Spiking Pattern of the Mouse Developing Inner Hair Cells Is Mostly Invariant Along the Tonotopic Axis

During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This activity at the pre-hearing stage allows the IHCs to autonomously excite the auditory nerve fibers and hence, represents an efficient mechanism to shape the tonotopic organization along the ascending auditory pathway. Using calcium imaging, we show that the activity in the developing cochlea consists of calcium waves that propagate across the supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, consistent with bursts of action potentials recorded in patch-clamp. In addition, adjacent auditory hair cells tend to have a synchronized [Ca2+]i activity, irrespective of their location along the base-to-apex gradient of the cochlea. Finally, we show that the mechanical ablation of the inner phalangeal cells (IPCs), a class of supporting cells, reduces the synchronized [Ca2+]i activity between neighboring sensory cells. These findings support the hypothesis that the tonotopic map refinement in higher auditory centers would depend on the synchronization of a discrete number of auditory sensory cells

Thyroid-Hormon Abhängigkeit des Reifprozesses von Bändersynapsen in Haarzellen

In dieser Arbeit untersuchten wir die Organisation, molekulare Zusammensetzung und Funktion von Bändersynapsen innerer Haarzellen (IHC) in einem hypo-thyroiden Mausmodell (Pax-8-/-). In Messungen exozytotischer Kapazitätsveränderungen in IHCs hypo-thyroider Tiere fanden wir eine ineffiziente Kopplung zwischen Ca2+ Strom und Exozytose. Quantifizierung der Anzahl der Bändersynapsen durch Immunhistochemie und Konfokalmikroskopie offenbarte eine höhere Zahl an Synapsen in Pax-8-/--Tieren im Vergleich zu Wildtyp- Tieren. Beide Eigenschaften weisen auf einen Verbleib hypo-thyroider IHCs in einem Entwicklungsstadium funktioneller und morphologischer Unreife hin. Durch Thyroxin-Ersatztherapie kann in diesen Zellen eine normale Entwicklung induziert werden. Ebenso fanden wir in hypo-thyroiden Tieren weitere Anzeichen für eine unvollständige Entwicklung innerer Haarzellen, wie fehlende Expression von BK-Typ K+ Kanälen, sowie verlängerte Präsenz der efferenten Innervation und der Aktionspotential-Generierung. Auf der molekularen Ebene erforschten wir in hypo-thyroiden Mäusen die Expression von zwei präsynaptischen Proteinen (SNAP 25 und Synaptobrevin 1) und stellten Abnahmen der jeweiligen mRNA Konzentrationen fest. Zusätzlich präsentiert unsere Studie ein Mausmodell zum Studium der auditorischen Funktion bei Menschen mit durch Thyroxin-Mangel hervorgerufener Taubheit. Wir schließen daraus, dass Thyroxin ein wichtiges Signal für die korrekte Entwicklung der inneren Haarzellen darstellt

Spatiotemporal pattern of action potential firing in developing inner hair cells of the mouse cochlea

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Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse

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Remodeling of the Inner Hair Cell Microtubule Meshwork in a Mouse Model of Auditory Neuropathy AUNA1

International audienceAuditory neuropathy 1 (AUNA1) is a form of human deafness resulting from a point mutation in the 5' untranslated region of the Diaphanous homolog 3 (DIAPH3) gene. Notably, the DIAPH3 mutation leads to the overexpression of the DIAPH3 protein, a formin family member involved in cytoskeleton dynamics. Through study of diap3-overexpressing transgenic (Tg) mice, we examine in further detail the anatomical, functional, and molecular mechanisms underlying AUNA1. We identify diap3 as a component of the hair cells apical pole in wild-type mice. In the diap3-overexpressing Tg mice, which show a progressive threshold shift associated with a defect in inner hair cells (IHCs), the neurotransmitter release and potassium conductances are not affected. Strikingly, the overexpression of diap3 results in a selective and early-onset alteration of the IHC cuticular plate. Molecular dissection of the apical components revealed that the microtubule meshwork first undergoes aberrant targeting into the cuticular plate of Tg IHCs, followed by collapse of the stereociliary bundle, with eventual loss of the IHC capacity to transmit incoming auditory stimuli

VGLUT3-p.A211V variant fuses stereocilia bundle and elongates synaptic ribbons in the human deafness DFNA25

DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied the phenotype of the mouse harboring the p.A221V mutation in human (corresponding to p.A224V in mouse). Using auditory brainstem response and distortion products of otoacoustic emissions, we showed that VGLUT3 A224V/A224V mouse replicates the DFNA25 progressive hearing loss with intact cochlear amplification. Scanning electron microscopy examinations demonstrated fused stereocilia bundle of the inner hair cells (IHCs) as the primary cause for DFNA25. In addition, the IHC ribbon synapses undergo structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons associated with an increase in the rate of the sustained releasable pool of exocytosis. These results indicate that the primary defect in DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3 A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment of DFNA25

VGLUT3‐p.A211V variant fuses stereocilia bundles and elongates synaptic ribbons

International audienceDFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons and patch-clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25. KEY POINTS: The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs). The VGLUT3-p.A211V variant is associated with human deafness DFNA25. Mutant mice carrying the VGLUT3-p.A211V variant show progressive hearing loss. IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres. These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages