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.

Cofilin overactivation improves hippocampus-dependent short-term memory.

Many living organisms of the animal kingdom have the fundamental ability to form and retrieve memories. Most information is initially stored as short-term memory, which is then converted to a more stable long-term memory through a process called memory consolidation. At the neuronal level, synaptic plasticity is crucial for memory storage. It includes the formation of new spines, as well as the modification of existing spines, thereby tuning and shaping synaptic efficacy. Cofilin critically contributes to memory processes as upon activation, it regulates the shape of dendritic spines by targeting actin filaments. We previously found that prolonged activation of cofilin in hippocampal neurons attenuated the formation of long-term object-location memories. Because the modification of spine shape and structure is also essential for short-term memory formation, we determined whether overactivation of hippocampal cofilin also influences the formation of short-term memories. To this end, mice were either injected with an adeno-associated virus expressing catalytically active cofilin, or an eGFP control, in the hippocampus. We show for the first time that cofilin overactivation improves short-term memory formation in the object-location memory task, without affecting anxiety-like behavior. Surprisingly, we found no effect of cofilin overactivation on AMPA receptor expression levels. Altogether, while cofilin overactivation might negatively impact the formation of long-lasting memories, it may benefit short-term plasticity.

Recovering object-location memories after sleep deprivation-induced amnesia.

It is well established that sleep deprivation after learning impairs hippocampal memory processes and can cause amnesia. It is unknown, however, whether sleep deprivation leads to the loss of information or merely the suboptimal storage of information that is difficult to retrieve. Here, we show that hippocampal object-location memories formed under sleep deprivation conditions can be successfully retrieved multiple days following training, using optogenetic dentate gyrus (DG) memory engram activation or treatment with the clinically approved phosphodiesterase 4 (PDE4) inhibitor roflumilast. Moreover, the combination of optogenetic DG memory engram activation and roflumilast treatment, 2 days following training and sleep deprivation, made the memory more persistently accessible for retrieval even several days later (i.e., without further optogenetic or pharmacological manipulation). Altogether, our studies in mice demonstrate that sleep deprivation does not necessarily cause memory loss but instead leads to the suboptimal storage of information that cannot be retrieved without drug treatment or optogenetic stimulation. Furthermore, our findings suggest that object-location memories, consolidated under sleep deprivation conditions and thought to be lost, can be made accessible again several days after the learning and sleep deprivation episode, using the clinically approved PDE4 inhibitor roflumilast.

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.

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.

Elucidating the role of protein synthesis in hippocampus-dependent memory consolidation across the day and night.

It is widely acknowledged that de novo protein synthesis is crucial for the formation and consolidation of long-term memories. While the basal activity of many signaling cascades that modulate protein synthesis fluctuates in a circadian fashion, it is unclear whether the temporal dynamics of protein synthesis-dependent memory consolidation vary depending on the time of day. More specifically, it is unclear whether protein synthesis inhibition affects hippocampus-dependent memory consolidation in rodents differentially across the day (i.e., the inactive phase with an abundance of sleep) and night (i.e., the active phase with little sleep). To address this question, male and female C57Bl6/J mice were trained in a contextual fear conditioning task at the beginning or the end of the light phase. Animals received a single systemic injection with the protein synthesis inhibitor anisomycin or vehicle directly, 4, 8 hr, or 11.5 hr following training, and memory was assessed after 24 hr. Here, we show that protein synthesis inhibition impaired the consolidation of context-fear memories selectively when the protein synthesis inhibitor was administered at the first three time points, irrespective of timing of training. Even though the basal activity of signaling pathways regulating de novo protein synthesis may fluctuate across the 24-hr cycle, these results suggest that the temporal dynamics of protein synthesis-dependent memory consolidation are similar for day-time and night-time learning.

Upregulation of Alzheimer's Disease Amyloid-ß Protein Precursor in Astrocytes Both in vitro and in vivo.

