Figures and data
5-HT levels exhibit ultraslow oscillations during NREM and WAKE.
A. Histology and experimental protocol. Left: expression of GRAB5-HT3.0 sensor (in green) in dorsal CA1 with optic fiber track above. Right: methodology for dual implantation surgeries. AAV9-hSyn-5HT3.0 was first injected into the right dorsal CA1. In the same surgery, an optic fiber was implanted above the injection site. After three weeks of viral expression, a silicon probe was implanted above the left dorsal CA1. Simultaneous recording of the GRAB5-HT3.0 sensor activity (fiber photometry) and electrophysiology was performed. B-E. Example dual fiber photometry-electrophysiology recording with times shown in E. B. Labeled sleep states resulting from automated sleep-scoring and intracranial EMG trace. C. Spectrogram (Stockwell transform) showing normalized power of a hippocampal LFP channel during awake and sleep states. D. Z-scored 5-HT trace. E. Spectrogram (Stockwell transform) of the 5-HT trace shown in D. F. Left: Mean 5-HT level by state, across all experiments (total n=6 mice, 12 recording sessions of 1.5-3 hours). Right: p-values from a multiple comparisons test applied after fitting a Bayesian linear mixed effects model to the data. G. Pie chart showing proportion of time spent in different behavioral states, averaged across all experiments. H. Top: examples of ultraslow 5-HT oscillations in NREM and WAKE. Bottom: Power spectrum of 5-HT signals in WAKE vs. NREM sleep. I. Control fluoxetine and saline injection experiments. A significant difference between the post-injection period of saline-injected and fluoxetine-injected animals (shaded in red) was observed (Wilcoxon ranked-sum test, p<0.001, n= 3 mice).
Ripples occur time-locked to ultraslow 5-HT oscillations.
A. Schematic showing the convolutional neural network used for ripple detection. 8-channel x 400 ms-LFP chunks were used as input. The bottom four channels (cyan) were taken from the dorsal CA1 and contained ripples, and the top four channels (magenta) were chosen from a non-adjacent part of the neocortex above the dorsal CA1. The model consisted of four convolution blocks (”Conv2d ”), each block comprising a 2D convolutional layer, a ReLU activation function, and batch normalization. Two dense layers with dropout and batch normalization (”Dense ”) followed and produced the final output, a 400 ms vector with values between 0-1, indicating the probability of a ripple occurring during the course of the input chunk. B. Example model output given the four LFP chunk inputs shown. First row: true positives. Second row: fast oscillations and movement artifacts not detected as ripples by the model. C. Spectrogram from a ripple detected by the model, 0-1 normalized. D. Characteristics of detected ripples. Ripples from all experiments were included, and probability distributions are shown. Top left: distribution of duration. Top right: distribution of z-score normalized ripple power. Bottom: distribution of ripple frequency. Ripple duration and normalized ripple power follow a log-normal distribution (duration: X2 (df = 7, N = 49,458) = 1.398e+03, p < .0001, normalized ripple power: X2 (df = 7, N = 49,458) = 422.1862, p < .0001). E. Example 5-HT trace and computed power in the ripple band (120-250 Hz). F. Same example 5-HT trace and individual detected ripples. G. Example of ripple cluster extraction. Ripple clusters were defined as having a minimum of 10 ripple events and an inter-ripple interval of less than 3 seconds. Note the few ripples occurring during the rising phase of 5-HT ultraslow oscillations in F are excluded from extracted ripple clusters in G. From these ripple clusters, the first (orange) and last (black) ripples in a cluster were extracted. H1-2. Ripple-triggered 5-HT in NREM (H1) and WAKE (H2) states. The first rows of H1 and H2 show all 50 s 5-HT segments centered around the ripple peak for different combinations of ripples (columns). In the first column, all ripples in the given state were included. The second and third columns used only the first or last ripple in extracted ripple clusters, respectively. The second rows of H1 and H2. show the mean ripple-triggered 5-HT traces (blue) and randomly shifted traces (orange) for each group of ripples. The orange traces were obtained by randomly shifting the ripple times for each condition and averaging the resulting 5-HT 50 s segments centered around those shifted times.
Ripples occurrence and power vary by the phase of ultraslow 5-HT oscillations.
