Homeostatic Control of Spontaneous Activity in the Developing Auditory System.

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dwight Bergles ( dbergles@jhmi.edu ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Both male and female mice of postnatal days (P) 6 - P15 were used for all experiments and randomly allocated to experimental groups. All mice were healthy and were only used for experiments detailed in this study. Transgenic breeders were crossed to female FVB mice to improve litter survival and, thus, progeny were not maintained on a pure C57BL/6 background. Mice were housed on a 12 hour light/dark cycle and were provided food ad libitum. All experiments and procedures were approved by the Johns Hopkins Institutional Care and Use Committee.

Transgenic Animal Models

Snap25-T2A-GCaMP6s and VGLUT3 −/− (Vglut3 KO) have been described previously (Madisen et al., 2016; Seal et al., 2008 ). Method Details Installation of cranial windows Inhalation anesthesia was induced with vaporized isoflurane (4% for 5 minutes, or until mice are non-responsive to toe-pinch) and lowered during the procedure (1–2%) to maintain a stable respiration rate of 80 breaths per minute. A midline incision beginning posterior to the ears and ending just anterior to the eyes was made. Two subsequent cuts were made to remove the dorsal surface of the scalp. A headbar was secured to the head using super glue (Krazy Glue). Fascia and neck muscles overlying the interparietal bone were resected and the area bathed in sterile, HEPES-buffered artificial cerebrospinal fluid that was replaced as necessary throughout the surgery. Using a 28G needle and microblade, the sutures circumscribing the interparietal bone were cut and the interparietal bone was removed to expose the midbrain. The dura mater was removed using fine scissors and forceps, exposing the colliculi and extensive vasculature. If the animal was to be imaged under the two-photon microscope, astrocytes were labeled by applying 1 μΜ sulfarhodamine 101 (SR101) in HEPES-ACSF for five minutes to the surface of the brain. A 5 mm coverslip (CS-5R; Warner Instruments) was then placed over the craniotomy, the surrounding bone was dried using a Kimwipe, and super glue was placed along the outer edges of the coverslip for adhesion to the skull. Replacement 0.9% NaCl solution was injected IP and a local injection of lidocaine was given to the back of the neck. Animals were weaned off isoflurane, placed under a warming lamp, and allowed to recover for a minimum of 1 hour prior to imaging. As reported previously ( Ackman et al., 2012 ), spontaneous activity was not seen in deeply anesthetized animals and emerged ~30 minutes after recovery from isoflurane exposure. For simultaneous imaging of IC and AC, mice were anesthetized as described above. The scalp was resected and a head-bar affixed to the skull with superglue. A cranial window over the auditory cortex (AC, ~3.5 mm lateral to lambda) was made using a micro-blade to shave away the bone overlying a region encompassing AC starting from the lateral suture and moving medially (~3 to 4 mm diameter). The AC window area was covered with HEPES-ACSF throughout the procedure. The dura was carefully removed before a glass cover slip (CS-4R; Warner Instruments) was glued over the window. IC cranial windows were made and animals recovered as described above. Delivery of agents to the round window niche Prior to cranial window installation, a postauricular incision was made and the cervical musculature retracted to expose the auditory bulla. Using microscissors, the posterior region of the bulla, just ventral to the facial nerve, was removed and the middle ear exposed. To visualize the round window niche and window, extraneous mesenchyme was removed using dental absorbent paper points. Gelfoam soaked in specific pharmacological agents (made up in isotonic, 5% mannitol solution; NBQX 50 mM) was placed within the round window niche against the semi-permeable round window membrane. After Gelfoam placement, the overlying muscles were moved back into place and the incision was closed with Vetbond. Animals were imaged within 1.5 hours after drug application. Auditory cortex ablation Prior to cranial window installation, an incision in the skull was made just posterior to the right auditory cortex. Auditory cortex tissue was then aspirated via a glass pipette tip coupled to a suction line. Gelfoam was packed into the volume removed and the wound closed with super glue (Vetbond). Following imaging, animals were perfused with 4% PFA and brains harvested. Brains were embedded in 2% agarose and sectioned for post-hoc analysis of cortex removal. In vivo calcium imaging After 1 hour of post-surgical recovery from anesthesia, pups were moved into a swaddling 15 mL conical centrifuge tube. The top half of this tube was removed to allow access to the headbar and visualization of the craniotomy. Pups were head-fixed and maintained at 37°C using a heating pad and temperature controller (TC-1000; CWE). During the experiments, pups were generally immobile; however, occasional limb and tail twitching did occur. For wide field epifluorescence imaging, images were captured at 10 Hz using a Hamamatsu ORCA-Flash4.0 LT digital CMOS camera coupled to a Zeiss Axio Zoom.V16 stereo zoom microscope. For midbrain imaging, a 4 × 4 mm field of view was illuminated continuously with a mercury lamp (Zeiss Illuminator HXP 200C) and visualized through a 1X PlanNeoFluar Z 1.0× objective at 17× zoom. For simultaneous imaging of cortex and midbrain, a 6 × 6 mm field of view was imaged at 11× zoom. Images were captured at a resolution of 512 × 512 pixels (16-bit pixel depth) after 2 × 2 binning to increase sensitivity. Each recording consisted of uninterrupted acquisition over 10 minutes. A two-photon microscope (Bergamo II; Thorlabs) was used to capture high resolution images of spontaneous activity in the dorsal aspect of the central nucleus of the inferior colliculus (IC). Two photon excitation was achieved using a Ti:sapphire laser (Chameleon Ultra II; Coherent) tuned to 920 nm. A 533 × 533 μm field of view was visualized using a 25× Nikon objective (N25X-APO-MP). Images were collected at a resolution of 512 × 512 pixels (16-bit pixel depth) at 30 Hz using a galvo-resonant scanner and subsequently averaged to produce a final framerate of 10 Hz. Each recording consisted of uninterrupted acquisition over 10 minutes.

