DOI: 10.5061/dryad.2rbnzs80g
title: GABAergic synapses between auditory efferent neurons and type II spiral ganglion afferent neurons in the mouse cochlea

description: Cochlear outer hair cells (OHCs) are electromotile and implicated in
 amplification of responses to sound that enhance sound sensitivity and
 frequency tuning. They send afferent information through glutamatergic
 synapses onto type II spiral ganglion neurons (SGNs). These synapses are
 weaker than those from cochlear inner hair cells (IHC) onto type I SGN,
 suggesting that type II SGNs respond only to intense sound levels. OHCs
 also receive efferent innervation from medial olivocochlear (MOC) neurons.
 MOC neurons are cholinergic yet inhibit OHCs due to the functional
 coupling of alpha9/alpha10 nicotinic acetylcholine receptors (nAChRs) to
 calcium-activated SK potassium channels. The resulting hyperpolarization
 reduces OHC activity-evoked electromotility and is implicated in cochlear
 gain control, protection against acoustic trauma, and attention. MOC
 neurons also label for markers of GABA and GABA synthesis. GABAB
 autoreceptor (GABABR) activation on MOC terminals has been demonstrated to
 reduce ACh release, confirming important negative feedback roles. However,
 the full complement of GABAergic activity in the cochlea is not currently
 understood, including mechanisms of GABA release from MOC axons, whether
 GABA diffuses from MOC axons to other postsynaptic cells, and the location
 and function of GABAA receptors (GABAARs). We used optical
 neurotransmitter detection, immunohistochemistry, and patch-clamp
 electrophysiology to demonstrate that in addition to presynaptic GABAB
 autoreceptor activation, MOC efferent terminals release GABA onto type II
 SGN afferent dendrites with postsynaptic activity mediated by GABAARs.
 This synapse may have roles including developmental regulation of cochlear
 innervation, fine tuning of OHC activity, or providing feedback to the
 brain about MOC and OHC activity.
description: <em>Mice and ethical approval</em>
 Animal procedures followed National Institutes of Health
 guidelines, as approved by the National Institute of Neurological
 Disorders and Stroke/National Institute on Deafness and Other
 Communication Disorders Animal Care and Use Committee. Male and female
 mice were used in experiments. Mouse lines included wildtype (WT) C57BL/6J
 (Jax Cat No: 000664), ChAT-IRES-Cre (Jax Cat No: 028861), ChAT-IRES-Cre
 Δneo (Jax Cat No: 031661), Ngn-Cre<sup>ERT2</sup> (Jax Cat No:
 008529), Ai14 tdTomato reporter mice (Jax Cat No: 007914), and Bhlhb5-Cre
 (on Sv/129, C57BL/6J mixed background). Mice were housed on a 12/12 hr
 light/dark cycle, with continuous availability of food and water. Mice
 were anesthetized by carbon dioxide inhalation at a rate of 20% of
 anesthesia chamber volume per minute and then killed by
 decapitation.
 <em>Immunohistochemistry</em> Mice
 aged postnatal day (P)5- 20 were euthanized as above and used for
 immunohistochemical experiments in cochlear whole mount preparations.
 Temporal bones were immediately removed and fixed via perfusion of 1 ml
 cold 4% PFA (Electron Microscopy Science) in 1X PBS through the oval and
 round windows, with 20-30 min post-fix in cold 4% PFA. Cochleae were
 washed with 1X PBS perfused through the round and oval window, then washed
 3x 10-15 min in 1X PBS. Cochleae were either fully or partially dissected,
 followed by blocking non-specific labeling (10% normal goat/donkey serum
 in 0.5% Triton X-100/1X PBS) for 1-2 hr, and washed for 15-20 min in 1X
 PBS. For experiments using an anti-GAD antibody (antibodies listed in
 <strong>Table M1</strong>), cochleae underwent an additional
 block in M.O.M. buffer (3-4 drops in 2.5 ml 1X PBS; Vector MKB-2213-1) at
 room temperature (RT) for 1 hr. After a 15–20-min wash, cochlear turns
 were incubated in primary antibody in blocking solution
 (<strong>Table M1</strong>) at 4ºC for 2-48 hr. Tissues were
 washed 3x 20 min in 0.25% TX-100/1X PBS, then incubated in secondary
 antibody in 5% normal goat/donkey serum and 0.25% TX-100/1X PBS for 1-2 hr
 at RT. Tissues were washed for 15 min in 0.25% TX-100/1X PBS with DAPI
 (1:5000), washed 2x 10 min in 0.25% TX-100/1X PBS, then washed 15-20 min
 in 1X PBS.  Antibody-labeled cochlear whole mounts were
 imaged on a Nikon A1R inverted microscope with 40X or 60X objectives,
 using Nikon Elements software v5.30. 
