method-for-selective-rem-sleep-deprivation-using-selective-nose

Method for Selective REM Sleep Deprivation using The Selective Nose Flicking for Forgetting REM (SNiFFer)

Sleep is broadly categorized into Rapid Eye Movement (REM) and non-REM stages, and various sleep deprivation techniques have been developed to study their ...

university of tsukuba

In anticipation of the massive burden of neurodegenerative disease within super-aged societies, great efforts have been made to utilize neural stem and progenitor cells for regenerative medicine. The capacity of intrinsic neural stem and progenitor cells to regenerate damaged brain tissue remains unclear, due in part to the lack of knowledge about how these newly born neurons integrate into functional circuitry. As sizable integration of adult-born neurons naturally occurs in the dentate gyrus region of the hippocampus, clarifying the mechanisms of this process could provide insights for applying neural stem and progenitor cells in clinical settings. There is convincing evidence of functional correlations between adult-born neurons and memory consolidation and sleep; therefore, we describe some new advances that were left untouched in our recent review.

Memory consolidation during sleep and adult hippocampal neurogenesis.

The occurrence of dreaming during rapid eye movement (REM) sleep prompts interest in the role of REM sleep in hippocampal-dependent episodic memory. Within the mammalian hippocampus, the dentate gyrus (DG) has the unique characteristic of exhibiting neurogenesis persisting into adulthood. Despite their small numbers and sparse activity, adult-born neurons (ABNs) in the DG play critical roles in memory; however, their memory function during sleep is unknown. Here, we investigate whether young ABN activity contributes to memory consolidation during sleep using Ca imaging in freely moving mice. We found that contextual fear learning recruits a population of young ABNs that are reactivated during subsequent REM sleep against a backdrop of overall reduced ABN activity. Optogenetic silencing of this sparse ABN activity during REM sleep alters the structural remodeling of spines on ABN dendrites and impairs memory consolidation. These findings provide a causal link between ABN activity during REM sleep and memory consolidation.

Sparse Activity of Hippocampal Adult-Born Neurons during REM Sleep Is Necessary for Memory Consolidation.

Adult-born neurons (ABNs) in the dentate gyrus bestow unique cellular plasticity to the mammalian brain. We recently found that the activity of ABNs during sleep is necessary for memory consolidation. Here, we describe our method for Ca imaging of ABN activity using a miniaturized fluorescent microscope and sleep recordings. As preparatory surgery and post-recording data processing can be major obstacles, we provide detailed descriptions and problem-solving tips. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2020).

Calcium imaging of adult-born neurons in freely moving mice.

Metabolites underlying brain function and pathology are not as well understood as genes. Here, we applied a novel metabolomics approach to further understand the mechanisms of memory processing in sleep. As hippocampal dentate gyrus neurons are known to consolidate contextual fear memory, we analyzed real-time changes in metabolites in the dentate gyrus in different sleep-wake states in mice. Throughout the study, we consistently detected more than > 200 metabolites. Metabolite profiles changed dramactically upon sleep-wake state transitions, leading to a clear separation of phenotypes between wakefulness and sleep. By contrast, contextual fear memory consolidation induced less obvious metabolite phenotypes. However, changes in purine metabolites were observed upon both sleep-wake state transitions and contextual fear memory consolidation. Dietary supplementation of certain purine metabolites impaired correlations between conditioned fear responses before and after memory consolidation. These results point toward the importance of purine metabolism in fear memory processing during sleep.

Metabolic fingerprints of fear memory consolidation during sleep.

The mammalian hippocampal dentate gyrus is a unique memory circuit in which a subset of neurons is continuously generated throughout the lifespan. Previous studies have shown that the dentate gyrus neuronal population can hold fear memory traces (i.e., engrams) and that adult-born neurons (ABNs) support this process. However, it is unclear whether ABNs themselves hold fear memory traces. Therefore, we analyzed ABN activity at a population level across a fear conditioning paradigm. We found that fear learning did not recruit a distinct ABN population. In sharp contrast, a completely different ABN population was recruited during fear memory retrieval. We further provide evidence that ABN population activity remaps over time during the consolidation period. These results suggest that ABNs support the establishment of a fear memory trace in a different manner to directly holding the memory. Moreover, this activity remapping process in ABNs may support the segregation of memories formed at different times. These results provide new insight into the role of adult neurogenesis in the mammalian memory system.

Remapping of Adult-Born Neuron Activity during Fear Memory Consolidation in Mice.

We developed a real-time sleep stage classification system with a convolutional neural network using only a one-channel electro-encephalogram source from mice and universally available features in any time-series data: raw signal, spectrum, and zeitgeber time. To accommodate historical information from each subject, we included a long short-term memory recurrent neural network in combination with the universal features. The resulting system (UTSN-L) achieved 90% overall accuracy and 81% multi-class Matthews Correlation Coefficient, with particularly high-quality judgements for rapid eye movement sleep (91% sensitivity and 98% specificity). This system can enable automatic real-time interventions during rapid eye movement sleep, which has been difficult due to its relatively low abundance and short duration. Further, it eliminates the need for ordinal pre-calibration, electromyogram recording, and manual classification and thus is scalable. The code is open-source with a graphical user interface and closed feedback loop capability, making it easily adaptable to a wide variety of end-user needs. By allowing large-scale, automatic, and real-time sleep stage-specific interventions, this system can aid further investigations of the functions of sleep and the development of new therapeutic strategies for sleep-related disorders.

Real-time, automatic, open-source﻿﻿ sleep stage classification system using single EEG for mice.

