A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

The overall goal of this procedure is to dose groups of fish accurately and efficiently with ethanol for assessing the effects of repeated dosing on withdrawal behavior. This is accomplished by first dosing the zebra fish, using a new method that has been developed to allow for the simultaneous drug exposure of whole groups of fish for precise amounts of time. The second step is to continue dosing the fish on a schedule that permits testing for specific effects of ethanol withdrawal.

Next, during withdrawal, fish are individually tested in a light dark task using motion tracking software to record movement. The final step is to analyze the data in order to investigate the effects of ethanol withdrawal on zebra fish behavior. Ultimately repeated ethanol dosing using the described procedure results in transient but marked effect on the behavior of zebra fish following two days of withdrawal.

The main advantage of this technique is that it allows for simultaneous drug administration to large groups of fish, and it's ideal for studies involving repeated drug, drug, or toxin exposure over long periods of time. This method can help answer key questions in the field of neuropharmacology, such as the quantification of withdrawal related behaviors. We first had the idea for this method when we tried to administer ethanol to multiple fish at the same time and found that netting was both an inaccurate and imprecise method for administering drugs.

To begin. Prepare an administration schedule such that the animals are dosed in the same environment and during the same time of day to avoid any confounds of time or biasing to visual stimuli. Next, obtain as many identical 1.5 liter clear polypropylene spawning tanks as necessary for the number of group sizes to allow for two groups of fish to be tested per day.

Use groups of eight fish per tank, one holding tank and one dosing tank is necessary per group. Therefore, the number of tanks is two times the number of groups. Then place 400 micron spawning inserts in all holding tanks.

Fill the tanks with habitat, water, or reverse osmosis water at the appropriate temperature here. 25 to 28 degrees Celsius is used for zebra fish. Ensure that the tanks are in a neutral environment to avoid conditioning, fish to external visual stimuli during dosing.

Next, prepare the drug solution by mixing the appropriate amount of the drug with habitat water in the dosing tanks here, a 0.2%ethanol solution and a 1.4%ethanol solution are prepared by combining high grade ethanol with water in the tank. Carefully net fish from their habitat tanks and transfer into the appropriate holding tank containing the spawning insert. Ideally, house the fish in the spawning insert to eliminate netting altogether with all fish in their respective holding tanks.

Gently lift the spawning insert out of the holding tank and place it into the appropriate drug solution tank. If possible, have assistance help with the transfer of all groups to the drug solution simultaneously to ensure precise dosing time. Record the dosing time as required.

30 minutes in the ethanol solution is used for the procedure described here at the end of the requisite dosing period. Remove the fish from the ethanol solution by carefully lifting the spawning insert out of the drug solution and placing it gently back into the holding tank. Obtain a light dark arena nine and one half centimeters wide by 55 centimeters long and nine and one half centimeters deep with a white waterproof floor.

A fix white and black waterproof non-reflective paper to the inside walls of the arena using Velcro with half of the arena covered in white and half covered in black, fill the arena to a depth of five centimeters with habitat water at a temperature of 25 to 28 degrees Celsius. Maintain this temperature throughout testing minimalize external visual stimuli by constructing a white three-sided enclosure for the arena to be placed in. Ensure the testing area has diffuse overhead.

Lighting that does not cause reflections on the water surface yet is sufficiently bright for the movement tracking software or post hoc manual quantification from video images. Then set the recording and movement analysis parameters of the behavior tracking software with a trial duration of five to 15 minutes, depending on the research question, transport the group of fish to be tested to the research area in the habitat tank and place them outside of the arena.Enclosure. Allow the fish to acclimate for 10 minutes.

Gently net a fish from the appropriate group and place in the center of the light dark arena releasing each fish when it is positioned parallel to the long axis of the arena. To avoid biasing the fish to the light or dark zone, begin recording behavior immediately after the animal is released. Watch for any software problems with tracking the fish or for fish jumping or freezing after the trial has ended, gently net and remove the fish from the arena to a holding tank or habitat tank.

Rotate the arena 180 degrees after half of the subjects have been tested to prevent any confounds due to biases resulting from which end of the arena is oriented toward the open end of the enclosure. Begin analysis by examining the time spent in light versus dark zones for each group and for each fish, obtain the relative time spent in the light and dark zones and analyze using a one sample T-test to determine if groups significantly prefer one area over the other. To compare preferences, calculate a preference index by subtracting the time spent in the light zone from the time spent in the dark zone, and compare differences between groups.

Using T-test, compare multiple groups with a one-way analysis of variance utilizing two key's, honest significant difference post hoc test, where necessary for non-parametric data sets. Use the resco Wallace test with Dunn's multiple comparison post hoc test, then compare velocity, number of zone transitions, meandering and immobility across groups. Using one-way analysis of variance, again, utilize two key's, honest significant difference post hoc test where necessary.

The administration of ethanol on either a weekly binge or daily moderate schedule resulted in altered anxiety levels as measured with a light dark test when compared to controls. Shown here are representative track plots of a single control zebra fish movement path throughout the five minute light dark trial for each condition. The same zebra fish track plot is represented as a heat map, which is a colored representation of zebrafish movement throughout the trial.

Based on the time the fish spent in the location represented by each pixel. When tested two days after the last dosing, zebrafish in the control group displayed the expected pattern of behavior with control fish spending significantly more time on the dark side of the arena. Similar to naive zebra fish in other studies, fish in the weekly binge group showed no preference for either the light or dark zones in the task when tested two days after their last ethanol administration.

Interestingly, fish in the daily moderate group significantly preferred the light site of the arena. In contrast to the control group, the preference index indicated a significant difference between control and daily moderate groups. There were no significant differences in swimming speed or immobility across groups, and therefore this effect was not due to a motor deficit in the fish.

While attempting this procedure, it's important to remember to administer the pharmacological compound of interest at the same time of day before or after feeding the fish. So following this procedure done with ethanol or other pharmacological compounds, other zebrafish tests of behavior can be done to assess things like addiction and withdrawal. After watching this video, you should have good understanding of how to dose groups of zebrafish efficiently and accurately for behavioral testing.

Following this procedure, particularly using the spawning inserts is a great way to ensure precision when dosing fish.