 
 <strong>Antibody</strong> 
 <strong>Manufacturer / Part
 Number</strong> 
 <strong>Dilution</strong> 
 <strong>Secondary
 Antibody</strong> 
 Anti-GAD65  
 *Note: used with M.O.M buffer (MKB-2213-1):  3-4 drops in 2.5 ml
 PBS Millipore MAB351R
 1:500-1:1000 
 Fisher A-21131: AlexaFluor® 488 Goat Anti-Mouse IgG2a
 (1:800) or Fisher AB_2340862: AlexaFluor® 647 AffiniPure Donkey Anti-Mouse
 IgG (1:800) 
 GABA<sub>A</sub>β3R 
 Novus Bio / NB300-199   
 1:250-1:600 
 Fisher: A-21206 AlexaFluor® 488 Donkey anti-Rabbit IgG (H+L),
 (1:800) 
 Synapsin 
 Millipore AB1543P 
 1:600 
 Fisher: A-21206 AlexaFluor® 488 Donkey anti-Rabbit IgG (H+L),
 (1:800) 
 Myosin VIIa Proteus
 Bioscience 25-6790 
 1:500 Jackson
 Immunoresearch 711-605-152, Donkey Anti-Rabbit 647, (1:800)
 
 <strong> </strong>
 <strong>Table M1</strong>. Antibodies used in
 immunohistochemistry experiments. <em>Patch-clamp
 recordings</em> For patch-clamp recordings from
 outer hair cells (OHCs) and type II spiral ganglion neuron (SGN) afferent
 dendrites,  the cochleae of P11-13 (OHC) or P5-10 (type II SGN) mice were
 dissected and placed under an insect pin affixed to a round glass
 coverslip with Sylgard (Dow Chemicals, Midland, MI, USA). Tissue was
 initially visualized using a Nikon Eclipse NI-E microscope with a QI-Click
 camera or FN-1 microscope with a pco.edge camera, with a 4X air objective
 for positioning of stimulation electrodes, drug application pipettes, or
 suction electrodes. Recordings were then performed using 40X or 60X water
 immersion objectives with DIC optics and an additional 1.25-2X
 magnification. For experiments with visualization of fluorescent cells,
 tissue was visualized under epifluorescent illumination using a Sola or
 Aura II lamp (Lumencor, Beaverton, OR, USA). For OHC
 recordings, evoked inhibitory postsynaptic currents (IPSCs) were obtained
 from the first row of OHCs in the whole-cell voltage-clamp recording
 configuration in response to extracellular electrical stimulation of the
 MOC fibers <sup>54,55</sup>. For type II SGN dendrite
 recordings, fibers under OHCs were exposed by removal of 4-6 OHCs using a
 glass pipette with a 10-15 µm diameter-tip. Patch-clamp recordings were
 performed using 1 mm diameter glass micropipettes (WPI: World Precision
 Instruments, Sarasota, FL, USA) pulled (Sutter P1000, Sutter Instrument
 Company, Novato, CA, USA) to resistances of 3-6 MΩ (OHC recordings) or
 6-10 MΩ (type II SGN dendrite recordings). Dissection and recording
 ‘extracellular’ solution contained (in mM): 5.8 KCl, 155 (OHC recordings)
 or 150 (all else) NaCl, 0.9 MgCl<sub>2,</sub> 1.3
 CaCl<sub>2,</sub> 0.7
 NaH<sub>2</sub>PO<sub>4,</sub> 5.6 glucose, and 10
 HEPES. The pH was 7.4, adjusted with 1N NaOH, and osmolarity = ~315 mOsm.
