Fig. 5: Differential Ca²⁺ dynamics of LS and PVT neurons that target histaminergic neurons in the sleep-wake cycle.

a Experimental scheme for labeling and recording histaminergic neuron-projecting LS neurons. rAAV-EF1α-DIO-mCherry-F2A-TVA mixed with rAAV-EF1α-DIO-N2cG, and CVS-EnvA-ΔG-GCaMP6s were injected into the TMN of HDC-CreERT2 mice. Optical fiber was implanted into mouse brains above the LS for calcium signal recording. b, c Representative images of virus expression in TMN (b) and LS (c). d Representative EEG, EMG, and LS fiber photometry of tested mice. The signals of 5 trails are illustrated in the heatmap (from 2 mice); color scale indicates ΔF/F and warmer colors indicate higher fluorescence signal. Data are presented as mean values ± SEM. e Experimental scheme for labeling and recording histaminergic neuron-projecting PVT neurons. rAAV-EF1α-DIO-mCherry-F2A-TVA mixed with rAAV-EF1α-DIO-N2cG, and CVS-EnvA-ΔG-GCaMP6s were injected into the TMN of HDC-CreERT2 mice. Optical fiber was implanted into mouse brains above the PVT for calcium signal recording. f, g Representative images of virus expression in TMN (f) and PVT (g). h Representative EEG, EMG, and PVT fiber photometry of tested mice. The signals of 5 trails are illustrated in the heatmap (from 2 mice); color scale indicates ΔF/F and warmer colors indicate higher fluorescence signal. Data are presented as mean values ± SEM. i–l Analysis of area under the curve before and after state transitions of wake to NREM sleep (i), NREM sleep to wake (j), NREM sleep to REM sleep (k), and REM sleep to wake (l) in the calcium signal recording of LS. n = 5 (derived from different mice). Statistical analyses were conducted using two-tailed Student’s t-tests without adjustment for multiple comparisons. Significance levels were presented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001. The exact p-values were as follows: 0.0008 (i), 0.0008 ( j), 0.0020 (k), and 0.0025 (l). m–p Analysis of area under the curve before and after state transitions of wake to NREM sleep (m), NREM sleep to wake (n), NREM sleep to REM sleep (o), and REM sleep to wake (p) in the calcium signal recording of PVT. n = 5 (derived from different mice). Statistical analyses were conducted using two-tailed Student’s t-tests without adjustment for multiple comparisons. Significance levels were presented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001. The exact p-values were as follows: 0.0460 (m), 0.0004 (n), 0.0007 (o), and 0.0191 (p). LS Lateral septal nucleus, NREM non-rapid eye movement, PVT Paraventricular nucleus of the thalamus, REM rapid eye movement, TMN Tuberomammillary nucleus.