The extracellular solution consisted of (in mM): 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH = 7.8). Recording micropipettes were made from borosilicate glass capillaries (BF120-69-15, WPI). The internal solution consisted of (in mM): 110 K-gluconate, 6 NaCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 EGTA (pH 7.3). The equilibrium potential of chloride ion (ECl−) was about −60 mV

according to the Nernst equation. In vivo whole-cell recording PLX4032 solubility dmso of Mauthner cells (M-cells), loose-patch recording of VIIIth nerves, and cell-attached recording of caudal hypothalamic (HC) neurons, retinal ganglion cells, optic tectum neurons, and hindbrain neurons were all made under visual guidance. For whole-cell recording on M-cells, as described in a previous study (Han et al., 2011), a tiny cut for breaking the skin was made

at the dorsal part ∼100 μm caudally to the location of M-cells. A recording micropipette (∼10 MΩ, tip diameter <2 μm) filled with the internal solution was inserted into the brain through this cut, and rostraventrally approached to M-cells with a persistent positive pressure for keeping tip clean. After the contact of micropipette tip with M-cell membrane, giga-ohm seal was formed by removing the positive pressure and applying a slight negative pressure. Whole-cell recording was achieved by delivering a few brief electrical zaps (25 μs to 2 ms) to break the cell membrane beneath the micropipette tip. For loose-patch recording of VIIIth nerves and cell-attach recording Ku-0059436 chemical structure of HC neurons, micropipettes with a resistance of ∼8 MΩ were used. VIIIth nerve bundles, which are laterally to M-cell lateral dendrites, were visible under the infrared microscope (FN-S2N, Nikon). A slight and persistent positive pressure was applied before reaching the nerves. After removing the positive pressure and applying a slight negative pressure, loose-patch recoding of VIIIth nerves was formed. Sound-evoked spike trains were readily recorded from VIIIth Linifanib (ABT-869) nerves. Each spike within sound-evoked

spike trains was phase-locked to the peak or valley of sound waves, and typically with amplitudes ranging from 0.5 to 3 mV. In paired recordings of the VIIIth nerve and M-cell, both the spontaneous and sound-evoked spikes of VIIIth nerves were correlated with postsynaptic currents of M-cells. For HC neuron recordings, loose-patch recoding was visually guided by GFP signal in ETvmat2:GFP larvae and followed the similar strategy as the VIIIth nerve recording. Recordings were made with a patch-clamp amplifier (MultiClamp 700B, Axon Instruments), and signals were filtered at 5 kHz and sampled at 10 kHz by using Clampex 10.2 (Molecular Devices). The total integrated charge of compound synaptic currents (CSCs) of M-cells within the first 20 ms after the onset was calculated.