 Extracellular solution at RT was perfused through the recording chamber at
 a rate of ~1-2 ml/min. Internal solutions for type II SGN recordings
 contained (in mM): 50 KCl, 80 K-methanesulfonate, 5
 MgCl<sub>2,</sub> 0.1 CaCl<sub>2,</sub> 5 EGTA, 5
 HEPES, 5 Na<sub>2</sub>ATP, 0.3 Na<sub>2</sub>GTP,
 and 5 Na<sub>2</sub>-phosphocreatine. The pH was adjusted to
 7.2 with KOH. 0.01 mM AlexaFluor hydrazide 488 or 594 was added during
 some experiments to allow visualization of cell morphology to confirm
 neuron identity. The osmolarity = ~290 and the liquid junction potential =
 -9 mV. Internal solutions for OHC recordings contained (in mM): 140 KCl,
 3.5 MgCl<sub>2</sub>, 0.1 CaCl<sub>2</sub>, 5
 EGTA, 5 HEPES, and 2.5 Na<sub>2</sub>ATP. The pH was adjusted
 to 7.2 with KOH and osmolarity = 283-290 mOsm. Drug application was
 performed using either a large bore gravity-fed glass pipette positioned
 close to the cochlear tissue (GABA or ACh application) or via addition to
 the re-circulating bath solution. Drugs were obtained from Sigma-Aldrich,
 Inc. (St. Louis, MO, USA), Alomone Labs (Jerusalem, Israel), and Thermo
 Fisher Scientific (Waltham, MA, USA). Patch-clamp
 recordings were performed in voltage-clamp using a Multiclamp 700B
 amplifier with 1550B or 1322A Digidata controlled by Multiclamp Commander
 v11.2 and pClamp v11.2 (Molecular Devices, Silicon Valley, CA, USA) or a
 HEKA EPC 10 amplifier controlled by PatchMaster v2.91 (HEKA Instruments
 Inc., Holliston, MA, USA). Recordings were sampled at 50 kHz and lowpass
 filtered at 10 kHz. OHCs were held at -40 mV for recordings of MOC to OHC
 synaptic activity. Type II SGN dendrites were held at -80 mV for most
 recordings unless stated otherwise. To test the reversal potential of
 GABAergic currents, the type II SGN dendrite membrane potential was
 stepped to holding voltages from -80 to +20 mV in 20 mV increments for 10
 seconds prior to GABA application. In all patch-clamp experiments, GABA
 application was performed once per 10 min.
 <em>Electrical stimulation of MOC
 axons</em> Endogenous neurotransmitter release
 from MOC axons was evoked using electrical axon stimulation via ~10-20 µm
 diameter-glass micropipettes (Sutter) containing extracellular solution
 described above and placed ~20-30 µm modiolar to the inner spiral bundle
 (ISB). For OHC recordings, the stimulation electrode was positioned below
 the IHC aligned with the OHC under study. For optimal stimulation,
 cochlear supporting cells were gently removed by applying negative
 pressure through a glass pipette with a broken tip. The MOC axon
 stimulation was applied using an electrical stimulus isolation unit (Iso
 Stim, AMPI or SIU isolation unit A-M System). Stimulation timing and rate
 (for imaging experiments: 50 Hz, 1 second train duration) was controlled
 by the PClamp software during both patch-clamp and optical
 experiments. <em>Estimation of the quantal
 content of transmitter release</em>. Under the
 assumption that evoked synaptic events in synapses with a low release
 probability follow a Poisson distribution, the quantal content of
 transmitter release (<em>m</em>) upon electrical stimulation
 of the MOC fibers was estimated by the “method of failures”: <em>m =
 ln (N/N<sub>0</sub>)</em><sup>56</sup>,
 where <em>N</em> is the total number of stimuli and
 <em>N<sub>0</sub></em> the number of failures.