Sleep stage-specific intervention is widely used to elucidate the functions of sleep and their underlying mechanisms. For this intervention, it is imperative to accurately classify rapid-eye-movement (REM) sleep. However, the proof of fully automatic real-time REM sleep classification in vivo has not been obtained in mice. Here, we report the in vivo implementation of a system that classifies sleep stages in real-time from a single-channel electroencephalogram (EEG). It enabled REM sleep-specific intervention with 90 % sensitivity and 86 % precision without prior configuration to each mouse. We further derived systems capable of classification with higher frequency sampling and time resolution. This attach-and-go sleep staging system provides a fully automatic accurate and scalable tool for investigating the functions of sleep.

Fully automatic REM sleep stage-specific intervention systems using single EEG in mice.

We developed CaliAli, a comprehensive suite designed to extract neuronal signals from one-photon calcium imaging data collected across multiple sessions in free-moving conditions in mice. CaliAli incorporates information from blood vessels and neurons to correct inter-session misalignments, making it robust against non-rigid brain deformations even after substantial changes in the field of view across sessions. This also makes CaliAli robust against high neuron overlap and changes in active neuron population across sessions. CaliAli performs computationally efficient signal extraction from concatenated video sessions that enhances the detectability of weak calcium signals. Notably, CaliAli enhanced the spatial coding accuracy of extracted hippocampal CA1 neuron activity across sessions. An optogenetic tagging experiment showed that CaliAli enhanced neuronal trackability in the dentate gyrus across a time scale of weeks. Finally, dentate gyrus neurons tracked using CaliAli exhibited stable population activity for 99 days. Overall, CaliAli advances our capacity to understand the activity dynamics of neuronal ensembles over time, which is crucial for deciphering the complex neuronal substrates of natural animal behaviors.

A comprehensive suite for extracting neuron signals across multiple sessions in one-photon calcium imaging.

Rapid eye movement (REM) sleep is primarily regulated by the brainstem pons. In particular, the sublaterodorsal tegmentum (SubLDT) in the dorsal pons contains neurons whose activity is selective to REM sleep. Elucidation of the precise identities of these neurons and their roles in REM sleep regulation is challenging, however, due to the functional and molecular heterogeneity of the SubLDT. A recent study revealed that corticotropin-releasing hormone-binding protein ()-positive neurons in the SubLDT projecting to the medulla play a crucial role in REM sleep regulation and that loss of these -positive neurons underlies sleep deficits observed in Parkinson's disease. The firing patterns of these neurons during sleep/wake, however, remained unknown. Here, we used an opto-tagging method and conducted cell-type-specific recordings from -positive neurons using a glass pipette microelectrode in unanesthetized male mice. We recorded 58 -positive neurons and found that many of these neurons are REM sleep-active neurons (41.4%) and that the remaining neurons are mostly either wake-active, wake/REM sleep-active, or non-REM sleep-active. In addition, projection-specific recordings revealed that the medulla-projecting -positive neurons are mostly REM sleep-active neurons (75.0%). Based on clustering analysis and spike waveform analysis, REM sleep-active -positive neurons can be further divided into different subtypes according to their electrophysiological properties, suggesting that -positive neurons play diverse roles in REM sleep regulation.

Diverse Firing Profiles of -positive Neurons in the Dorsal Pons Suggestive of Their Pleiotropic Roles in REM Sleep Regulation in Mice.

While memory consolidation is widely believed to require memory reactivation synchronized with theta oscillations during REM sleep, direct causal evidence linking specific neuronal ensembles to this process has been lacking. Strong theta oscillations arise in the hippocampal dentate gyrus, where a small population of principal neurons is continuously generated throughout adulthood. Although these adult-born neurons (ABNs) are known to modulate hippocampal circuits for memory, the causality between their specific information content and memory-related behavior was unknown. Here, we show that REM sleep reactivation of memory ensembles comprising as few as ~3 ABNs is necessary for fear memory consolidation. Crucially, we found that the synchronization of ABN activity with a specific theta phase is essential for associative memory consolidation. Our findings thus provide causal evidence that consolidation critically depends on both the reactivation of minimal neuronal populations and precise neuronal coordination within theta-defined temporal windows.

Article	Year
Memory consolidation during sleep and adult hippocampal neurogenesis. Neural regeneration research\| PubMed ID: 30531064	2019
Sparse Activity of Hippocampal Adult-Born Neurons during REM Sleep Is Necessary for Memory Consolidation. Neuron\| PubMed ID: 32502462	2020
Calcium imaging of adult-born neurons in freely moving mice. STAR protocols\| PubMed ID: 33458703	2021
Metabolic fingerprints of fear memory consolidation during sleep. Molecular brain\| PubMed ID: 33568175	2021
Remapping of Adult-Born Neuron Activity during Fear Memory Consolidation in Mice. International journal of molecular sciences\| PubMed ID: 33808976	2021
Real-time, automatic, open-source sleep stage classification system using single EEG for mice. Scientific reports\| PubMed ID: 34045518	2021
Fully automatic REM sleep stage-specific intervention systems using single EEG in mice. Neuroscience research\| PubMed ID: 36206953	2023
A comprehensive suite for extracting neuron signals across multiple sessions in one-photon calcium imaging. Nature communications\| PubMed ID: 40216771	2025
Diverse Firing Profiles of -positive Neurons in the Dorsal Pons Suggestive of Their Pleiotropic Roles in REM Sleep Regulation in Mice. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 40550695	2025
Transient reactivation of small ensembles of adult-born neurons during REM sleep supports memory consolidation in mice. Nature communications\| PubMed ID: 40764488	2025

Iyo Koyanagi

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