Periodic Reporting for period 1 - DynaHear (Solving the dynamic range problem of hearing: deciphering and harnessing cochlear mechanisms of sound intensity coding)
Okres sprawozdawczy: 2023-01-01 do 2025-06-30
Our sense of hearing processes stimuli that differ in sound pressure by more than six orders of magnitude. Yet, while the presynaptic inner hair cells (IHCs) cover this wide dynamic range, each postsynaptic spiral ganglion neuron (SGN) encodes only a fraction and the intensity information is then reconstructed by the brain. This so-called “dynamic range problem” of hearing is known for decades, but how sound intensity information is decomposed into different neural pathways remains elusive.
In vivo recordings report major functional SGN diversity and ensembles of such diverse neurons collectively encode intensity for a given sound frequency. Recently, a major heterogeneity of afferent SGN synapses with IHCs as well as different molecular SGN profiles have been discovered. How these relate to the diverse sound coding properties of SGNs remains to be elucidated.
DynaHear sets out to close this gap by testing the hypothesis that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound intensity information. This is enabled by innovative approaches to cochlear structure and function, some of which we have recently established, while others will be developed in DynaHear. We will combine electrophysiology, optogenetics, molecular labelling and tracing, multiscale and multi-modal imaging, with computational modeling. We will elucidate the molecular underpinnings of afferent synaptic heterogeneity, decipher mechanisms establishing it, and relate them to functional SGN diversity.
DynaHear promises to fundamentally advance our understanding of sound intensity coding and to contribute to solving the dynamic range problem. Moreover, it will help to better understand synaptic hearing impairment, assist hearing rehabilitation, and pave the way for innovative therapeutic approaches such as gene therapy and optogenetic hearing restoration.
In vivo recordings report major functional SGN diversity and ensembles of such diverse neurons collectively encode intensity for a given sound frequency. Recently, a major heterogeneity of afferent SGN synapses with IHCs as well as different molecular SGN profiles have been discovered. How these relate to the diverse sound coding properties of SGNs remains to be elucidated.
DynaHear sets out to close this gap by testing the hypothesis that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound intensity information. This is enabled by innovative approaches to cochlear structure and function, some of which we have recently established, while others will be developed in DynaHear. We will combine electrophysiology, optogenetics, molecular labelling and tracing, multiscale and multi-modal imaging, with computational modeling. We will elucidate the molecular underpinnings of afferent synaptic heterogeneity, decipher mechanisms establishing it, and relate them to functional SGN diversity.
DynaHear promises to fundamentally advance our understanding of sound intensity coding and to contribute to solving the dynamic range problem. Moreover, it will help to better understand synaptic hearing impairment, assist hearing rehabilitation, and pave the way for innovative therapeutic approaches such as gene therapy and optogenetic hearing restoration.
The work within DynaHear is progressing well. The advances made towards the three objectives are described below.
Obj. 1: Elucidate molecular underpinnings of heterogeneous presynaptic Ca2+ influx
We established MINFLUX imaging of IHC presynaptic active zones (AZs), allowing us to start early with the work to map out the abundance and localization of AZ proteins in relation to their position within the IHC (originally planned to start in year 3). Some of these data are already available as a preprint (Kapooor et al., 2025). Detailed analysis of CaV channel subunits has started and immunolocalization of CaVbeta subunits has been performed. Analysis of the role of the CaVa2d2 subunit has started with experiments in Cacna2d2tm1Svi mice. A mouse line carrying a Halo-tagged CaV1.3 subunit has been generated and is currently evaluated.
Obj. 2: Decipher signaling mechanisms establishing heterogeneous properties of afferent IHC synapses
We have imported the GPR156 and Gai1/3 DKO mouse lines and are breeding them in order to obtain animals with the correct genotype, first immunohistochemical characterizations have been performed. Following the sanitation of this mouse line, we have started first electrophysiological characterization. We have cross-bred conditional Runx1-KO mice with Bhlbh5-Cre animals and have performed experiments. To examine the role of efferent signaling, we have decided to focus on genetic differentiation due to the lower burden placed on the experimental animals and the better reliability of experiments compared to surgical ablation. We have generated a mouse line by crossing a conditional SNAP-25 KO line with Urocortin-Cre mice to silence efferent neurons from the lateral olivocochlear system. Experiments have started and initial results indicate that the genetic disruption of SNAP-25 expression in efferent olivocochlear synapses was successful.
