GABAergic Synapses Between Auditory Efferent Neurons and Type II Spiral Ganglion Afferent Neurons in the Mouse Cochlea

Background Introduction

The complexity and precision of the auditory system have always been important topics in neuroscience research. As a key component of the auditory system, the interactions between cells and neurons within the cochlea are crucial for the realization of auditory functions. Outer hair cells (OHCs) amplify sound signals through their electromotile properties, thereby enhancing auditory sensitivity and frequency tuning. OHCs not only receive cholinergic feedback from medial olivocochlear (MOC) efferent neurons but also transmit information to the brainstem via type II spiral ganglion neurons (SGNs). However, the mechanisms of interaction among neurons in the OHC region are not yet fully understood, especially the role of GABA (gamma-aminobutyric acid).

Previous studies have shown that MOC neurons may release not only acetylcholine (ACh) but also GABA. However, the specific functions and mechanisms of GABA in the cochlea remain unclear. This study aims to explore whether there is a GABAergic synapse between MOC neurons and type II SGNs and reveal its role in cochlear function.

Source of the Paper

This paper was co-authored by Julia L. Bachman, Siân R. Kitcher, Lucas G. Vattino, et al., with the research team coming from the National Institute on Deafness and Other Communication Disorders (NIDCD) under the National Institutes of Health (NIH), as well as the National Scientific and Technical Research Council (CONICET) of Argentina, among other institutions. The paper was published in the Proceedings of the National Academy of Sciences (PNAS) on February 18, 2025, titled “GABAergic synapses between auditory efferent neurons and type II spiral ganglion afferent neurons in the mouse cochlea.”

Research Process

1. Experimental Design and Methods

This study comprehensively analyzed the GABAergic synapse between MOC neurons and type II SGNs by combining optical neurotransmitter detection, immunohistochemistry, and patch-clamp electrophysiology techniques. The specific procedures are as follows:

a) Immunohistochemistry Experiment

The researchers used Chat-IRES-Cre; tdTomato mouse models, labeling the GABA synthesis enzyme (glutamic acid decarboxylase, GAD) and presynaptic markers (synapsin) through immunostaining to determine whether MOC neurons possess GABAergic characteristics. The experimental subjects were cochlear tissues from mice aged postnatal day 5 (P5) to postnatal day 20 (P20). Imaging was performed using a 40x or 60x objective lens on a Nikon A1R inverted microscope and analyzed using Nikon Elements software.

b) Patch-Clamp Electrophysiology Experiment

The researchers isolated OHCs from the apical turn of the cochlea of P11-13 mice and conducted whole-cell voltage-clamp recordings. By electrically stimulating MOC neuron axons, they recorded postsynaptic currents (PSCs) in OHCs and evaluated the regulatory effects of GABA B receptors (GABA BR) on neurotransmitter release. Additionally, the researchers performed patch-clamp recordings on the dendrites of type II SGNs from P3-10 mice to study responses mediated by GABA A receptors (GABA AR).

c) Optical Neurotransmitter Detection

The researchers injected an adeno-associated virus (AAV) into the posterior semicircular canal to express the GABA optical indicator (iGABASnFR) in type II SGNs. They detected GABA released from MOC neuron axons using confocal microscopy and studied the dynamics of GABA release combined with electrical stimulation techniques.

2. Experimental Results

a) GABAergic Characteristics of MOC Neurons

Immunohistochemical experiments showed that MOC neuron axon terminals exhibited GAD immunopositivity at postnatal day 7 (P7) and remained stable before and after the onset of hearing (P9-P20). The colocalization of GAD with the presynaptic marker synapsin indicates that MOC neurons possess GABAergic characteristics.

b) Regulation of MOC Neurotransmitter Release by GABA B Receptors

Patch-clamp experiments demonstrated that GABA reduces ACh release by activating GABA B receptors on MOC axon terminals, decreasing calcium influx through P/Q-type voltage-gated calcium channels (VGCCs). This mechanism is similar to the regulatory mechanism at the MOC-IHC (inner hair cell) synapse.

c) Release of GABA from MOC Neurons to Type II SGNs

Optical detection experiments showed that electrical stimulation of MOC neuron axons induced an increase in iGABASnFR fluorescence signals in type II SGNs, indicating that GABA is released from MOC axons and diffuses to type II SGNs. This phenomenon exists both before and after the onset of hearing.

d) Responses Mediated by GABA A Receptors in Type II SGNs

Patch-clamp recordings showed that exogenous GABA could evoke current responses in type II SGNs, and these responses could be blocked by the GABA A receptor antagonist gabazine. Current-voltage relationship experiments further confirmed that GABA acts through chloride ion channels mediated by ionotropic GABA A receptors.

3. Research Conclusions

This study provides the first confirmation of functional GABAergic synapses between MOC neurons and type II SGNs. GABA not only regulates neurotransmitter release from MOC neurons via GABA B receptors but also directly affects type II SGN activity via GABA A receptors. This discovery reveals the complex neuronal network within the cochlea, offering new insights into the fine-tuning of the auditory system.

Significance and Highlights of the Study

1. Scientific Significance

This study is the first to reveal GABAergic synapses between MOC neurons and type II SGNs, filling a gap in the research on the mechanisms of neuronal interactions in the cochlea. This discovery provides important clues for understanding the neural regulatory network within the cochlea.

2. Application Value

The presence of GABAergic synapses may be significant for the development and regulation of auditory functions. For example, GABAergic signaling may play a critical role during the development of cochlear neurons or affect auditory sensitivity by regulating OHC activity. Furthermore, this discovery may provide new targets for the treatment of auditory diseases.

3. Research Highlights

• Novel Experimental Methods: Combining optical neurotransmitter detection, immunohistochemistry, and patch-clamp electrophysiology techniques to comprehensively analyze the function of GABAergic synapses.
• Important Discoveries: First confirmation of GABAergic synapses between MOC neurons and type II SGNs, revealing their roles in cochlear function.
• Broad Application Prospects: Provides new directions for research on the development, functional regulation, and related diseases of the auditory system.

Other Valuable Information

The experimental data, immunohistochemistry images, and optical GABA indicator images from this study have been made publicly available and can be accessed through the Dryad database (DOI: 10.5061/dryad.2rbnzs80g). Additionally, the research team thanks the Genie project team at Janelia Research Campus for providing the iGABASnFR plasmids and Alan Hoofring from the NIH Medical Arts Branch for drawing the synaptic schematic diagram.

Through the comprehensive application of multidisciplinary techniques, this study reveals the GABAergic synapse between MOC neurons and type II SGNs in the cochlea, opening up new fields in auditory system research.