DOI: 10.5061/dryad.v41ns1s70
title: Data from: Microprism-based two-photon imaging of the mouse inferior colliculus reveals novel organizational principles of the auditory midbrain

description: To navigate real-world listening conditions, the auditory system relies on
 the integration of multiple sources of information. However, to avoid
 inappropriate cross-talk between inputs, highly connected neural systems
 need to strike a balance between integration and segregation. Here, we
 develop a novel approach to examine how repeated neurochemical modules in
 the mouse inferior colliculus lateral cortex (LC) allow controlled
 integration of its multimodal inputs. The LC had been impossible to study
 via imaging because it is buried in a sulcus. Therefore, we coupled
 two-photon microscopy with the use of a microprism to reveal the
 first-ever sagittal views of the LC to examine neuronal responses with
 respect to its neurochemical motifs under anesthetized and awake
 conditions. This approach revealed marked differences in the acoustic
 response properties of LC and neighboring nonlemniscal portions of the
 inferior colliculus. In addition, we observed that the module and matrix
 cellular motifs of the LC displayed distinct somatosensory and auditory
 responses. Specifically, neurons in modules demonstrated primarily offset
 responses to acoustic stimuli with enhancement in responses to bimodal
 stimuli, whereas matrix neurons showed onset responses to acoustic stimuli
 and suppressed responses to bimodal stimulation. Thus, this new approach
 revealed that the repeated structural motifs of the LC permit functional
 integration of multimodal inputs while retaining distinct response
 properties.
description: <strong>Animal subjects:</strong>
 Male mice of 8-12 weeks of age were used. Males were used as our
 preliminary data suggest male/female differences in DC organization
 (Ibrahim, Shinagawa, Xiao, Asilador, & Llano, 2022) and will be
 published separately. GAD67-GFP (GAD1GFP) knock-in mice (developed and
 shared with permission from Dr. Yuchio Yanagawa at Gunma University and
 obtained from Dr. Douglas Oliver at the University of Connecticut), where
 GFP is exclusively expressed in GABAergic cells (Tamamaki et al., 2003),
 were used to visualize the GABAergic cells and LC modules. To
 simultaneously monitor calcium signals and visualize the GFP+ cells,
 Tg(Thy1-jRGECO1a)GP8.20Dkim/J mice (Jackson Laboratory, Stock# 030525)
 were crossed with GAD67-GFP knock-in mice to generate
 GAD67-GFPxThy1-jRGECO1a hybrid mice. The mice were housed and bred at the
 animal research facility of The Beckman Institute for Advanced Science and
 Technology. The animal care facilities are approved by the Association for
 Assessment and Accreditation of Laboratory Animal Care (AAALAC). To
 phenotype the GFP-positive animals, a single-photon fluorescence
 microscope was used for the transcranial examination of the pups at
 postnatal day 4 using excitation: 472/30 nm and emission 520/35 nm filters
 along with dichroic 505 nm long pass at low power magnification
 (2.5x/0.08, Olympus objective, MPlanFL N, Japan). The positive pups
 exhibited a green fluorescence in the cerebellum, the cerebral cortex, and
 the olfactory bulb and were only kept with their parents until weaning.
 The genotyping for jRGECO1a was done using a probe composed of a forward
 primer: GCCGCCGAGGTCAAGA & a reverse primer:
 TCCAACTTGATGTCGACGATGTAG) by Transnetyx™ (Transnetyx, USA). Samples of the
 mice's tail snips around one month of age were used for genotyping.
 All applicable guidelines for the care and use of animals were followed.
 All procedures were approved by the Institutional Animal Care and Use
 Committee (IACUC). 
 <strong>Surgery</strong> The
 general procedures of craniotomy were previously described (Goldey et al.,
 2014), with modifications to place craniotomy over the IC. Before surgery,
 mice were anesthetized with a mixture of ketamine, xylazine, and
 acepromazine (100 mg/kg, 3 mg/kg, and 3mg/kg, respectively) delivered
 intraperitoneally. The anesthesia was maintained during the surgery and
 imaging using only ketamine (100 mg/kg). To prevent neural edema during or
 after the craniotomy, an intramuscular injection of dexamethasone sodium
 (4.8 mg/kg) was given just before the surgery using an insulin syringe.