Image processing

For wide field imaging, raw images were imported into the ImageJ environment and corrected for photobleaching by fitting a single exponential to the fluorescence decay and subtracting this component from the signal (Bleach Correct function, exponential fit). Images were then imported into MATLAB (Mathworks) and intensities were normalized as ΔF/F o values, where ΔF = F - F o and F o was defined as the fifth percentile value for each pixel. Ovoid regions of interest (ROIs) encompassing the entire left and right inferior colliculi were drawn (see Figure 1D ). Across all conditions, the size of the ROIs was invariant, however, due to small differences in the imaging field between animals, the ROIs were placed manually for each imaging session. Peaks in the signals were detected in MATLAB using the built-in peak detection function (findpeaks) using a fixed value threshold criterion; because fluorescence values were normalized, this threshold was fixed across conditions (2% ΔF/F o ). Occasionally, large events in the cortex or superior colliculus would result in detectable fluorescence increases in the IC. These events broadly activated the entire surface of the IC and did not exhibit the same spatially-confined characteristics as events driven by the periphery. These events were not included in the analysis (see Figure S7 ). For two-photon imaging, images were imported into ImageJ for registration. The red channel (SR101) was registered (MultiStackReg; translation) and the transformation matrices applied to the green channel (GCaMP6s). Registered images were imported into MATLAB and intensities were normalized as ΔF/F o values as described above. For analysis of neurons and neuropil, ten random x-y locations were generated in MATLAB and ROIs manually drawn around the nearest neuron and surrounding neuropil. Peaks in signals were detected in MATLAB using the built-in peak detection function (findpeaks) using a fixed value threshold criterion (mean + 3 standard deviations for each cell). For determining periods of animal movement during simultaneous imaging of AC and IC, a small section of the image with prominent movement artifacts was used as the basis for movement detection. This image was subject to image registration (dftregistration function, Matlab; Guizar-sicairos et al., 2008 ) and the transformation matrices used to align the images were stored. We then took the magnitude (norm) of the transformation matrix to determine periods of movement. When the image is stable, the magnitude of the transformation matrix equates to 1. When the image shifts, the magnitude of the transformation matrix exceeds 1. Periods of movement were defined as extended periods (> 3 frames) where the magnitude of the transformation matrix was greater than 1.

