Article Figures & Data
Figures
- Figure 1.
GCaMP6m transduction of IC neurons and experimental setup. A, Coronal sections at 150 μm intervals showing widespread transduction of neurons throughout most of the dorsal parts of the IC. Scale bar, 1 mm. B, Higher-magnification image of one of the sections (A, white box) with the approximate locations of the DCIC, CNIC, and external cortex of the IC (ECIC) marked. Scale bar, 300 μm. C, Higher-magnification image (B, white box) showing individual IC cell bodies. Scale bar, 25 μm. D, Photograph of glass window (outlined by dotted lines) inserted into the skull over the IC. Scale bar, 500 μm. E, Experimental setup used for sound delivery and two-photon calcium imaging.
- Figure 2.
Tone-evoked calcium responses of IC neurons in one imaging area. A, Fluorescence traces of all 27 neurons that were identified in this imaging area. B, Micrograph of the imaging area (200 × 200 μm) with ROIs drawn as dotted ellipsoids. ROIs marked in white indicate nonresponsive neurons. All other ROIs are color-coded according to the results of a SVM classification (see main text; blue, CNIC; red, DCIC). All CNIC neurons were found to the left of the dashed vertical line, all DCIC neurons were found to the right of the line. FRAs are depicted next to each responsive neuron. Scale bar, 10 μm. C, Fluorescence traces of neuron 19 ordered according to sound frequency and level. The nine traces associated with the nine repeats of each stimulus are plotted in gray. The average calcium signals are plotted in black. Scale bars indicate 2 ΔF/F and 1 s, respectively. D, FRA corresponding to C. E, F, Fluorescence traces and FRA for neuron 4, plotted as in C and D. R, Rostral; M, medial.
- Figure 3.
Functional distribution of BFs within the IC. A, The relative spatial locations of 322 tone-responsive neurons from one animal were reconstructed in 3D space and color-coded according to each neuron's BF. Blue asterisk marks the coordinates of a laser ablation made at the end of this experiment. B, DAPI staining of the histological section that includes the site of the laser lesion (see inset). Scale bar, 50 μm. All neurons from A are superimposed onto this section, color-coded according to their BFs. C, Two representative imaging areas from this animal, one putatively DCIC (dark red), the other putatively CNIC (blue), were used to train a SVM. D, Using this SVM to classify the rest of the neurons from this animal segregates the data into putative DCIC (dark red) and CNIC (blue) areas. E, The same SVM classifier constructed using data from this animal was also used on all other animals. Representative examples from three other animals show the spatial distribution of neurons' BFs (top) and the results of the SVM classification (bottom). R, rostral; L, lateral. Axis arrow length, 200 μm. “Depth from pia” indicates the depth in micrometers relative to the tip of the IC. Note that due to the curvature of the IC the actual distance to the pia may be smaller for imaging areas at some x–y distance away from the tip.
- Figure 4.
Tonotopic arrangement in the DCIC and upper CNIC. A, Histogram of BFs for upper CNIC neurons. B, Histogram of BFs for DCIC neurons. C, Location of upper CNIC neurons in one example animal color-coded by BF and collapsed onto the same coronal plane. D, BF plotted against distance along the dorsoventral axis indicated by the arrow in C. To improve visibility data points have been jittered in the x and y dimension by up to 15 μm and 0.125 octaves, respectively. Red line shows linear fit to nonjittered data. Four outliers with BFs of >10 kHz were excluded from C and D. E, Location of DCIC neurons in one example animal color-coded by BF and collapsed onto the same coronal plane to show variation in BF with depth. F, Location of DCIC neurons from one example animal color-coded by BF and collapsed onto the same horizontal plane. G, BFs plotted against distance along the tonotopic axis indicated by the arrow in F. H, Location of DCIC neurons from another example animal color-coded by BF and collapsed onto the same horizontal plane. I, BFs plotted against distance along the tonotopic axis indicated by the left arrow in H. J, BFs versus the distance along the tonotopic axis indicated by the right arrow in H. K, Directions of tonotopic gradients found in the DCIC of different animals (in one case, shown in H, two opposing frequency gradients were found). The arrow length is proportional to the R value of the projection. The line style indicates the P index (not corrected for multiple comparisons): solid (p < 10−5), dashed (p < 10−4), dotted (p < 0.001), and sparsely dotted (p < 0.01). C, Caudal; R, rostral; L, lateral; M, medial. Scale bars, 50 μm.
- Figure 5.