 After placing the animal in the stereotaxic apparatus (David Kopf
 Instruments, USA), both eyes were protected by applying Optixcare
 lubricant eye gel (Aventix Animal Health, Canada). The hair on the scalp
 was then removed by massaging the scalp with a depilatory cream (Nair)
 using a cotton-tipped applicator and leaving the cream on the scalp for
 4-5 minutes. The cream was then removed by a thin plastic sheet (flexible
 ruler) to leave a hair-free area on the scalp. The remaining tiny hairs
 were then removed by alcohol swab and the area was then sterilized by
 applying 10% Povidone Iodine (Dynarex, USA) using a sterile cotton-tipped
 applicator. The medial incision was made with a scalpel blade #10, and 0.2
 ml of 0.5% lidocaine was injected intradermally into the scalp. The skin
 extending from the medial line to the temporalis muscle was completely
 removed using a pair of microscissors to produce a wide skinless area
 above the skull. Number 5/45 forceps were used to remove any remaining
 periosteum. The remaining dried or firmly attached pieces of periosteum
 were removed with a scalpel blade #10. The skull was cleaned with sterile
 saline and dried with gently pressurized air. Using the stereotaxic
 apparatus, a wide area of ~3 x 4 mm above the left IC was made. A micro
 drill bit (size #80, Grainger, USA) was used to drill through the skull
 starting from the rostrolateral (Figure 1A, yellow circle) region to
 lambda (Figure 1A, black circle) following the border in a clockwise
 direction (Figure 1A, dotted black rectangle). To prevent overheating of
 the superficial brain tissues and to mitigate the occasional spurts of
 skull bleeding during the drilling, ice-cold sterile saline was used to
 intermittently irrigate the surface. A stream of pressurized air was also
 applied during the drilling procedures to prevent overheating and remove
 the debris produced by the drilling. Caution was taken not to pierce the
 dura when performing the craniotomy while crossing the sagittal or the
 lambdoid sutures to avoid damaging the underlying sinuses. After drilling,
 the skull was irrigated in sterile saline and the bone flap (the undrilled
 bone over the craniotomy area) was gently examined for complete separation
 from the rest of the skull (Figure 1B). Using a pair of no. 5/45 forceps,
 the bone flap was gently removed. To control the bleeding if it occurred,
 a piece of sterile hemostatic gel (Adsorbable Gelatin Sponge USP,
 Haemosponge, GBI, India) which was pre-soaked in ice-cold saline, was
 applied to the bleeding spot. Once the bleeding ceased, the brain was kept
 covered in sterile saline. In some surgeries, the dura was peeled off from
 the surface of the IC while removing the bone flap. In the surgeries where
 the dura remained intact, a Bonn microprobe (F.S.T, Germany, Item #
 10032-13) was used to pierce the dura in an area that is not above the IC
 and devoid of cortical vasculature (e.g., a part of exposed cerebellum).
 After piercing, the dura was carefully and gently lifted, and a pair of
 no. 5/45 forceps were used to grab the dura to gently tear it to the
 extent of the transverse sinus to avoid bleeding of the major venous
 structures.  Using the stereotaxic apparatus, a 30
 degree diamond knife (F.S.T, Germany, Item # 10100-30) was placed in its
 vertical position. From the medial line, the knife was placed at the
 lateral horizon of the IC. The knife was then inserted 1.5 mm ventrally.