Generation of spatial correlation maps

For generation of spatial correlation maps within and across IC (as seen in Figure 2E , 4H , and S2E ), ROIs (10 × 10 pixels) were placed across the tonotopic axis as indicated. These signals served as spatial correlation seeds and were used to generate spatial correlation maps. This was done by computing the correlation value between the seed signal and every pixel in the image. Each seed generated a single map which was then placed into a distinct color channel. The aggregate of all spatial correlation maps, each within its own color channel, results in the panels shown. Because of prominent photon scattering and background fluorescence, correlation maps appear more diffuse and contain more gray values calculated using epifluorescence images ( Figure 4H and S2E ) than in correlation maps produced using two-photon excitation ( Figure 2E ). For generation of spatial correlation maps between IC and auditory cortex (as seen in Figure 4B ), an ROI was manually drawn around the IC and correlations were calculated for each pixel across the entire image. Pixels with an r value greater than 0.8 were defined as auditory cortex. A similar approach was used to delineate boundaries of the visual cortex with signals in the superior colliculus. Pearson’s correlation coefficients were calculating using the normalized fluorescence intensity.

Generation of auditory cortex maps

ROIs were placed across the future tonotopic axis of the IC and fluorescent signals normalized as described above and mean subtracted. For each IC ROI, peaks of minimum height 3% ΔF/F o were detected and the channel with the greatest ΔF/F o was used to determine the location of the peak. AC activity corresponding to those peaks was averaged (weighted by IC peak height). The AC map for each animal was generated by taking each average AC image (corresponding to activity at different tonotopic areas in the IC), and placing them into a distinct color channel (blue representing the most medial single bands, future low frequency zones, and red representing the most lateral bands, future higher frequency zones). Responding areas were delimited by thresholding the average image at 80% maximum response. The average tonotopic map across animals was generated by a threshold of 50% across the smoothed average image of thresholded maps.

Auditory stimulation

Sounds were presented using a free-field speaker (MF1; Tucker-Davis Technologies) placed 10 cm from the left ear. For wide field imaging, animals were placed in a custom-made sound isolation chamber that attenuated outside noises by 40 dB SPL. Calibration of noise levels was performed using an ultrasonic probe (Sokolich). Stimuli consisted of sinusoidal amplitude modulated tones (1 s, 10 Hz modulation) from 3kHz to 96kHz. All stimuli were cosine-squared gated (5 ms) and played in a random order at 5 second intervals. Intensity levels were not flat across the entire range of the speaker ( Figure S3 ) and no level correction was applied. Because thresholds are very high in animals just after hearing onset, all tones were presented at 0 dB attenuation. Tones were generated within the RPvdsEx software, triggered using the microscope’s frame out signal, and delivered through the RZ6 Audio Processor (Tucker-Davis technologies). During imaging, vocalizations and ambient noise were captured using the Sokolich ultrasonic probe and digitized at 196kHz through the RZ6 Audio Processor.

Electrophysiology

For inner supporting cell recordings, apical segments of the cochlea were acutely removed from P6-P8 control or Vglut3 KO mice and used within 2 hours of the dissection. For spiral ganglion neuron recordings, middle segments of the cochlea were acutely removed from P5-P7 control or Vglut3 KO mice and used immediately, or were placed into culture media (DMEM as described below) and used within 4 hours. Cochleae were then moved into a recording chamber and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (1.5 – 2 mL/min) consisting of the following (in mM): 115 NaCl, 6 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Solutions were superfused at near physiological temperature (32–34°C) using a feedback-controlled in-line heater (Warner Instruments) for all experiments except those indicated as 24°C in Figure 7 .

Whole-cell recordings of inner supporting cells

(ISCs) and spiral ganglion neurons (SGNs) were made under visual control using differential interference contrast microscopy (DIC). Electrodes had tip resistances between 3.5–4.5 MW when filled with internal consisting of (in mM): 134 KCH 3 SO 3 , 20 HEPES, 10 EGTA, 1 MgCl 2 , 0.2 Na-GTP, pH 7.3. Spontaneous currents were recorded with ISCs held at −80mV. Errors due to the voltage drop across the series resistance and the liquid junction potential were left uncompensated. Recordings that displayed more than 10% increase in access resistance were discarded. Spontaneous currents were recorded with pClamp 10 software using a Multiclamp 700B amplifier, low pass filtered at 2 kHz, and digitized at 5 kHz with a Digidata 1322A analog-to-digital converter (Axon Instruments). Voltage responses to current injections in SGNs were recorded were low pass filtered at 20 kHz, and digitized at 50 kHz.

Analysis of intrinsic membrane properties

Series of current steps were injected into SGNs with start and end points not exceeding −100 mV and −20 mV steady state membrane potentials. Input resistances were calculated by taking the peak (min or max of signal through first 20% of current step) or the steady state voltage change (last 100 ms of step). Tau was calculated by fitting a single exponential curve between 10 and 90% maximum voltage change to a −2.5 pA current injection. Current threshold for action potential generation was measured by increasing steps in 5 pA increments until an action potential occurred.

