236
placed in the experimental tank at the position the fi sh head normally occupies,
amplifi ed (Nexus amplifi er) and signal averaged by the Spike 2 script to determine
sound pressure levels in dB rms re: 1 μPa.
AEPs recorded via the sub-dermal electrodes were differentially amplifi ed and
band-pass fi ltered (DP-301, Warner Instruments), and then digitized on a CED
Micro 1401 analog to digital interface run by Spike 2 software. A total of 2000
repetitions were averaged for each sound intensity and frequency, and power spectra
(FFT, 512 or 1024 points) of these averaged waveforms were calculated to examine
peaks at twice the stimulus frequency that result from the opposed orientation of
hair cells and non-linearities in the auditory system. Thresholds were defi ned as the
lowest sound level to show a repeatable AEP waveform above background noise
and an FFT peak at twice the stimulus frequency. AEP recordings obtained here
were similar to those reported previously for this species using identical experimental setups (Maruska et al. 2007 ).
2.4 Single Neuron Recordings in the Auditory Hindbrain
and Midbrain
Single cell extracellular auditory neuron recordings from the hindbrain and midbrain previously measured in A. abdominalis for a separate study (Maruska and
Tricas 2009b ) were used here for comparison with the newly generated saccular
potential and AEP recording data. These recordings were performed in the auditory
medulla and midbrain torus semicircularis, and full methodological details can be
found in Maruska and Tricas ( 2009b ). Briefl y, immobilized fi sh were positioned in
an acrylic head holder above an underwater speaker (UW-30) in an experimental
tank (30 cm diameter; fi sh positioned 10 cm above speaker) on a vibration isolation
table inside a sound isolation chamber (Industrial Acoustics). Fish were ventilated
continuously with seawater (23–25 °C) pumped through the mouth and over the
gills during the experiments. The brain was exposed by dorsal craniotomy and the
cranial cavity fi lled with Fluorinert fl uid (FC-75, 3M) to enhance clarity, prevent
drying, and reduce bleeding.
Extracellular single neuron recordings were made with carbon fi ber (Carbostar-1,
Kation Scientifi c, Inc., 400–800 kΩ) or glass (15–35 MΩ, fi lled with 4 M sodium
chloride) microelectrodes advanced through the midbrain torus semicircularis (TS)
or octaval nuclei of the hindbrain (primarily descending octaval nucleus) as an
auditory search stimulus was presented (100–200 Hz at 124–126 dB rms re: 1 μPa).
Neural action potentials were amplifi ed (500×–10,000×) and band-pass fi ltered
(100–5000 Hz) with a Neurolog system (Digitimer, Inc.) and then converted to digital fi les with a CED power 1401 system run by Spike 2 software. Acoustic stimuli
were generated by the CED digital to analog interface controlled by Spike 2 software, attenuated, and amplifi ed before being sent to the underwater speaker.
Stimulus characteristics were similar to those described above for AEP experiments
except that 100 repetitions of 40 ms (10 ms rise and fall, 20 ms plateau) were used
K.P. Maruska and J.A. Sisneros
placed in the experimental tank at the position the fi sh head normally occupies,
amplifi ed (Nexus amplifi er) and signal averaged by the Spike 2 script to determine
sound pressure levels in dB rms re: 1 μPa.
AEPs recorded via the sub-dermal electrodes were differentially amplifi ed and
band-pass fi ltered (DP-301, Warner Instruments), and then digitized on a CED
Micro 1401 analog to digital interface run by Spike 2 software. A total of 2000
repetitions were averaged for each sound intensity and frequency, and power spectra
(FFT, 512 or 1024 points) of these averaged waveforms were calculated to examine
peaks at twice the stimulus frequency that result from the opposed orientation of
hair cells and non-linearities in the auditory system. Thresholds were defi ned as the
lowest sound level to show a repeatable AEP waveform above background noise
and an FFT peak at twice the stimulus frequency. AEP recordings obtained here
were similar to those reported previously for this species using identical experimental setups (Maruska et al. 2007 ).
2.4 Single Neuron Recordings in the Auditory Hindbrain
and Midbrain
Single cell extracellular auditory neuron recordings from the hindbrain and midbrain previously measured in A. abdominalis for a separate study (Maruska and
Tricas 2009b ) were used here for comparison with the newly generated saccular
potential and AEP recording data. These recordings were performed in the auditory
medulla and midbrain torus semicircularis, and full methodological details can be
found in Maruska and Tricas ( 2009b ). Briefl y, immobilized fi sh were positioned in
an acrylic head holder above an underwater speaker (UW-30) in an experimental
tank (30 cm diameter; fi sh positioned 10 cm above speaker) on a vibration isolation
table inside a sound isolation chamber (Industrial Acoustics). Fish were ventilated
continuously with seawater (23–25 °C) pumped through the mouth and over the
gills during the experiments. The brain was exposed by dorsal craniotomy and the
cranial cavity fi lled with Fluorinert fl uid (FC-75, 3M) to enhance clarity, prevent
drying, and reduce bleeding.
Extracellular single neuron recordings were made with carbon fi ber (Carbostar-1,
Kation Scientifi c, Inc., 400–800 kΩ) or glass (15–35 MΩ, fi lled with 4 M sodium
chloride) microelectrodes advanced through the midbrain torus semicircularis (TS)
or octaval nuclei of the hindbrain (primarily descending octaval nucleus) as an
auditory search stimulus was presented (100–200 Hz at 124–126 dB rms re: 1 μPa).
Neural action potentials were amplifi ed (500×–10,000×) and band-pass fi ltered
(100–5000 Hz) with a Neurolog system (Digitimer, Inc.) and then converted to digital fi les with a CED power 1401 system run by Spike 2 software. Acoustic stimuli
were generated by the CED digital to analog interface controlled by Spike 2 software, attenuated, and amplifi ed before being sent to the underwater speaker.
Stimulus characteristics were similar to those described above for AEP experiments
except that 100 repetitions of 40 ms (10 ms rise and fall, 20 ms plateau) were used
K.P. Maruska and J.A. Sisneros