Circuit mechanism for suppression of frontal cortical ignition during NREM sleep.

Conscious perception is greatly diminished during sleep, but the underlying circuit mechanism is poorly understood. We show that cortical ignition-a brain process shown to be associated with conscious awareness in humans and non-human primates-is strongly suppressed during non-rapid-eye-movement (NREM) sleep in mice due to reduced cholinergic modulation and rapid inhibition of cortical responses. Brain-wide functional ultrasound imaging and cell-type-specific calcium imaging combined with optogenetics showed that activity propagation from visual to frontal cortex is markedly reduced during NREM sleep due to strong inhibition of frontal pyramidal neurons. Chemogenetic activation and inactivation of basal forebrain cholinergic neurons powerfully increased and decreased visual-to-frontal activity propagation, respectively. Furthermore, although multiple subtypes of dendrite-targeting GABAergic interneurons in the frontal cortex are more active during wakefulness, soma-targeting parvalbumin-expressing interneurons are more active during sleep. Chemogenetic manipulation of parvalbumin interneurons showed that sleep/wake-dependent cortical ignition is strongly modulated by perisomatic inhibition of pyramidal neurons.

An Excitatory Circuit in the Perioculomotor Midbrain for Non-REM Sleep Control.

The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.

Suv39h1 regulates memory stability by inhibiting the expression of Shank1 in hippocampal newborn neurons.

Adult newborn neurons are involved in memory encoding and extinction, but the neural mechanism is unclear. We found the adult newborn neurons at 4 weeks are recruited by learning and subjected to epigenetic regulations, consequently reducing their ability to be re-recruited later. After removal of the epigenetic blockage, Suv39h1 KO mice showed an increased recruiting number of aged newborn neurons and enhanced flexibility in learning tasks. Besides NRXN1, we found SHANK1, the synaptic scaffold protein, is one of the major targets of Suv39h1, regulating memory stability. Expression of Shank1 is transiently engaged to enhance synaptogenesis during learning and is strongly suppressed by Suv39h1 from 5 h after learning. Exogenously overexpression of Shank1 in dentate gyrus increased the density of mushroom spines and decreased the persistency of old memories. Our study indicated the activity-regulated epigenetic modification in newly matured newborn neurons in hippocampus insulates temporally distinct experiences and stabilizes old memories.

In vivo imaging of immediate early gene expression reveals layer-specific memory traces in the mammalian brain.

The dynamic processes of formatting long-term memory traces in the cortex are poorly understood. The investigation of these processes requires measurements of task-evoked neuronal activities from large numbers of neurons over many days. Here, we present a two-photon imaging-based system to track event-related neuronal activity in thousands of neurons through the quantitative measurement of EGFP proteins expressed under the control of the EGR1 gene promoter. A recognition algorithm was developed to detect GFP-positive neurons in multiple cortical volumes and thereby to allow the reproducible tracking of 4,000 neurons in each volume for 2 mo. The analysis revealed a context-specific response in sparse layer II neurons. The context-evoked response gradually increased during several days of training and was maintained 1 mo later. The formed traces were specifically activated by the training context and were linearly correlated with the behavioral response. Neuronal assemblies that responded to specific contexts were largely separated, indicating the sparse coding of memory-related traces in the layer II cortical circuit.

Microglia regulate sleep through calcium-dependent modulation of norepinephrine transmission.

Sleep interacts reciprocally with immune system activity, but its specific relationship with microglia-the resident immune cells in the brain-remains poorly understood. Here, we show in mice that microglia can regulate sleep through a mechanism involving Gi-coupled GPCRs, intracellular Ca2+ signaling and suppression of norepinephrine transmission. Chemogenetic activation of microglia Gi signaling strongly promoted sleep, whereas pharmacological blockade of Gi-coupled P2Y12 receptors decreased sleep. Two-photon imaging in the cortex showed that P2Y12-Gi activation elevated microglia intracellular Ca2+, and blockade of this Ca2+ elevation largely abolished the Gi-induced sleep increase. Microglia Ca2+ level also increased at natural wake-to-sleep transitions, caused partly by reduced norepinephrine levels. Furthermore, imaging of norepinephrine with its biosensor in the cortex showed that microglia P2Y12-Gi activation significantly reduced norepinephrine levels, partly by increasing the adenosine concentration. These findings indicate that microglia can regulate sleep through reciprocal interactions with norepinephrine transmission.

