DOI: 10.5061/dryad.4tmpg4fq5
title: iGABASnFR data from: Co-release of GABA and ACh from medial olivocochlear neurons as a fine regulatory mechanism of cochlear efferent inhibition

description: During development, inner hair cells (IHCs) in the mammalian cochlea are
 unresponsive to acoustic stimuli but instead exhibit spontaneous activity.
 During this same period, neurons originating from the medial olivocochlear
 complex (MOC) transiently innervate IHCs, regulating their firing pattern
 which is crucial for the correct development of the auditory pathway.
 Although the MOC-IHC is a cholinergic synapse, previous evidence indicates
 the widespread presence of gamma-aminobutyric acid (GABA) signaling
 markers, including presynaptic GABAB receptors (GABABR). In this study, we
 explore the source of GABA by optogenetically activating either
 cholinergic or GABAergic fibers. The optogenetic stimulation of MOC
 terminals from GAD;ChR2-eYFP and ChAT;ChR2-eYFP mice evoked synaptic
 currents in IHCs that were blocked by α-bungarotoxin. This suggests that
 GABAergic fibers release ACh and activate α9α10 nicotinic acetylcholine
 receptors (nAChRs). Additionally, MOC cholinergic fibers release not only
 ACh but also GABA, as the effect of GABA on ACh response amplitude was
 prevented by applying the GABAB-R blocker (CGP 36216). Using optical
 neurotransmitter detection and calcium imaging techniques, we examined the
 extent of GABAergic modulation at the single synapse level. Our findings
 suggest heterogeneity in GABA modulation, as only 15 out of 31 recorded
 synaptic sites were modulated by applying the GABABR specific antagonist,
 CGP (100-200 µM). In conclusion, we provide compelling evidence that GABA
 and ACh are co-released from at least a subset of MOC terminals. In this
 circuit, GABA functions as a negative feedback mechanism, locally
 regulating the extent of cholinergic inhibition at certain efferent-IHC
 synapses during an immature stage.
description: # iGABASnFR data from: Co-release of GABA and ACh from medial
 olivocochlear neurons as a fine regulatory mechanism of cochlear efferent
 inhibition Dataset DOI:
 [10.5061/dryad.4tmpg4fq5](https://doi.org/10.5061/dryad.4tmpg4fq5) ##
 Description of the data and file structure We have submitted our raw
 iGABASnFR optical detection data to support the Castagnola et al 2025
 manuscript. **Notes:** Data and File Structure: All raw data files
 contained in the .zip file “Castagnola_et_al_iGABASnFR.zip”  Excel file
 “Castangola et al iGABASnFR file list final” 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 .nd2 LIM images” file type. .nd2 files can be viewed in Nikon
 Elements viewers or ImageJ. Sharing / Access information: Please contact
 [catherine.weisz@nih.gov](mailto:catherine.weisz@nih.gov) with any
 questions. Folder: “Castagnola et al 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, 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 (MOC axon
 electrical stimulation) is provided in research methods. **File naming
 conventions:** All files start with iGABASnFR to denote the experiment
 type (iGABASnFR “GABA Sniffer” optical GABA detection), followed by two
 initials (LT = Lester Torres Cadenas, SK = Sian Kitcher, the
 experimenters), followed by a number to indicate the cell ID in the
 dataset. Some files also have “control”, “0Ca” (zero calcium external
 solution), or “wash” (return to normal calcium external solution) in the
 file name to indicate the experimental condition. **Folder Structure:** 
 “Castagnola et al iGABASnFR” folder contains the Excel file “Castagnola et
 al iGABASnFR file list final.xlxs”, and the same file in .csv format. It
 also contains 14 subfolders, one per experiment, with the same naming
 conventions as the imaging files (iGABASnFR LT1-7, or iGABASnFR SK1-7).
 The LT folders each contain three files, one each for the control, zero
 calcium (0Ca), or wash conditions, indicated at the end of the file name.
 **Variables and abbreviations:** 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 GABA = gamma aminobutyric acid, an inhibitory
 neurotransmitter iGABASnFR = “GABA Sniffer”, an optical GABA indicator
 introduced to cochlear spiral ganglion neurons via AAV injection into the
 cochlea mM = milli Molar concentration **Code / Software** No additional
 code was generated for this work.