A brief Illustration of the surgical procedures.

(A) A surface view of the mouse’s skull showing the landmarks of the craniotomy over the IC. (B) An image of the bone flap after finishing the drilling. (C-E) A cartoon and two images of the brain surface showing the insertion and the pocket made by the diamond knife at the lateral surface of the left IC. (F-H) A cartoon and two images of the brain surface showing the insertion of the microprism into its pocket to directly face the LC after displacing the blade of the diamond knife. (I-K) A cartoon and two images of the brain surface showing the final setup of the microprism for two different animals. (L) An image of the brain surface showing the placement of a glass window for the surface imaging of the DC. (M) A cartoon of the coronal section of the IC demonstrating the different fields of views taken along the mediolateral axis parallel to the curvature of the IC surface at different depths (1-5) from the pial surface. Cereb: Cerebellum, Ctx: Cerebral cortex, DC: Dorsal cortex of the IC, IC: Inferior colliculus, ICC: Central nucleus of the IC, Lam. Lambdoid, LC: Lateral cortex of the IC, Sag. Sagittal, SS: Sigmoid sinus, Surf: Surface, TS: Transverse sinus.

The distribution of GABAergic cells on the LC imaged via microprism, and DC imaged from the dorsal surface.

All images were taken for the GFP signals exclusively expressed in GABAergic cells. (A-B) Low magnification of fluorescence images for the GFP signals obtained from the IC surface with or without the microprism, respectively. The dotted white line in B represented the medial and lateral horizons of the IC. (C-E) The 2P images of the GABAergic cells on the LC imaged via microprism showing the GABAergic modules across multiple animals. (D and F) The 2P images of the GABAergic cells on either the dorsolateral surface of the DC imaged directly from its dorsal surface showing the GABAergic modules across multiple animals (red boxes) or its dorsomedial surface showing a homogenous distribution of GABAergic cells of different sizes with no signs of the GABAergic modules across multiple animals (blue boxes). (G) A low magnification fluorescence image of the IC surface with the microprism showing a laser lesion spot made through the microprism to validate the position of the microprism relative to the IC. (H) A histological coronal section at the level of the IC showing that the laser lesion made by the microprism was located at the sagittal surface of the LC imaged via microprism. (I-J) Low-magnification fluorescence images of the IC surface from the dorsal surface show a laser lesion spot made on the dorsolateral surface of the LC on the GABAergic module. The dotted white lines represent the alignment of the laser lesion (dotted red circle) to the location of the GABAergic module at the dorsolateral surface. (K) A histological coronal section at the level of the IC showing that the laser lesion made on the IC surface targeted the GABAergic module at the dorsolateral portion of the LC. All solid irregular white lines were made at the border of the GABAergic modules; D: Dorsal, IC: Inferior colliculus, L: Lateral, R: Rostral. Cereb: Cerebellum, TS: Transverse sinus.

2P expression of jRGECO1a along with evoked signals in the DC imaged from the dorsal surface and LC imaged via microprism.

(A-B) The 2P images of the jRGECO1a expression on the DC imaged from the dorsal surface and the LC imaged via a microprism, respectively. (C-D) The time traces of the evoked calcium signals by either different frequencies and amplitudes of pure tones or different overall amplitudes and rates of AM of broadband noise, respectively, which were obtained from two different cells on the DC (top traces) or the LC imaged via microprism (bottom traces); The length of the colored bars (top) indicates the intensity of the stimulus, while the color indicates the tone or AM frequency depending on the color scale (bottom). AM-noise: Amplitude modulated broadband noise, D: Dorsal, DC: Dorsal cortex, L: Lateral, LC: Lateral cortex, m.prism: Microprism, R: Rostral.

The auditory cellular organization of the LC imaged via microprism vs. DC imaged from the dorsal surface.

(A and C) The 2P images of GFP (green) and jRGECO1a expression (red) on either the LC imaged via the microprism or the DC imaged from the dorsal surface, respectively. (B and D) The pseudocolor images show the responsive cells to the pure tone of different combinations of frequencies and levels on the surface of the LC imaged via the microprism or the DC imaged from its dorsal surface, respectively. (E) The cumulative distribution function of the best tone frequencies of all cells collected either from the LC imaged via microprism (blue line) or the DC imaged directly from its dorsal surface (red line). D: Dorsal, L: Lateral, R: Rostral. (*p<0.05, LC vs. DC).

The cellular response to pure tones and AM-noise of the LC imaged via microprism vs. DC imaged from the dorsal surface.