Activation of locus coeruleus noradrenergic neurons rapidly drives homeostatic sleep pressure.

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep, but the underlying mechanism remains unclear. Optogenetic activation of locus coeruleus noradrenergic neurons immediately increased sleep propensity following transient wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused rapid declines of locus coeruleus calcium activity and noradrenaline release. This suggests that functional fatigue of noradrenergic neurons, which reduces their wake-promoting capacity, contributes to sleep pressure.

Control of Non-REM Sleep by Midbrain Neurotensinergic Neurons.

The periaqueductal gray (PAG) in the midbrain is known to coordinate behavioral and autonomic responses to threat and injury through its descending projections to the brainstem. Here, we show that neurotensin (NTS)-expressing glutamatergic neurons in the ventrolateral PAG (vlPAG) powerfully promote non-rapid eye movement (NREM) sleep partly through their projection to the caudal medulla. Optogenetic and chemogenetic activation of vlPAG NTS neurons strongly enhanced NREM sleep, whereas their inactivation increased wakefulness. Calcium imaging and optrode recording showed that they are preferentially active during NREM sleep. The NREM-promoting effect of vlPAG NTS neurons is partly mediated by their projection to the caudal ventromedial medulla, where they excite GABAergic neurons. Bidirectional optogenetic and chemogenetic manipulations showed that the medullary GABAergic neurons also promote NREM sleep, and they innervate multiple monoaminergic populations. Together, these findings reveal a novel pathway for NREM sleep generation, in which glutamatergic neurons drive broad GABAergic inhibition of wake-promoting neuronal populations.

Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation.

Epigenetic mechanisms regulate the formation, consolidation and reconsolidation of memories. However, the signaling path from neuronal activation to epigenetic modifications within the memory-related brain circuit remains unknown. We report that learning induces long-lasting histone modifications in hippocampal memory-activated neurons to regulate memory stability. Neuronal activity triggers a late-onset shift in Nrxn1 splice isoform choice at splicing site 4 by accumulating a repressive histone marker, H3K9me3, to modulate the splicing process. Activity-dependent phosphorylation of p66α via AMP-activated protein kinase recruits HDAC2 and Suv39h1 to establish repressive histone markers and changes the connectivity of the activated neurons. Removal of Suv39h1 abolished the activity-dependent shift in Nrxn1 splice isoform choice and reduced the stability of established memories. We uncover a cell-autonomous process for memory preservation in which memory-related neurons initiate a late-onset reduction of their rewiring capacities through activity-induced histone modifications.

The role of epigenetic regulation in learning and memory.

The formation of long-term memory involves a series of molecular and cellular changes, including gene transcription, protein synthesis and synaptic plasticity dynamics. Some of these changes arise during learning and are subsequently retained throughout life. 'Epigenetic' regulation, which involves DNA methylation and histone modifications, plays a critical role in retaining long-term changes in post-mitotic cells. Accumulating evidence suggests that the epigenetic machinery might regulate the formation and stabilization of long-term memory in two ways: a 'gating' role of the chromatin state to regulate activity-triggered gene expression; and a 'stabilizing' role of the chromatin state to maintain molecular and cellular changes induced by the memory-related event. The neuronal activation regulates the dynamics of the chromatin status under precise timing, with subsequent alterations in the gene expression profile. This review summarizes the existing literature, focusing on the involvement of epigenetic regulation in learning and memory. We propose that the identification of different epigenetic regulators and signaling pathways involved in memory-related epigenetic regulations will provide mechanistic insights into the formation of long-term memory.