, 2003 and Oltedal et al., PD0332991 chemical structure 2007). Furthermore, direct access to presynaptic boutons via the patch pipette should not only allow one to control the presynaptic membrane potential but also to measure and manipulate the presynaptic Ca2+ concentration by direct loading of synthetic Ca2+ dyes and Ca2+-caging compounds. Until now, these types of experiments were only possible in large

synapses such as the calyx of Held. In summary, we anticipate that the combined application of HPICM-assisted patch-clamp recordings, together with previously described electrophysiological and imaging methods to image vesicular release and Ca2+-dynamics in individual synaptic boutons (e.g., Ariel and Ryan, 2010, Ermolyuk et al., 2012, Hoppa et al., 2012, Li et al., 2011 and Li and Tsien, 2012), will provide answers

to these and other questions relating to the behavior of small central synapses. Hippocampal neurons were isolated from P1–P2 rat pups and cultured in Neurobasal-based medium either on an astrocyte feeder layer or on poly-D-lysine-treated coverslips. All recordings were conducted KU-55933 at ambient temperature (23°C–26°C) 12–19 days after plating. The standard extracellular solution Olopatadine used in all experiments contained 125 mM NaCl, 2.5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 30 mM glucose, 0.01 mM NBQX, 0.05 mM APV, and 25 mM HEPES (pH 7.4). Active synapses were labeled with 20 μM (bath concentration) FM1-43 (Invitrogen) or 200 μM SynaptoRed C1 (SRC1,

Biotium) by incubation in the extracellular solution, with 90 mM NaCl replaced by 90 mM KCl for 90 s followed by a 10–15 min wash in the original solution. Tetrodotoxin (1 μM) was added to the extracellular solution in some experiments to slow down spontaneous destaining of the FM dyes. HPICM topographic images were obtained using a custom-modified SICM sample scanner ICNano-S (Ionscope) and custom software as described previously (Novak et al., 2009). Briefly, the scan head consisted of a PIHera P-621.2 X-Y Nanopositioning Stage (Physik Instrumente [PI]) with 100 × 100 μm travel range that moved the sample and a LISA piezo actuator P-753.21C (PI) with travel range 25 μm for pipette positioning along the z axis. Coarse sample positioning was achieved with translation stages M-111.2DG (x-y directions) and M-112.1DG (z axis) (PI). The z piezo actuator was driven by a 200 W peak power high-voltage PZT amplifier E-505 (PI), while the x-y nanopositioning stage was driven by 3 × 14 W amplifier E-503 (PI).