(A and C) Bar graphs showing the difference in the cellular response to pure tone (tone) vs. AM-noise on the LC imaged via microprism or the DC imaged directly from the dorsal surface, respectively (*p<0.05, pure tone vs. AM-noise). (B) A bar graph showing the difference in the cellular response to the pure tone (tone) and AM-noise of the matrix after normalization against modules imaged via microprism (*p<0.05, matrix vs. modules). (D and E) Bar graphs showing the percentage of spectrally (two-way ANOVA: f(1, 4, 9) = 50.3, 0.0, and 4.0 - p = 7.1×10-7, 1.0, and 0.01 for the region, sound level, and interaction, respectively, Fisher post hoc, p = 5.1×10-6, 4.9×10-4, 0.02, 0.058, and 0.14 for modules vs. matrix at 40, 50, 60, 70, and 80 dB, respectively) or temporally (two-way ANOVA: f(1, 4, 9) = 19.3, 0.0, and 1.2 - p = 7.1×10-7, 1.0, and 0.32 for the region, sound level, and interaction, respectively, Fisher post hoc, p = 2.7×10-7 for modules vs. matrix) modulated cells, respectively, across different sound levels in modules (green bars) vs. matrix (blue bars) imaged via microprism. (*p<0.05, matrix vs. modules). (F and G) box plots showing the mean (black lines) and the distribution (colored dots) of the SMI (two-way ANOVA: f(1, 4, 14583) = 48.4, 210.4, and 21.3 - p < 0.001 for the region, sound level, and interaction, respectively, Fisher post hoc test, p = 6.2×10-4, 4.2×10-5, <0.001, and < 0.001 for LC vs. DC at 40, 60, 70, and 80 dB respectively) and TMI (two way ANOVA: f(1, 4, 16139) = 431.4, 59.1, and 8.3 - p < 0.001 for the region, sound level, and interaction, respectively, Fisher post hoc test, p = 0.002, <0.001, < 0.001, <0.001, and <0.001 for LC vs. DC at 40, 50, 60, 70, and 80 dB, respectively), respectively, across different sound levels in LC imaged via microprism (blue) vs. DC imaged directly from the dorsal surface (red) (*p<0.05, LC vs. DC). LC: Lateral cortex, DC: Dorsal cortex.

The waveforms of the somatosensory vs. acoustic responses of the LC imaged via microprism.

(A-B) The time traces of the evoked calcium signals by the beginning of auditory or somatosensory stimuli, respectively (onset responses). (C-D) The time traces of the evoked calcium signals by the termination of auditory or somatosensory stimuli, respectively (offset responses).

Types of somatosensory and auditory responses from the LC neurons imaged via microprism.

(A) The 2P images of GFP (green) and jRGECO1a expression (red) in the LC imaged via microprism showing the modules within white irregular lines. (B, D, and F) The pseudocolor images show the auditory or somatosensory responsive cells of the LC imaged via microprism as well as the merged response, respectively (C and E) The pseudocolor images show onset (ON), offset (OFF), or onset/offset (ON/OFF) responsive cells of the LC imaged via microprism to auditory and somatosensory stimuli, respectively. (G) The pseudocolor images show enhanced and suppressive response of the auditory responsive cells of the LC imaged via microprism following the bimodal stimulation based on their response index (RI). (H and I) Bar graphs showing the percentage of responsive cells to auditory (Aud.), somatosensory (Som.), or both stimuli in modules and matrix imaged via microprism, respectively – Modules: one-way ANOVA: f(2, 11) = 38.1 - p = 4.0×10-5, Fisher posthoc test, p = 1.9×10-5 or 7.6×10-5 for the percentage of responsive cells to sound only vs. the percentage of responsive cells to somatosensory stimulation only or to both, respectively and p = 0.23 for the percentage of responsive cells to somatosensory stimulation only vs. both, Matrix: one-way ANOVA: f(2, 11) = 23.9 - p = 2.4×10-4, Fisher posthoc test, p = 1.7×10-4 or 2.4×10-4 for the percentage of responsive cells to sound only vs. the percentage of responsive cells to somatosensory stimulation only or to both, respectively - p = 0.77 for the percentage of responsive cells to somatosensory stimulation only vs. both) (*p<0.05, vs. Aud.). (J) A box plot showing the normalized cell fraction of auditory (Aud.) (matrix median = 1.05) or somatosensory (Som.) (matrix median = 0.14) responsive cells in the matrix normalized to those in modules imaged via microprism (*p<0.05, matrix vs. modules). (K) A box plot showing the cell fraction of onset (ON. Aud.) (matrix median = 2.2) or offset (OFF. Aud.) auditory (matrix median = 0.08) responsive cells in the matrix normalized to those in modules imaged via microprism (*p<0.05, matrix vs. modules). (L) A box plot showing the cell fraction of the responsive cells of enhanced (Enh. resp.) or suppressive (Supp. resp.) response in the matrix normalized to those in modules imaged via microprism following bimodal stimulation (*p<0.05, matrix vs. modules). D: Dorsal, R: Rostral.

