Neurons of the avian cochlear nucleus magnocellularis (NM) receive glutamatergic inputs from the spiral ganglion cells via the auditory nerve and feedback GABAergic inputs primarily from the superior olivary nucleus. We investigated regulation of Ca(2+) signaling in NM neurons with ratiometric Ca(2+) imaging in chicken brain slices. Application of exogenous glutamate or GABA increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) in NM neurons. Interestingly, GABA-induced Ca(2+) responses persisted into neuronal maturation, in both standard and energy substrate enriched artificial cerebrospinal fluid. More importantly, we found that electrical stimulation applied to the glutamatergic and GABAergic afferent fibers innervating the NM was able to elicit transient [Ca(2+)](i) increases in NM neurons, and the amplitude of the Ca(2+) responses increased with increasing frequency and duration of the electrical stimulation. Antagonists for ionotropic glutamate receptors significantly blocked these [Ca(2+)](i) increases, whereas blocking GABA(A) receptors did not affect the Ca(2+) responses, suggesting that synaptically released glutamate but not GABA induced the Ca(2+) signaling in vitro. Furthermore, activation of GABA(A) receptors with exogenous agonists inhibited synaptic activity-induced [Ca(2+)](i) increases in NM neurons, suggesting a role of GABA(A) receptors in the regulation of Ca(2+) homeostasis in the avian cochlear nucleus neurons.