FIGURE SUMMARY
Title

Neural Circuits Underlying Visually Evoked Escapes in Larval Zebrafish

Authors
Dunn, T.W., Gebhardt, C., Naumann, E.A., Riegler, C., Ahrens, M.B., Engert, F., Del Bene, F.
Source
Full text @ Neuron

Looming-Specific Neurons in the Optic Tectum Encode Critical Size

(A) Left panel is a schematic of the larval zebrafish brain indicating the positions of the optic tectum (OT) with its (neuropil (NP) and cell body layers (stratum periventriculare, or SPV), the pretectum/thalamus (PT/TH), and the midbrain tegmental region (MB). The right panels depict a transverse average intensity projection of a 5-dpf Tg(elavl3:GCaMP2) larval brain (used as an anatomical reference) and accompanying sagittal view. TH, thalamus; PT, pretectum. Dotted lines denote the position of each eye: r, rostral; c, caudal; d, dorsal; and v, ventral.

(B) Left panels depict single planes showing anatomy (gray) and activity (blue) in the dorsal and ventral OT, PT/TH, and MB. Individual region of interest (ROI) numbers (ROIs shown as colored circles) correspond to the traces on the right, which illustrate the general pattern of activity observed across midbrain visual areas in response to looming stimuli. Neurons in the dorsal OT (1 and 2) respond weakly to looming stimuli. Neurons in the ventral OT (3 and 4) show more varied responses but typically respond strongly to and favor looming stimuli. Neurons in PT/TH (5 and 6) respond to both looming and flashed stimuli. Neurons in MB (7 and 8) were typically active spontaneously and non-stimulus-locked. Boxes represent stimulus presentation periods. Traces are re-ordered and concatenated from longer recordings.

(C) The middle panels show trial-averaged normalized ΔF/F evoked by looming and flashed stimuli from 1,613 active neurons across 12 fish. Each neuron is sorted according to its selectivity index (see Experimental Procedures) in descending order (1 = looming exclusive, -1 = flash exclusive). The left-most panel shows the corresponding anatomical location of each neuron, color-coded as in (B). The right-most panel shows the average normalized ΔF/F binned across 100 neurons from the sorted list. Traces are normalized to [0 1] after trial averaging. Normalization values were skewed to the right (maximum 586.24%ΔF/F, minimum 21.02%ΔF/F, and skewness 1.28). Dotted lines and boxes represent stimulus presentation periods, with start times indicated by arrowheads.

(D) The top panel depicts all neurons from (C) mapped to a reference brain and colored according to selectivity index. Arrowhead shows the preponderance of looming-selective neurons in the ventral OT. Differences in the number of OT neurons between the left and right hemispheres reflects a sampling bias (data from stimuli presented in the left and right visual fields were pooled); most OT imaging was unilateral. The middle panel shows bar plots quantifying mean selectivity (left) and responsive cell density (right) across the OT (n = 60 imaging planes), PT (n = 34), and MB (n = 44). In the bottom panel, histograms show the distribution of looming selectivity across neurons in the OT (n = 973 cells), PT (n = 279), and MB (n = 361). **p < 10-5, *p < 0.01, permutation test. N = 12 larvae. r, rostral; c, caudal; d, dorsal; and v, ventral.

(E) Top shows the responses of 110 OT neurons in one fish to looming stimuli simulating approach at three different velocities (R/V 545 ms, 1,090 ms, and 2,730 ms, top to bottom). The stimulus size (angle) over time for each condition, convolved with a calcium impulse response function (CIRF) (τ  = 962 ms), is shown in red. Bottom shows the first temporal principal component (TPC1, ± SEM across fish) averaged over eight fish, 1,533 neurons. The spots above each trace schematize the size of the looming stimulus before the TPC1 threshold (dark spot). Stimuli continue to expand (ellipses) until the end of the stimulus epoch.

(F) Quantifications of average convolved stimulus visual angle (left) and edge velocity (right) at TPC1 threshold times (81% TPC1 response after normalization to maximum across all stimulus conditions) for all three velocity conditions. The colored curves show the convolved stimulus size and edge velocity for each condition, fanned to show TPC1 dynamics for individual fish. Each color value corresponds to the normalized amplitude of TPC1 activity during expansion. Crosses show the average value of each stimulus variable at the TPC1 threshold for each velocity condition. Error bars are SEM for TPC1 threshold timing (horizontal) and respective stimulus variables (vertical) across fish. Dotted lines represent the mean visual angle and edge velocity at TPC1 threshold across all fish and conditions.