 Protocols of 100 stimuli at a frequency of 1 Hz were used to estimate
 <em>m</em>. At this stimulation frequency, neither the
 kinetics nor the amplitudes of the evoked responses changed throughout the
 protocol. To test the effect of drugs or toxins on the quantal content of
 evoked ACh release from the MOC synaptic terminals, <em>m</em>
 was first assessed 2-3 times in order to establish a mean control value
 (<em>m</em><sub>c</sub>) before incubating the
 tissue with the drug or toxin to evaluate changes in neurotransmitter
 release.  The tissue was then incubated for the time necessary to reach a
 plateau in the compound’s effect (ranging from 5 to 15 minutes, depending
 on the compound), after which the stimulation protocol was repeated (2-3
 times).   <em>Posterior
 semi-circular canal (PSC) adeno-associated virus (AAV)
 injections</em> Posterior semicircular canal
 (PSC) injections to introduce adeno-associated virus (AAV) particles into
 the cochlea were performed as described in <sup>57</sup>,
 using aseptic procedures. In brief, neonatal pups (P0-2) were
 hypothermia-anaesthetized for ~5 min until they did not respond to
 stimulation, and then remained on an ice pack for the duration of the
 procedure. A postauricular incision was made using micro-scissors and the
 skin was retracted. The PSC was identified under a surgical microscope and
 a glass micropipette pulled to a fine point was positioned using a
 micro-injector (WPI). For each mouse only one ear was injected with ~1.2
 µL AAV solution containing gene sequences encoding optical GABA indicator
 (iGABASnFR) variants: iGABASnFR.F102G or FLEX.iGABASnFR2.0. The incision
 was closed using a drop of surgical glue. About 4-5 pups per litter were
 injected. Pups were recovered to normal body temperature on a warming pad,
 while receiving manual stimulation to aid recovery. To increase the
 likelihood of the dam accepting the pups post-surgery, each pup was gently
 rubbed with bedding from the home cage and, if possible, urine that was
 collected from the dam using a cotton-tipped applicator, before returning
 to their home cage. In addition, mineral oil was applied to the dam’s nose
 to block detection of any surgical or human odors on the pups prior to
 being returned to the home cage. <em>iGABASnFR
 imaging</em> For fluorescence imaging of
 iGABABSnFR-transduced cells in acutely dissected cochlear preparations,
 the euthanasia, dissections, extracellular solutions, drug application,
 and electrical stimulation of MOC axons were as above for patch-clamp
 recordings. iGABASnFR.F102G was obtained as plasmids from Addgene.org
 (Watertown, MA, USA). The plasmids were then packaged by SignaGen
 (Frederick, MD, USA) into AAV particles with the PHP.eB serotype and human
 synapsin (hSyn) promotor, the combination of which results in expression
 specifically in neurons, although a few hair cells also exhibited
 expression. iGABASnFR2-containing plasmids were kindly gifted by the GENIE
 Project Team at the Janelia Research Campus, Howard Hughes Medical
 Institute (Ashburn, VA, USA), and similarly packaged into AAV
 particles. iGABASnFR imaging experiments were carried
 out using non-Cre-dependent virus injected into C57BL/6J mice (P14-15) or
 Cre-dependent (FLEX) virus injected into Ngn1-CreERT2; tdTomato or
 Bhlhb5-Cre; tdTomato mice (P8-11), which result in iGABASnFR expression
 specifically in neurons. There was no difference in the baseline
 fluorescence of FLEX.iGABASnFR2.0 between Bhlhb5-Cre or Ngn1CreERT2 mice
 (Bhlhb5-Cre mean baseline fluorescence: 36.4 ± 16.4 arbitrary units (AU),
 n = 14, Ngn1CreERT2 mean baseline fluorescence 40.6 ± 11.3 A.U., n = 3
 preparations, One-way ANOVA p = 0.92262), so results were pooled between
 these two genotypes for the P8-11 mouse experiments. The iGABASnFR or
 tdTomato fluorescence was localized to cells with the clear morphology of
 type II SGN dendrites. In experiments in which OHCs were also labeled, the
