The precise timing of neuronal spikes may lead to changes in synaptic connectivity and is thought to be crucial for learning and memory. However, the effect of spike timing on neuronal connectivity in the intact brain remains unknown. Using closed-loop optogenetic stimulation in CA1 of freely moving mice, we generated unique spike patterns between presynaptic pyramidal cells (PYRs) and postsynaptic parvalbumin (PV)-immunoreactive cells. The stimulation led to spike transmission changes that occurred together across all presynaptic PYRs connected to the same postsynaptic PV cell. The precise timing of all presynaptic and postsynaptic cell spikes affected transmission changes. These findings reveal an unexpected plasticity mechanism, in which the spike timing of an entire cell assembly has a more substantial impact on effective connectivity than that of individual cell pairs.