Spontaneous synchronized bursts of activity play an essential role in the maturation and plasticity of neuronal networks. To investigate the cellular properties that enable spontaneous network activity we have used dissociated cultures of hippocampal neurons that express prolonged network activity bursts. Acute exposure to a low concentration of NMDA caused an increase in spontaneous firing rates and intracellular calcium concentration ([Ca²⁺]i). However, in the course of a chronic (longer than one day) exposure to NMDA, [Ca²⁺]i recovered back to normal baseline levels, and only sporadic asynchronous calcium transients were detected. Spontaneous network bursts were still absent one hour after the removal of NMDA, indicating a persistent downregulation of network activity, which did recover eventually 2 days later. This effect of NMDA was activity-dependent as it was blocked by co-application of tetrodotoxin (TTX). The chronic NMDA-treated neurons expressed normal morphology and active membrane properties as well as spontaneous mEPSCs and postsynaptic reactivity to glutamate. However, in response to trains of afferent stimulation in paired recordings, the treated neurons expressed synaptic depression as opposed to synaptic potentiation seen in control cells. Also, treated neurons did not respond to low intensity electrical field stimulation as did control cells. In addition, western blot analysis revealed that chronic exposure to NMDA altered presynaptic but not postsynaptic protein expression patterns, suggesting a presynaptic locus of effect. Thus, a long lasting increase in activity downregulates neurotransmitter release in order to prevent over-excitation of the network, and consequently@ blocks the generation of network bursts.