All time points were significantly less than 100% in the wild-type neurons; the 1, 3, and 5 min time points were significantly less than 100% in thesynGAP+/neurons; and only the 5 min time point was significantly less than 100% in thesynGAP/neurons (p< 0

All time points were significantly less than 100% in the wild-type neurons; the 1, 3, and 5 min time points were significantly less than 100% in thesynGAP+/neurons; and only the 5 min time point was significantly less than 100% in thesynGAP/neurons (p< 0.05, two-tailed, one-samplettest). also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 mNMDA brought on transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion ofsynGAPlacked the transient regulation by the NMDA receptor. Depressive disorder of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices fromsynGAP+/mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton. Keywords:cytoskeleton, actin, spines, LTD, Ras, Rac == Introduction == Excitatory neurons in the CNS form synaptic connections onto dendritic protrusions called spines. The shapes and sizes of MG-115 spines are amazingly plastic (Fischer et al., 1998;Lscher et al., 2000). They can emerge, recede, and switch their morphology in seconds by remodeling the underlying actin cytoskeleton (Halpain, 2000;Matus, 2000). The morphology of spines is related to synaptic strength; for example, the number of AMPA receptors in the MG-115 postsynaptic membrane correlates with spine size (Harris and Stevens, 1989;Nusser et al., 1998). Changes in spine morphology are thus an important aspect of synaptic plasticity (Okamoto et al., 2004;Kramr et al., 2006;Chen et al., 2007). A complex of proteins known as the postsynaptic density (PSD) resides below the postsynaptic surface of a spine (Kennedy, 1997,2000;Kim and Sheng, 2004). It contains transmembrane receptors, scaffold proteins, and signaling molecules with numerous attachments to actin filaments. NMDA-type glutamate receptors (NMDA receptors) are at the core of one signaling complex in the PSD that includes rasGRF1 and synGAP (Kennedy et al., 2005). Activation of Ras by NMDA receptors in hippocampal neurons is usually mediated by RasGRF (Krapivinsky et al., 2003;Li et al., MG-115 2006). SynGAP, however, inactivates Ras by stimulating Ras-GTP hydrolysis (Chen et al., 1998;Kim et al., 1998). Thus, the relative activities of RasGRF and synGAP shape the timing and magnitude of NMDA receptor-mediated Ras signaling. In the present study, we show that 50% reduction of the level of synGAP is sufficient to cause a significant increase in large, MG-115 mushroom spines on hippocampal pyramidal neurons in brains of adults heterozygous for any synGAP deletion, consistent with our previous studies in culturedsynGAP+/neurons (Vazquez et al., 2004). Because spine morphology is determined by the underlying actin cytoskeleton (Markham and Fifkova, 1986), our observations indicate that synGAP is usually involved in regulation of actin in spines. Indeed, we found that steady-state levels of activity at several points in the actin regulating pathway that controls phosphorylation/inactivation of the actin-severing protein cofilin (Carlisle and Kennedy, 2005) were elevated in forebrains from adultsynGAP+/mice. Levels of Ras- and Rac-GTP and activated PAK were elevated; as was the level of phospho-cofilin, its inactive form. Transient dephosphorylation and activation of cofilin in response to NMDAR activation was also abolished in the absence of synGAP. We found that this dephosphorylation is usually impartial of Rac and the PAK13 kinase cascade. These findings demonstrate that synGAP contributes to control of spine morphology by regulating both steady-state phosphorylation and transient NMDA receptor-dependent dephosphorylation of cofilin. Because synGAP contributes to regulation of the actin cytoskeleton and AMPA receptor insertion through Ras-related pathways Rabbit Polyclonal to ADAMTS18 (Rumbaugh et al., 2006), our results suggest that synGAP is usually a critical common network element through which two of the fundamental molecular changes underlying synaptic plasticity are controlled. == Materials and Methods == == == == == == Mouse strains. == Generation of the synGAP knock-out collection is usually explained byVazquez et al. (2004). The mutant collection is usually maintained in a 129 and C57BL6 mixed background because the mutant collection breeds poorly when it is fully out-crossed to C57BL6. Homozygous green fluorescent protein (GFP) line-M transgenic mice (Feng et al., 2000) in a C57BL6 background (a kind gift from Dr. Joshua Sanes, Harvard University or college, Cambridge, MA) were crossed withsynGAP+/mice to visualize dendritic spines in area CA1 of the hippocampus. == Analysis of spines. == ThreeGFP+//synGAP+/+andGFP+//synGAP+/littermate pairs, 36 months in age, were perfused transcardially with fixative (4% formaldehyde,15%.