Sustained synaptic activity was achieved using a protocol that was verified to increase synaptic vesicle release, as demonstrated by FM1-43 dye labeling. After such a treatment, immunostaining showed a decrease in the levels of surface GluA1 and GluA2/3 subunits of the AMPAR in syn-YFP-apposed synapses relative to synapses with terminals from nontransfected neurons. The authors showed that AMPAR internalization was increased under the conditions of persistent UV-driven synaptic activation. Homeostatic plasticity has been shown to change levels of other synaptic components; however, in the conditions employed by Hou and colleagues, no change was seen in the levels of the NMDAR

subunit GluN1 or scaffolding protein PSD-95. The synapse-specific downregulation of postsynaptic AMPARs was find more then characterized in mechanistic detail. Sodium channel blocker TTX, pan-NMDAR antagonist D-AP5, and a Ca2+-free extracellular solution all blocked the decrease in AMPARs, but AMPAR antagonist GYKI was ineffective. This indicated that action potential-generated synaptic vesicle release leading to NMDAR activation and subsequent Ca2+ influx through the channel were important but that AMPAR activity was dispensable. Importantly, the selleck products authors differentiated

this reduction in AMPARs from Hebbian LTD by using inhibitors of consensus signaling pathways for LTD induction (Collingridge et al., 2010). The calcineurin inhibitor FK-506, GluN2B antagonist Ifenprodil, and CaMKII inhibitor KN62 had no effect on the UV-induced AMPAR reduction but were effective against an NMDA-induced AMPAR downregulation, a chemically-induced model of Hebbian LTD. Furthermore,

NMDA treatment did not occlude the UV-induced reduction in AMPAR abundance, arguing that the Hebbian LTD and UV-induced AMPAR downregulation are mechanistically distinct. The loss of total GluA2/3 at persistently activated synapses prompted Hou and colleagues to look for changes in GluA protein why turnover as an additional mechanism for AMPAR downregulation. The UV-induced scaling was robust even when protein synthesis was inhibited by anisomycin, arguing that a decrease in AMPAR subunit synthesis was not involved. An alternative explanation could be an increase in degradation. Indeed, the authors saw that the UV-induced reduction in total GluA2/3 was prevented by the proteasome inhibitor MG-132, although the lysosome inhibitor chloroquine was ineffective. Consistent with this, immunostaining of AMPAR-specific E3 ligase Nedd4 and ubiquitin in synapses with UV-activated terminals was increased relative to control synapses. Importantly, this synaptic scaling down of postsynaptic AMPARs appears to be a result of increased activity of local proteasomes near the activated synapses, because the authors found that MG132-sensitive, UV-induced degradation of AMPARs was persistent even in the dendritic branches that had been severed from the soma.