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Regulation of intracellular pH (pH(i)) in neurons is crucial to maintain their physiological function. In the current study, newly-developed polydimethylsiloxane (PDMS) microfluidic devices were used to independently investigate pH(i) regulation in neuronal soma and neurites. Embryonic cortical neurons were cultured in PDMS microfluidic devices with soma growing in one chamber (seeded) and neurites extending through a set of perpendicular microchannels into the opposite parallel chamber (non-seeded). Neurons in the microchambers were characterized by the vital dye calcein-red, polarized mitochondria, and expression of neuronal specific beta-tubulin (type-III), axonal Tau-1 protein, dendritic microtubule associated protein (MAP-2), and Na(+)/H(+) exchanger isoform 1 (NHE-1). Neurites exhibited higher resting pH(i) than soma (7.16 +/- 0.09 vs. 6.90 +/- 0.15). The neurites had a proton extrusion rate 3.7-fold faster than in soma following NH(4)Cl prepulse-mediated acidification (p < 0.05). The difference in the pH(i) regulation rates between neurites and soma can be accounted for by the larger surface area to volume ratio in the neurites. Interestingly, pharmacological inhibition of NHE-1 activity blocked the pH(i) regulation in soma and in neurites by approximately 70% (p < 0.05). Taken together, our study demonstrated that the microfluidic devices provide a useful tool to study neuronal pH(i) regulation in soma and their neurites. We conclude that NHE-1 plays an important role in regulation of pH(i) in both compartments.