 Caution was taken to avoid hitting any major blood vessels to avoid
 bleeding. From its position, the knife was retracted 0.3-0.5 mm medially
 to create an initial pocket for the insertion of the microprism (Figures
 1C-E). The microprism (1.5 mm-silver coated, Tower Optical Corporation)
 was then inserted, so one of its sides was parallel to the lateral surface
 of the diamond blade, the other side was facing up, and its hypotenuse was
 facing ventrally (Figures 1F and G). The wooden piece of a cotton swab was
 carefully driven down to push the microprism until its top surface was at
 the same level as the IC dorsal surface. Keeping the microprism in its
 place with the wooden end of a swab, the diamond knife was carefully
 retracted upward leaving the microprism behind (Figure 1H). If the
 retraction of the diamond knife pulled the prism up, the wooden piece
 would be used to push it down simultaneously with the knife movement. To
 control the bleeding, a piece of sterile hemostatic gel was used as
 before. The tip of the diamond knife was used to secure the microprism in
 its position for at least 10 minutes (Figure J). During that time, the
 solution of the 1% agarose was prepared for use. A pair of forceps were
 then used to gently place a sterile 5 mm cover glass #1 (Thomas
 Scientific, USA) over the skull covering the opened area above the IC. The
 cover glass was secured by a wooden trimmed piece of sterile cotton swab
 by gently pressing the cover glass from the top. Since the surface of the
 IC was located below the surfaces of both the cerebellum and cerebral
 cortex, the cover glass usually traps about a 0.7-1.0 mm thick sheet of
 sterile saline. The movement of the liquid medium caused by heart
 pulsation was found to be a source of motion artifact during imaging, so
 the gap between the surface of the IC and the cover glass was filled with
 1% agarose gel that was made in saline at a temperature range of 33- 35 °C
 (Figure 1K). The extra agarose gel pieces were cut off and removed by the
 scalpel blade, and the wet skull was then dried out using pressurized air.
 A titanium headpost as described before (Goldey et al., 2014) was glued
 carefully on the top of the skull to be at the same level as the cover
 glass. Following the manufacturer's instructions, the C&B
 Metabond (Parkell, Japan) was used to secure the headpost in its place.
 Excluding the steps of the microprism insertion, the same procedures were
 followed for DC imaging from the surface (Figure 1L). For awake
 preparations, the same procedures were followed under isoflurane
 anesthesia starting with 4% of isoflurane as an induction dose with 1-2%
 isoflurane during the surgery. The animal was transferred under the
 microscope objective and left to recover from isoflurane anesthesia for 1
 hour before imaging. To ensure the animal’s recovery, the animal was
 visually inspected for its breathing rate (Ewald, Werb, & Egeblad,
 2011) and ongoing limb movements.
 <strong>Acoustic stimulation:</strong>
 Using a custom-made MATLAB (The MathWorks, Natick, MA, USA) code,
 either 500 ms pure tone or AM-noise was generated. Thirty-five (5x7) pure
 tones with a series of sound pressure levels (40, 50, 60, 70, 80 dB SPL)
 and a series of carrier frequencies (5000-40000 Hz with a half-octave gap)
 were presented with a cosine window. Forty-five (5x9) 100% AM white noise
 bursts with a series of sound pressure levels (40, 50, 60, 70, 80 dB SPL)
 and modulation frequencies (0, 2, 4, 8, 16, 32, 64, 128, 256 Hz) were
 generated. The stimuli of either pure tone or AM-noise combinations were
 played in random sequence to the mice with a 600 ms interstimulus interval
 (ISI) by a TDT RP2.1 processor (Tucker-Davis Technologies, US) and
 delivered by a TDT ES1 speaker (Tucker-Davis Technologies, US). In other
 sets of experiments, 500 ms of unmodulated broadband noise was presented 5
 times with ISI of 600 ms at 80 dB SPL to examine the acoustic activity
 between modules and matrix compared to somatosensory stimulation.
   The output of the TDT ES1 speaker was calibrated
 using a PCB 377A06 microphone, which feeds a SigCal tool to generate a
 calibration file for all tested frequencies (5–40 kHz). To enable the
 custom-made MATLAB code to read this calibration file, the values were
 first processed by MATLAB signal processing toolbox (sptool) to generate a
 256-tap FIR filter to apply the calibration using the following parameters
 [arbitrary magnitudes, least square, order: 256, sampling rate: 97,656.25,
 frequency vector (5–40 kHz), amplitude vector (40–80 dB SPL), and weight
 vector ones (1128)].<br>Somatosensory stimulation: 
 The somatosensory stimulation was done by deflecting the right
 whiskers of the animals at an average excursion of 1.2 mm with a 50 Hz
 rate over 500 ms to match the acoustic stimulus. A total of five stimuli
 were presented with 600 ms ISI. The whisker deflection was performed using
 a Brüel & Kjær vibrator (V203 10/32 UNF- CE, United Kingdom) that
 was driven by a wave generator (4063, BK precision, USA) after amplifying
 the signals with an SLA1 100 W power amplifier (ART, USA). 
