During development of CNS, young neurons experience various stimuli, and thereafter differentiate to mature neurons in an activity-dependent manner. Membrane depolarization acts as an inducer of excitability and various signals in the neurons, which can be used as a model of neuronal activity. However, the mechanisms of the influence of membrane depolarization on neuronal differentiation have not been fully understood. Therefore, we investigated the effect of membrane depolarization on morphology of spines and generation of valid electrical activity. Using rat hippocampal cultures treated from the plating day with or without high KCl (35 mM, termed HK), we directly observed living neurons transfected with green fluorescence protein-expressing plasmid through a two-photon laser scanning confocal microscope and electrophysiological recording using a patch-clamp technique. Compared with controls, the neurons cultured with HK for 3 days in vitro (DIV) showed marked filopodia-like protrusions as well as an increase in the number of spines, but those cultured with HK for 6 DIV profoundly lost these spines, resulting in a small number of fine filopodia-like protrusions proximally and on the cell body, and a smooth surface of distal dendrites. Electrophysiological recordings showed no spontaneous responses in 6 DIV HK-treated neurons. Moreover, addition of an N-methyl-D-aspartate receptor (NMDAR) antagonist to HK-treated neurons blocked the shrinkage and decrease in the number of filopodia-like protrusions significantly. These findings suggest that membrane depolarization of developing neurons induces synaptogenesis in the early stages of development but chronic treatment with HK causes pathological changes through NMDAR, and that there may be alternative mechanisms for the physiological differentiation of neurons in later developmental stages.