234
of Washington. Fish were then transferred to holding tanks containing seawater at
20–22 °C and allowed to acclimate for at least 24 h prior to use in experiments. Fish
were maintained on a 12 h light:dark cycle and fed daily with fi sh fl akes or frozen
squid/fi sh. Auditory threshold tuning curves were determined from 8 saccular
potential recordings in 7 Hawaiian sergeant fi sh (3 males, 4 females; SL = 130.0 ±0.5
SD mm; BM = 93.2 ± 12.4 SD g).
Methods for recording saccular potentials from the Hawaiian sergeant fi sh were
adapted from those used on the plainfi n midshipman fi sh (Sisneros 2007 ). Briefl y,
fi sh were anesthetized with benzocaine and immobilized by an intramuscular injection of pancuronium bromide. The saccule of the inner ear was exposed by dorsal
craniotomy, and the cranial cavity was fi lled with teleost Ringer’s solution to prevent drying and enhance clarity. Fish were positioned so that the saccule was 10 cm
above the surface of an underwater loudspeaker (UW-30) that was embedded in
sand on the bottom of a 30 cm diameter, 24 cm high Nalgene experimental tank. The
tank was positioned on a vibration isolation table and housed within an acoustic
isolation chamber (Industrial Acoustics Co.), while all recording and stimulus generation equipment was located outside the chamber. Fish were ventilated continuously with seawater (22–24 °C) pumped through the mouth and over the gills during
the experiments.
Acoustic stimuli were generated by the reference output signal of a lock-in
amplifi er (Stanford Research Systems SR830) that was input to an audio amplifi er
and underwater speaker (UW-30). The frequency response of the underwater
speaker was measured with a mini-hydrophone (Bruel and Kjaer 8103) in the position normally occupied by the fi sh head. Relative sound pressure measurements
were then made with a spectrum analyzer (Stanford Research Systems SR780), calibrated by peak-to-peak voltage measurements on an oscilloscope, and then adjusted
with Matlab software so that the sound pressures at all tested frequencies (75–385
Hz) were of equal amplitude (within ±2 dB). Auditory stimuli consisted of 8–10
repetitions of single 500 ms duration tones with rise and fall times of 50 ms. Each
repetition was presented at a rate of 1 every 1.5 s. Pure tone stimuli were presented
at 10 Hz increments from 75 to 145 Hz and 20 Hz increments from 165 to 385 Hz.
To determine threshold tuning responses, pure tone stimuli were presented at sound
pressures from 100 to 145 dB re: 1 μPa in incremental steps of 3 dB.
Saccular potentials were recorded with glass microelectrodes (tip diameter, 1–2
μm) fi lled with 3 M KCl (1–10 MΩ). Electrodes were visually guided and placed
into the endolymph of the saccule close to the sensory macula. Analog saccular
potentials were preamplifi ed (100×), input to a digital signal processing lock-in
amplifi er, and then stored on a PC computer running a custom data acquisition
Matlab software control program. The lock-in amplifi er yields a DC RMS voltage
output signal that is proportional to the component of the signal whose frequency is
exactly locked to the reference frequency. The reference frequency was set to the
second harmonic of the stimulation frequency signal (i.e., twice the fundamental
frequency) since the maximum evoked potential from the saccule of teleost fi shes
occurs at twice the stimulus sound frequency due to the presence of nonlinear and
oppositely oriented hair cell populations within the saccule (Cohen and Winn 1967 ;
K.P. Maruska and J.A. Sisneros
of Washington. Fish were then transferred to holding tanks containing seawater at
20–22 °C and allowed to acclimate for at least 24 h prior to use in experiments. Fish
were maintained on a 12 h light:dark cycle and fed daily with fi sh fl akes or frozen
squid/fi sh. Auditory threshold tuning curves were determined from 8 saccular
potential recordings in 7 Hawaiian sergeant fi sh (3 males, 4 females; SL = 130.0 ±0.5
SD mm; BM = 93.2 ± 12.4 SD g).
Methods for recording saccular potentials from the Hawaiian sergeant fi sh were
adapted from those used on the plainfi n midshipman fi sh (Sisneros 2007 ). Briefl y,
fi sh were anesthetized with benzocaine and immobilized by an intramuscular injection of pancuronium bromide. The saccule of the inner ear was exposed by dorsal
craniotomy, and the cranial cavity was fi lled with teleost Ringer’s solution to prevent drying and enhance clarity. Fish were positioned so that the saccule was 10 cm
above the surface of an underwater loudspeaker (UW-30) that was embedded in
sand on the bottom of a 30 cm diameter, 24 cm high Nalgene experimental tank. The
tank was positioned on a vibration isolation table and housed within an acoustic
isolation chamber (Industrial Acoustics Co.), while all recording and stimulus generation equipment was located outside the chamber. Fish were ventilated continuously with seawater (22–24 °C) pumped through the mouth and over the gills during
the experiments.
Acoustic stimuli were generated by the reference output signal of a lock-in
amplifi er (Stanford Research Systems SR830) that was input to an audio amplifi er
and underwater speaker (UW-30). The frequency response of the underwater
speaker was measured with a mini-hydrophone (Bruel and Kjaer 8103) in the position normally occupied by the fi sh head. Relative sound pressure measurements
were then made with a spectrum analyzer (Stanford Research Systems SR780), calibrated by peak-to-peak voltage measurements on an oscilloscope, and then adjusted
with Matlab software so that the sound pressures at all tested frequencies (75–385
Hz) were of equal amplitude (within ±2 dB). Auditory stimuli consisted of 8–10
repetitions of single 500 ms duration tones with rise and fall times of 50 ms. Each
repetition was presented at a rate of 1 every 1.5 s. Pure tone stimuli were presented
at 10 Hz increments from 75 to 145 Hz and 20 Hz increments from 165 to 385 Hz.
To determine threshold tuning responses, pure tone stimuli were presented at sound
pressures from 100 to 145 dB re: 1 μPa in incremental steps of 3 dB.
Saccular potentials were recorded with glass microelectrodes (tip diameter, 1–2
μm) fi lled with 3 M KCl (1–10 MΩ). Electrodes were visually guided and placed
into the endolymph of the saccule close to the sensory macula. Analog saccular
potentials were preamplifi ed (100×), input to a digital signal processing lock-in
amplifi er, and then stored on a PC computer running a custom data acquisition
Matlab software control program. The lock-in amplifi er yields a DC RMS voltage
output signal that is proportional to the component of the signal whose frequency is
exactly locked to the reference frequency. The reference frequency was set to the
second harmonic of the stimulation frequency signal (i.e., twice the fundamental
frequency) since the maximum evoked potential from the saccule of teleost fi shes
occurs at twice the stimulus sound frequency due to the presence of nonlinear and
oppositely oriented hair cell populations within the saccule (Cohen and Winn 1967 ;
K.P. Maruska and J.A. Sisneros