 <strong>Two-Photon imaging: </strong>
 Immediately after surgery, the anesthetized animal was taken and
 secured under the microscope objective by clamping the arms of the head
 post to two perpendicular metal posts mounted on the microscope stage. A
 custom-built 2P microscope was used. The optical and the controlling
 components were supplied from Bruker, Olympus, and Thorlabs. The imaging
 of the DC was made using a 20x water-immersion objective (LUMPlanFI/IR,
 20X, NA: 0.95, WD: 2 mm; Olympus Corporation, Tokyo, Japan), while the
 lateral surface of the IC was imaged by the long working distance 16x
 water-immersion objective (N16XLWD-PF - CFI LWD Plan Fluorite Objective,
 16X, NA: 0.8, WD: 3.0 mm; Nikon, Tokyo, Japan) to be able to reach the
 focal point of the microprism at its hypotenuse. For imaging both the GFP
 or jRGECO1a signals, the excitation light was generated by InSight X3
 laser (Spectra-Physics Lasers, Mountain View, CA, USA) tuned to a
 wavelength of 920 or 1040 nm, respectively. A layer of a 1:1 mixture of
 wavelength multipurpose ultrasound gel (National Therapy, Canada) with
 double-deionized water was used to immerse the objective. This gel was
 able to trap the water and reduce its evaporation during imaging. The
 emitted signals were detected by a photomultiplier tube (Hamamatsu
 H7422PA-4, Japan) following a t565lp dichroic and a Chroma barrier
 et525/70m filter for GFP and et595/50m filter for jRGECO1a signals. Images
 (512x512 pixels) were collected at a frame rate of 29.9 Hz in the resonant
 galvo mode. Data were collected from the dorsal surface of the IC by
 scanning the surface of the IC based on the GFP and jRGECO1a signals
 through the medial and lateral horizons of the IC. Generally, the scanning
 was started by moving the 20x objective to the most ventromedial position,
 where there is no ability to see any cells (Figure 2B, medial horizon).
 Then, the objective was driven to slowly move dorsolaterally to get cells
 in focus (Figure 1M), where the average depth of the first field of view
 was 169 m. The lateral movement of the objective was intended to be
 aligned and parallel to the curvature of the DC surface indicated by
 getting the superficial cells in focus to avoid any possibility of imaging
 the upper layers of the central nucleus of the IC, so the average depth of
 the most superficial point of the imaging was 92 m. The scanning was
 ended by moving the objective to the most ventrolateral position of the
 IC, where there is no ability to see GFP laterally (Figure 2B, lateral
 horizon), where the last field of view was collected at an average depth
 of 225 m. Generally, each field of view was selected based on the
 expression of GFP signals as an indicator for the cells in focus and being
 acoustically active by using a search stimulus that was 500 ms broadband
 noise with zero modulation at 80 dB SPL. The z-axis of the objective was
 determined relative to the pia surface. The relative position of each
 region was tracked through the micromanipulator (MP-285, Sutter
 Instruments) that controlled the microscope objective. The positions of
 imaged areas were further aligned across animals to a common coordinate
 using the midline, lateral extremes, and the vasculature landmarks taken
 via imaging of the GFP signals of the cranial window with low
 magnification (4x Olympus objective, 4x4 binning, 50 ms frame time) using
 a Lumen 200 bulb (Prior Scientific Inc, USA) and CoolSNAP MYO camera
 (Photometrics, USA, excitation: 488 nm and emission: 515–550 nm). Imaging
 of the lateral surface of the IC via microprism was done similarly. In
 both cases, the frame timing of the scanner and the sound stimuli were
 both digitized and time-locked using a Digidata 1440A (Molecular Devices,
 Sunnyvale, CA, USA) with Clampex v. 10.3 (Molecular Devices, Sunnyvale,
 CA, USA).  <strong>Laser
 lesion:</strong> The laser lesion was produced
 using a 720 or 820 nm laser beam at 74-80 mW power at the level of the
 objective for 100 to 250 seconds depending on the size of the lesioned
 area, the speed of laser scanning (frame rate), and the depth of the
 lesion (z-axis length). In brief, the area of interest and the dimension
 of the laser lesion were initially determined using 920 nm as indicated by
 the GFP signals through scanning one of the GABAergic modules. The depth
 of the GABAergic modules was then determined by counting the optical
 slices starting from their superficial surface. The tunable laser was then
 switched from 920 nm to the shorter wavelength (720 or 820 nm) to scan the
 same area and depth at a rate (1-2 frames/ second).