BACKGROUND: The amyloid cascade hypothesis of Alzheimer's disease (AD) posits that amyloid-ß (Aß) protein accumulation underlies the pathogenesis of the disease by leading to the formation of amyloid plaques, a pathologic hallmark of AD. Aß is a proteolytic product of amyloid-ß protein precursor (AßPP; APP), which is expressed in both neurons and astrocytes. Although considerable evidence shows that astrocytes may play critical roles in the pathogenesis of AD, the longitudinal changes of amyloid plaques in relationship to AßPP expression in astrocytes and cellular consequences are largely unknown. OBJECTIVE: Here, we aimed to investigate astrocyte-related pathological changes of Aß and AßPP using immunohistochemistry and biochemical studies in both animal and cell models. METHODS/RESULTS: We utilized 5XFAD transgenic mice and found age-dependent upregulation of AßPP in astrocytes demonstrated with astrocytic reactive properties, which followed appearance of amyloid plaques in the brain. We also observed that AßPP proteins presented well-defined punctate immuno reactivity in young animals, whereas AßPP staining showed disrupted structures surrounding amyloid plaques in older mice. Moreover, we utilized astrocyte cell models and showed that pretreatment of Aß42 resulted in downstream astrocyte autonomous changes, including up regulation in AßPP and BACE1 levels, as well as prolonged amyloidogenesis that could be reduced by pharmacological inhibition of BACE1. CONCLUSION: Collectively, our results show that age-dependent AßPP up regulation in astrocytes is a key feature in AD, which will not only provide novel insights for understanding AD progression, but also may offer new therapeutic strategies for treating AD.

Phosphodiesterase inhibitors roflumilast and vardenafil prevent sleep deprivation-induced deficits in spatial pattern separation.

Sleep deprivation (SD) is known to impair hippocampus-dependent memory processes, in part by stimulating the phosphodiesterase (PDE) activity. In the present study, we assessed in mice whether SD also affects spatial pattern separation, a cognitive process that specifically requires the dentate gyrus (DG) subregion of the hippocampus. Adult male mice were trained in an object pattern separation (OPS) task in the middle of the light phase and then tested 24 hr thereafter. In total, we conducted three studies using the OPS task. In the first study, we validated the occurrence of pattern separation and tested the effects of SD. We found that 6 hr of SD during the first half of the light phase directly preceding the test trial impaired the spatial pattern separation performance. As a next step, we assessed in two consecutive studies whether the observed SD-induced performance deficits could be prevented by the systemic application of two different PDE inhibitors that are approved for human use. Both the PDE4 inhibitor roflumilast and PDE5 inhibitor vardenafil successfully prevented SD-induced deficits in spatial pattern separation. As a result, these PDE inhibitors have clinical potential for the prevention of memory deficits associated with loss of sleep.

A brief period of sleep deprivation leads to subtle changes in mouse gut microbiota.

Not getting enough sleep is a common problem in our society and contributes to numerous health problems, including high blood pressure, diabetes and obesity. Related to these observations, a wealth of studies has underscored the negative impact of both acute and chronic sleep deprivation on cognitive function. More recently it has become apparent that the gut microbiota composition can be rapidly altered, modulates brain function and is affected by the aforementioned health problems. As such, changes in the microbiota composition may contribute to the behavioural and physiological phenotypes associated with sleep deprivation. It is unclear, however, whether a brief period of sleep deprivation can also negatively impact the gut microbiota. Here, we examined the impact of 5 hr of sleep deprivation on gut microbiota composition of male C57Bl6/J mice. Despite the fact that the overall microbial composition did not change between the control- and sleep-deprived groups, the relative abundance of the Clostridiaceae and Lachnospiraceae were slightly altered in sleep-deprived animals compared to controls. Together, these data suggest that depriving mice of sleep for 5 hr leads to subtle changes in the gut microbiota composition.

Sleep deprivation-induced impairment of memory consolidation is not mediated by glucocorticoid stress hormones.

