Forced expression of Lmx1b enhances differentiation of mouse ES cells into serotonergic neurons

The LIM homeodomain transcription factor Lmx1b is a key factor in the specification of the serotonergic neurotransmitter phenotype. Here, we explored the capacity of Lmx1b to direct differentiation of mouse embryonic stem (mES) cells into serotonergic neurons. mES cells stably expressing human Lmx1b were generated by lentiviral vector infection. Clones expressing Lmx1b at a low level showed increased neurogenesis and elevated production of neurons expressing serotonin, serotonin transporter, Tryptophan hydroxylase 2, and transcription factor Pet1, the landmarks of serotonergic differentiation. To explore the role of Lmx1b in the specification of the serotonin neurotransmission phenotype further, a conditional system making use of a floxed inducible vector targeted into the ROSA26 locus and a hormone-dependent Cre recombinase was engineered. This novel strategy was tested with the reporter gene encoding human placental alkaline phosphatase, and demonstrated its capacity to drive transgene expression in nestin+ neural progenitors and in Tuj1+ neurons. When it was applied to the inducible expression of human Lmx1b, it resulted in elevated expression of serotonergic markers. Treatment of neural precursors with the floor plate signal Sonic hedgehog further enhanced differentiation of Lmx1b-overexpressing neural progenitors into neurons expressing 5-HT, serotonin transporter, Tryptophan hydroxylase 2 and Pet1, when compared to Lmx1b-non expressing progenitors. Together, our results demonstrate the capacity of Lmx1b to specify a serotonin neurotransmitter phenotype when overexpressed in mESC-derived neural progenitors

Control of IsAHP in mouse hippocampus CA1 pyramidal neurons by RyR3-mediated calcium-induced calcium release.

In several neuronal preparations, the ryanodine-sensitive calcium store was reported to participate in the generation of slow afterhyperpolarization currents (IsAHP) involved in spike frequency adaptation. We show that calcium release from the ryanodine-sensitive calcium store is a major determinant of the triggering of IsAHP in mouse CA1 pyramidal neurons. Whole-cell patch clamp recordings in hippocampus slices show that the intracellular calcium stores depletion using an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase (5 muM cyclopiazonic acid), as well as the specific blockade of ryanodine receptors (100 muM ryanodine) both reduced the IsAHP by about 70%. Immunohistology, using an anti-RyR3 specific antibody, indicates that RyR3 expression is particularly enriched in the CA1 apical dendrites (considered as the most important site for sAHP generation). We show that our anti-RyR3 antibody acts as a functional RyR3 antagonist and induced a reduction in IsAHP by about 70%. The additional ryanodine application (100 mu M) did not further affect IsAHP, thus excluding RyR2 in IsAHP activation. Our results argue in favor of a specialized function of RyR3 in CA1 pyramidal cells in triggering IsAHP due to their localization in the apical dendrite

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use