Transmitted light imaging

Cochlear segments were imaged with a 40× water immersion objective and recorded using MATLAB and a USB capture card (EZ Cap). Difference movies were generated by subtracting frames at time t n and t n+5 seconds using ImageJ software to generate an index of transmittance change over time. To quantify transmittance changes, a threshold of three standard deviations above the mean was applied to the values. To calculate the frequency of these events, the whole field was taken as an ROI and peaks were detected using MATLAB (findpeaks function). To calculate area of these events, a Gaussian filter (sigma = 2.0) was applied to the image after thresholding and the borders detected using MATLAB (bwlabel function). The area was then calculated as the number of pixels within the border multiplied by the area scaling factor (μm/pixel) 2 .

Cochlear explant culture

Cochleae were dissected from P5 control (Snap25-T2A-GCaMP6s) and Vglut3 KO mice (Snap25-T2A-GCaMP6s; VGLUT3T −/− ), as described previously ( Tritsch et al., 2007 ; Zhang-Hooks et al., 2016 ). The cochleae were acutely dissected in ice-cold, sterile-filtered HEPES-buffered ACSF consisting of the following (in mM): 130 NaCl, 2.5 KCl, 10 HEPES, 1 NaH 2 PO 4 , 1.3 MgCl 2 , 2.5 CaCl 2 , and 11 D-Glucose. Explants were mounted onto Cell-Tak (Corning) treated coverslips and incubated at 37°C for 24 hours in Dulbecco’s modified Eagle’s medium (F-12/DMEM; Invitrogen) supplemented with 1% fetal bovine serum (FBS) and 10U/mL penicillin (Sigma).

Confocal imaging of cochlear explants

After one day in vitro , cochleae were moved into a recording chamber and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (1.5 – 2 mL/min) consisting of the following (in mM): 115 NaCl, 6 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Imaging was performed at near physiological temperature (32–34°C) using a feedback-controlled in-line heater (Warner Instruments). Images were captured at 1 frame per second using a Zeiss laser scanning confocal microscope (LSM 710, Zeiss) through a 20X objective (Plan APOCHROMAT 20×/1.0 NA) at 512 × 512 pixel (354 × 354 μm; 16-bit depth) resolution. Sections were illuminated with a 488nm laser (maximum 25mW power). NBQX (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[ f ]quinoxaline-7-sulfonamide, 50 μM, Tocris) and benzbromarone (BBE, 20 μM, Sigma) were applied by addition to the superfusing ACSF. To elicit depolarization and visualization of all ganglion neurons at the end of the experiment, explants were exposed to high potassium ACSF consisting of the following (in mM): 80 NaCl, 40 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Only cells which increased their fluorescence by 25% over baseline in response to high potassium were used for analysis.

Analysis of calcium transients in SGNs

Images were imported into ImageJ and image registration (MultiStackReg) was used to correct for slight drifts in the imaging field. Regions of interest were drawn around SGNs that exhibited increases in fluorescence in response to high potassium external solution. Fluorescence changes were normalized as ΔF/F o values, where ΔF = F - F o and F o was defined as the fifth percentile value for each pixel. Peaks in the signals were detected in MATLAB using the built-in peak detection function (findpeaks) with a fixed value threshold criterion (mean + 3 standard deviations for each cell). Across all cells, events were specified when three or more cells displayed peaks in fluorescence within a one second time window. For correlation analysis, the five nearest and farthest cells to a target cell were calculated using the center of mass of the ROI. Correlations on these groups of cells were calculated using normalized fluorescence signals.

Immunohistochemistry

Mice were deeply anesthetized with isoflurane and perfused with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed for 45 minutes at room temperature and stored at 4°C until processing. Bra ins were embedded in 2% agarose gel and 50 μm sections were cut with a vibratome and mounted onto Superfrost Plus glass slides (Fisher). Sections were incubated overnight with primary antibodies against GFP (pChicken; 1:4000, Aves) and NeuN (pMouse; 1:500, Millipore). Sections were then rinsed three times with PBS and incubated for two hours at room temperature with secondary antibodies raised in donkey (Alexa-488 and Alexa-546; 1:2000, Life Technologies). Slides were washed three times in PBS, allowed to dry, and sealed using Aqua Polymount (Polysciences, Inc.). Images were captured using a laser scanning confocal microscope (LSM 710 Meta, Zeiss).

