276
loudspeaker was measured using a calibrated HTI-96-MIN hydrophone (High Tech
Inc.) and a B&K 4524 triaxial accelerometer (Brüel and Kjær) positioned at the
location of the fi sh’s head during the experiments. Relative sound pressure and particle motion were calculated using an oscilloscope and adjusted with the attenuator
to ensure that the sound pressure and particle motion at all frequencies were of
equal amplitude (±2 dB) (Radford and Mensinger 2014 ).
Single and multiunit recordings were amplifi ed (x1000; Dagan Ex-1), fi ltered
(300 Hz–3 kHz), recorded for up to 7 days after implant, stored on a portable computer using Chart5 software and analyzed offl ine with CED Spike2 software.
Although microwires often yielded multiunit activity, neuron discrimination was
usually limited to one or two units that yielded the greatest amplitude and had
clearly distinguishable waveforms above the noise level. To verify that the same
afferent(s) was consistently recorded during an experiment, individual fi bers were
distinguished using waveform analysis in addition to spike amplitude. All statistical
analysis was performed using GraphPad Software (San Diego, CA, USA) or
SigmaStat for Windows version 3.10 (Systat Software, Inc., Richmond, CA, USA).
All data represent mean values ± 1 S.E.M. unless otherwise indicated.
2.2 Data Analysis
Neural responses to tones were quantifi ed for vector strength (VS or synchronization coeffi cient, R ) and evoked spikes rates across the entire stimulus cycle. Spike
rates for directional responses were expressed as the maximum evoked spike rate
minus the mean resting rate for each neuron (e.g., peak-DC) (Goldberg and Brown
1969 ). VS varies from zero (random distribution; no phase locking) to one (all
spikes in the same bin; strong phase locking) and has been determined to be a better
predictor for auditory frequency encoding among vertebrates than maximum
evoked spike rates for frequencies ≤1 kHz (Fay 1978 , 1982 , 1994 ; Javel and Mott
1988 ; Sisneros and Bass 2003 ). The signifi cance of phase locking was determined
by the calculation of the Rayleigh statistic, Z , which is defi ned as R
2 × N , where R is
the coeffi cient of synchronization (or vector strength) and N is the total number of
spikes sampled. Responses with Z ≥ 4.5 ( P = 0.01, utricle) or Z ≥ 6.9 ( P = 0.001, lateral line) were considered signifi cantly phase locked (Batschelet 1981 ). Threshold
was defi ned at the lowest intensity to evoke an increase in spike rate above spontaneous activity, or a signifi cant Z value as described in other studies (Lu and Fay
1993 ; Maruska and Tricas 2009 ) and determined from 80 to 400 Hz. Directional
responses for each individual neuron were calculated at the same supra-threshold
stimulus strength (~5–10 dB above threshold) at each of the eight different stimulus
orientations and examined as both spike rate (spikes/sec) and vector strength.
A.F. Mensinger
loudspeaker was measured using a calibrated HTI-96-MIN hydrophone (High Tech
Inc.) and a B&K 4524 triaxial accelerometer (Brüel and Kjær) positioned at the
location of the fi sh’s head during the experiments. Relative sound pressure and particle motion were calculated using an oscilloscope and adjusted with the attenuator
to ensure that the sound pressure and particle motion at all frequencies were of
equal amplitude (±2 dB) (Radford and Mensinger 2014 ).
Single and multiunit recordings were amplifi ed (x1000; Dagan Ex-1), fi ltered
(300 Hz–3 kHz), recorded for up to 7 days after implant, stored on a portable computer using Chart5 software and analyzed offl ine with CED Spike2 software.
Although microwires often yielded multiunit activity, neuron discrimination was
usually limited to one or two units that yielded the greatest amplitude and had
clearly distinguishable waveforms above the noise level. To verify that the same
afferent(s) was consistently recorded during an experiment, individual fi bers were
distinguished using waveform analysis in addition to spike amplitude. All statistical
analysis was performed using GraphPad Software (San Diego, CA, USA) or
SigmaStat for Windows version 3.10 (Systat Software, Inc., Richmond, CA, USA).
All data represent mean values ± 1 S.E.M. unless otherwise indicated.
2.2 Data Analysis
Neural responses to tones were quantifi ed for vector strength (VS or synchronization coeffi cient, R ) and evoked spikes rates across the entire stimulus cycle. Spike
rates for directional responses were expressed as the maximum evoked spike rate
minus the mean resting rate for each neuron (e.g., peak-DC) (Goldberg and Brown
1969 ). VS varies from zero (random distribution; no phase locking) to one (all
spikes in the same bin; strong phase locking) and has been determined to be a better
predictor for auditory frequency encoding among vertebrates than maximum
evoked spike rates for frequencies ≤1 kHz (Fay 1978 , 1982 , 1994 ; Javel and Mott
1988 ; Sisneros and Bass 2003 ). The signifi cance of phase locking was determined
by the calculation of the Rayleigh statistic, Z , which is defi ned as R
2 × N , where R is
the coeffi cient of synchronization (or vector strength) and N is the total number of
spikes sampled. Responses with Z ≥ 4.5 ( P = 0.01, utricle) or Z ≥ 6.9 ( P = 0.001, lateral line) were considered signifi cantly phase locked (Batschelet 1981 ). Threshold
was defi ned at the lowest intensity to evoke an increase in spike rate above spontaneous activity, or a signifi cant Z value as described in other studies (Lu and Fay
1993 ; Maruska and Tricas 2009 ) and determined from 80 to 400 Hz. Directional
responses for each individual neuron were calculated at the same supra-threshold
stimulus strength (~5–10 dB above threshold) at each of the eight different stimulus
orientations and examined as both spike rate (spikes/sec) and vector strength.
A.F. Mensinger