Receptive field properties of DCIC and CNIC neurons. A, Histogram of BW20. Inset, The mean BW20 as a function of the neurons' BF. B, Histogram of BWmax. Inset, The mean BWmax as a function of the neurons' BF. C, Histogram of reliability coefficients. Upper fluorescence traces (red) show the responses evoked by nine presentations of the best stimulus for a DCIC neuron (same as in Fig. 2E,F). Reliability coefficient = 0.671. Upper fluorescence traces (blue) show the responses evoked by nine presentations of the best stimulus for a CNIC neuron (same as in Fig. 2C,D). Reliability coefficient = 0.740. D, Pie chart of FRA shapes of CNIC neurons. The slices indicate (clockwise) the proportion of O-shaped, I-shaped, and V-shaped FRAs. The small pie chart indicates the proportions of FRA shapes for neurons with BFs of ≤8.4 kHz. The proportions were calculated separately for half-octave wide BF bins and then averaged. E, Same as D for DCIC neurons. Examples for each type of FRA are also plotted next to the corresponding region of the pie charts. Vertical lines above histograms indicate means.
- Figure 6.
Imaging corticocollicular projections. A, Coronal section through the auditory cortex showing neurons transfected with GCaMP6m. Scale bar, 100 μm. B, Coronal section through the IC showing corticocollicular axons in this animal. Scale bar, 200 μm. C, Magnified section from B. Scale bar, 50 μm. D, Large-scale in vivo view from above of corticocollicular axons and terminals. Scale bar, 100 μm. E, Example imaging area. Scale bar, 10 μm. F, Same imaging area as in E with nonresponsive ROIs indicated in gray and responsive ROIs color-coded according to BF. G, Example traces of a corticocollicular bouton response ordered according to frequency and level. The nine traces associated with the nine repeats of a particular stimulus are plotted in gray. The average trace is plotted in black. Scale bars: 1 ΔF/F and 1 s, respectively. H, FRA corresponding to G. I, Cumulative distributions of correlation coefficients between fluorescence traces of pairs of boutons. The median (dotted vertical line) correlation coefficient for all pairs of responding boutons in the dataset is close to zero (gray), suggesting that nonconnected pairs dominate. The median correlation coefficient for pairs of boutons from the same axon is much higher (black). Pairs of boutons belonging to the same axon were identified by visual inspection of structural images such as that shown in E. J, Correlation coefficient matrix of all boutons in one imaging area. Correlated boutons were assigned to clusters of terminals likely belonging to the same axon/neuron (Petreanu et al., 2012) to estimate the number of distinct axons per imaging area. The criterion used for assigning boutons to a cluster was an R value of at least 0.25, which is equivalent to p = 0.05 on the “same-axon pairs” distribution shown in I. D, Dorsal; M, medial; R, rostral.
- Figure 7.
Tonotopic organization of corticocollicular boutons. A, Location of corticocollicular boutons from one example animal color-coded by best frequency (BF) and collapsed onto the horizontal plane. B, BFs of the boutons plotted against distance along the tonotopic axis indicated by the arrow in A. To improve visibility, data points were jittered in the y dimension by up to 0.125 octaves. C, Three of 10 animals showed evidence of a relationship between BF and bouton location. Directions of gradients (black) plotted on top of DCIC gradients (gray) from Figure 4K. The arrow length is proportional to the R value of the projection. The line style indicates the P index (not corrected for multiple comparisons): solid (p < 10−5), dashed (p < 10−4), dotted (p < 0.001), and sparsely dotted (p < 0.01). D, E, Location of corticocollicular boutons color-coded by BF and collapsed onto the same horizontal plane for two other example animals that did not show evidence for tonotopic order. M, Medial; R, rostral. Scale bars, 50 μm.
- Figure 8.
Response properties of corticocollicular boutons. A, Histogram of BFs. Gray broken lines show histogram of BFs of DCIC neurons (from Fig. 4B). B, Histogram of BW20. Results for DCIC neurons are replotted in red (from Fig. 5A). C, Histogram of BWmax. Results for DCIC neurons are replotted in red. (from Fig. 5B). D, Histogram of reliability coefficients. Results for DCIC neurons are replotted in red (from Fig. 5C). Green traces show the responses evoked by nine presentations of the best stimulus of the bouton whose traces and FRA are shown in Figure 6G,H. Reliability coefficient = 0.163. Red traces show the responses evoked by nine presentations of the best stimulus for a DCIC neuron (replotted from Fig. 5C). Reliability coefficient = 0.671. E, Pie chart showing relative proportions of FRA shapes. The slices indicate (clockwise) the proportion of O-shaped, I-shaped, and V-shaped FRAs, together with representative examples of each FRA type. The small pie chart indicates the proportions of FRA shapes for DCIC neurons (from Fig. 5E). Vertical lines above histograms indicate means.
