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

Summary

We present a novel protocol for the selective deprivation of Rapid Eye Movement (REM) sleep in rodents.

Abstract

Sleep is broadly categorized into Rapid Eye Movement (REM) and non-REM stages, and various sleep deprivation techniques have been developed to study their functions. Traditional methods—such as mechanical stimulation or dropping the animal during REM sleep—often lead to rapid habituation and unintended stress effects, making interpretation difficult. Here, we introduce a novel, mild, and gentle method for REM sleep deprivation in rodents: SNiFFer. Rodents are known to be highly sensitive to nasal stimuli. In this method, a soft paintbrush gently strokes the animal's nose during REM sleep, reliably triggering arousal. To selectively apply this technique during REM sleep, we utilized an AI-based real-time Electroencephalography (EEG) sleep-stage classification system. The intervention was triggered only during REM episodes, and a yoked control group was included, receiving the same stimulation pattern regardless of sleep state. We further applied SNiFFer in a Trace Fear Conditioning paradigm to test whether REM sleep deprivation within 6 h after learning interferes with memory consolidation. Compared to yoked controls, SNiFFer-treated animals showed a significant decrease in total REM episode and REM episode duration, indicating the method's specificity and efficacy. In conclusion, the SNiFFer technique offers a highly selective, minimally invasive tactile approach for REM sleep deprivation in mice. It enables precise control of experimental conditions and opens new possibilities for investigating REM sleep's role in memory and cognition—with a ticklish twist.

Introduction

Rapid eye movement (REM) sleep plays a pivotal role in cognitive processes such as memory consolidation^1,2. To investigate the functional significance of REM sleep in the brain, REM sleep deprivation is commonly employed as an experimental approach. Currently, four primary methods are widely used to induce REM sleep deprivation in rodents: the rotating disk method¹, the flowerpot method³, the air-puff method⁴, and physical stimulation techniques^5,6. While the rotating disk and flow....

Protocol

All animal experiments were approved by the University of Tsukuba Institutional Animal Care and Use Committee. Male and female C57BL/6J mice (11–13 weeks old, 20–35 g) were used in this study. The complete list of materials and instruments used in this study is provided in the Table of Materials.

1. Surgical implantation of EEG electrodes¹⁷

1. Anesthesia and preparation of the animal
   1. Induce anesthesia with 4% isoflurane mixed with air in 3 min. Adequate anesthesia was confirmed by the absence of the pedal withdrawal reflex and muscle tone, along with ....

Results

Surgical implantation of EEG electrodes
Mice should be fully anesthetized during the surgery. If they exhibit signs of failure of anesthesia, such as struggling, they should be removed from the protocol. Similarly, if bleeding lasts for more than 10 s, it may cause severe damage to the mouse’s brain, and removal from experimentation is recommended. After the surgery, most mice typically wake up within 1 h. Their gait and eye movement should return to normal, similar to pre-surgical conditions.

Discussion

This protocol enables selective REM sleep deprivation in mice with yoked control mice. Unlike traditional methods such as the flowerpot and rotating disk techniques, this system allows the implementation of finely matched control groups, in which animals receive identical stimulation at the same circadian time. Furthermore, this approach minimizes variability in stimulation location, a common limitation of conventional gentle handling and air puff methods. As a result, this technique may permit a more precise isolation o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5% glucose	Otsukba	35061410
6-pin header	Hirose	172-0989
Adhesive	Konishi	16351
Anaesthetic machine	Shinano Seisakusho	SN-487-0T
Crimp housing	Hirose	688-9095
Crimp socket	Hirose	688-8982
Freezeflame4	Med Associates	RRID:SCR_014429	Version 4.104
GraphPad Prism	GraphPad	RRID:SCR_002798	Version 10.5.0
Ibuprofen	Tokyo Chemical Industry	I0415
Isoflurane	Viatris	901036504
Loctite454	Henkel	UFI:  MAK2-7WRP-G202-F235
Mouse (C57BL/6J)	The Jackson Laboratory	RRID: IMSR_JAX:000664	11.0 - 13.0 week of age at fear conditioning session
Paintbrush	Namurataiseidou	4943668000448	SDflat No.8, Tip length: 8.2 mm; width: 18.8 mm
Provinice liquid	Shofu	21400BZZ00451000	250 mL
Provinice powder	Shofu	21400BZZ00451000	250 g
SleepSignRecorder	KISSEI COMTEC	RRID:SCR_018200	Version 1.2.10
Stainless steel screw	Yamazaki	N/A	φ1.0 × 2.0
Standard Stereotaxic Instruments	ALA Scientific	68038
Sunflower oil	Sigma-Aldrich	S5007-250ML
White petrolatum	Taiyo Pharmaceutical	K09-TS