Isolation, culture optimization and functional characterization of stem cell neurospheres from mouse neonatal olfactory bulb and epithelium

Abstract

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.

Remifentanil reduces glutamate toxicity in rat olfactory bulb neurons in culture

“…There is strong evidence that N-Methyl-D-Aspartate (NMDA) receptors play a major role in the central sensitization processes associated with hyperalgesia. 10 Recent animal 8 , 11 and human studies 12 , 13 , 14 , 15 , 16 confirmed this hypothesis, providing evidence that blocking NMDA receptors can prevent opioid-induced hyperalgesia. Studies by Guntz et al 21 showed remifentanil has no direct effect on NMDA receptors but increases NMDA current mediated by μ-opioid receptor activation, probably through an intracellular pathway.…”

“…The collapsed cells were suspended with fresh medium (Neurobasal medium, FBS 10%, B27 2%, and antibiotic 0.01%) and then the cells were seeded in 24-well plates (Corning, USA). The plate was stored in an incubator (5% CO 2 ; 37 °C) 13 , 14 ( Fig. 1 ).…”

Neurospheres: a potential in vitro model for the study of central nervous system disorders

“…Firstly, it is necessary to extract the nervous tissue, in this case the brain, and secondly the region of interest, which may be SVZ or SGZ. Then, the tissue needs to be dissociated enzymatically using enzymes such as trypsin/EDTA, papain, accutase and collagenase, followed by mechanical dissociation [11,34,35]. The cell pellet generated is cultivated on non-adherent substrate using a serum-free growth medium with mitogenic growth factors in a humidified incubator at 37 °C and 5% CO 2 [11].…”

Should we use remifentanil in every dose and every case?

“…Cerebellum cell cultures were obtained from the department of medical pharmacology of the Ataturk University (Erzurum, Turkey). Briefly, the cells after being centrifuged in 1200 rpm for 5 min were sown in 24 well plate (Corning, USA) by fresh medium (Neurobasal medium, FBS %10, B27 %2 and antibiotic %0,01) and stored at incubator (5% CO 2 ; 37°C) [11,12] (Figure 1).…”