Obj. 3: Relate afferent synaptic heterogeneity and SGN diversity
We analyzed CaV1.3A749G mutant mice and confirmed that the voltage range of AZ Ca2+ influx determines the spontaneous and evoked SGN firing phenotype (Karagulyan et al., 2025). Initial results of in vivo electrophysiology recordings from the auditory nerve of Runx1-cKO mice (see Obj. 2) indicate that, contrary to expectations, spontaneous firing rates are significantly lower than in WT. We have now started experiments for AAV-mediated overexpression of Runx1 in SGNs. We established advanced lightsheet imaging of cleared cochlear tissue and use this to examine the distribution and connectivity of afferent SGN types as well as ribbon synapses and localize them in relation to the IHC (Aakhte et al., 2025), aided by newly developed Deep-Learning tools to segment the 3D microscopy data (Muth et al., 2025). These will also be used with Netrin-G1 tdTomato mice which we employ for the identification of SGN subtypes. A model of spontaneous and sound-evoked firing of SGNs has been prepared (Karagulyan et al., 2025) and is updated and refined based on the results of ongoing experiments.
Finally, we have directly measured the pre- and postsynaptic activity of the IHC – SGN synapse (Jaime Tobón & Moser, 2023) and analyzed it based on the position (modiolar / pillar side) of the targeted synapse in paired electrophysiological recordings, confirming that synapses with high spontaneous rate of release are found predominantly on the IHC's pillar side (Jaime Tobón & Moser, 2024), indicating that synaptic heterogeneity in IHCs directly contributes to the diversity of spontaneous and sound-evoked firing of SGNs.
References
Aakhte M et al. 2025. Isotropic, aberration-corrected light sheet microscopy for rapid high-resolution imaging of cleared tissue. [preprint] bioRxiv. doi: 10.1101/2025.02.21.639411
Bastille I et al. 2025. Combinatorial transcriptional regulation establishes subtype-appropriate synaptic properties in auditory neurons. Cell Rep. doi: 10.1016/j.celrep.2025.115796.
Jaime Tobón LM, Moser T. 2023. Ca2+ regulation of glutamate release from inner hair cells of hearing mice. PNAS. doi: 10.1073/pnas.2311539120
Jaime Tobón LM, Moser T. 2024. Bridging the gap between presynaptic hair cell function and neural sound encoding. eLife. doi: 10.7554/eLife.93749.3
Kapoor R et al. 2025. Charting the nanotopography of inner hair cell synapses using MINFLUX nanoscopy. [preprint] bioRxiv. doi: 10.1101/2025.04.22.649963
Karagulyan N et al. 2024. Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity. [preprint] eLife . doi: 10.7554/eLife.103481.2
Karagulyan N et al. 2025. Gating of hair cell Ca2+ channels governs the activity of cochlear neurons. Sci Adv. doi: 10.1126/sciadv.adu7898
Moser T et al. 2023. Diversity matters - extending sound intensity coding by inner hair cells via heterogeneous synapses. EMBO J. doi: 10.15252/embj.2023114587
Obj. 1: Elucidate molecular underpinnings of heterogeneous presynaptic Ca2+ influx
We established MINFLUX imaging of IHC presynaptic active zones (AZs), allowing us to start early with the work to map out the abundance and localization of AZ proteins in relation to their position within the IHC (originally planned to start in year 3). Some of these data are already available as a preprint (Kapooor et al., 2025). Detailed analysis of CaV channel subunits has started and immunolocalization of CaVbeta subunits has been performed. Analysis of the role of the CaVa2d2 subunit has started with experiments in Cacna2d2tm1Svi mice. A mouse line carrying a Halo-tagged CaV1.3 subunit has been generated and is currently evaluated.
