Synaptic scaffolding proteins from membrane-associated guanylate kinases (MAGUK) family are implicated in synapse formation and functioning. To better understand the role of one of the proteins of this family, SAP97, we studied with electron microscopy the effects of its overexpression on spine and synapse morphology in CA1 pyramidal neurons of rat organotypic hippocampal slice cultures. Dramatic spine enlargement induced by SAP97 overexpression was accompanied by marked morphological changes, with spines enwrapping and engulfing presynaptic terminals. The size and complexity of the PSD was also significantly increased. Similar to PSD-95, SAP97 promoted formation of multi-innervated spines (MIS). In addition, both MAGUK proteins induced multiple excitatory contacts on dendritic shafts suggesting a mechanism for shaft synapse formation. Formation of MIS and shaft synapses was blocked by the nitric oxide synthase (NOS) inhibitor L-NAME. Immunochemistry revealed that overexpression of SAP97 was associated with overexpression of PSD-95 and recruitment of nNOS to the synapse. These data provide evidence for both common and distinct structural alterations produced by overexpression of SAP97 and PSD-95 and demonstrate strong interactions between these two proteins to regulate contact formation through nitric oxide signaling. (c) 2010 Wiley-Liss, Inc.