Acute brain slices represent a “workhorse” model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organisation and functioning in acute brain slices. The comprehensive assesment of microglial states in acute brain slices demonstrated time-dependent microglial phenotype changes that are influenced by P2Y12R and CX3CR1 signalling in an experimentally relevant timeframe. By using a recently developed, highly sensitive fluorescent ATP sensor, we reveal that acute injury induces complex ATP dynamics consisting of a sudden rise in extracellular ATP followed by spontaneously emerging focal ATP events, which recruit microglial processes in a P2Y12R- and CX3CR1-dependent manner. Paralel to these changes, downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses. Importantly, the lack of microglia or microglial P2Y12R or CX3CR1 function results in markedly impaired ripple activity ex vivo, as confirmed by microglia depletion in vivo. Collectively, our data suggest that microglia are inherent modulators of complex neuronal networks with indispensable roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.