, 2009). These data suggest that the proteasome is a key downstream mediator of localized activity-dependent Caspase cleavage neuronal signaling and therefore may play a role in activity-dependent spinogenesis. In this study, we used pharmacological and genetic manipulations in combination with time-lapse two-photon microscopy and two-photon glutamate uncaging to investigate the role of the proteasome in new spine growth on dendrites of hippocampal pyramidal neurons. We show that acute inhibition of the proteasome rapidly reduces the rate

of spine outgrowth. Synaptic activity, NMDA receptors, and CaMKII, but not PKA, are upstream regulators of proteasome-mediated spine outgrowth, which is dependent upon interaction between CaMKII and the GluN2B subunit of the NMDA receptor. The S120 residue of the Rpt6 proteasomal subunit is critical for proteasome-dependent spine outgrowth in individual neurons, indicating that the proteasome acts postsynaptically and in a cell-autonomous manner to regulate spine outgrowth. Our data support a model in which synaptic

activity promotes spine outgrowth via an NMDA receptor- and CaMKII-mediated regulation of local proteasomal degradation. To determine whether the proteasome plays a role in regulating the growth of new dendritic spines, we used pharmacological manipulations and time-lapse two-photon microscopy to measure the effect of acute inhibition of the proteasome on CYTH4 the rate of spine outgrowth. Hippocampal pyramidal neurons in organotypic slice cultures Selleckchem Dabrafenib were transfected with enhanced green fluorescent protein (EGFP) and imaged using a two-photon microscope. Dendrites of EGFP-expressing CA1 neurons were imaged at 15 or 20 min intervals before and after treatment with drugs or vehicle (Figure 1A). Treatment with the proteasome inhibitor MG132 (10 μM) reduced the rate of spine outgrowth to half (52% ± 12%) that of vehicle-treated control cells (100% ± 13%; p < 0.05; Figure 1B).

Because MG132 inhibits the activity of calpains as well as the proteasome, we confirmed our findings with the proteasome-specific inhibitor lactacystin. Treatment with lactacystin (10 μM) reduced the rate of spine outgrowth by 68% (32% ± 7%) as compared to vehicle control (100% ± 13%; p < 0.001; Figure 1C). Similar reductions in spine outgrowth were observed for both apical and basal dendrites (see Figure S1A available online). The reduction in spine outgrowth due to lactacystin was not significantly different than that due to MG132 (p = 0.2), suggesting that reduced spine outgrowth in the presence of MG132 is specifically due to inhibition of the proteasome. To ensure that the effect of proteasome inhibition was saturated, we doubled the concentration of lactacystin in the bath.