Figures and data
Infraslow calcium oscillation of granule cells during NREM sleep.
A. Representative recording session showing infraslow oscillation (ISO) during NREM sleep. From top to bottom: brain states, EEG power spectrogram (0-25 Hz), EMG amplitude, photometric signal. B. Left, Schematic representation of the recording setup. Right, a fluorescence image showing the expression of GCaMP6s (green) in the granule cell layer and the optic fiber placement (dashed line) in a Dock10-Cre mouse. Scale bar, 500 µm. C. Quantification of calcium activity in granule cells (13 recording sessions in 5 Dock10-Cre mice, n.s. – no significance, ** - P<0.01, paired t-test) in wake (W), NREM sleep (N), and REM sleep (R). D. Left: oscillation peak frequency during NREM sleep and wake based on Fourier transformation of the photometry signal. Right: Quantification of calcium oscillations in GCs (13 sessions in 5 mice). E. A representative example showing the coincidence of calcium troughs with MAs. F. Percentage of outcome of brain states following calcium drops during NREM sleep. G. Peri-stimulus time histogram (PSTH) in one recording session showing calcium signal aligned with the onset of MAs. Bottom left: parameters of the calcium signal used for quantification. H. Quantification of the latency (t) and magnitude of the calcium trough (Drop) during MAs (13 sessions from 5 Dock10-Cre mice).
Infraslow calcium oscillation of mossy cells during NREM sleep.
A. Representative recording session showing infraslow calcium oscillation during NREM sleep in a Drd2-Cre+/- mouse injected with AAV-FLEX-GCaMP6s. From top to bottom: brain states, EEG power spectrogram (0-25 Hz), EMG amplitude, photometric signal. B. Fluorescence images showing the expression of GCaMP6s (green) in the mossy cells in the area marked with the white rectangle. Blue, DAPI. Scale bars, 500 µm and 50 µm. C. Quantification of calcium activity in mossy cells during different brain states (12 recording sessions in 4 Drd2-Cre+/- mice; n.s. – no significance, ** - P<0.01, paired t-test). D. Left: oscillation peak frequency during NREM sleep based on Fourier transformation of the photometry signal. Right: quantification of calcium oscillations during NREM sleep. E. A representative example showing the coincidence of calcium troughs with MAs (represented with vertical green lines). F. Percentage of outcome of brain states following calcium troughs during NREM sleep. G. PSTH from one recording session showing calcium signals aligned with the onset of MAs. H. Quantification of the latency (t) and the magnitude of calcium troughs (Drop) during the MAs (12 sessions from 4 Drd2-Cre mice).
Two-photon calcium imaging of DG activity in sleeping mice.
A. Left, schematic layout of two-photon imaging and EEG recording setup. Right, representative images showing putative granule cells (pGCs) and putative mossy cells (pMCs). Scale bars, 50 µm. B-C Representative two-photon recording sessions of field of views containing putative pGCs pMCs. From top to bottom: brain states (gray – awake, orange – NREM), EEG spectrogram (0-25 Hz), EMG, velocity (vel.), and calcium traces in individual cells. Red traces - upregulated cells, blue traces –downregulated cells, gray traces – non-significant cells. D-E. Left, Percentage of Up-, downregulated and non-significant cells from the entire recorded cell populations. Middle, quantification of calcium activity in different brain states in up-, down-regulated cells (pGCs: P<0.05 in up-regulated cells between NREM and MA, P=0.77 in down-regulated cells between NREM and MA; pMCs: P<0.001 in up-regulated cells between NREM and MA, P=0.63 in down-regulated cells between NREM and MA, paired t-test). Right, Averaged activity in up-, down-regulated pGCs and pMCs during microarousals (MA). Time 0 indicates the MA onset. Putative GCs: 369 cells from 3 C57BL/6J mice; putative MCs: 269 cells from 2 C57BL/6J mice.
Phasic release of 5-HT in the DG during NREM sleep.
A. Left, schematic of experimental design. Right, expression of 5-HT sensor in the hippocampus. B. A representative example of 5-HT signals during different brain states. From top to bottom: brain states, EEG power spectrogram (0-25 Hz), EMG signal, photometric signal. The dashed-box enlarged below in panel B. Note the coincidence of 5-HT release with MAs during NREM sleep (black arrows and vertical green lines). C. Outcome of brain states following 5-HT release during NREM sleep (averaged data from 6 mice). D. Quantification of 5-HT signals in the DG during different brain states (14 sessions from 6 C57BL/6J mice, ** - P<0.01, *** - P<0.001, paired ttest). Data were normalized to Z scores in each recording session. E. Quantification of oscillatory cycles of 5-HT signals in the DG (14 recording sessions from 6 C57BL/6J mice).
Correlation between DG oscillation and activity of 5-HT neurons during NREM sleep.
A. Left, Schematic representation of the 2-site photometry experimental design. Right, Expression of CaMKII-GCaMP6s and fiber placement in the DG and Raphe nuclei. B. A representative example of concurrent recording of DG and Raphe 5-HT neurons in a Sert-Cre+/- mouse during sleep. From top to bottom: brain states, EEG power spectrogram (0-25 Hz), EMG amplitude, photometric calcium signals (CaMKII-G6s) in DG and in dorsal Raphe. C. Correlation analysis of calcium activity between DG and Raphe 5-HT neurons during NREM sleep and wakefulness in one re-cording session. D. Quantification of correlation coefficient between DG activity and Raphe activity during different brain states (11 sessions from 3 Sert-Cre+/- mice, *** - P<0.001, paired ttest).
Genetic knockdown of 5-HT1a receptors in DG impairs ISO and memory performance.
A. Schematic representation of the experimental design. A mix of AAV9-CamKII-GCaMP6s and AAV1-hSyn-Cre was injected into the DG of 5-HT1aflox/flox mice. B. Representative example showing photometry and EEG recordings in the DG of a control mouse injected with AAV9-CaMKII-GCaMP6s alone. Right, Fourier transformation of calcium activity during wake (blue) NREM sleep (red). C. A representative example showing photometry and EEG recordings in the DG of a mouse injected with AAV9-CaMKII-GCaMP6s and AAV1-hSyn-Cre. Right, Fourier transformation of calcium activity during wake (blue) NREM sleep (red). D. Left, Quantification of the relative power of the calcium oscillation in the range of 1-2 cycles/min in the Cre and control groups (16 sessions from 5 mice for Cre, 16 sessions from 6 mice for control). Right, Quantification of calcium oscillation amplitudes in the Cre and control groups. Calcium signals in each mouse were normalized to Z scores. ** - P<0.01, *** - P<0.001, unpaired t-test. E, Schematic representation of the CFC experimental design. F, Left, Contextual fear recall tests showing percentage of freezing in one minute time bins for 5-HT1aflox+/+ mice bilaterally injected with AAV9-CaMKII-Cre-GFP (Cre) or AAV9-CaMKII-GFP (GFP). Right, Quantification of freezing behavior over 5-minute interval during contextual recall tests in Cre and GFP groups (N=11 for Cre, N=12 for GFP, *, P<0.05, ***, P<0.001, un-paired t-test).
