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Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3′-iminodipropionitrile ototoxicity in the mouse

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Abstract

The cellular and molecular events that precede hair cell (HC) loss in the vestibular epithelium during chronic ototoxic exposure have not been widely studied. To select a study model, we compared the effects of sub-chronic exposure to different concentrations of 3,3′-iminodipropionitrile (IDPN) in the drinking water of two strains of mice and of both sexes. In subsequent experiments, male 129S1/SvImJ mice were exposed to 30 mM IDPN for 5 or 8 weeks; animals were euthanized at the end of the exposure or after a washout period of 13 weeks. In behavioral tests, IDPN mice showed progressive vestibular dysfunction followed by recovery during washout. In severely affected animals, light and electron microscopy observations of the vestibular epithelia revealed HC extrusion towards the endolymphatic cavity. Comparison of functional and ultrastructural data indicated that animals with fully reversible dysfunction did not have significant HC loss or stereociliary damage, but reversible dismantlement of the calyceal junctions that characterize the contact between type I HCs (HCI) and their calyx afferents. Immunofluorescent analysis revealed the loss of calyx junction proteins, Caspr1 and Tenascin-C, during exposure and their recovery during washout. Synaptic uncoupling was also recorded, with loss of pre-synaptic Ribeye and post-synaptic GluA2 puncta, and differential reversibility among the three different kinds of synaptic contacts existing in the epithelium. qRT-PCR analyses demonstrated that some of these changes are at least in part explained by gene expression modifications. We concluded that calyx junction dismantlement and synaptic uncoupling are early events in the mouse vestibular sensory epithelium during sub-chronic IDPN ototoxicity.

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Acknowledgements

This study was supported by grants BFU2015-66109-R (MINECO/FEDER, EU), and 2017 SGR 621 (Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya), and a Minor Research Grant from the Ménière’s Society, UK. E.A.G. was supported by the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (FI-DGR 2015 Program) and by the Ministerio de Educación, Cultura y Deporte de España (FPU 2015). The electron and confocal microscopy studies were performed at the Scientific and Technological Centers of the University of Barcelona (CCiT-UB). We thank Dr. Benjamı́n Torrejon-Escribano, Josep M. Rebled, Rosa Rivera and Adriana Martínez Gené for technical assistance and Natacha F. Kolar for animal care. We also thank Dr. Judit Homs and the students Meritxell Deulofeu, Sílvia Prades, Marc Bosch-Mola, Bertrán Álvarez-Pérez, Aina Espinosa, Judith Lloret, Júlia Valor, and Maria Capdevila for their contributions to the study.

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  1. Departament de Ciències Fisiològiques, Institut de Neurociènces, Universitat de Barcelona, Feixa Llarga s/n, 08907, L’Hospitalet de Llobregat, Catalunya, Spain

    Erin A. Greguske, Maria Carreres-Pons & Jordi Llorens

  2. Institut d’Investigació Biomèdica de Bellvitge, IDIBELL, L’Hospitalet de Llobregat, 08907, Catalunya, Spain

    Erin A. Greguske & Jordi Llorens

  3. Departament d’Infermeria Fonamental i Medicoquirúrgica, Institut de Neurociències, Universitat de Barcelona, Feixa Llarga s/n, 08907, L’Hospitalet de Llobregat, Catalunya, Spain

    Blanca Cutillas

  4. Research Group of Clinical Anatomy, Embryology and Neuroscience (NEOMA), Departament de Ciències Mèdiques, Facultat de Medicina, Universitat de Girona, 17003, Girona, Catalunya, Spain

    Pere Boadas-Vaello

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  1. Erin A. Greguske
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Greguske, E.A., Carreres-Pons, M., Cutillas, B. et al. Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3′-iminodipropionitrile ototoxicity in the mouse. Arch Toxicol 93, 417–434 (2019). https://doi.org/10.1007/s00204-018-2339-0

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