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Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent y...

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Autores principales: Voelker, Johannes, Voelker, Christine, Engert, Jonas, Goemann, Nikolas, Hagen, Rudolf, Rak, Kristen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8534573/
https://www.ncbi.nlm.nih.gov/pubmed/34685782
http://dx.doi.org/10.3390/cells10102802
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author Voelker, Johannes
Voelker, Christine
Engert, Jonas
Goemann, Nikolas
Hagen, Rudolf
Rak, Kristen
author_facet Voelker, Johannes
Voelker, Christine
Engert, Jonas
Goemann, Nikolas
Hagen, Rudolf
Rak, Kristen
author_sort Voelker, Johannes
collection PubMed
description Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.
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spelling pubmed-85345732021-10-23 Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells Voelker, Johannes Voelker, Christine Engert, Jonas Goemann, Nikolas Hagen, Rudolf Rak, Kristen Cells Article Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration. MDPI 2021-10-19 /pmc/articles/PMC8534573/ /pubmed/34685782 http://dx.doi.org/10.3390/cells10102802 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Voelker, Johannes
Voelker, Christine
Engert, Jonas
Goemann, Nikolas
Hagen, Rudolf
Rak, Kristen
Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title_full Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title_fullStr Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title_full_unstemmed Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title_short Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells
title_sort spontaneous calcium oscillations through differentiation: a calcium imaging analysis of rat cochlear nucleus neural stem cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8534573/
https://www.ncbi.nlm.nih.gov/pubmed/34685782
http://dx.doi.org/10.3390/cells10102802
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