 <strong>Data processing:</strong>
 Data collection: The data were collected as separate movies
 (512x512 pixels) for each pure tone or AM-noise runs in a resonant galvo
 mode. Depending on the amplitude and frequency combinations for each type
 of acoustic stimulus, 40- or 52-second periods were assigned as a movie’s
 length for pure tone (35 stimulus combinations), or AM-noise (45 stimulus
 combinations), respectively. Using ImageJ software, the z-projection was
 used to compute one single image representing either the sum, the standard
 deviation, or the median of all the image sequences in the movie. Based on
 these single images, the region of interest (ROI) was manually drawn
 around each detectable cell body. Figures 3A and B represent the sum of
 the image sequences of the movie collected from the dorsal surface, or the
 lateral surface of the IC via microprism, respectively. For the subsequent
 processing steps, Python open-source libraries were used (listed
 below). Motion Correction and Filtering: The imread
 function from the OpenCV library was used in grayscale mode to import
 images into a numpy array from a single folder containing TIFF images. The
 array was then exported as a single TIFF stack into a temporary folder
 using the mimwrite function from the imageio library, and the process was
 repeated for each folder. The NoRMCorre algorithm (Pnevmatikakis &
 Giovannucci, 2017) embedded in the CaImAn library (Giovannucci et al.,
 2019) was used to apply motion correction to each of the TIFF files. The
 data were then imported into a numpy array, rounded, and converted to
 16-bit integers. The images were filtered using a 2D Gaussian filter with
 a sigma value of 1 (surface View) / 2 (prism View) in each direction, then
 a 1D Gaussian temporal filter with a sigma value of 2 was applied using
 the ndimage.gaussian_filter and ndimage.gaussian_filter1d function from
 the scipy library, respectively. Data Extraction: The
 ROI sets, which were manually created using ImageJ, were imported using
 the read_roi_zip function from the read_roi library. The sets were then
 used to create two masks; one mask was used as a replica of the ROIs and
 the second mask was made around the original ROI (roughly four times
 larger in area). The smaller mask was applied to find the average pixel
 value within each ROI, while the larger mask was applied to find the
 average pixel value of the neuropil. The neuropil correction was applied
 using the following equation (Akerboom et al., 2012; Kerlin, Andermann,
 Berezovskii, & Reid, 2010);<br>Corrected value=Date
 value-(0.4 X Neuropil value). To identify the calcium signals, df/f was
 calculated by using the following equation. <br>Δf/f=((Date
 value-Background value))/(Background value )   where the background value
 is the slope estimating the background levels with fluorescence bleaching
 factored in. The data were then reorganized so that all segments with the
 same stimulus frequency and stimulus amplitude were grouped. The area
 under the curve (AUC) of the calcium signals of the excitatory responses
 only was then used as a metric to determine the magnitude of the response
 and for subsequent analysis (Berens et al., 2018; Overk, Rockenstein,
 Florio, Cheng, & Masliah, 2015). Cell Flagging:
 The correlation coefficient between each of the trials was calculated
 using the stats. Pearson function from the scipy library. The average
 correlation coefficient was calculated for each stimulus frequency and
 stimulus amplitude. Similar to previous work (A. B. Wong & Borst,
 2019), if the average correlation coefficient was above the threshold of
 0.6, the cell was flagged as being responsive to that stimulus combination
 of frequency and amplitude, and the best tone and modulation frequencies
 were calculated for every cell (Barnstedt et al., 2015). Knowing BTF and
 the best modulation frequency (BMF) for each neuron enabled us to
 calculate the SMI and TMI at each sound level following these equations;
  <br>SMI<br>=(Response to BTF-Response to unmodulated
 noise)/(Response to BTF+Response to unmodulated noise)<br>The SMI
 has values ranging from (-1 to 1). While neurons with values closer to 1
 are more responsive to pure tone, neurons with values closer to -1 are
 more responsive to noise with no
 modulation.<br>TMI<br>=(Response to BMF-Response to
 unmodulated noise)/(Response of BMF+Response to unmodulated
 noise)<br>Similarly, the TMI has values ranging from (-1 to 1).
