The release-ready subset of these vesicles can be rapidly exocytosed and supports transmission during sporadic AP firing ( Murthy et al., 2001; Schikorski and Stevens, 2001), whereas the remainder is recruited during sustained activity ( Rizzoli and

Betz, 2005). The second major pool—the so-called resting pool—contains all vesicles that are incapable of AP-evoked exocytosis. Pool definitions are operational; they depend on the measurement procedure. Glutamate released to the synaptic cleft, the most relevant parameter for synaptic function, cannot be compared reliably to the total vesicular glutamate reserve on a single synapse level. As a proxy, staining and destaining of vesicles by lipophilic FM dyes has been used. This method labels vesicles that collapse fully in the bouton membrane and remain exposed to the extracellular space for several seconds, until the membrane patch is recycled

by clathrin-mediated www.selleckchem.com/products/tenofovir-alafenamide-gs-7340.html endocytosis. If, however, some vesicles release transmitter without full collapse, they are not detectable by FM staining ( Harata et al., 2006). To detect all vesicles that were in contact with the extracellular space, no matter how briefly, optical monitoring of vesicular pH is the method of choice. Genetically encoded pH indicators have been developed to measure the activity of hundreds of boutons simultaneously ( Burrone et al., 2006; Fredj and Burrone, 2009; Granseth et al., 2006; Miesenböck et al., 1998; Sankaranarayanan Resminostat and Ryan, 2000). In dissociated culture, pH-based methods consistently report a resting pool of about 50% ( Fernandez-Alfonso and Ryan, Osimertinib in vitro 2008; Fredj and Burrone, 2009; Kim and Ryan, 2010; Li et al., 2005), whereas FM dye staining results in even larger values, up to 85% ( Harata et al., 2001). The function of such a large nonreleasable pool is highly controversial. The resting pool could act as a reservoir of vesicles

that can be mobilized after periods of synaptic disuse ( Kim and Ryan, 2010), it might be accessed during NMDA-receptor-dependent presynaptic potentiation ( Ratnayaka et al., 2012), it could support spontaneous vesicle release ( Fredj and Burrone, 2009; Hua et al., 2011; Ramirez et al., 2012), or it could act as a local protein reservoir to buffer cytosolic proteins necessary for vesicle cycling ( Denker et al., 2011b). Synaptic properties change during development (Feldmeyer and Radnikow, 2009; Mozhayeva et al., 2002), a process that has been studied in detail at the calyx of Held, a giant synapse in the auditory brain stem (Borst and Soria van Hoeve, 2012). The immature calyx contains a large fraction of nonreleasable vesicles (de Lange et al., 2003), is unreliable, and shows pronounced short-term depression (Taschenberger and von Gersdorff, 2000). After hearing onset (P12–P14), it becomes very reliable and fast, driving the postsynaptic cell at high frequencies (Lorteije et al., 2009; Sonntag et al., 2011).