ABSTRACT
The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.
Subject(s)
Cell Culture Techniques/methods , Cerebrospinal Fluid Proteins/metabolism , Neuroepithelial Cells/cytology , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cerebrospinal Fluid Proteins/isolation & purification , Cerebrospinal Fluid Proteins/physiology , Chick Embryo , Immunohistochemistry/methods , Neurogenesis , Organ Culture Techniques/methods , Tegmentum Mesencephali/cytology , Tegmentum Mesencephali/embryologyABSTRACT
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.