The somatosensory vs. auditory responses over the DC neurons.

(A) A low magnification of the image obtained from the IC surface. (B-D) The top row: The 2P images of GFP (green) and jRGECO1a expression (red) of three main regions (dorsolateral, dorsal, and dorsomedial) of the DC surface imaged from the dorsal surface showing the modules within irregular white lines in the dorsolateral region, the bottom row: The pseudocolor images show the somatosensory responsive cells after somatosensory stimulations. (E) The pseudocolor images show the responsive cells of the LC imaged from the dorsal surface to auditory and somatosensory stimulations. (F) The pseudocolor images show onset (ON), offset (OFF), or onset/offset (ON/OFF) responsive cells of the LC imaged from the dorsal surface to auditory stimulation. (G) The pseudocolor images show the enhanced and suppressive response of the auditory responsive cells of the LC imaged from the dorsal surface following the bimodal stimulation based on their response index (RI). (H) A box plot shows the percentage of somatosensory responsive cells in LC imaged either from the dorsal surface (LC (dors.)) or via microprism (LC (m. p.)) as well as the DC - one-way ANOVA: f(2, 11) = 6.7 & p = 0.02, Fisher posthoc test, p = 0.01 or 0.01 for the percentage of somatosensory responsive cells on the DC vs. LC imaged by microprism or LC imaged from the dorsal surface, respectively - p = 0.95 for the percentage of somatosensory responsive cells on LC imaged via microprism vs. LC imaged from the dorsal surface) (*p<0.05, vs. DC). (I) A box plot showing the cell fraction of auditory (Aud.) or somatosensory (Som.) responsive cells in the matrix normalized to those in modules imaged from the dorsal surface (*p<0.05, matrix vs. modules). (J) A box plot showing the cell fraction of onset (ON. Aud.) or offset (OFF. Aud.) auditory responsive cells in the matrix normalized to those in modules imaged from the dorsal surface (*p<0.05, matrix vs. modules). (K) A box plot showing the cell fraction of the responsive cells of enhanced (Enh. Resp.) or suppressed (Supp. Resp.) responses in the matrix normalized to those in modules imaged from the dorsal surface following bimodal stimulation (*p<0.05, matrix vs. modules). Cereb: Cerebellum, Ctx: Cerebral cortex, M: medial, R: Rostral, TS: Transverse sinus.

The acoustic response from LC imaged via microprism and DC imaged from the dorsal surface in awake animals.

(A and C) The 2P images of GFP (green) and jRGECO1a expression (red) on either the LC surface imaged via the microprism or the DC imaged directly from the dorsal surface, respectively showing the modules within irregular white lines. (B and D) The pseudocolor images show the responsive cells to the pure tone of different combinations of frequencies and levels of the LC imaged via the microprism or the DC imaged directly from the dorsal surface, respectively. (E) The cumulative distribution function of the best tone frequencies of all cells collected either from the LC imaged via microprism (blue) or the DC (red). (F) A bar graph showing the percentage of responsive vs. nonresponsive cells to pure tone or AM-noise within the LC imaged via microprism or the DC (*p<0.05, pure tone vs. AM-noise). (G) A bar graph showing the percentage of responsive cells to pure tone or AM-noise for LC imaged via microprism vs. DC (*p<0.05, LC vs. DC). (H) A bar graph showing the percentage of responsive cells to pure tone or AM-noise for modules vs. matrix imaged via microprism(*p<0.05, matrix vs. modules). D: Dorsal, L: Lateral, Non-resp.: Non-responsive, Resp.: Responsive, R: Rostral.

The somatosensory response from LC imaged via microprism in awake animals.

(A, C, and E) The pseudocolor images show the auditory or somatosensory responsive cells of the LC imaged via microprism to auditory and somatosensory stimulations as well as the merged response, respectively. (B and D) The pseudocolor images show onset (ON), offset (OFF), or onset/offset (ON/OFF) responsive cells of the LC imaged via microprism to auditory and somatosensory stimulations, respectively. (F) The pseudocolor images show the enhanced or suppressed responses of the auditory responsive cells following the bimodal stimulation based on their response index (RI). (G) A bar graph showing the cell fraction of auditory (Aud.) or somatosensory (Som.) responsive cells in the matrix vs. modules imaged via microprism (*p<0.05, matrix vs. modules). (H) A bar graph showing the cell fraction of the responsive cells of suppressed (Supp. resp.) or enhanced (Enh. resp.) response in the matrix vs. modules imaged via microprism following bimodal stimulation (*p<0.05, low vs. high response). D: Dorsal, R: Rostral.