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 µM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 µM), PPADS (10 µM) and MRS2179 (10 µM), but not by NF023 (10 µM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 µM) 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.