Midbrain and forebrain patterning delivers immunocytochemically and functionally similar populations of neuropeptide Y containing GABAergic neurons.

Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 µM), noradrenaline (30 µM), acetylcholine (30 µM) and dopamine (30 µM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 µM) and CGP55845 (1 µM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 µM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 µM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.

P2X2, P2X4 and P2Y1 receptors elevate intracellular Ca2+ in mouse embryonic stem cell-derived GABAergic neurons.

BACKGROUND AND PURPOSE: Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs. EXPERIMENTAL APPROACH: mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies. KEY RESULTS: Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors. CONCLUSIONS AND IMPLICATIONS: GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation.