Volume 26, Issue 6, Pages 743-754 (March 2016) Functional Profiles of Visual-, Auditory-, and Water Flow-Responsive Neurons in the Zebrafish Tectum  Andrew W. Thompson, Gilles C. Vanwalleghem, Lucy A. Heap, Ethan K. Scott  Current Biology  Volume 26, Issue 6, Pages 743-754 (March 2016) DOI: 10.1016/j.cub.2016.01.041 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Experimental Setup for Calcium Imaging of Neural Responses to Stimuli across Three Modalities (A) A schematic of the experimental setup. Zebrafish larvae were exposed to four visual stimuli, two lateral line stimuli, and an auditory stimulus while neuronal activity was simultaneously imaged using SPIM. (B) Pan-neuronal GCaMP5G was imaged in the tectum from a dorsal perspective, orthogonal to the illumination plane. Individual cells (yellow) were automatically segmented using custom-written code in MATLAB. (C) A raster plot of the change in fluorescence over time for each cell in a single experimental trial. Defined patterns of activity were observed in response to the presentation of the various sensory stimuli (vertical bar, red; horizontal bar, purple; full-field flash, blue; small spot, green; water flow, light green; auditory tone, orange). See also Movie S1 and Figures S1 and S2. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Different Visual and Non-visual Stimuli Selectively Activate Different Parts of the Tectal Neuropil (A) Kaede expression in retinal ganglion cell axons in the tectal neuropil (dotted outline) of a 6-dpf Atho7:Gal4;UAS:Kaede larva at each imaging depth. The neuropil layers are identified by the fluorescence profile across the axis outlined by the yellow line in each panel. SO, stratum opticum; SFGS, stratum fibrosum et griseum superficiale; SGC, stratum griseum centrale; SAC/SPV, stratum album centrale/stratum periventriculare. Scale bars, 50 μm. (B) The average correlation between the presentation of each stimulus and the GCaMP5G responses in the tectal neuropil is shown for each depth. Visual responses in the neuropil are seen to have higher correlations to the stimulus presentation than non-visual responses, with apparent separation of the regions responsible for processing different visual stimuli. Each panel represents the average correlation of 11 fish. See also Figure S6. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Responses of Cells in the Tectal PVL to Both Visual and Non-visual Stimuli (A) An example ensemble of neurons (yellow) with functionally similar response profiles defined by PCA-promax. (B) Classification of different functional clusters by linear regression to the presentation of each of the six sensory stimuli. Four vertical bar-responsive, three horizontal bar-responsive, three full-field flash-responsive, five small spot-responsive, two water flow-responsive, and two auditory tone-responsive clusters were identified. (C) The average (black) and SD (gray) of the responses of the different functional clusters produced by PCA-promax, ordered sequentially by peak response. Clusters are assigned names (top right of each panel) indicating the stimulus to which they respond and the chronological order among clusters responsive to the same stimulus. Colors represent the presentations of stimuli as per Figure 1C. See also Figures S2, S3, S4, and S5. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Clusters of Neurons Are Responsive to Salient Stimulus Features (A–F) Average overall response traces for cells in each cluster, separated by stimulus. (A) Responses to the moving vertical bar occur either early (blue) or late (red) to the rostral-moving bar, and either early (green) or late (pink) to the caudal-moving bar. Each cluster has a significant preference for a particular direction of stimulus movement (A′). (B and B′) Similar preferences were observed for direction selectivity to the ventral (blue) and dorsal (red) moving horizontal bar, with a cluster also responsive to the offset of the stimulus (green) also observed. (C and C′) Significant on/off selectivity was observed respectively for the first (blue) and third (green) clusters responsive to a full-field flash. (D and D′) Clusters show significant preference for either of the two rostral (blue and green) or caudal movements (red and orange) of the small spot, while SS4 (pink) had no significant direction selectivity. SS4 responses peaked when the spot was in the rostral part of the visual field. (E and E′) Water flow stimulation resulted in two distinct responses that corresponded either to the onset of each water puff (red trace), or slowly to only the first water puff (blue). (F and F′) Significant on/off selectivity was also observed for the first (blue) and second (red) clusters responsive to the auditory tone. Black bar, stimulus presentation; >, rostral; <, caudal; v, ventral; ˄, dorsal movement; error bars, SEM; ∗p < 0.05, Kolmogorov-Smirnov test. See also Figures S6 and S7. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Highly Variable Ensembles Are Anchored by Reliably Responding Core Cells (A) The average pairwise matching index across all trials for each ensemble of cells is low but significantly higher than that of a simulated ensemble with the same local distribution of cells. The AUD2 cluster provides one exception (∗p < 0.01, Wilcoxon signed-rank test; error bars, SEM). (B) An example ensemble of cells from SS4 cluster (small spot-responsive) in three repeated trials. The cellular composition of the ensemble is more varied between trial 1 and 2 (matching index [MI] = 0.59) than between trials 2 and 3 (MI = 0.70). Core cells present in at least 80% of trials are red; non-core cells are yellow. (C) A significantly higher proportion of core cells exist in all ensembles from visual-responsive clusters and WF2 than expected by chance (∗p < 0.01, Wilcoxon signed-rank test; error bars, SEM). The AUD2 cluster contained no core cells. (D) The core cells within an ensemble are significantly more compact in all visual stimuli-responsive clusters and the second water flow-responsive cluster than a simulated core within each ensemble (∗p < 0.01, Wilcoxon signed-rank test; error bars, SEM). (E) The average response of core cells is broadly similar to that of the whole cluster, although there is a trend toward stronger responses to the salient stimulus feature. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Ensembles and Cores Show Relatively Little Overlap and Correlation (A) Matching indices between cells belonging to ensembles from different clusters in the same animal are indicated. The increase in matching index for temporally adjacent clusters is likely due to the slow kinetics of GCaMP5G. (B) Matching indices between cells belonging to the cores of ensembles from different functional clusters in the same fish. AUD2 has no core, resulting in black squares. (C) Average cross-correlation between cells in different ensembles within the same experimental trial. Average correlation between different cells within the same ensemble is expected to be high but still less than one. (D) Average cross-correlation between core cells of different ensembles. Black squares result when core cells from both ensembles are never present within the same imaging plane. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Tectal Cells Show Reduced Visual Responses When Presented Simultaneously with Auditory or Water Flow Stimuli (A) Visually responsive cells have reduced activity when auditory or water flow stimuli are presented simultaneously with the visual stimulus. n = 3,450 cells from ten fish. The thick gray line indicates stimulus start. The second peak in blue trace is a visual “off” response to the removal of the bar 1 s after the cessation of vertical bar movement. The magnitude of the “off” visual response appears greatly reduced by prior presentation of non-visual stimuli. (B) Auditory-responsive cells have increased activity when visual or water flow stimuli are presented simultaneously with the auditory stimulus. n = 714 cells from ten fish. (C) Water flow-responsive cells show no significant change in activity when visual or auditory stimuli are presented simultaneously with the water flow stimulus. n = 3,654 cells from ten fish. (D) The peak fluorescence during visual responses is significantly reduced when auditory or water flow stimuli are presented simultaneously. Peak auditory responses are significantly elevated with co-presentation of visual or water flow stimuli. Bars represent mean peak response, and error bars show SEM. ∗p < 0.01, Wilcoxon signed-rank test. Current Biology 2016 26, 743-754DOI: (10.1016/j.cub.2016.01.041) Copyright © 2016 Elsevier Ltd Terms and Conditions