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Synchronized and sequential activity patterns in neural networks have been proposed as a mechanism underlying many complex behaviors, such as procedural memories and decision-making. Hebb (1949) suggested that neuronal assemblies were formed by the strengthening of synaptic connections between cells that often fire together. However, the role of short-term synaptic dynamics in network activity patterns and sequence formation has not been widely studied in living neural networks. We used optical and electrophysiological recordings to describe synaptic properties that accompany co-operative firing and sequential activity patterns of groups of neurons in cultures of mouse cortical cells (>15DIV). Spontaneous activity and connectivity was studied in whole cell patch clamp recordings from a total of 42 neuronal pairs. Oscillatory firing activity was recorded in 26 cells and connectivity rate was 52% (22/42 pairs). Some of the pairs had nearly synchronous up states (starting within <10ms) whereas others were more out of phase. When short term plasticity was tested in these pairs, cells firing together mainly displayed short-term synaptic depression (n=5, i.e., high release probability), whereas cells firing in sequence, mostly displayed short-term synaptic facilitation (n=6, i.e., low release probability). To further investigate the impact of short-term synaptic dynamics in cell assembly behavior we performed calcium imaging with single cell resolution in groups of several hundred cells. The release probability was modified by changing the extracellular calcium concentration. At low extracellular calcium concentrations (1mM) synaptic connections were mainly facilitating (n= 5/7 pairs) and the transitions of activity among groups of neurons were increased showed by calcium imaging (n=6 dishes). On the contrary, at a higher extracellular calcium concentration (4mM) we observed short-term depression in all the connections (n=7 pairs) and a reduced variability of cell assemblies (n=6 dishes). These results suggest a direct relationship between neuronal groups firing together and short-term synaptic plasticity in living neural networks, and suggest that a given microcircuit might be reprogrammed by the targeted modification of the excitatory synaptic dynamics due to the presence of neuromodulators. [ABSTRACT FROM AUTHOR]