In these GluN2B KI mice, activity-dependent association of CaMKII with the NMDA receptor is abrogated without altering CaMKII-T286 autophosphorylation,

indicating that stimulus-induced activation of CaMKII is normal (Halt et al., 2012). Thus, activity-dependent new spine growth could depend upon the local concentration of active proteasomes rather than stimulation of global proteasomal activity. What target proteins are degraded by the proteasome in order to stimulate new spine growth? Due to the rapid reduction in spinogenesis observed with proteasome inhibition, we expect that the immediate protein targets normally act to inhibit spine outgrowth and are rapidly turned over in an activity- and proteasome-dependent selleck inhibitor manner. Although a large number of proteins have been shown to regulate spine morphology and dynamics, the most promising candidates for regulating new spine outgrowth are those associated with regulation

of the spine actin cytoskeleton (Tashiro and Yuste, 2004 and Tolias et al., 2011). One promising candidate could be Ephexin5, a RhoA guanine nucleotide exchange Selumetinib supplier factor shown to negatively regulate excitatory synapse formation (Margolis et al., 2010). Another prime candidate could be Rap2, a member of the Ras family of GTPases. Rap2 overexpression causes a reduction in dendritic spine density (Ryu et al., 2008), suggesting that it may be a negative regulator of spinogenesis. Both of these candidates are plausible targets of activity-induced upregulation of proteasomal activity; in each case, enhanced second degradation would be expected to lead to increased spine density. Malfunction of the proteasome (Tai and Schuman, 2008) and alterations in dendritic spine morphologies and densities (Bhatt et al., 2009) have independently been associated with neurological disorders resulting in mental retardation. Our data support a direct role for the proteasome in the activity-induced spinogenesis thought to be critical for normal brain function and for learning and memory. Disruption of proteasome-mediated degradation would be expected to interrupt activity-induced spine outgrowth during

experience-dependent circuit modifications, suggesting one plausible mechanism by which proteasomal dysfunction may lead to neurological dysfunction. Hippocampal slices were prepared from postnatal day (P) 5–P7 Sprague-Dawley rats or wild-type or GluN2B KI mice (Halt et al., 2012), as described (Stoppini et al., 1991). Genes were delivered 2–5 days (EGFP alone) or 3–4 days (EGFP and Rpt6-WT or Rpt6-S120A) prior to imaging using biolistic gene transfer (180 PSI), as described (Woods and Zito, 2008). We coated 1.6 μm gold beads with 10 μg of EGFP (Clontech) or with 10 μg EGFP and 25 μg HA-tagged Rpt6-WT or Rpt6-S120A (Djakovic et al., 2012). EGFP-transfected pyramidal neurons (5–12 days in vitro [DIV]) were imaged at 29°C in ACSF using a custom two-photon microscope (Woods et al., 2011; Supplemental Experimental Procedures).