Retinotectal Processing of Looming Stimuli

(A) Left panel is a representative imaging plane from a 5-dpf Tg(Isl2b:Gal4;UAS:SyGCaMP6s) fish, which specifically expresses GCaMP6s in the axon terminals of RGCs. Scale bar, 20 µm. Right panel shows traces from the four ROIs indicated on the left (yellow circles), with convolved looming stimulus time courses for slow (R/V = 2,730 ms), medium (R/V = 1,090 ms), and fast (R/V = 545 ms) stimuli shown on top for reference. Gray intervals denote stimulus duration.

(B) Left panel is a raster plot after regression cluster analysis of normalized ΔF/F responses for n = 5,023 RGC terminal ROIs across N = 6 fish, sorted according to the number of individual traces assigned to each respective cluster. Start frames for each stimulus are denoted by the dotted white, vertical lines. Different clusters are separated by horizontal lines. The right panel shows mean traces of the four main clusters (containing at least 2% of the total ROIs from at least five fish, top to bottom: clusters 1–4, N = 3,420, 788, 413, and 192 ROIs, respectively).

(C) The left panel shows a representative imaging plane from a 5-dpf Tg(elavl3:H2B-GCaMP6s;Isl2b:Gal4;UAS-SyGCaMP6s) fish, which labels SINs. Scale bar, 20 µm. The right panel shows traces from the four ROIs indicated on the left (yellow circles).

(D) Left panel is a raster plot after regression cluster analysis of normalized ΔF/F responses for n = 541 SIN neurons across N = 11 fish, sorted according to the number of individual traces assigned to each respective cluster. Right panel shows mean traces of the three main clusters (containing at least 2% of the total neurons from at least eight fish, top to bottom: clusters 1-3, N = 163, 101, and 44 neurons, respectively).

Laser Ablation of the Mauthner System Alters Escape Trajectory and Reduces Initial Bend Angle

(A) Schematic of the zebrafish brain and hypothesized information flow from the eye, through the contralateral OT, to the contralateral hindbrain Mauthner system (M-system) comprising the Mauthner cell (M-cell) and homologs in rhombomeres 4-6.

(B) Two-photon micrographs showing an example of the M-cell (left), MiD2 (center), and MiD3 (right) pre- and post-unilateral ablation. For each fish, the M-cell and MiD2/MiD3 clusters were ablated together. Ablations were specific to targeted neurons and did not affect nearby cells. Cells were backfilled with dextran-conjugated dye. Scale bar is 20 µm.

(C) The average maximum turn angle derived from orientation change, see Experimental Procedures, during escapes is significantly altered for maneuvers contralateral to the ablated side, with the largest change evident at the first (maximum) bend. The average escape ipsilateral to the ablated side remained unchanged. All traces are aligned to the time point of the first bend. Error is SEM across all events, N = 8 fish. Pre, non-ablated, n = 60 events; pre, ablated, n = 73; post, non-ablated, n = 88; post, ablated, n = 66. p = 0.002, permutation test.

(D) Left panel shows escape trajectories pre- and post-ablation (green and pink, respectively) elicited by stimuli ipsilateral to the ablated side. Right panels shows escape trajectories pre- and post-ablation (green and black, respectively) elicited by stimuli contralateral to the ablated side.

(E) Top panel shows stick diagrams representing fish position 40 ms after escape initiation for the ablated and non-ablated sides, pre- and post-ablation. The shift in escape trajectory post-ablation is more apparent on this timescale. Bottom panel is an angular histogram of the maximum turn angle pre- and post-ablation for the ablated and non-ablated sides.

(F) Left panel shows quantification of maximum turn angle across fish pre- and post-ablation for the ablated and non-ablated sides. *p = 0.002, n.s., p = 0.093, permutation test. Right panel shows that this change is also apparent in histograms of maximum turn angle across all events; **p < 10-5 ablated side, p = 0.175 non-ablated side, permutation test across all events. Error is bootstrapped SEM.

(G) Left panel shows quantification of escape duration. No significant change is apparent on either side after ablation. Non-ablated, p = 0.073. Ablated, p = 0.101, permutation test. Error bars are SEM across fish. Right panel shows the distribution of spontaneous turns does not change after ablation, providing further evidence of ablation specificity; pre-ablation n = 744, post-ablation n = 911 spontaneous swim events; n.s., p = 0.725, permutation test. Error is bootstrapped SEM.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Neuron