Brain-wide Organization of Neuronal Activity and Convergent Sensorimotor Transformations in Larval Zebrafish

Abstract

Simultaneous recordings of large populations of neurons in behaving animals allow detailed observation of high-dimensional, complex brain activity. However, experimental approaches often focus on singular behavioral paradigms or brain areas. Here, we recorded whole-brain neuronal activity of larval zebrafish presented with a battery of visual stimuli while recording fictive motor output. We identified neurons tuned to each stimulus type and motor output and discovered groups of neurons in the anterior hindbrain that respond to different stimuli eliciting similar behavioral responses. These convergent sensorimotor representations were only weakly correlated to instantaneous motor activity, suggesting that they critically inform, but do not directly generate, behavioral choices. To catalog brain-wide activity beyond explicit sensorimotor processing, we developed an unsupervised clustering technique that organizes neurons into functional groups. These analyses enabled a broad overview of the functional organization of the brain and revealed numerous brain nuclei whose neurons exhibit concerted activity patterns.

Figure 1.. Whole-brain recordings of neuronal activity.

(a) Schematic of experimental setup for fictive swimming combined with light-sheet imaging (see Methods). (b) Illustration of functional dataset format. Whole-brain volumes were imaged at ~2 volumes/sec for ~50 min (2.11 ± 0.21 volumes/second; 6,800 ± 470 time frames, n = 18 fish). The activity traces of individual neurons were automatically extracted for 8.0×10⁴ ± 1.6×10⁴ cellular ROI’s per animal (n = 18). Scale bar, 50 μm. (c) Illustration of visual stimuli presented during functional imaging. Four stimulus paradigms from left to right: phototactic stimulus (phT), moving stripes (Optomotor response or OMR), expanding dot (looming or visual escape response), and dark flashes. (d) Example neuronal activity of single neuron ROI’s within an imaging plane (right inset) in the tectal region in the midbrain (cell activity is z-score normalized). The stimulus (phototactic stimuli alternating with white background) is illustrated with a bar above the calcium traces, and the recorded fictive behavior is plotted at the bottom of the panel, with three plots indicating left turns, forward swims and right turns from top to bottom. (e) Image stacks were registered to the Z-brain reference brain atlas (Randlett et al., 2015) containing ~300 labels of anatomical regions. Scale bar, 50 μm.

Figure 2.. Identification of sensory related neurons.

(a) Stimulus regression, using phototactic stimulus as example. Fish were shown a periodic stimulus during imaging that consists of leftwards and rightwards phototactic stimuli separated by a whole-field bright background. The stimulus regressors (black) are constructed by convolving a binary step function with an impulse kernel of GCaMP6. The colored traces show the ΔF/F (mean±SD for all ROI’s with Pearson’s correlation coefficient r>0.5) for a single fish. (b-e) Average stimulus response maps across multiple fish for (b) phototactic stimuli (phT), (c) moving stripes (optomotor response, or OMR), (d) expanding dots (looming or visual escape response), and (e) whole-field dark versus bright (dark-flash response). (f) Extraction of stimulus-locked component of neural activity. The stimulus presentation (top) is periodic. The neural activity (middle) can be averaged over the stimulus period to extract the stimulus-locked, periodic component (bottom). (g) Cells can be ranked by the magnitude (percent of variance explained) of their stimulus-locked component. Three example cell activity traces are shown in order of decreasing periodicity during OMR stimulus presentations (h-k) Functional cell types for cells tuned to different stimulus types (see Fig. S2 legend for detailed descriptions. h: phT, i: OMR, j: Looming, k: Dark Flash. The cells with the most highly periodic activity for each stimulus type were selected and sorted into clusters using k-means clustering (Methods). Left panels show average activity for each cluster. Right panels show anatomical location of cells within clusters. Scale bars, 50 μm. OTec: optic tectum. aHB: anterior hindbrain. Cb: cerebellum. pTec: pretectum. PO: preoptic area. Dien: Diencephalon. Pa: pallium. Hb: habenula.

Figure 3.. Identification of motor-related neurons.