Quantification and statistical analyses

All statistics were performed in the MATLAB (Mathworks) programming environment. All statistical details, including the exact value of n, what n represents, and which statistical test was performed, can be found in the figure legends. Unless otherwise noted, data are presented as mean ± standard error of the mean. All datasets were tested for Gaussian normality using the D’Agostino’s K 2 test. If datasets were normal, two-tailed paired t-tests were used. Otherwise, the nonparametric Mann-Whitney test was used. For single comparisons, significance was defined as p 3 frames) where the magnitude of the transformation matrix was greater than 1.

Generation of spatial correlation maps

For generation of spatial correlation maps within and across IC (as seen in Figure 2E , 4H , and S2E ), ROIs (10 × 10 pixels) were placed across the tonotopic axis as indicated. These signals served as spatial correlation seeds and were used to generate spatial correlation maps. This was done by computing the correlation value between the seed signal and every pixel in the image. Each seed generated a single map which was then placed into a distinct color channel. The aggregate of all spatial correlation maps, each within its own color channel, results in the panels shown. Because of prominent photon scattering and background fluorescence, correlation maps appear more diffuse and contain more gray values calculated using epifluorescence images ( Figure 4H and S2E ) than in correlation maps produced using two-photon excitation ( Figure 2E ). For generation of spatial correlation maps between IC and auditory cortex (as seen in Figure 4B ), an ROI was manually drawn around the IC and correlations were calculated for each pixel across the entire image. Pixels with an r value greater than 0.8 were defined as auditory cortex. A similar approach was used to delineate boundaries of the visual cortex with signals in the superior colliculus. Pearson’s correlation coefficients were calculating using the normalized fluorescence intensity.

Generation of auditory cortex maps

ROIs were placed across the future tonotopic axis of the IC and fluorescent signals normalized as described above and mean subtracted. For each IC ROI, peaks of minimum height 3% ΔF/F o were detected and the channel with the greatest ΔF/F o was used to determine the location of the peak. AC activity corresponding to those peaks was averaged (weighted by IC peak height). The AC map for each animal was generated by taking each average AC image (corresponding to activity at different tonotopic areas in the IC), and placing them into a distinct color channel (blue representing the most medial single bands, future low frequency zones, and red representing the most lateral bands, future higher frequency zones). Responding areas were delimited by thresholding the average image at 80% maximum response. The average tonotopic map across animals was generated by a threshold of 50% across the smoothed average image of thresholded maps.

Auditory stimulation

Sounds were presented using a free-field speaker (MF1; Tucker-Davis Technologies) placed 10 cm from the left ear. For wide field imaging, animals were placed in a custom-made sound isolation chamber that attenuated outside noises by 40 dB SPL. Calibration of noise levels was performed using an ultrasonic probe (Sokolich). Stimuli consisted of sinusoidal amplitude modulated tones (1 s, 10 Hz modulation) from 3kHz to 96kHz. All stimuli were cosine-squared gated (5 ms) and played in a random order at 5 second intervals. Intensity levels were not flat across the entire range of the speaker ( Figure S3 ) and no level correction was applied. Because thresholds are very high in animals just after hearing onset, all tones were presented at 0 dB attenuation. Tones were generated within the RPvdsEx software, triggered using the microscope’s frame out signal, and delivered through the RZ6 Audio Processor (Tucker-Davis technologies). During imaging, vocalizations and ambient noise were captured using the Sokolich ultrasonic probe and digitized at 196kHz through the RZ6 Audio Processor.

Electrophysiology

For inner supporting cell recordings, apical segments of the cochlea were acutely removed from P6-P8 control or Vglut3 KO mice and used within 2 hours of the dissection. For spiral ganglion neuron recordings, middle segments of the cochlea were acutely removed from P5-P7 control or Vglut3 KO mice and used immediately, or were placed into culture media (DMEM as described below) and used within 4 hours. Cochleae were then moved into a recording chamber and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (1.5 – 2 mL/min) consisting of the following (in mM): 115 NaCl, 6 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Solutions were superfused at near physiological temperature (32–34°C) using a feedback-controlled in-line heater (Warner Instruments) for all experiments except those indicated as 24°C in Figure 7 .

