Axoscope software

After the pairing phase was completed, fish were temporarily separated, and behavioral testing was performed on a single fish following the protocol described elsewhere (Issa et al., 2011 (link)). Each fish was placed in a testing chamber (dimensions: 11 × 4 × 3 cm). A pair of conductive electrodes placed on either side of the chamber recorded the electric field potentials. Bare electrodes were 1 mm in thickness with 3–5 mm metal exposure. Electrodes were connected to an AC differential amplifier (AM-Systems model 1700, Carlsborg, WA, United States), and signals were amplified 1,000-fold. Electrical signals were low-pass filtered at 300 Hz and high-pass filtered at 1 KHz. Electrical field potentials are generated by muscle contractions when the fish moves (Issa et al., 2011 (link)). These signals were digitized using a Digidata-1322A digitizer then stored using Axoscope software (Molecular Devices, Inc., Sunnyvale, CA, United States). The experimental animals were acclimatized for 30 min before behavioral testing was initiated. Swimming behavior was recorded immediately following acclimation. Immediately after, startle escape responses were recorded.

The surgical techniques used were identical to those previously described in the studies by Marsat et al. (2009) (link) and Marsat and Maler (2010) (link). Cells of the lateral segment (LS) of the ELL were targeted. A. albifrons brain anatomy is very similar to that of A. leptorhynchus, so major landmark blood vessels described in the study by Maler et al. (1991) (link) and electrode depth served as an adequate guide to locate LS pyramidal cells (see Histology). In vivo recordings were made via metal-filled extracellular electrodes (Frank and Becker, 1964 ) and amplified with a Model 1700 Amplifier (A-M Systems). Data were recorded (Axon Digidata 1500 data acquisition system and AxoScope software, Molecular Devices) at a 20 kHz sampling rate. ON and OFF cells were identified using known response properties, particularly responses to sinusoidal stimulation and spike-triggered average waveforms calculated from responses to 0–60 Hz noise (Saunders and Bastian, 1984 (link)).

We used custom-made suction electrodes to record extracellularly the activity of the 5A nerves, unless stated otherwise. To record the pro-mesothoracic connective, we used a hook electrode. Recordings started 5 min before and lasted for at least 40 min after pilocarpine bath-application. Data were acquired and stored on the computer for off-line analysis using two four-channel differential amplifiers (Model 1700, A-M Systems, USA) and Axon Digidata 1440A A-D board with Axo-Scope software (Molecular Devices, Sunnyvale, CA, USA).