 While neurons with values closer to 1 are more responsive to AM-noise,
 neurons with values closer to -1 are more responsive to noise with no
 modulation.<br>For the comparison between somatosensory and auditory
 responses, the threshold of 0.4 was used as a cutoff threshold to
 determine the responsive cells to each stimulation. The average response
 of each somatosensory, acoustic (500 ms of unmodulated noise), or
 simultaneous somatosensory and acoustic (bimodal) stimulation was then
 calculated for each responsive cell. The correlation method was validated
 as a tool to determine the responsive cells to somatosensory stimulation
 by generating an ROC curve comparing the automated method to a blinded
 human interpretation. The AUC of the ROC curve was 0.88. This high AUC
 value indicates that the correlation method can rank the random responsive
 cells better than the random nonresponsive cells. At the correlation
 coefficient (0.4), which was the cutoff value to determine the responsive
 cells for somatosensory stimulation, the specificity was 87% and the
 sensitivity 72%, the positive predictive value was 73%, and the negative
 predictive value was 86%. Additionally, all the false-responsive cells
 were excluded from the analysis. To examine the effect of somatosensory
 stimulation on auditory responses, the response index (RI) was calculated
 for the auditory-responsive cells using the following
 equation; <br>RI  =(Auditory response to bimodal
 stimulation)/(Auditory response to auditory stimulation only) Reurons with
 RI values &gt; 1 have enhanced or higher responses induced by
 simultaneous auditory and somatosensory stimulation, while neurons with RI
 values &lt; 1 have downregulated or suppressed auditory responses
 induced by auditory and simultaneous somatosensory
 stimulation.<br>Map Generation: The average radius for all ROIs was
 calculated to ensure that all cells on the tonotopic map had uniform
 radii. A color key was also generated for every corresponding map. A blank
 canvas was generated using the Image module from the pillow library, and a
 circle was created for each cell and filled with a corresponding color to
 its value. Receptive field sum (RFS) analysis: The
 binary RFS was calculated for every cell by determining whether the cell
 was responsive to a particular combination of frequency and amplitude
 (Bowen et al., 2020). A value of 1 was given to the responsive cell, if it
 responds to a single frequency/amplitude combination. In contrast, a value
 of 1 was added to the tally for each responsive combination if the cell
 was broadly responsive. Accordingly, the higher and lower values of RFS
 indicate a broad and narrow RF, respectively. To examine the differences
 between the RFS of the cells at different areas under different
 preparations, the cumulative distribution function was used. 