(a) Left: anatomical map of all cellular ROI’s with r>0.5 to either leftwards (red) or rightwards (cyan) motor regressors during OMR stimulus blocks (example fish). (b) Average motor regression maps for OMR stimulus blocks (n=11 fish). Analogous maps for other stimuli shown in Fig. S3e-h. Inset: activity in the proximity of the hindbrain spinal projection neurons that control turning (RoV3, MiV1 and MiV2 neurons); masks from Z-Brain Atlas. (c) Motor regression: motor regressors (for left, right turns, respectively) were constructed by convolving the processed fictive swimming traces with an impulse kernel. Brain activity from all cellular ROI’s is regressed against these regressors. The dF/F traces show the mean±SD for all ROI’s with r>0.5 for a single fish. (d) Example decomposition of the motor output into stimulus-driven (motor avg.) and stimulus-independent (motor res.) components. (e) Dissection of brain-wide motor-related activity. Lower left panel: Scatter plot of regression coefficients for all cells with respect to motor avg. (y axis) and motor res. (x axis) regressors, for an example fish in relation to the left-side motor output. Maps were calculated using all stimulus blocks together (similar analysis for individual stimulus blocks shown in Fig. S3m). Top left, purple box: stimulus-driven motor map showing cells ranking in the top 2% for motor avg. regression only. Bottom right, green box: independent motor map showing cells ranking in the top 2% for motor res. regression only. Top right, blue box: map showing cells ranking in the top 2% for both motor avg. and motor res. regressions. (f) Cells ranking in the top 2% for forward swimming (bilateral) regression only (calculated using all stimulus blocks together). Note the bilateral and dense cluster of cells at the red arrow locations in rhombomere 5, close to the MiD2 reticulospinal neurons (according to ZBrain). The bilateral swimming regressor is the average of left and right motor regressors (Fig. S3k). (g) Cells ranking in the top 2% for turning (unilateral) regressions only (calculated using all stimulus blocks together). The lateral swimming regressors are the right and left motor regressors minus the bilateral regressor (Fig. S3k). Dotted yellow lines: boundary between rhombomeres 2 and 3. Note the contralateral activity (arrowheads). Scale bars, 50 μm. pTec: pretectum. nMLF: nucleus of the medial longitudinal fasciculus. aHB: anterior hindbrain. pHB: posterior hindbrain. vSPN: ventral spinal projection neurons. rh: rhombomere. aHB(1-2): anterior hindbrain rhombomeres 1,2. ARTR: anterior rhombencephalic turning region. IO: inferior olive.

Figure 4.. Multi-stimulus integration.

(a) Average map across fish for the intersection of phototactic responsive cells and OMR responsive cells (top 2% of cells by rank). Multi-stimulus responsive cells are concentrated in rh. 1 and the medial stripes of rh.2. (b) Whole-brain regressions were performed to a set of regressors that include phT only, OMR only, phT&OMR in the same direction (congruent), and phT&OMR in opposite directions (incongruent). Cells were classified by their best regressor. Average functional activity of cells associated with each regressor is plotted (ΔF/F, mean±SD). (c) Multi-stimulus convergence maps for all stimulus pairs (top 5% of cells by rank). Cells are colored based on their direction tuning and congruence. For directional stimuli (phT, OMR, loom), congruent cells are tuned to both stimuli in the same direction, while incongruent are tuned to opposite directions. Incongruent cells were labeled according to the direction of the stimulus listed first. For pairs involving the dark flash stimulus, congruent cells were defined as tuned to whole-field dark (which elicit large angle turns), while incongruent cells were tuned to whole-field bright. (d) Quantification of the number of convergent cells for each stimulus pair. Number and shading indicate proportion of cells that are highly tuned (top 5%) to either stimulus. (e) Illustration of two alternative hypotheses for convergence of multiple stimulus pathways. Left - direct motor convergence: information from non-overlapping visual representations (e.g. phT, OMR) directly feeds into premotor systems, which then compete to produce different behaviors. Right – sensory convergence: different visual representations first feed into a behavior-centric visual representation before affecting motor circuits. The present results support the second model, with the anterior hindbrain containing the behavior-centric, convergent visual representations. (f) Distribution of convergence cells for different brain regions along the anterior-posterior axis. Percentages are normalized to all convergence cells, shown for all stimulus pairs. Significant numbers of convergent cells are found in the diencephalon, midbrain, and anterior hindbrain rhomobomeres 1&2 (aH(1-2)). For phT/OMR and OMR/DF stimulus pairs, the average density of convergent cells is highest in rhombomere 1. Telen: telencephalon. (g) Scatter plot of convergent activity in a 2-dimensional sensory-motor space (Methods). Red points: convergent cells, defined as the intersection of the top 5% of cells ranked by phT and OMR regression. Blue points: top cells ranked by sensory component (same number of points as red). Green points: top cells ranked by motor component (same number of cells as red). Top and right: histograms of motor and sensory components, comparing distribution of convergent cells (red) to the most motor or sensory-related cells, respectively. (h) Cellular activity for 3 example neurons shown in (g) during phT and OMR stimulus blocks. (i) Average anatomical maps (n = 11 fish) showing location of the cells represented in (e): top-ranking convergent cells (left map, red), sensory-related cells (middle map, blue) and motor-related cells (middle map, green), and merge (right map, overlap between sensory-related and convergent appears purple. Note significant overlap in the anterior hindbrain (appears purple). Boundaries for rhombomeres 1,2, and 3 are overlaid. Scale bars, 50 μm.