Obj. 2: Decipher signaling mechanisms establishing heterogeneous properties of afferent IHC synapses
We have imported the GPR156 and Gai1/3 DKO mouse lines and are breeding them in order to obtain animals with the correct genotype, first immunohistochemical characterizations have been performed. Following the sanitation of this mouse line, we have started first electrophysiological characterization. We have cross-bred conditional Runx1-KO mice with Bhlbh5-Cre animals and have performed experiments. To examine the role of efferent signaling, we have decided to focus on genetic differentiation due to the lower burden placed on the experimental animals and the better reliability of experiments compared to surgical ablation. We have generated a mouse line by crossing a conditional SNAP-25 KO line with Urocortin-Cre mice to silence efferent neurons from the lateral olivocochlear system. Experiments have started and initial results indicate that the genetic disruption of SNAP-25 expression in efferent olivocochlear synapses was successful.
Obj. 3: Relate afferent synaptic heterogeneity and SGN diversity
We analyzed CaV1.3A749G mutant mice and confirmed that the voltage range of AZ Ca2+ influx determines the spontaneous and evoked SGN firing phenotype (Karagulyan et al., 2025). Initial results of in vivo electrophysiology recordings from the auditory nerve of Runx1-cKO mice (see Obj. 2) indicate that, contrary to expectations, spontaneous firing rates are significantly lower than in WT. We have now started experiments for AAV-mediated overexpression of Runx1 in SGNs. We established advanced lightsheet imaging of cleared cochlear tissue and use this to examine the distribution and connectivity of afferent SGN types as well as ribbon synapses and localize them in relation to the IHC (Aakhte et al., 2025), aided by newly developed Deep-Learning tools to segment the 3D microscopy data (Muth et al., 2025). These will also be used with Netrin-G1 tdTomato mice which we employ for the identification of SGN subtypes. A model of spontaneous and sound-evoked firing of SGNs has been prepared (Karagulyan et al., 2025) and is updated and refined based on the results of ongoing experiments.
Finally, we have directly measured the pre- and postsynaptic activity of the IHC – SGN synapse (Jaime Tobón & Moser, 2023) and analyzed it based on the position (modiolar / pillar side) of the targeted synapse in paired electrophysiological recordings, confirming that synapses with high spontaneous rate of release are found predominantly on the IHC's pillar side (Jaime Tobón & Moser, 2024), indicating that synaptic heterogeneity in IHCs directly contributes to the diversity of spontaneous and sound-evoked firing of SGNs.
References
Aakhte M et al. 2025. Isotropic, aberration-corrected light sheet microscopy for rapid high-resolution imaging of cleared tissue. [preprint] bioRxiv. doi: 10.1101/2025.02.21.639411
Bastille I et al. 2025. Combinatorial transcriptional regulation establishes subtype-appropriate synaptic properties in auditory neurons. Cell Rep. doi: 10.1016/j.celrep.2025.115796.
Jaime Tobón LM, Moser T. 2023. Ca2+ regulation of glutamate release from inner hair cells of hearing mice. PNAS. doi: 10.1073/pnas.2311539120
Jaime Tobón LM, Moser T. 2024. Bridging the gap between presynaptic hair cell function and neural sound encoding. eLife. doi: 10.7554/eLife.93749.3
Kapoor R et al. 2025. Charting the nanotopography of inner hair cell synapses using MINFLUX nanoscopy. [preprint] bioRxiv. doi: 10.1101/2025.04.22.649963
Karagulyan N et al. 2024. Probing the role of synaptic adhesion molecule RTN4RL2 in setting up cochlear connectivity. [preprint] eLife . doi: 10.7554/eLife.103481.2
Karagulyan N et al. 2025. Gating of hair cell Ca2+ channels governs the activity of cochlear neurons. Sci Adv. doi: 10.1126/sciadv.adu7898
Moser T et al. 2023. Diversity matters - extending sound intensity coding by inner hair cells via heterogeneous synapses. EMBO J. doi: 10.15252/embj.2023114587
We have started to elucidate the mechanisms establishing pre- and postsynaptic heterogeneity in the inner ear, which were so far unknown. Next to data indicating which synaptic proteins are differently regulated at the IHC AZ, we have begun showing how these proteins are arranged at the nanoscale. Our findings, not least those on the role of Ca2+ channels in regulating the activity of SGNs, have implications for the underlying mechanisms and potential treatment of noise-induced hearing loss. Finally, we expect them to aid in the development of gene therapeutic approaches in the inner ear and of optogenetic hearing restoration, which is pursued in our laboratory.