Poulsen et al., 2021 - Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system. Current biology : CB   31(9):1977-1987.e4 Full text @ Curr. Biol.

Fig. 1 (A) Schematic of the imaging and experimental setup used to observe brain activity from zebrafish larvae, showing the mini-speaker attached to the back coverslip of the chamber. (B) Summary of the analysis workflow used to identify and categorize auditory-responsive neurons across the brain. (C) Top: a schematic of the stimulus train used, including stimuli with a range of different forms, frequencies, and amplitudes. Bottom: the mean response of all the auditory-responsive ROIs in the resulting dataset is shown. (D and E) All auditory-responsive ROIs across the brain are shown with the strength of the response represented by the color and the correlation to the linear regression model (r2 value) represented as the size of the sphere. The strength of the response is between 2 (the threshold for inclusion) and 12 SD. Brain regions containing a high proportion of auditory-responsive ROIs are identified spatially (inset, D). Cb, cerebellum; HB, remaining hindbrain; ON, octavolateralis nucleus; Tec, tectum; Teg, tegmentum; Tel, telencephalon; Th, thalamus; TS, torus semicircularis. C, caudal; D, dorsal; R, rostral; V, ventral here and in subsequent figures. Scale bar applies to both (D) and (E). Particle motion and sound pressure recordings of the chamber can be found in Figure S1.

Fig. 2 K-means clustering revealed 6 clusters of ROIs that respond to particular properties of acoustic stimuli. (A and B) Two broadly tuned clusters were detected that were predominately located in the ON. (C) A pure-tone-specific cluster was present in the medial HB, lateral cerebellum, TS, and pretectum. (D) A third cluster, selective for pure tones, was detected with ROIs in the TS, ON, and lateral cerebellum. (E) An onset cluster was present in the TS, the lateral cerebellum, and the medial HB. (F) The final cluster revealed white noise volume ramp-selective ROIs located in the pallial region of the telencephalon, the lateral cerebellum, and sparsely in the HB. Scale bar applies to all panels. Response strengths and r2 values are represented as in Figure 1.

Fig. 3 (A–C) The mean responses (A) of all ROIs selectively responding to each frequency in our stimulus train (top) and (B) their positions in the brain from a dorsal view and (C) a lateral view. Scale bar applies to both (B) and (C). (D) Using a one-dimensional kernel density estimation (KDE) computation, we looked at the density of frequency-selective ROIs at the whole-brain level, from rostral to caudal. Because of the overlapping responses and similar spatial distributions of ROIs responding to 2.5-kHz, 3-kHz, and 4-kHz tones, we combined these into a single category for (C) and (D). (E–N) Dorsal views showing ROIs of each frequency used in the stimulus train that passed the supervised analysis thresholding. The relevant frequency is indicated in each panel. Although the sound level to both ears is equal, there is asymmetry in the ROIs passing threshold due to the fish being illuminated from the front and one side. The right side, which receives a brighter and sharper SPIM plane, generally yields more ROIs that pass our inclusion criteria.

Fig. 4 (A) The fraction of ROIs per frequency at the whole-brain (WB) and regional levels. Remaining brain regions now shown here can be found in Figure S2. (B–G) The spatial distribution of ROIs from the dorsal (top left) and lateral (top right) views are shown. Bar graphs (bottom left) show the proportions of nearest neighbors for ROIs of a given frequency preference (separate bars for ROIs responding to different frequencies), with same-frequency neighbors indicated by a black box. For comparison, data from the spatially randomized dataset are shown in the last bar. Matrices (bottom right) of the neighbor frequency index show pairs of frequencies that are overrepresented (blue, bar in B) or underrepresented (red) as neighbors. Significant results versus the randomized dataset are indicated (⋅p < 0.05; ⋅⋅p < 0.01; ⋅⋅⋅p < 0.001; ⋅⋅⋅⋅p < 0.0001). See Figure S3 for p values. Kruskal-Wallis tests with Dunn’s correction for multiple comparisons were performed to establish p values. The k-nearest neighbor heatmap legend is shown in (B) and applies to (B)–(G). The same information for the remaining brain regions can be found in Figure S2, and all p values are presented in Figure S3. Figure S4 shows additional whole-brain responses to logarithmic frequencies from 251 to 1,998 Hz at the original volume and 3 lower volumes, −6, −12, and −18 dB below. Scale bars represent 50 μm.

Acknowledgments:
ZFIN wishes to thank the journal Current biology : CB for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Curr. Biol.