 focal plane of imaging was set to image only type II SGNs, or in the case
 of curved tissue that had focal planes containing both OHCs and type II
 SGN fibers, only the type II SGN-containing regions-of-interest (ROIs)
 were analyzed (for analysis see <em>Image Processing</em>,
 below). Although unlikely, we cannot completely exclude the possibility
 that iGABASnFR was aberrantly expressed in MOC neurons due to spread of
 AAV particles via cerebrospinal fluid (CSF). However, analysis was
 restricted to regions with clear type II SGN morphology, and we did not
 detect expression in MOC axon terminals. iGABASnFR
 imaging was performed on a Nikon A1R upright confocal microscope using
 resonant scanning in both red (568 nm, for tdTomato imaging in cochlear
 neurons from transgenic mice) and green (488 nm, for iGABASnFR variants)
 channels. In a subset of experiments with exogenous application of GABA or
 ACh, but not in experiments with electrical stimulation of MOC axons, the
 Nikon ‘denoise’ function was utilized during imaging, which improves image
 quality by estimating and removing the Poisson-distributed shot noise,
 without changing signal intensity. In some experiments, a DIC-like image
 was simultaneously collected using the transmitted light detector, which
 converts the laser signal into a greyscale 3D image. Imaging settings
 included line averaging of 4-16 lines, bi-directional scanning, 512-1024
 resolution, and frame rates of ~7-15 frames per second.
 <em>Image processing</em>
 Fluorescence intensity changes were measured in
 iGABASnFR-transduced neurons using ImageJ (NIH, Bethesda, MD, USA). A
 maximum intensity projection of the green (iGABASnFR) image stack was
 generated and then thresholded to set the regions to be used for ROI
 selection. Thresholds were set to 121, but manually adjusted in the case
 of tissue with brighter or dimmer fluorescence background (mean = 119 ±
 20). The ‘analyze particles’ function was used to automatically draw ROIs
 around the iGABASnFR-expressing structures of interest, and each ROI was
 classified by eye as type I or type II SGNs from either fluorescence or
 transmitted detector images. It was not possible to identify individual
 neurons from either tdTomato or iGABASnFR images, so ROIs likely contain
 multiple neuronal segments. These ROIs were then used to measure
 fluorescence intensities in the original image stack for each frame.
 Fluorescence intensity values per ROI and per frame were imported into
 Origin v2021 (Origin Lab, Northhampton, MA, USA). The fluorescence
 baseline was measured for 1 second prior to stimulus onset (electrical MOC
 axon stimulation) or 10 seconds prior to drug application (exogenous GABA
 or ACh application). The iGABASnFR responses were determined for the 30
 seconds of exogenous ACh or GABA application compared to the 10 seconds
 prior to drug application. For experiments utilizing electrical
 stimulation of neurotransmitter release from MOC axons, we made a binary
 determination of whether each ROI had a positive ‘response’ to the axon
 stimulation. First, we measured the mean and standard deviation of the
 baseline fluorescence (1 second prior to MOC axon stimulation). Then, we
 measured the maximum fluorescence following MOC axon stimulation. To
 prevent a noisy fluorescence signal from giving an artificially high
 ‘maximum’ intensity, we used a rolling average (5 frames before, 5 frames
 after) to smooth the trace, and determined the fluorescence maximum from
 this rolling average trace. An ROI had a positive ‘response’ if the
 maximum fluorescence following axon stimulation was greater than two
 standard deviations above the baseline mean (mean +2SDs). Next, to
 determine the magnitude of the response to MOC axon stimulation we
 calculated the ΔF/F of the fluorescence. This was defined as the maximum
 fluorescence following MOC axon stimulation minus the mean baseline
 fluorescence, divided by the mean baseline fluorescence.
