In that sense, chemically fixed preparations reveal an underlying organization of the active zone that is missed in the cryo-EM studies, and the two EM approaches—EM on chemically fixed and on unfixed preparations—provide complementary Epacadostat ic50 insights in the organization of active zones. How then can we interpret the results obtained with EM studies of mutant synapses? For example, in Munc13-1 KO mice synaptic vesicle docking appears to be normal as analyzed by EM of chemically fixed synapses (Augustin et al., 1999) but impaired as analyzed by cryo-EM of unfixed samples (Siksou et al., 2009). A possible interpretation of this finding is that

docking analyzed with fixed samples is prone to artifacts, but the situation is not as straightforward as it seems. If in chemically fixed samples even nondocked vesicles always appear docked, no mutation should cause a loss of docking as analyzed by this method. However, in chemically fixed RIM mutant synapses,

vesicles are at least partially undocked (Kaeser et al., 2011). The fundamental problem here LY294002 in vitro is that docking as defined by EM is not a functional definition, and both EM approaches may provide a technique-dependent limited view, with neither allowing the claim of absolute conclusions. Short-term synaptic plasticity can increase or decrease the strength of a synaptic signal several-fold (Fioravante and Regehr, 2011). This change dramatically alters the size of a postsynaptic response elicited by a train of presynaptic action potentials. Many different mechanisms of short-term synaptic plasticity almost were identified, nearly all of which involve the active zone, although in different ways. At the most basic level, short-term plasticity of release

is due to the interplay between the buildup of residual Ca2+ and the loss of releasable vesicles from one action potential to the next. Ca2+ entering the active zone via Ca2+ channels is buffered away quickly, leading to Ca2+-transients of less than 1 ms (Meinrenken et al., 2003). However, during repeated action potentials, especially at frequencies of >10 Hz, residual Ca2+ accumulates because the decay of Ca2+-transients decelerates as Ca2+-buffers become saturated, resulting in an increase of the release probability and thus facilitation. At the same time, vesicles undergoing exocytosis need to be replenished. Although the precise rate of vesicle replenishment differs among synapses and is also regulated by Ca2+ (see discussion below), replenishment of release-ready vesicles can be rate-limiting during action-potential trains, leading to synaptic depression. Thus, short-term plasticity due to the interplay of residual Ca2+ and vesicle depletion depends on synapse-specific factors such as available Ca2+-buffers, the size of the readily releasable pool of vesicles, and the basal release probability.