Figure 3. Tonotopic remapping within the cortical deafferentation zone revealed by chronic mesoscale and cellular calcium imaging.

(A) Top, approach for widefield calcium imaging using a tandem lens epifluorescence microscope in awake Thy1-GCaMP6s × CBA/CaJ mice that express GCaMP6s in pyramidal neurons (PyrNs). Bottom, schematic depicts the typical arrangement of individual fields within the ACtx based on tonotopic best frequency (BF) gradients (as detailed in Romero et al., 2019). (B) Chronic widefield BF maps in example sham and trauma mice (top and bottom, respectively) show BF remapping within the deafferented high-frequency regions throughout the ACtx after acoustic trauma. (C) Left, approach for chronic two-photon calcium imaging of layer 2/3 PyrN’s along the A1 tonotopic gradient. Right, two-photon imaging field-of-view superimposed on the widefield BF map measured in another example mouse. (D) Top, example of tone-evoked GCaMP transients measured as the fractional change in fluorescence and deconvolved activity. Bottom, peak deconvolved amplitudes for tones of varying frequencies and levels are used to populate the complete frequency-response area and derive the BF (downward arrow) and threshold (leftward arrow) for each neuron. (E) BF arrangements in L2/3 PyrNs measured at three times over the course of a month in representative sham and trauma mice. A support vector machine (SVM) was trained to bisect the low- and high-frequency zones of the A1 BF map (LF [<16 kHz] and HF [≥16 kHz], respectively). The dashed line represents the SVM-derived boundary to segregate the LF and HF regions. The SVM line is determined for each mouse on day −4 and then applied to the same physical location for all future imaging sessions following alignment. (F) Timeline for chronic two-photon imaging and cochlear function testing in each sham and trauma mouse. (G) Individual PyrNs are placed into five distance categories based on their Euclidean distance to the SVM line and the BF of each category is expressed as the median ± bootstrapped error. Following trauma, BFs in the HF zone are remapped to sound frequencies at the edge of the cochlear lesion. (H) Across all tone-responsive PyrNs measured at three time points, the percent of neurons with BFs corresponding to edge frequencies (11.3–16 kHz) was greater in trauma mice (N = 4 mice, n = 1749 PyrNs) than sham (N = 4 mice, n = 1748 PyrNs), was greater in the deafferented HF region than the intact LF region, and increased over time in trauma mice compared to sham controls (three-way analysis of variance [ANOVA] with Group, Region, and Time as factors: main effect for Group, F = 34.29, p = 5 × 10^–9; Group × Region interaction term, F = 7.42, p = 0.007; Group × Time interaction term, F = 10.17, p = 0.00004). (I) Competitive expansion of edge frequency BFs in the deafferented HF zone was not accompanied by a change in neural response threshold (three-way ANOVA: main effect for Group, F = 0.8, p = 0.37; Group × Region interaction term, F = 0.93, p = 0.33; Group × Time interaction term, F = 1.33, p = 0.27).