Zebrafish: A Predictive Model for Assessing Seizure Liability - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 162

zebrafish research, consists of a high-resolution black and white video camera
(Phillips LT385) that can scan a video image in 40 ms, that is, at the rate of 25
video images per second, and a customized zebrafish behavior chamber that holds
microplates (up to 96 wells) containing individual zebrafish. The VideoTrack System
uses bright light to view zebrafish in microwells and to monitor movement contin-
uously, and an infrared light to track zebrafish in a dark chamber. The video camera
analog signal is digitized to 8 bits with luminosity values from0 (black) to 255 (white).
From digitized video images, the VideoTrack System can analyze multiple
movement parameters including time spent in peripheral versus central area of each
well, number of movements per unit of time, number and angle of rotations of each
movement, and D at low, medium, or high speed.
For these experiments, the motion tracker recorded activity from 20 wells in
24-well microplates, the maximum number of wells that can be imaged due to the
format of the video camera (NTSC standard). The signal threshold setting for each
well, which ranged from 20 to 140, was adjusted as follows. Briefly, after clicking on
the full-scale image icon, 20 circles were oriented over each well and movement of
single zebrafish was recorded. To reduce reflection or other shadows that could affect
thresholding, area size analyzed in each well corresponded as closely as possible to
the actual diameter of each well. After adjusting the size of each well, “draw areas”
were deselected. Then, the first well to be analyzed was selected using the “tiles”
button. To ensure that zebrafish were identified, we used the detection threshold
window that displayed number of red pixels over the body of each zebrafish. If a
zebrafish was identified in the well, the movement threshold window was used to
examine each animal on the screen. If a small white dot was visible on the body of
the zebrafish, then the instrument was ready to track movement. If no white dot was
identified or a number of lines appeared in a well with no zebrafish movement, the
size of the recording area was adjusted to exclude any dark areas.

13.2.3 Compounds and Treatment Conditions

To validate the zebrafish seizure assay, we tested nine compounds known to cause
seizures in mammals and zebrafish, including PTZ, 4-AP, picrotoxin, strychnine
hemisulfate, methoxychlor, amoxapine, aminophylline hydrate, bicuculline methio-
dide, and enoxacin (ATSDR, 1994; Winter et al., 2008), and lidocaine as a negative
control compound. Fish water with 0.1% DMSO was used as carrier control.
Compounds, mechanisms of action, and concentrations assessed are shown in
Table 13.1. Stock drug solutions were made in 100% DMSO and stored at

20 C.

On the day of the experiments, drug solution was diluted to a 2 concentration in fish

water (5mMNaCl, 0.17mMKCl, 0.33mMCaCl 2 , 0.33mMMgSO 4 , 10mMHEPES,
pH7.5). For treatment with each compound concentration, a single 6dpf zebrafishwas
deposited in each well of 24-well plates containing 250

m L fish water. Then,

immediately before image acquisition, we added 250

drug solution. Final DMSO concentration was 0.1% and final fluid volume in each
well was 500

m L fish water containing 2

m L. For this study, initial test concentrations were selected based on