Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness

Abstract

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Approaches to Study Gap Junctional Coupling

“…Background labeling of endogenous biotin Huang et al, 1992Livnah et al, 1993 Biocytin 375 (Strept-/) Avidin conjugates Permeable through all astrocytic Cx Background labeling of endogenous biotin Horikawa andArmstrong, 1988 Livnah et al, 1993 2-NBDG 342 Imaging of glucose distribution Degradation to non-fluorescent derivative Yoshioka et al, 1996Yamada et al, 2000 6-NBDG 342 Imaging of glucose distribution Speizer et al, 1985 LY, Lucifer Yellow; NB, neurobiotin; Cx, connexin; NBDG, deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose; AF, Alexa Fluor. Augustin et al, 2016;Eitelmann et al, 2019Eitelmann et al, , 2020. Using peroxidases produces a light-stable product that is not sensitive to photo-bleaching (Konietzko and Muller, 1994;D'Ambrosio et al, 1998).…”

“…The latter ones can be linked to either fluorophores or enzymes. Using a fluorophore allows the combination with further immunohistochemical processing of the tissue ( Schools et al, 2006 ; Augustin et al, 2016 ; Eitelmann et al, 2019 , 2020 ). Using peroxidases produces a light-stable product that is not sensitive to photo-bleaching ( Konietzko and Muller, 1994 ; D’Ambrosio et al, 1998 ).…”

“…The anisotropy of tracer spreading originates from astrocyte topography ( Anders et al, 2014 ; Augustin et al, 2016 ; Ghezali et al, 2018 ; Wadle et al, 2018 ). Interestingly, in the lateral superior olive—a conspicuous nucleus in the auditory brainstem—absence of spontaneous cochlea-driven neuronal activity leading to disturbed neuronal circuitry ( Hirtz et al, 2012 ; Muller et al, 2019 ) causes altered astrocyte topography followed by altered orientation of tracer-coupled networks ( Eitelmann et al, 2020 ). In recent years, several different approaches were developed to analyze the anisotropy of tracer-coupled networks ( Figure 4 ).…”

“…However, anisotropic tracer diffusion will be visualized only if the tracer is injected into an astrocyte that is not located in the center ( Houades et al, 2008 ; Condamine et al, 2018a ) as the “Vector sum” approach is not capable to detect the anisotropy of tracer-coupled networks if they are symmetric with respect to a point ( Eitelmann et al, 2019 ). The second vector-based approach calculates the “Vector means” in four 90°sectors and the ratio of tracer extension is calculated ( Figure 4B 6 ; Eitelmann et al, 2019 , 2020 ). However, analyzing the anisotropy of tracer spreading using only two fixed axes might result in falsified results.…”

Molecular Mechanisms of Sensorineural Hearing Loss and Development of Inner Ear Therapeutics

“…On a different note, the paper by Eitelmann and co-workers [ 9 ] reported the altered gap junction network topography in mouse models of human hereditary deafness. Mutations in various genes, such as those coding for the voltage-gated calcium channel CaV1.3 or the calcium sensor otoferlin in the inner hair cells of the cochlea, cause hereditary deafness.…”