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

Simultaneous imaging and manipulation of a genetically defined neuronal population can provide a causal link between its activity and function. Here, we designed a miniaturized microscope (or 'miniscope') that allows fluorescence imaging and optogenetic manipulation at the cellular level in freely behaving animals. This miniscope has an integrated optical connector that accepts any combination of external light sources, allowing flexibility in the choice of sensors and manipulators. Moreover, due to its simple structure and use of open source software, the miniscope is easy to build and modify. Using this miniscope, we demonstrate the optogenetic silencing of hippocampal CA1 neurons using two laser light sources-one stimulating a calcium sensor (i.e., jGCaAMP7c) and the other serving as an optogenetic silencer (i.e., Jaws). This new miniscope can contribute to efforts to determine causal relationships between neuronal network dynamics and animal behavior.

Miniaturized microscope with flexible light source input for neuronal imaging and manipulation in freely behaving animals.

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.

Fear generalization is a symptom of anxiety-related disorders, including acute stress disorder and post-traumatic stress disorder. Using a contextual fear conditioning paradigm, we found that mice exposed to a similar neutral context but not a different neutral context soon after training showed fear generalization immediately after contextual fear memory consolidation (i.e., 6 h after training). This fear generalization was reflected by a change not only in the total amount but also the pattern of freezing between conditioned and generalized contexts. These results provide insight into the factors that influence fear generalization and can facilitate future studies investigating the underlying pathophysiological mechanisms of anxiety-related disorders.

Fear generalization immediately after contextual 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.

Inducing fear memory extinction by re-presenting a conditioned stimulus (CS) is the foundation of exposure therapy for post-traumatic stress disorder (PTSD). Investigating differences in the ability of different CS presentation patterns to induce extinction learning is crucial for improving this type of therapy. Using a trace fear conditioning paradigm in mice, we demonstrate that spaced presentation of the CS facilitated the extinction of a strong fear memory to a greater extent than continuous CS presentation. These results lay the groundwork for developing more effective exposure therapy techniques for PTSD.

Spaced conditioned stimulus presentation facilitates the extinction of strong fear memory 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.

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
Miniaturized microscope with flexible light source input for neuronal imaging and manipulation in freely behaving animals. Biochemical and biophysical research communications\| PubMed ID: 31376934	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
Fear generalization immediately after contextual fear memory consolidation in mice. Biochemical and biophysical research communications\| PubMed ID: 33906108	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
Spaced conditioned stimulus presentation facilitates the extinction of strong fear memory in mice. Biochemical and biophysical research communications\| PubMed ID: 38735136	2024
A comprehensive suite for extracting neuron signals across multiple sessions in one-photon calcium imaging. Nature communications\| PubMed ID: 40216771	2025
Transient reactivation of small ensembles of adult-born neurons during REM sleep supports memory consolidation in mice. Nature communications\| PubMed ID: 40764488	2025

Toshie Naoi

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