On CA1 pyramidal neurons, the majority of excitatory synapses are located on dendritic spines. Previous experiments demonstrated that the induction of LTP can modify spine numbers and morphology. However, there was no direct proof if and when the newly grown spines are contacted by a presynaptic terminal and potentially form a functional synapse. To address this, the extent of colocalization of newly grown spines with antibody staining for either synapsin, a marker of mature presynaptic

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... s or GluR2, a subunit of postsynaptic AMPA receptors common at functional synapses was determined. Growth of dendritic spines was induced by extracellular local high frequency stimulation or by using a "chemical LTP" induction protocol. Overall, the number of spines that colocalized with synapsin puncta seemed to increase with the age of the spine. This indicates that new spines grown upon LTP induction initially lack presynaptic innervation suggesting that new spines do not emerge from pre-existing shaft synapses but protrude towards an existing presynaptic contact. Most of the newly grown spines are GluR2 negative, suggesting that these spines do not contain a fully functional synapse within the first six hours of existence. Because LTP leads to spine growth, the question was if LTD induces the retraction of previously existing spines. Using two-photon time-lapse microscopy, it was observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, while theta-burst stimulation led to the formation of new spines, as reported previously. Thus, spines on CA1 pyramidal neurons from organotypic slice cultures can undergo bidirectional morphological plasticity; spines can be formed and eliminated in an activity-dependent way.