Figure 5.. Whole-brain functional clustering.

(a) A diverse collection of automatically identified functional clusters from one example fish. Clustering was performed on activity data from all stimulus blocks together. After whole-brain clustering, the most dissimilar clusters were selected based on their hierarchical ranking for visualization. a1: functional activity profiles: normalized ΔF/F for each cell is plotted along the horizontal axis. a2: corresponding anatomical map. (b) Full set of individual clusters as z-projections (from the same fish). All clusters are shown in batches of 6 on duplicate z-projections for clarity. (c) Clusters for an example fish ranked from stimulus-related (periodic, as in Fig. 3c, red) to not stimulus-related (aperiodic, purple). (d) Clusters for an example fish ranked from motor-related (high regression coefficient to motor res., as in Fig. 3f, red) to not motor-related (low regression coefficient, purple). (e) Histogram of size distribution of all clusters, pooled across fish (mean±SEM). (f) Histogram of average correlation between cells within clusters, pooled across fish (mean±SEM). (g) Correlation matrix of all clusters from a single fish, ordered so that most similar clusters are adjacent. Submatrix indicated by red bars highlights putative olfactory bulb neurons (see Fig. S6f). (h) Visualization of all clusters within one fish with t-SNE. For color assignment, the total of 139 clusters (6,499 cells) were ordered by hierarchical clustering, and adjacent shades of hsv colors are assigned based on the resulting leaf order. Gray points represent cells that did not pass the clustering criterion i.e. not assigned to any cluster (1:10 down-sampled for clarity). (i) Two-fold cross-validation (as in Fig. S5e) between multiple sets of stimuli within each fish (n = 6 fish), with scores indicating the fraction of cells in matched clusters over the total number of cells. Each fish in the analysis has been presented with all 5 different stimuli. The “all” category uses the combined data from all 5 stimulus periods. (j) Results of the automatic clustering algorithm applied to only the 'spontaneous' condition (no stimulus presented), for an example fish. Only cells that passed the two-fold cross-validation test are shown. (k) Histogram of average within-cluster anatomical distance, pooled across fish (mean±SEM). (l) Examples of clusters that are anatomically isolated. (m) Examples of anatomically dispersed clusters that are difficult to identify based on anatomical location, as they are intermixed with other clusters. (n) Average map of clusters conserved in anatomical space. Each of these clusters are selected for having anatomically corresponding clusters in at least 6 other fish (out of 18 fish, see Methods). Clusters are ranked and colored within fish as in (k). (o) Putative abducens nucleus (ABD) network for the control of eye-movements. Arrows: anterior and posterior clusters of the ABD map to rhombomeres 5 and 6, respectively. Arrowheads: oculomotor nucleus (OCM) clusters. (p) Illustration of the eye-control circuit. Red/blue indicates control of rightward/leftward eye movement. (q-r) Two-dimensional sensory/eye-movement mapping (similar to Figure 3f, except the average response of the ABD were used as eye-movement regressors instead of the tail-movement motor output). q: Analysis of the sensory and eye-movement activity of one example fish. Horizontal axis: stimulus component (square root of variance explained by periodic component of activity); vertical axis: motor component (regression coefficient with left eye-movement res., as in Fig. 3d); Top 1000 motor-ranked cells are plotted in colors, corresponding to (r). r: Anatomical map of cells shown in (q). See Fig. S6g for corresponding figures for right motor res. Scale bars, 50 μm. Dien: Diencephalon. OTec: optic tectum. aHB: anterior hindbrain. ABN: abducens nucleus. CN-X: cranial nerve X. ARTR: anterior rhombencephalic turning region. pTec: pretectum.