Revealing the Role of Postnatal Theta Rhythm in Establishing Memory Consolidation Mechanism during REM Sleep

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K06419
• Research Institution: University of Tsukuba
• Principal Investigator: Kumar Deependra 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (20747696)
• Co-Investigator(Kenkyū-buntansha): 船戸 弘正 筑波大学, 国際統合睡眠医科学研究機構, 客員教授 (90363118)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: Sleep / memory / Theta / hippocampus / neonates / TRAP2 / Dreamless

Outline of Annual Research Achievements

We crossed dreamless (NalcnDrl/+) mice with TRAP2 (FOS2A-iCreER) and Ai27 (RCL-hChR2(H134R)/tdT) to label activated (TRAped) cells during contextual fear conditioning. Neonatal (P26) dreamless and wild-type mice displayed a normal freezing behavior during the conditioning period and the next day memory recall. However, memory test 15 days later showed significant decline in freezing behavior (p=0.035) in dreamless. Moreover, a comparison of freezing on day one and day 15 indicates significant decrease (p=0.008), reflecting rapid memory decay in dreamless. The number of cells (TARPed) participating in encoding in the hippocampus and Anterior Cingulate Cortex was comparable in dreamless and wild-type.We established tetrode implantation in neonates. They showed normal learning behavior.

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

Various transgenic mice required for this project are established. Ai95 (RCL-Gcamp6f) will be crossed with TRAP2 and dreamless to image learning-tagged cell activity during memory consolidation in sleep. For theta wave enhancement, dreamless will be crossed with ChAT-cre and Ai27 and receive laser light in the medial septum during REM sleep. The lens implantation for imaging and optic fiber implantation for optogenetics is yet to be established.

Strategy for Future Research Activity

Analysis of sleep and hippocampal LFP: Neonatal (P17) triple transgenic (Ai27 X TRAP2 X NalcnDrl/+) mice will be implanted with LFP/EEG/EMG electrodes. After one week of the recovery period, baseline sleep and LFP recording will be performed. At P26, they will undergo contextual fear conditioning (CFC) followed by sleep/LFP recording. Sleep/LFP recording will be continued for 15 days to find a correlation between REM sleep and theta changes with freezing behavior.

Imaging learning tagged cells during sleep: Neonatal (P17) triple transgenic mice (Ai95 X TRAP2 X NalcnDrl/+) will be implanted with Lens/EEG/EMG. At P26, they will undergo CFC and tamoxifen treatment to tag learning activated cells with Gcamp6f. Tagged cell activity will be imaged to study memory reactivation during REM sleep.

Causes of Carryover

We need to procure consumables items for LFP/EEG/EMG surgery. For example, platinum-iridium wire for preparing tetrode, miniature EEG screw for neonates, dental cement and solution, IC sockets, etc.
The imaging experiment requires a sufficient number of green lenses (inscopix) and baseplates, which are not reusable. Up to three dummy microscopes are needed for habituation purposes.
Antibodies (RFP, FOS, GFP, DCx, and ChAT) for immunohistochemistry and Tamoxifen for tagging activated cell is required. Several types of primers for genotyping various transgenic mice are needed.

Research Products

• [Presentation] Learning and memory deficit in adult dreamless mice (2022)
  • Author(s): Deependra Kumar, Tomoyuki Fujiyama, Takeshi Kanda, Chika Miyoshi, Miyo Kakizaki, Masanori Sakaguchi, Hiromasa Funato, Masashi Yanagisawa
  • Organizer: 99th Annual Meeting of the Physiological Society of Japan-JST-CREST “Opt Bio” / WPI-IIIS Joint Symposium
  • Int'l Joint Research