Increased brain
ammonium (NH4(+)/NH3) plays a central role in the manifestation of
hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+)/NH3 on the
calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+)/NH3-evoked
calcium changes, employing fluorescence imaging with
Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+)/NH3 for 30 minutes caused a transient
calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90%) experienced a persistent
calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+)/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+)/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of
glutamine synthetase resulted in significantly larger
calcium increases in response to NH4(+)/NH3, indicating that
glutamine accumulation was not a primary cause.
Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of
voltage-gated sodium channels,
sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of
sodium/
calcium exchange (NCX),
AMPA- or mGluR5-receptors did not dampen NH4(+)/NH3-induced
calcium increases. They were, however, significantly reduced by inhibition of
NMDA receptors and depletion of intracellular
calcium stores. Taken together, our measurements show that sustained exposure to NH4(+)/NH3 causes a sustained increase in intracellular
calcium in astrocytes in situ, which is partly dependent on
NMDA receptor activation and on release of
calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte
calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.