A. Schematic showing one period of a slow 5-HT oscillation. The rising phase of the oscillation occurs from -180° to 0°, and the falling phase occurs from 0° to 180°. B. Mean z-scored inter-ripple interval (IRI) by 5-HT phase angle during NREM (left) and WAKE (right). C. Mean rising phase IRI - mean falling phase IRI, plotted by session and mouse level in WAKE (left) and NREM (right). Red point with error bar indicates predicted difference and confidence interval after fitting a general linear mixed effects model to the data. P-values shown were derived from a post-hoc multiple comparisons test on the fitted model. (n=6 mice, 12 sessions). D. Example 5-HT trace (top) and corresponding 5-HT phase angles and ripples (bottom) for NREM (left) and sleep (right). The peak of the slow oscillation (0°) is indicated by the dashed purple line. E1. Schematic polar plot showing one period for a slow 5-HT oscillation. The falling phase of the oscillation occurs from 0° to 180°, and the rising phase occurs from 180° to 0°. E2. Phase of all NREM ripples relative to the ultraslow 5-HT oscillation. E4. Phase of all WAKE ripples relative to the ultraslow 5-HT oscillation. E3. Mean phase vector of NREM and WAKE ripples. F. Z-scored ripple power by 5-HT phase angle during NREM (left) and WAKE (right). Red vertical dashed lines delineate analyzed phase segments: ‘center ‘(-90° to 90°) vs. ‘side ‘(-180° to -90° and 90° to 180°). Representative ripples from each phase grouping are shown above. G. Mean center phase ripple power - mean side phase ripple power, plotted by session and mouse level in WAKE (left) and NREM (right). Red point with error bar indicates predicted difference and confidence interval after fitting a general linear mixed effects model to the data. P-values shown were derived from a post-hoc multiple comparisons test on the fitted model.
EMG and MAs vary by the phase of ultraslow 5-HT oscillations.
A.-D2. Relationship between microarousal (MA) occurrence and the phase of slow 5-HT oscillations. A. Example trace showing 5-HT, EMG, and MAs during a NREM bout. B. Example trace showing extracted 5-HT phase angle and MAs. C. MA occurrence according to 5-HT phase angle. D1. MA-triggered 5-HT across all MA events. D2. Mean MA-triggered 5-HT trace (blue) plotted with mean of randomly shifted 5-HT trace (orange). The orange trace was derived by randomly shifting all MA times and averaging the resulting 5-HT segments around those shifted times. E.-G. Relationship between the EMG signal and phase of slow 5-HT oscillations. E. Example traces showing extracted 5-HT phase angle and the EMG signal during NREM (left, blue) and WAKE (right, black) states. F. Mean z-scored EMG signal by 5-HT phase angle during NREM and WAKE states. G. Mean rising phase EMG - mean falling phase EMG, plotted by session and mouse level. Red point with error bar indicates predicted difference and confidence interval after fitting a general linear mixed effects model to the data. P-values shown were derived from a post-hoc multiple comparisons test on the fitted model.
Coherence varies by the phase of ultraslow 5-HT oscillations
A. Schematic showing representative hippocampal and cortical traces used for coherence calculations. B. Mean z-scored hippocampal-cortical coherence by frequency for NREM (left) and WAKE (right). C. Mean coherence by 5-HT phase angle for delta (1-5 Hz.), theta (6-10 Hz.), slow gamma (30-60 Hz.), fast gamma (60-100Hz.) and high frequency oscillation (HFO, 100-150 Hz.) bands in NREM (left column) and WAKE (right column). D. Mean rising phase coherence - mean falling phase coherence, plotted by session and mouse level for different frequency bands (rows) and states (columns). Red point with error bar indicates predicted difference and confidence interval after fitting a general linear mixed effects model to the data. P-values shown were derived from a post-hoc multiple comparisons test on the fitted model.
Relationship between ripple incidence and 5-HT levels depends on time-scale
A. Ripple incidence by behavioral state shows an inverted-U dose response relationship, with a peak at intermediate 5-HT levels (see Figure 1F). B. Within states, ripple incidence depends on the phase of the ultraslow 5-HT oscillation. At the same absolute 5-HT level (e.g. green dots), therefore, different ripple incidences are observed.
