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      Voltage-Activated Calcium Channels as Functional Markers of Mature Neurons in Human Olfactory Neuroepithelial Cells: Implications for the Study of Neurodevelopment in Neuropsychiatric Disorders

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          Abstract

          In adulthood, differentiation of precursor cells into neurons continues in several brain structures as well as in the olfactory neuroepithelium. Isolated precursors allow the study of the neurodevelopmental process in vitro. The aim of this work was to determine whether the expression of functional Voltage-Activated Ca 2+ Channels (VACC) is dependent on the neurodevelopmental stage in neuronal cells obtained from the human olfactory epithelium of a single healthy donor. The presence of channel-forming proteins in Olfactory Sensory Neurons (OSN) was demonstrated by immunofluorescent labeling, and VACC functioning was assessed by microfluorometry and the patch-clamp technique. VACC were immunodetected only in OSN. Mature neurons responded to forskolin with a five-fold increase in Ca 2+. By contrast, in precursor cells, a subtle response was observed. The involvement of VACC in the precursors’ response was discarded for the absence of transmembrane inward Ca 2+ movement evoked by step depolarizations. Data suggest differential expression of VACC in neuronal cells depending on their developmental stage and also that the expression of these channels is acquired by OSN during maturation, to enable specialized functions such as ion movement triggered by membrane depolarization. The results support that VACC in OSN could be considered as a functional marker to study neurodevelopment.

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          Most cited references55

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          Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

          1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
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            CNS stem cells express a new class of intermediate filament protein.

            Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.
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              Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.

              In cultures of embryonic striatum, we previously reported that EGF induces the proliferation of single precursor cells, which give rise to spheres of undifferentiated cells that can generate neurons and glia. We report here that, in vitro, these embryonic precursor cells exhibit properties and satisfy criteria representative of stem cells. The EGF-responsive cell was able to generate the three major phenotypes of the mammalian CNS--neurons, astrocytes, and oligodendrocytes. Approximately 90% of both primary spheres and secondary expanded clones, derived from the primary spheres, contained all three cell types. The increase in frequency of EGF-generated spheres, from 1% in primary culture to close to 20% in secondary culture, and the large number of clonally derived secondary spheres that could be generated from a single primary sphere indicate that EGF induces both renewal and expansion of the precursor cell itself. In population studies, the EGF-responsive cells were carried through 10 passages, resulting in a 10(7)-fold increase in cell number, without losing their proliferative and multilineage potential. Thus, this study describes the first demonstration, through clonal and population analyses in vitro, of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                14 June 2016
                June 2016
                : 17
                : 6
                : 941
                Affiliations
                [1 ]Laboratorio de Neurofarmacología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, Mexico City 14370, Mexico; hecsolch@ 123456imp.edu.mx (H.S.-C.); mvaldes@ 123456imp.edu.mx (M.V.-T.)
                [2 ]Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; edgarfloressoto@ 123456yahoo.com.mx (E.F.-S.); reyes.garcia.jorge@ 123456gmail.com (J.R.-G.); lmmr@ 123456unam.mx (L.M.-M.)
                [3 ]Departamento de Neurobiología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, Mexico City 14370, Mexico; ecalixto@ 123456imp.edu.mx
                Author notes
                [* ]Correspondence: bekin@ 123456imp.edu.mx ; Tel.: +52-55-4160-5097
                Article
                ijms-17-00941
                10.3390/ijms17060941
                4926474
                27314332
                f292e448-5519-4577-a4e8-aa45d9dcc6f2
                © 2016 by the authors; 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 ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 04 February 2016
                : 07 June 2016
                Categories
                Article

                Molecular biology
                calcium flux,differentiation process,human precursors,neuronal excitability,voltage-gated ca2+ channels,neuropsychiatric disorders

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