First published online March 21, 2005
Journal of Experimental Biology 208, 1363-1372 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01534
Development of the acoustically evoked behavioral response in zebrafish to pure tones
David G. Zeddies1,* and
Richard R. Fay1,2,
1 Parmly Hearing Institute, Loyola University – Chicago, Chicago,
Illinois, USA
2 Department of Psychology, Loyola University – Chicago, Chicago,
Illinois
View larger version (51K):
[in a new window]
|
Fig. 1. Equipment setup used to evoke responses in larval zebrafish. A standard,
24-well culture dish was secured to a  inch thick, translucent,
plastic platform using screw-down fasteners. The platform was securely mounted
onto a Bruel & Kjaer Mini-shaker Type 4810 so that the platform could be
vertically displaced. Another plastic platform was mounted on the non-moving
body of the shaker and illuminated obliquely to provide diffuse, uniform
illumination to the underside of the moving platform. An accelerometer was
mounted on the moving platform and a video camera was placed above. The same
set up was used for adult fish, except that a single plastic well was mounted
in place of the 24-well culture dish.
|
|
View larger version (21K):
[in a new window]
|
Fig. 2. The RMS acceleration of the platform for the 24-well culture dish with the
central eight wells filled to the standard depth. (A) The RMS acceleration of
the platform for the test frequencies as a function of the output voltage from
the Tucker-Davis System 3. (B) The same acceleration data plotted as a
function of frequency.
|
|
View larger version (20K):
[in a new window]
|
Fig. 3. Calibration of the probe tube microphone. A custom-made probe tube was
attached to the microphone of a Bruel & Kjaer Type 2235 Precision Sound
Level Meter. The tip of the probe tube was placed near a calibrated Bruel
& Kjaer 8103 hydrophone and the sound pressure level (SPL) of both the
hydrophone and sound level meter with the probe were measured for different
frequencies at a stimulus level of 1 volt RMS applied to a UW 30 speaker in a
cylindrical tank. Measurements were made at three different locations in the
tank (Trials A, B and C). Probe attenuation, the SPL difference between the
hydrophone and the sound level meter with probe, was used to calibrate the
probe for use under water.
|
|
View larger version (16K):
[in a new window]
|
Fig. 4. Sound pressure level (SPL) in the wells of the 24-well culture dish. The
probe tube was used to measure the SPL in the wells for the frequencies and
levels used in experiments (see text). Note that approximately 6 dB separates
the lines because each line represents a doubling of voltage to the
shaker.
|
|
View larger version (27K):
[in a new window]
|
Fig. 5. Sound pressure time waveforms (right insets), examples of two cycles (left
insets), and the spectra for three frequencies (100, 600 and 1200 Hz) at the
highest stimulus levels used in experiments.
|
|
View larger version (41K):
[in a new window]
|
Fig. 6. Examples of video frame subtraction. (A,B) Show the subtraction of two
different consecutive frames for the fish in quiet. In A, no movement occurs
between frames and the resulting difference image (column 3) contains only
noise. In B, two fish move resulting in two darker patches in the difference
image. C shows several fish moving in the presence of the stimulus. Note that
in areas where no movement takes place, the noise is small and consistent in
the difference images. Fish are 4.5 mm (16 dpf).
|
|
View larger version (12K):
[in a new window]
|
Fig. 7. Example of a typical histogram showing the movement of larval fish in
quiet. For a data set consisting of 56 trials, the first 2.32 s of each trial
were frame subtracted and the number of pixels above the noise threshold was
determined for a moving window of three frames. The frequency of occurrence of
the number of pixels above the noise threshold is plotted as a histogram, and
a single exponential equation was fit to the histogram (dotted line; goodness
of fit  =0.1012). In this case, a positive response would be registered
if the cumulative number of pixels above the noise threshold when the tone was
on (frames 60 to 62) was >91 (P<0.0001; arrowhead).
|
|
View larger version (17K):
[in a new window]
|
Fig. 8. AEBR thresholds in larval fish for cosine-squared gated, 120 ms, 400 Hz
tones as a function of the rise–fall time.
|
|
View larger version (31K):
[in a new window]
|
Fig. 9. AEBR thresholds in larval fish of different ages shown as SPL (top) and
acceleration (bottom). The mean ± S.D. for the 5–26
dpf fish is plotted as thick black lines. An experiment for a group of animals
consisted of two sets of trials (see Materials and methods). Therefore, each
group has two plotted curves using the same symbols but connected with
different lines (solid for trial set 1, and dashed for trial set 2). Two
different groups of 5 dpf fish were tested, so 5 dpf appears twice (with
different symbols).
|
|
View larger version (17K):
[in a new window]
|
Fig. 10. AEBR thresholds for adult and larval fish in a single large well. The top
panel is SPL, the bottom panel is the same data plotted as a function of
acceleration.
|
|
View larger version (112K):
[in a new window]
|
Fig. 11. Images of 4 and 5 dpf zebrafish larvae. The swimbladder has inflated and is
clearly visible in 5 dpf animals (arrows), but not in the 4 dpf animals. Scale
bar, 1 mm.
|
|
View larger version (22K):
[in a new window]
|
Fig. 12. AEBR thresholds for larval fish in which the swimbladder was deflated. For
comparison, the mean ± S.D. of the 5 to 26 dpf intact fish
(from Fig. 9) is also
shown.
|
|
© The Company of Biologists Ltd 2005