 <strong>Assessment of tonotopic
 organization.</strong> The tonotopy was assessed
 by examining the best fit of linear or nonlinear quadratic polynomial
 regressions between the BTFs of cells and their locations along different
 anatomical axes. For the LC(microprism), the regression fit was examined
 along the main four axes such as dorsal to ventral, rostral to caudal,
 dorsocaudal to ventrorostral, and dorsorostral to ventrocoudal. Based on
 the previous reports showing the tonotopic organization over the DC
 (Barnstedt et al., 2015; A. B. Wong & Borst, 2019), the tonotopy
 was examined along the rostromedial to caudolateral axis. The x2 and y2
 coordinates of each cell were determined. At the starting point of each
 axis, the x1 and y1 coordinates were assigned values as (0, 0). Then the
 distance (d) between each cell and the starting point at each axis was
 calculated based on the following
 equation; <br>d=√((x2-x1)^2+(y2-y1)^2 )<br>For each 50 um (bin
 size), the geometric average of the BTFs of all cells as well as their
 distances from the starting point at each axis were computed. The linear
 or quadratic polynomial regressions were then examined between the
 geometric averages of BTFs and the geometric averages of distances of
 cells for each animal. <strong>Detecting the
 spontaneous activity from the nonresponsive
 cells:</strong> Movies have been recorded from
 the nonresponsive cells of the LC(microprism) when no stimulation was
 presented. The time trace of jRGECO1a signals was extracted from each
 cell. A polynomial fitting of the time trace was used to correct for
 photobleaching and to adjust for the baseline for the whole trace. The
 sliding average of the baseline was then calculated for 1 second (~29
 frames), and the z score was calculated for the subsequent frames. The
 spontaneous activity was detected when the z-score was &gt;
 3.0. <br>Statistics:<br>All statistical analyses and graphs
 were made using Origin Pro software. The normality of the distributions of
 the data was examined using a Kolmogorov-Smirnov test. The Mann-Whitney
 test as well as two-sample or paired t-test were used to examine the
 difference in data distribution or the difference between samples of
 non-normal and normal distribution, respectively. Either one-way or
 two-way ANOVA as well as Fisher as a post hoc test were used for the mean
 comparison between different groups of normal distribution.  The
 Chi-square () test or Kruskal-Wallis ANOVA tests were used to compare
 the samples based on cell numbers of their cellular population. The
 cumulative frequency function was computed to calculate the fraction of
 cells tuned at each frequency based on their best frequency. Differences
 were deemed significant when p-value &lt;0.05. The upper and lower
 borders of each box plot are at the 25th and 75th percentile, with
 whiskers at 95% spread.
 <strong>Histology:</strong> After
 the 2P imaging, the mouse was transcardially perfused with 4%
 paraformaldehyde (PFA) solution in PBS solution. The isolated brain was
 then placed overnight in PFA for a complete fixation. The brain was then
 moved from the PFA to a graded sucrose solution (10, 20, and 30%) until it
 sunk. 50 m coronal sections were taken at the level of the IC using a
 Leica cryostat. The sections were then mounted on a slide, coverslipped,
 and imaged using a confocal microscope for imaging the GFP signals as an
 indicator for the GABAergic cells in the GAD67-GFP knock-in mouse
 (excitation: 488 nm and emissions: 515–550 nm). <br>For the
 examination of jrgeco1a expression in the IC, the mouse was transcardially
 perfused with saline with a high concentration of potassium and a high
 ratio of calcium to magnesium [(in mM) 3.5 KCl, 25 glucose, 125 NaCl, 1.2
 KH2PO4, 2 CaCl2, 0.5 MgSO4] under ketamine anesthesia. The brain was then
 fixed by a subsequent perfusion of 4% PFA. Selected 40 m coronal sections
 at the level of the IC were immunostained for NeuN using (NeuN (D4G40) XP®
 Rabbit mAB, Cell Signaling, Cat# 62994, 1:100) primary antibody and (goat
 anti-rabbit IgG conjugated to Alexa 405, 1:200) secondary antibody
 following the immunostaining procedures that were previously described
 (Issa, Sekaran, & Llano, 2023; Vaithiyalingam Chandra Sekaran et
 al., 2021). Confocal images were obtained using a Confocal Zeiss LSM 710
 Microscope using 10 and 40x objectives and tile scan functions. For NeuN,
 Alexa 405 signals were obtained by a 405 nm laser and emission at 415-466
 nm. The GFP signals were obtained by a 488 nm laser and emission at
 501-551 nm. The jRGECO1a signals were obtained by 561 nm laser and
 emission at 600-700 nm. All cells with somas expressing a red fluorescence
 of jrgeco1a were quantified in 2P-imaged areas of the DC and LC. The
 density of the cells expressing jrgeco1a was then calculated based on the
 cells expressing NeuN.
