(1995). Zebrafish-handling procedures were approved by Institute of Neuroscience, Chinese Academy of Sciences. For in vivo electrophysiological recording and time-lapse two-photon imaging, learn more zebrafish aged at 3–6 or 15–20 dpf were immobilized with the neuromuscular junction blocker α-bungarotoxin (100 μg/ml), mechanically fixed by 1.0% low melting point agarose, and incubated in HEPES-buffered saline containing 134 mM NaCl, 2.9 mM KCl, 2.1 mM

CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). The lens of the eye was removed to expose the surface of the RGC layer (Figure 1B). In vivo perforated whole-cell recording of cells at the ganglion cell layer was made under visual control at room temperature (Zhang et al., 2010). In zebrafish larvae, displaced amacrine cells in the ganglion cell layer are rarely observed (Connaughton et al., 1999; Kay et al., 2001; Zhang et al., 2010), and most Selleckchem SP600125 of the cells at the ganglion cell layer are RGCs. The recording pipette was made from borosilicate glass capillaries (WPI), had a resistance in the range of 10–12 MΩ, and was tip filled with internal solution and then backfilled with internal solution containing amphotericin

B (200 μg/ml). The internal solution contained 110 mM K-gluconate, 6 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM EGTA (pH 7.3). Therefore, the ECl− was about −59.8mV. Recording was made with patch-clamp amplifiers (MultiClamp 700B; Axon Instruments). The whole-cell capacitance was fully compensated, and the series resistance (10–20 MΩ) was compensated at 70%–80% (lag 60 μs) and monitored during the experiment. The data were discarded if the series resistance varied by >20% during recording. Signals were filtered at 2 kHz

and sampled at 10 kHz using AxoScope software 10.0 (Axon Instruments). To electrically activate synapses formed by BCs on RGCs, BCs at the INL were extracellularly stimulated by focal extracellular stimulation (duration, 0.1 ms; intensity, 2–40 μA) with theta glass electrodes Adenosine (tip opening, 1–2 μm). Data obtained from RGCs, which only exhibited e-EPSCs, were collected and analyzed. In order to record mEPSCs of RGCs, TTX (1 μM) was included in the bath solution. For whole-field flash and MBSs, a mini LCD (Sony; PLM-A35) was mounted on the camera port of the microscope (BX51WI; Olympus), allowing projection of computer-generated images onto the retina of zebrafish larvae (Figure 1A). Visual responses were evoked by whole-field flashes with a step increase (2 s duration) in the light illumination or moving bars with rightward direction (width, 6 μm; speed, 0.1 μm/ms). All drugs were from Sigma-Aldrich. For in vivo two-photon calcium imaging of BC axon terminals, we used double-transgenic zebrafish Tg(Gal4-VP16xfz43,UAS:GCaMP1.6) larvae that were obtained by crossing Tg(UAS:GCaMP1.6) (gift from Dr.