iGABASnFR data from: Co-release of GABA and ACh from medial olivocochlear neurons as a fine regulatory mechanism of cochlear efferent inhibition
Data files
Oct 14, 2025 version files 3.25 GB
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Castagnola_et_al_iGABASnFR.zip
3.25 GB
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README.md
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Abstract
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.
Dataset DOI: 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 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.