Brain function operates through the coordinated activation of neuronal assemblies. Graph theory predicts that scale-free topologies, which include "hubs" (superconnected nodes), are an effective design to orchestrate synchronization. Whether hubs are present in neuronal assemblies and coordinate network activity remains unknown. Using network dynamics imaging, online reconstruction of functional connectivity, and targeted whole-cell recordings in rats and mice, we found that developing hippocampal networks follow a scale-free topology, and we demonstrated the existence of functional hubs. Perturbation of a single hub influenced the entire network dynamics. Morphophysiological analysis revealed that hub cells are a subpopulation of gamma-aminobutyric acid-releasing (GABAergic) interneurons possessing widespread axonal arborizations. These findings establish a central role for GABAergic interneurons in shaping developing networks and help provide a conceptual framework for studying neuronal synchrony.

The roles of protein kinase C and the MAP-kinase extracellular receptor kinase in structural changes associated with long-term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N-methyl-d-aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis-dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12-myristate 13-acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12-myristate 13-acetate. Both the conditioning medium and phorbol 12-myristate 13-acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long-term increase in network activity and functional synaptic connectivity among the cultured neurons.

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Despite widespread interest in dendritic spines, little is known about the mechanisms responsible for spine formation, retraction, or stabilization. We have now found that a brief exposure of cultured hippocampal neurons to a conditioning medium that favors activation of the NMDA receptor produces long-term modification of their spontaneous network activity. The conditioning protocol enhances correlated activity of neurons in the culture, in a process requiring an increase in [Ca(2+)](i) and is associated with both formation of novel dendritic spines and pruning of others. The novel spines are likely to be touched by a presynaptic terminal, labeled with FM4-64 dye, whereas the absence of such terminals increases the likelihood of spine pruning. These results indicate that long-term functional changes are correlated with morphological modifications of dendritic spines of neurons in a network.