 <strong>Artwork:</strong> The
 cartoon of the mouse brain in Figures 1 C, F, and I was obtained from
 Brain Explorer 3D viewer Allen Mouse Brain
 (https://connectivity.brain-map.org/3d-viewer?v=1) and modified to be
 suitable for illustration. All figures were designed and made using Adobe
 Illustrator (Adobe, San Jose, CA). To keep working within the Adobe
 environment to avoid losing the resolution of the figures, Adobe Photoshop
 (Adobe, San Jose, CA) was used to crop the borders of some images to save
 space. The color pallets were chosen to avoid the combination of the green
 and red colors for all maps, so they will be suitable for color-blind
 readers. To induce the same effect in the microscope images combining the
 red (jRGECO1a) and green (GFP) fluorescence, we kept the green signals of
 the GFP unchanged, but we substituted the red fluorescence of jRGECO1a
 with magenta as a pseudocolor.
description: # Microprism-based two-photon imaging of the mouse inferior colliculus
 reveals novel organizational principles of the auditory midbrain
 [https://doi.org/10.5061/dryad.v41ns1s70](https://doi.org/10.5061/dryad.v41ns1s70) ## Description of the data and file structure The data were collected after the 2P imaging of the craniotomy window of the mouse inferior colliculus either directly from the top surface or from the lateral surface using a microprism. After extracting the cellular responses, different parameters were calculated such as best tone frequency (BTF), best modulation frequency (BMF), spectral modulation index (SMI), and temporal modulation index (TMI). The basics of these calculations were discussed in the methods section. ### Files and variables #### File: Figure_4-figure_supplement_1-data_source_1.xlsx * **Description:**  These data are for 8 animals as shown in figure 4. Each sheet of the excel file is for one animal as shown by the name of the sheet. Each sheet shows the correlation between x and y variable as follows * X: Distance from the midline (um) (Column A) * Y: The log of the best tuned sound frequency of each cell obtained from each mouse (Asleep preparation) (Column B). #### File: Figure_5-data_source_1.xlsx * **Description:**  The distribution of the BTF at the levels of DC imaged directly from the top and LC imaged by microprism within asleep preparation. Within LC and DC, the data were organized through two columns. The first column shows the bin centers (Column A) and the second one shows the BTF (Column B). spell error in the data file- it's cumulative. #### File: Figure_5-data_source_2.xlsx * **Description:** Variables are the SMI of the cells obtained from the LC (Column A) vs DC (Column B) under asleep preparation. #### File: Figure_5-data_source_3.xlsx * **Description:**  Variables are the TMI of the cells obtained from the LC (Column A) vs DC (Column B) under asleep preparation. #### File: Figure_9-figure_supplement_1-data_source_1.xlsx * **Description:**  These data are for 6 animals as shown in figure 9. Each sheet of the excel file is for one animal as shown by the name of the sheet. Each sheet shows the correlation between x and y variable as follows; * X: Distance from the midline (um) (Column A) * Y: The log of the best tuned sound frequency of each cell obtained from each mouse (Asleep preparation) (Column B). #### File: Figure_10-data_source_1.xlsx * **Description:**  The distribution of the BTF at the levels of DC imaged directly from the top and LC imaged by microprism within awake preparation. Within LC and DC, the data were organized through two columns. The first column shows the bin centers (Column A) and the second one shows the BTF (Column B). #### File: Figure_10-data_source_2.xlsx * **Description:**  Variables are the SMI of the cells obtained from the LC (Column A) vs DC (Column B) under awake preparation. #### File: Figure_10-data_source_3.xlsx * **Description:**  Variables are the TMI of the cells obtained from the LC (Column A) vs DC (Column B) under awake preparation. #### File: Figure_10-figure_supplement_1-data_source_1.xlsx **Description:**  * The distribution of the receptive field sum (RFS) that indicate the width of the tuning curve of the neurons. The RFS was shown for the neurons of the DC imaged directly from the top and LC imaged by microprism within asleep vs awake preparations. Within each brain region (LC vs DC) and condition (asleep vs awake), the data were organized through two columns. The first column shows the bin centers and the second one shows the RFS. ## Code/software It is a custom made code used for analyzing the cellular response from the movies collected after imaging. The code is used for the following;  * Correction against motion artifact.  * Sorting the cellular data. * Detection of the responsive cells based on the correlation between the different imaging trials.  * Cell flagging based on the response.  * Mapping of each filed of view based on the cell flagging.  The figures included in the main article are included separately as te supplemental files to Zenodo under the CC-BY license. ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA