DRN neuronal activity increased during quiescence.

A. A schematic of our all-optical system that integrates tracking, dual-color volumetric fluorescence imaging, and optogenetic manipulation. B. Example zebrafish exhibited alternating locomotor and quiescent states during spontaneous behavior. C. Maximum intensity projection (MIP) of whole-brain imaging in a zebrafish, showing 30 s averaged neural activity during locomotor (left) and quiescent (middle) states; their difference is shown on the right. The red circle marks the dorsal raphe nucleus (DRN). Scale bar, 100 µm. D. Relationship between locomotor speed and neural activity in DRN (n = 3).

DRN 5-HT activation induces a quiescent but non-sleep-like state.

A. MIP of whole-brain data from a 7 dpf Tg(tph2:ChrimsonR-mKate2 × elav3:h2b-jGCaMP8s) zebrafish acquired by two-photon microscopy. Scale bar, 100 µm. B. Locomotor velocity changes in Tg(tph2:ChrimsonR) and control zebrafish during DRN 5-HT neuron activation (n = 6). Yellow shading marks optogenetic stimulation. C. Top: Body roll angle (rotation in the Y–Z plane) increases during natural sleep, indicating loss of postural stability. Body roll angle in control (n = 8), sleep-deprived (SD, n = 6), and Tg(tph2:ChrimsonR) zebrafish (n = 12). Yellow indicates optogenetic stimulation. In Tg(tph2:ChrimsonR) fish, light versus no-light conditions were compared with the Wilcoxon matched-pairs signed rank test. Tg(tph2:ChrimsonR) versus control and sleep-deprived versus Tg(tph2:ChrimsonR) were compared with the Mann–Whitney U test. ****p < 0.0001. D. Top: Experimental timeline over two light–dark cycles (14 h light/10 h dark). The first cycle was normal; in the second, optogenetic stimulation was applied during the first 6 h of the dark period. Average locomotor speed and sleep duration in the 4 h after stimulation were compared with the corresponding 4 h of the first dark period. Bottom: Differences in average locomotor speed and sleep duration between Tg(tph2:ChrimsonR) (n = 24) and control zebrafish (n = 24), analyzed with the Mann–Whitney U test.

DRN 5-HT activation modulates brain state.

A. Cumulative variance explained by demixed principal components (dPCs) related to optogenetic stimulation in Tg(tph2:ChrimsonR) zebrafish (n = 5) and controls (n = 5). B. Time course of whole-brain activity projected onto dPC1 in Tg(tph2:ChrimsonR) zebrafish (n = 5). Yellow shading marks optogenetic stimulation. C. Left: Histogram of brain-region weight distribution in dPC1. Yellow shading highlights high-weight regions (|weight| > 0.03, 272 regions). Right: Spatial distribution of these regions in the zebrafish brain. Scale bar, 100 µm. D. R2 between neural activity in dPC1 high-weight regions and locomotor behavior, compared with randomly selected regions (n = 272; Mann–Whitney U test, ****p<0.0001).

DRN 5-HT activation modulates motor circuits to reduce sound-evoked responses.

A. Experimental protocol for sound stimulus experiments. B. Probability of sound-evoked escape in Tg(tph2:ChrimsonR) and control zebrafish before, during, and after optogenetic stimulation. Yellow shading marks optogenetic activation. Wilcoxon matched-pairs signed rank test, **p = 0.0039. C. Population raster plot of simultaneously recorded neurons during DRN 5-HT activation and control. Blue lines mark sound onset. D. Schematic of sound, motor, and DRN activation subspaces identified by dPCA. Left and right MIPs show brain regions with high weights in each subspace in an example zebrafish (bottom right panel is the same as the panel shown in Figure 3C). E. Left: Similarity matrix of sound-evoked population responses during DRN activation vs. control. Right: Same analysis comparing awake and drug-induced sleep. F. Principal angle analysis shows the motor subspace is significantly aligned with the DRN activation subspace, while the sound subspace is nearly orthogonal (p-values from a nonparametric permutation test, 1000 iterations).

DRN 5-HT neuron activation exerts graded suppression on motor subspace.

A. Schematic of the linear regression analysis. B. Example neurons with low (top) and high (bottom) variability in regression coefficients. Left, middle, and right panels show regression coefficients, mean activity across bout types, and neuronal spatial locations. C. Two motor-related regions with distinct modulation after DRN 5-HT activation. Left, middle, and right panels show their spatial locations, activity in control and optogenetic trials, and trial-averaged activity. D. Spatial distribution of motor-correlated neurons differentially modulated by DRN 5-HT activation. Neural magnitude is quantified by variance. Neurons are color-coded by variance reduction (darker red, stronger suppression; lighter red, weaker effect), as in (Fig. 5E,F). E. Relationship between DRN 5-HT–induced modulation and the coefficient of variation (CV) of regression coefficients across motor-correlated neurons. F. Hyperbolic multidimensional scaling (HMDS) of neural correlation distances shown in a 3D Poincaré ball (left) and a 2D projection (right). Top: functional embedding during DRN 5-HT activation; bottom: control period.

DRN 5-HT activation suppresses locomotion and induces a quiescent state distinct from sleep.

A. Schematic of burst and tonic optogenetic stimulation paradigms. B. Top: locomotor speed of a zebrafish during burst stimulation. Bottom: locomotor speed of a zebrafish during tonic stimulation. C. Relationship between body roll angle and swimming speed in Tph2:ChrimsonR zebrafish. Each point represents one second; yellow points indicate periods of DRN 5-HT activation. D. Relationship between body roll angle and swimming speed in sleep-deprived zebrafish. E. Quiescence per hour in Tph2:ChrimsonR zebrafish. F. Quiescence per hour in control zebrafish.

DRN 5-HT activation did not alter neural dynamics within the sound-evoked subspace.

A. Temporal evolution of whole-brain neural activity projected onto the sound-evoked subspace and the motor-correlated subspace in an example Tg(tph2:ChrimsonR) zebrafish. Yellow shading indicates periods of optogenetic stimulation. B. Similarity matrices of sound-evoked neuronal population responses during DRN 5-HT activation and control periods for fish 2–4 (fish 1 shown in Fig. 4E). C. Same analysis as in panel B, but comparing the awake state with the drug-induced sleep state. D. Schematic of auditory stimulation paradigms with different sound intensities. E. Similarity matrices of sound-evoked neuronal population responses for strong (left) and weak (right) sound stimuli during DRN 5-HT activation and control periods.

DRN 5-HT activation exerts graded suppression on the motor subspace.

A. Relationship between dP C1motor weights and R2. Red points indicate the motor-correlated neurons included in the analysis. B-D. Relationship between DRN 5-HT activation–induced modulation of neural activity and the coefficient of variation (CV) of regression coefficients in motor-correlated neurons for fish 2–4 (fish 1 shown in Fig. 5D–E).

DRN 5-HT activation diversifies motor network activity.

A. Cosine distance matrices of motor-correlated neurons during DRN activation (left) and control (right) periods. Neural activity magnitude was quantified by the variance. Neurons were ranked by changes in variance between DRN 5-HT activation and control periods. B. Embedding dimension as a function of BIC, see Methods. C. Shepard diagram showing pairwise distances in the embedding space vs. data pairwise distances in A during DRN activation. Left: Euclidean embedding, BIC = -210054; Right: Hyperbolic embedding, BIC = -227165. The dimensionality d = 6. D. Same as C, but during control period.