properties, and identifies the mechanisms regulating E/I balance. These results provide a framework to explain the diverse, and in some cases, contradictory experimental observations on the E/I state in different brain states and conditions.