 <em>Quantification and Statistical
 Analyses</em> Statistics were performed in Origin
 v2021-2023 or GraphPad Prism 6 (RRID:SCR_002798, Boston, MA, USA). Data
 were tested for normality using a Shapiro-Wilk test. Statistical tests
 included a one-way ANOVA for parametric tests and one-way repeated
 measures ANOVA with post-hoc pairwise Scheffe tests. For non-parametric
 statistical analyses, with two groups, a Wilcoxon rank sum was applied,
 and for non-parametric comparisons of more than two groups, a Friedman
 test followed by a Dunn multiple-comparison test was used. Figures were
 prepared in Origin, ImageJ, and Adobe Illustrator (Adobe, San Jose, CA,
 USA). <strong>Methods
 References</strong>            Ballestero, J.
 <em>et al.</em> Short-term synaptic plasticity regulates the
 level of olivocochlear inhibition to auditory hair cells. <em>J.
 Neurosci.</em> <strong>31</strong>, 14763–74
 (2011).            Vattino, L. G., Wedemeyer, C.,
 Elgoyhen, A. B. & Katz, E. Functional Postnatal Maturation of the
 Medial Olivocochlear Efferent–Outer Hair Cell Synapse. <em>J.
 Neurosci.</em> <strong>40</strong>, 4842–4857
 (2020).            DEL CASTILLO, J. & KATZ, B.
 Quantal components of the end-plate potential. <em>J.
 Physiol.</em> <strong>124</strong>, 560–73
 (1954).            Isgrig, K. & Chien, W. W.
 Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in
 Neonatal Mouse. <em>J. Vis. Exp.</em> (2018)
 doi:10.3791/56648.
description: # GABAergic synapses between auditory efferent neurons and type II spiral
 ganglion afferent neurons in the mouse cochlea
 [https://doi.org/10.5061/dryad.2rbnzs80g](https://doi.org/10.5061/dryad.2rbnzs80g) ## Description of the data and file structure Access this data at: [https://doi.org/10.5061/dryad.2rbnzs80g](https://doi.org/10.5061/dryad.2rbnzs80g) We have submitted our raw Patch-Clamp Electrophysiology, Immunohistochemistry, and iGABASnFR optical detection data to support the Bachman, Kitcher et al 2025 manuscript. **Notes:** Data and File Structure: All raw data files contained in the .zip file “Bachman et al 2025”  Excel file “Bachman et al 2025 PNAS files” contains a list of file names, types, and additional details for the dataset. Nikon files are all Nikon LIM .nd2 file type, but are noted in the attached excel file as Nikon LIM file type. .nd2 files can be viewed in Nikon Elements viewers or ImageJ. Sharing / Access information: Please contact corresponding author [catherine.weisz@nih.gov](mailto:catherine.weisz@nih.gov) with any questions. ### Folder: GABA A R immunos Fig 4 Folder: “GABA A R immunos Fig 4” contains confocal imaging Z-stacks from dissected cochlear preparations from transgenic mice. ·        **ChAT Cre tdT P20 Row2sec2_stack_2** is a Nikon elements .nd2 file. The data is from post-natal day 20 ChAT-IRES-Cre (“CIJ”, Cre recombinase expressed in cholinergic neurons) x tdTomato (“tdTom” or “tdT” reporter mouse line in which the red fluorescent protein is produced in Cre-positive cells) mice. Immunohistochemistry procedures are detailed in the methods. The file contains multiple imaging planes that were combined into the projection images shown in the figure panels. For the manuscript, the image was cropped to include only the outer hair cell region. The imaging channels included green (anti-GABA A beta 3 receptor, converted to yellow for colorblind palette in published image), red (tdTomato reporter labeling in cholinergic MOC axons, converted to magenta), far-red (purple, anti-GAD label, converted to blue in final image). ·        **Ngn1 Cre tdt P20 r3s3_40x_crop** is a Nikon Elements .nd2 file. The data is from post-natal day 20 Neurogenin1-CreERT2 (Cre recombinase expressed in spiral ganglion neurons in the cochlea, random sparse expression in the absence of tamoxifen) x tdTomato (reporter mouse line in which the red fluorescent protein is produced in Cre-positive cells) mice. Immunohistochemistry procedures are detailed in the methods. The file contains multiple imaging planes that were combined into the projection images shown in the figure panels. For the manuscript, the image was cropped to include only the outer hair cell region. The imaging channels included green (anti-GABA A beta 3 receptor, converted to yellow for colorblind palette in published image), red (tdTomato reporter labeling in a random sparse subset of type II SGN, converted to magenta), far-red (purple, anti-GAD label, converted to blue in final image). ### Folder: GAD immunos devel Fig 1 Folder: “GAD immunos devel Fig 1” contains confocal imaging Z-stacks from dissected cochlear preparations from transgenic mice. ·        Five Nikon Elements .nd2 files that can be read in Nikon Elements viewers or ImageJ are contained in the folder. Immunohistochemistry procedures are detailed in the methods. The file contains multiple imaging planes that were combined into the projection images shown in the figure panels. For the manuscript, the image was cropped to include only the outer hair cell region. File naming uses our original file names. o   In four of the images:  **05JUN22 P7 CIJ x tdT GAD65_488__Myo7a_647 A1C1Med 60xCrop002 A_P9_Medial_60x_405Dapi_488GAD65_561tdTom_647Myo7a_Wu 21JUN22 P11 CIJxtdT GAD65_488__Myo7a_647 A2C1Med 60xCrop001 23JUN22 P13 CIJxtdT GAD65_488__Myo7a_647 A1C1Med 60xCrop2** mice were euthanized at different ages (P7, P9, P11, or P13) to examine both the development of MOC efferent axons in the cochlea (identified in ChAT-IRES-Cre (CIJ)  x tdTomato (tdTom or tdT) reporter labeling, red channel converted to magenta for colorblind palette in final figure), as well as the developmental pattern of their GABAergic nature as determined by immuno labeling for the GABA synthetic enzyme GAD (green in original image, converted to blue in published figure for colorblind palette). Localization of MOC axons relative to OHCs visible due to immunolabeling of the OHCs with anti-MyoVIIa antibody (far-red in originals, shown in greyscale in individual panels). For these images, the animal age is included in the file name. o   For the fifth image **Row1sec3_stack_1**, the data is from a P20 ChAT-IRES-Cre ; tdTomato mouse. Image channels are green (GAD, converted to blue in final image), red (tdTomato genetic label of MOC axons, converted to magenta), and far-red (anti-synapsin antibody, converted to yellow). ### Folder iGABASnFR Folder “iGABASnFR” contains Nikon Elements .nd2 files that can be opened in Nikon Elements, a Nikon Elements viewer, or ImageJ. For these experiments, the iGABASnFR optical GABA indicator (green channel) is imaged using confocal timelapse imaging at between 7 and 16 frames per second, all at the same Z-plane. In some experiments with transgenic animals, the tdTomato reporter was imaged in the red channel. In some images, the “transmitted detector (TD)” was used to provide a bright-field-like image to aid in focal plane determination. The timing of the stimulation (exogenous GABA or acetylcholine application, or MOC axon electrical stimulation) is provided in research methods. **Variables used:** ROI = region of interest, a biologically relevant area of an image selected for fluorescence analysis delF/F = delta F / F, the change in fluorescence compared to baseline fluorescence for a given ROI **File naming conventions:** year-month-day \_ preparation (cochlea#, equivalent to animal #, each day starts over with 1)_region of cochlea imaged (starts over with 1 for each prep) \_ 00# - repetition in this location (usually first repetition per region used, unless of experimental problem such as stimulator not turned on). **Folder Structure:** “iGABASnFR” contains three subfolders. “ACh exogenous iGABASnFR” contains 6 files in which ACh was exogenously applied during the time series imaging. “GABA exogenous iGABASnFR” contains 6 files in which GABA was exogenously applied during the time series imaging. “MOC stim iGABASnFR” contains two subfolders, “P8-11 MOC stim iGABA SnFR” contains 20 files from post-natal day 8 to 11 mice. The MOC axons were stimulated electrically during these experiments. “P14-15 MOC stim iGABA SnFR” contains 6 files from post-natal day 14-15 mice. The MOC axons were stimulated electrically during these experiments. ### Folder: type II SGN recordings Folder “type II SGN recordings” contains patch-clamp electrophysiology data for experiments and related results for figure 5. Data from 41 cells are included, with some of the cells being included in multiple sections of figure 5 (eg data included in both GABA application numbers and gabazine data). These data were recorded using either the Molecular Devices pClamp software version 11 or the HEKA Patchmaster EBC10 software. Files can be opened with various software: Molecular devices (.abf) files can be opened with Clampfit software. This can be downloaded