Functional expression of electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse cortical astrocytes is dependent on S255-257 and regulated by mTOR

The electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), is the major bicarbonate transporter expressed in astrocytes. It is highly sensitive for bicarbonate and the main regulator of intracellular, extracellular, and synaptic pH, thereby modulating neuronal excitability. However, despite these essential functions, the molecular mechanisms underlying NBCe1-mediated astrocytic response to extracellular pH changes are mostly unknown. Using primary mouse cortical astrocyte cultures, we investigated the effect of long-term extracellular metabolic alkalosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+ -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, and phosphoproteomic analysis. The results showed significant downregulation of NBCe1 activity following metabolic alkalosis without influencing protein abundance or surface expression of NBCe1. During alkalosis, the rate of intracellular H+ changes upon challenging NBCe1 was decreased in wild-type astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Alkalosis-induced decrease of NBCe1 activity was rescued after activation of mTOR signaling. Moreover, mass spectrometry revealed constitutively phosphorylated S255-257 and mutational analysis uncovered these residues being crucial for NBCe1 transport activity. Our results demonstrate a novel mTOR-regulated mechanism by which NBCe1 functional expression is regulated. Such mechanism likely applies not only for NBCe1 in astrocytes, but in epithelial cells as well

Characterization of primary neurospheres generated from mouse ventral rostral hindbrain

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons

Morphometric study of the root anatomy in furcation area of mandibular first molars

Furcation involvement in periodontal disease has been a challenge for the dentist. OBJECTIVE: The aim of this study was to investigate root dimensions in the furcation area of 233 mandibular first molars. MATERIAL AND METHODS: Digital photomicrographs were used to obtain the following measurements on the buccal and lingual surfaces of each tooth: root trunk height (RT), horizontal interadicular distance obtained 1 mm (D1) and 2 mm (D2) below the fornix and interadicular angle (IA). RESULTS: Mean± standard deviation of buccal and lingual furcation measurements were, respectively, 1.37±0.78 mm and 2.04±0.89 mm for RT; 0.86±0.39 mm and 0.71±0.42 mm for D1; 1.50±0.48 mm and 1.38±0.48 mm for D2; 41.68±13.20º and 37.78±13.18º for IA. Statistically significant differences were found between all measured parameters for buccal and lingual sides (p<0.05, paired t test). CONCLUSIONS: In conclusion, the lingual furcation of mandibular first molars presented narrower entrance and longer root trunk than the buccal furcation, suggesting more limitation for instrumentation and worse prognosis to lingual furcation involvements in comparison to buccal lesions