Whole-cell recordings of inner supporting cells

(ISCs) and spiral ganglion neurons (SGNs) were made under visual control using differential interference contrast microscopy (DIC). Electrodes had tip resistances between 3.5–4.5 MW when filled with internal consisting of (in mM): 134 KCH 3 SO 3 , 20 HEPES, 10 EGTA, 1 MgCl 2 , 0.2 Na-GTP, pH 7.3. Spontaneous currents were recorded with ISCs held at −80mV. Errors due to the voltage drop across the series resistance and the liquid junction potential were left uncompensated. Recordings that displayed more than 10% increase in access resistance were discarded. Spontaneous currents were recorded with pClamp 10 software using a Multiclamp 700B amplifier, low pass filtered at 2 kHz, and digitized at 5 kHz with a Digidata 1322A analog-to-digital converter (Axon Instruments). Voltage responses to current injections in SGNs were recorded were low pass filtered at 20 kHz, and digitized at 50 kHz.

Analysis of intrinsic membrane properties

Series of current steps were injected into SGNs with start and end points not exceeding −100 mV and −20 mV steady state membrane potentials. Input resistances were calculated by taking the peak (min or max of signal through first 20% of current step) or the steady state voltage change (last 100 ms of step). Tau was calculated by fitting a single exponential curve between 10 and 90% maximum voltage change to a −2.5 pA current injection. Current threshold for action potential generation was measured by increasing steps in 5 pA increments until an action potential occurred.

Transmitted light imaging

Cochlear segments were imaged with a 40× water immersion objective and recorded using MATLAB and a USB capture card (EZ Cap). Difference movies were generated by subtracting frames at time t n and t n+5 seconds using ImageJ software to generate an index of transmittance change over time. To quantify transmittance changes, a threshold of three standard deviations above the mean was applied to the values. To calculate the frequency of these events, the whole field was taken as an ROI and peaks were detected using MATLAB (findpeaks function). To calculate area of these events, a Gaussian filter (sigma = 2.0) was applied to the image after thresholding and the borders detected using MATLAB (bwlabel function). The area was then calculated as the number of pixels within the border multiplied by the area scaling factor (μm/pixel) 2 .

Cochlear explant culture

Cochleae were dissected from P5 control (Snap25-T2A-GCaMP6s) and Vglut3 KO mice (Snap25-T2A-GCaMP6s; VGLUT3T −/− ), as described previously ( Tritsch et al., 2007 ; Zhang-Hooks et al., 2016 ). The cochleae were acutely dissected in ice-cold, sterile-filtered HEPES-buffered ACSF consisting of the following (in mM): 130 NaCl, 2.5 KCl, 10 HEPES, 1 NaH 2 PO 4 , 1.3 MgCl 2 , 2.5 CaCl 2 , and 11 D-Glucose. Explants were mounted onto Cell-Tak (Corning) treated coverslips and incubated at 37°C for 24 hours in Dulbecco’s modified Eagle’s medium (F-12/DMEM; Invitrogen) supplemented with 1% fetal bovine serum (FBS) and 10U/mL penicillin (Sigma).

Confocal imaging of cochlear explants

After one day in vitro , cochleae were moved into a recording chamber and continuously superfused with bicarbonate-buffered artificial cerebrospinal fluid (1.5 – 2 mL/min) consisting of the following (in mM): 115 NaCl, 6 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Imaging was performed at near physiological temperature (32–34°C) using a feedback-controlled in-line heater (Warner Instruments). Images were captured at 1 frame per second using a Zeiss laser scanning confocal microscope (LSM 710, Zeiss) through a 20X objective (Plan APOCHROMAT 20×/1.0 NA) at 512 × 512 pixel (354 × 354 μm; 16-bit depth) resolution. Sections were illuminated with a 488nm laser (maximum 25mW power). NBQX (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[ f ]quinoxaline-7-sulfonamide, 50 μM, Tocris) and benzbromarone (BBE, 20 μM, Sigma) were applied by addition to the superfusing ACSF. To elicit depolarization and visualization of all ganglion neurons at the end of the experiment, explants were exposed to high potassium ACSF consisting of the following (in mM): 80 NaCl, 40 KCl, 1.3 MgCl 2 , 1.3 CaCl 2 , 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 11 D-glucose, and saturated with 95% O 2 / 5% CO 2 to maintain a pH of 7.4. Only cells which increased their fluorescence by 25% over baseline in response to high potassium were used for analysis.