as part of the pClamp software suite (pClamp, AxoScope, and Clampfit; free download, does not require dongle unless the amplifier is being used to collect new data) at: -        [https://support.moleculardevices.com/s/article/Axon-pCLAMP-11-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page](https://support.moleculardevices.com/s/article/Axon-pCLAMP-11-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page) -        In addition, various converters can be used to convert .abf files to Matlab or python formats (eg. [https://www.mathworks.com/matlabcentral/fileexchange/6190-abfload](https://www.mathworks.com/matlabcentral/fileexchange/6190-abfload)) HEKA files (.dat files are the primary data files, .bak, .mrk, .onl, .pul, and .sol files are companion files that automatically get opened with the associated .dat file that has the same name) can be opened with HEKA Patchmaster (not Patchmaster Next aka PMN) or HEKA Fitmaster: -        [https://www.heka.com/downloads/downloads_main.html#down_fitmaster](https://www.heka.com/downloads/downloads_main.html#down_fitmaster) -        [https://www.heka.com/downloads/downloads_main.html#down_patchmaster](https://www.heka.com/downloads/downloads_main.html#down_patchmaster) (acquisition software) -        Full functionality requires purchase of a software dongle, but software can be run in the ‘demo’ mode to open and view data. Note: opening of some files generates a “Too many sample points” error, use the “My risk” button to clear the error and proceed. Open from within the fitmaster software, and load the .dat file. -        File converters are available for Matlab and python, for example this python converter: [https://github.com/campagnola/heka_reader](https://github.com/campagnola/heka_reader) -        In addition, .dat files were exported as .txt files for opening in any text editor or in Microsoft Excel. For these files, each column represents a ‘sweep’ which can be either a single sweep from a different voltage step in an “IV” experiment, or a single sweep from a drug application experiment. The voltage command (V) data and corresponding current response (I) data are in adjacent columns. Column titles have titles that detail the type of experiment (eg. “TypeII_IV_Vclmp” is an “IV” type recording (see abbreviations below) from a type II neuron in the Vclamp or voltage-clamp configuration, with the numbers responding to the experiment number within the file, and the sweep number. All data from a single cell is included in the same .txt file, including IV curves, drug application, or longer single voltage steps. In all cells GABA (1 mM) is applied to the tissue during the recording from a local gravity-fed pipette, with the timing noted either by a “tag” within the file trace or by the digital output amplifier channel which was used to control valves to apply the GABA-containing solution. In some experiments, the GABA A receptor blocker gabazine was bath applied prior to GABA application, with the details of the gabazine application detailed in the attached excel file (“Bachman et al 2025”) contained within the .zip file structure. Most data were recorded at a holding potential of -80 mV, but in some experiments the membrane potential was stepped to a different holding potential prior to GABA application, detailed in the excel file. **File naming conventions:** All experiments are identified by a code containing letters (initials of experimenter: Julia Bachman or Cat Weisz) and a number code, original cell IDs and file names are being used. **Variables and abbreviations:** SGN = spiral ganglion neuron OHC = outer hair cell GABA = gamma aminobutyric acid, an inhibitory neurotransmitter mV = milli volts, a unit of electrical potential in neurons pA = pico-amps, unit of electrical measurement of currents in neurons mM = milli Molar concentration Vhold = membrane holding voltage, the voltage that the internal neuron state was set at using the patch-clamp amplifier IV = current voltage, shorthand for a type of voltage-clamp experiment in which the neuron membrane potential is set to different holding potentials from -110 to ~+20 mV in 10 mV increments while recording the resulting currents in the neuron Appl = shorthand for ‘application’ pipette, a gravity fed pipette used to apply drugs (usually GABA) focally to the tissue **Code / Software** No additional code was generated for this work.