The general consensus is that sleep promotes neuronal recovery and plasticity, whereas sleep deprivation (SD) impairs brain function, including cognitive processes. Indeed, a wealth of data has shown a negative impact of SD on learning and memory processes, particularly those that involve the hippocampus. The mechanisms underlying these negative effects of sleep loss are only partly understood, but a reoccurring question is whether they are in part caused by stress hormones that may be released during SD. The purpose of the present study is therefore to examine the role of glucocorticoid stress hormones in SD-induced memory impairment. Male C57BL/6J mice were trained in an object-location memory paradigm, followed by 6 hr of SD by mild stimulation. At the beginning of the SD mice were injected with the corticosterone synthesis inhibitor metyrapone. Memory was tested 24 hr after training. Blood samples taken in a separate group of mice showed that SD resulted in a mild but significant increase in plasma corticosterone levels, which was prevented by metyrapone. However, the SD-induced impairment in object-location memory was not prevented by metyrapone treatment. This indicates that glucocorticoids play no role in causing the memory impairments seen after a short period of SD.

A brief period of sleep deprivation causes spine loss in the dentate gyrus of mice.

Sleep and sleep loss have a profound impact on hippocampal function, leading to memory impairments. Modifications in the strength of synaptic connections directly influences neuronal communication, which is vital for normal brain function, as well as the processing and storage of information. In a recently published study, we found that as little as five hours of sleep deprivation impaired hippocampus-dependent memory consolidation, which was accompanied by a reduction in dendritic spine numbers in hippocampal area CA1. Surprisingly, loss of sleep did not alter the spine density of CA3 neurons. Although sleep deprivation has been reported to affect the function of the dentate gyrus, it is unclear whether a brief period of sleep deprivation impacts spine density in this region. Here, we investigated the impact of a brief period of sleep deprivation on dendritic structure in the dentate gyrus of the dorsal hippocampus. We found that five hours of sleep loss reduces spine density in the dentate gyrus with a prominent effect on branched spines. Interestingly, the inferior blade of the dentate gyrus seems to be more vulnerable in terms of spine loss than the superior blade. This decrease in spine density predominantly in the inferior blade of the dentate gyrus may contribute to the memory deficits observed after sleep loss, as structural reorganization of synaptic networks in this subregion is fundamental for cognitive processes.

The role of sleep in regulating structural plasticity and synaptic strength: Implications for memory and cognitive function.

Dendritic spines are the major sites of synaptic transmission in the central nervous system. Alterations in the strength of synaptic connections directly affect the neuronal communication, which is crucial for brain function as well as the processing and storage of information. Sleep and sleep loss bidirectionally alter structural plasticity, by affecting spine numbers and morphology, which ultimately can affect the functional output of the brain in terms of alertness, cognition, and mood. Experimental data from studies in rodents suggest that sleep deprivation may impact structural plasticity in different ways. One of the current views, referred to as the synaptic homeostasis hypothesis, suggests that wake promotes synaptic potentiation whereas sleep facilitates synaptic downscaling. On the other hand, several studies have now shown that sleep deprivation can reduce spine density and attenuate synaptic efficacy in the hippocampus. These data are the basis for the view that sleep promotes hippocampal structural plasticity critical for memory formation. Altogether, the impact of sleep and sleep loss may vary between regions of the brain. A better understanding of the role that sleep plays in regulating structural plasticity may ultimately lead to novel therapeutic approaches for brain disorders that are accompanied by sleep disturbances and sleep loss.

Soluble Gamma-secretase Modulators Attenuate Alzheimer's ß-amyloid Pathology and Induce Conformational Changes in Presenilin 1.

A central pathogenic event of Alzheimer's disease (AD) is the accumulation of the Aß42 peptide, which is generated from amyloid-ß precursor protein (APP) via cleavages by ß- and γ-secretase. We have developed a class of soluble 2-aminothiazole γ-secretase modulators (SGSMs) that preferentially decreases Aß42 levels. However, the effects of SGSMs in AD animals and cells expressing familial AD mutations, as well as the mechanism of γ-secretase modulation remain largely unknown. Here, a representative of this SGSM scaffold, SGSM-36, was investigated using animals and cells expressing FAD mutations. SGSM-36 preferentially reduced Aß42 levels without affecting either α- and ß-secretase processing of APP nor Notch processing. Furthermore, an allosteric site was identified within the γ-secretase complex that allowed access of SGSM-36 using cell-based, fluorescence lifetime imaging microscopy analysis. Collectively, these studies provide mechanistic insights regarding SGSMs of this class and reinforce their therapeutic potential in AD.