Analysis of calcium transients in SGNs

Images were imported into ImageJ and image registration (MultiStackReg) was used to correct for slight drifts in the imaging field. Regions of interest were drawn around SGNs that exhibited increases in fluorescence in response to high potassium external solution. Fluorescence changes were normalized as ΔF/F o values, where ΔF = F - F o and F o was defined as the fifth percentile value for each pixel. Peaks in the signals were detected in MATLAB using the built-in peak detection function (findpeaks) with a fixed value threshold criterion (mean + 3 standard deviations for each cell). Across all cells, events were specified when three or more cells displayed peaks in fluorescence within a one second time window. For correlation analysis, the five nearest and farthest cells to a target cell were calculated using the center of mass of the ROI. Correlations on these groups of cells were calculated using normalized fluorescence signals.

Immunohistochemistry

Mice were deeply anesthetized with isoflurane and perfused with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed for 45 minutes at room temperature and stored at 4°C until processing. Bra ins were embedded in 2% agarose gel and 50 μm sections were cut with a vibratome and mounted onto Superfrost Plus glass slides (Fisher). Sections were incubated overnight with primary antibodies against GFP (pChicken; 1:4000, Aves) and NeuN (pMouse; 1:500, Millipore). Sections were then rinsed three times with PBS and incubated for two hours at room temperature with secondary antibodies raised in donkey (Alexa-488 and Alexa-546; 1:2000, Life Technologies). Slides were washed three times in PBS, allowed to dry, and sealed using Aqua Polymount (Polysciences, Inc.). Images were captured using a laser scanning confocal microscope (LSM 710 Meta, Zeiss).

Supplementary Material 1 2 Movie S1: Spontaneous activity in the developing inferior colliculus, related to Figure 1 . Spontaneous activity in the inferior colliculus (IC) of awake, unanesthestized mice ( Snap25-T2A-GCaMP6s) before hearing onset (postnatal day 7). Images were acquired at 10 Hz; playback is 2× real time. 3 Movie S2: High resolution imaging of spontaneous activity in the inferior colliculus, related to Figure 2 . Spontaneous activity in the IC of awake, unanesthestized mice ( Snap25-T2A-GCaMP6s ) before hearing onset (postnatal day 7). Images were collected in the dorsal aspect of the central nucleus of the IC, 150 μm below the pial surface. Images were acquired at 10 Hz; playback is 2× real time. 4 Movie S3: Spontaneous activity in IC and AC is correlated before hearing onset, related to Figure 4 . Wide field imaging of spontaneous activity in the midbrain and posterior cortex of Snap25-T2A-GCaMP6s mice. Images were acquired at 10 Hz; playback is 2× real time. 5 Movie S4: AC received tonotopically-organized information from IC before hearing onset, related to Figure 4 . Wide field imaging of spontaneous activity in midbrain and posterior cortex of Snap25-T2A-GCaMP6s mice. IC fluorescence was increased 2× for clarity. Images were acquired at 10 Hz; playback is in real time. 6 Movie S5: Tonotopically organized events persists in mice lacking functional VGLUT3, related to Figure 5 . Spontaneous activity in the IC of Snap25-T2A-GCaMP6s; Vglut3 −/− mice before the onset of hearing. Images were acquired at 10 Hz; playback is 2× real time. 7 Movie S6: Correlated spiral ganglion neuron activity requires glutamatergic signaling, related to Figure 6 . Time-lapse imaging of spontaneous activity in SGNs in excised cochleae from Snap25-T2A-GCaMP6s mice (P5 + 1DIV). Images were acquired at 1 Hz, playback is 20× real time. NBQX was applied at 50 μΜ and high potassium was applied at 40 mM (all in ACSF). 8 Movie S7: Correlated SGN activity in Vglut3 KO mice requires K + release from supporting cells, related to Figure 7 . Time-lapse imaging of spontaneous activity in SGNs of Snap25-T2A-GCaMP6; Vglut3 −/− mice (P5 + 1DIV). Transient amplitude and degree of coordination are reduced compared to controls. Images were acquired at 1 Hz, playback is 20× real time. NBQX was applied at 50 μΜ, BBE at 20 μΜ and high potassium was applied at 40 mM (all in ACSF).