The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long‐term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short‐term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABA_A‐antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA‐stop/PAI‐1) significantly interfered with the activity‐induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor‐mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long‐term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short‐term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.