We investigated how blockade of spontaneous action potentials influenced the synaptogenesis by measuring the field population spike using hippocampal organotypic cultures. Although respective blockade of inhibitory and excitatory neurotransmission by picrotoxin and CNQX did not significantly induce cell death in all hippocampal area, sodium channel blocking drugs (tetrodotoxin or lidocaine) caused specific and severe damage and affected the formation of functional synapse in CA1 and the entorhinal cortex but not in CA3. It is suggested that the spontaneous action potentials would play a critical role during synaptogenesis.