Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models have many advantages and subsequently are being increasingly used in the biomedical sciences. From development to drug discovery and toxicology studies. Behavior of fish models are similarly being increasingly used for environmental and biomedical research.

Here it is particularly important to leverage decades of research experience in aquatic toxicology and behavioral ecology to advance the environmental biomedical sciences. Our method could be used to understand bioavtivities of chemicals and the benefits and hazards they may present. The main advantage of this protocol is it provides sensitive and rapid approach to understand diagnostically chemical activities of usefulness to the environmental and biomedical sciences.

The implications of this technique are intended to support the diagnosis of commercial bioavtivities and behavioral effects. Most of these chemicals often lack important toxicity information. However, it can also be applied to understand the effects of other chemicals such as, pharmaceuticals and pesticides.

This aspect is important to consider as comparative environmental pharmacology and toxicology data is often lacking for industrial compounds. In addition to measuring larval locomotor activity during altering light, dark photo periods, this protocol can be used to measure larval photomotor responses. Which effectively examine the magnitude of movement difference between light to dark and dark to light transitions.

To begin, dissolve caffeine in reconstituted hard water. Then perform serial dilutions to produce lower caffeine treatment levels. To prepare the individual exposure chambers pour 20 milliliters of solution into four 100 milliliter glass beakers for the zebrafish.

Pour 200 milliliters of solution into three 500 milliliter glass beakers for the fathead minnows. Next, use a transfer pipette to place 10 aged four to six hours post-fertilization zebrafish embryos into each of the beakers. Using a modified transfer pipette place 10 fathead minnow larvae aged within 24 hours of hatching into each of the exposure chambers.

Place the zebrafish and fathead minnow chambers in an incubator. After 96 hours load individual fish into separate wells of 48 and 24 well plates. Place a well plate with at least one larval fish in the recording chamber.

Then in the video tracking software, click file generate protocol. In the location count field of the dialogue box enter the number of individual wells of the well plate. And click OK.At the top of the screen, click view full screen.

To display a overhead camera view of the well plate. Then click the draw areas icon. Select the circle icon in the field labeled areas.

Using the cursor, delineate the circular video tracking area of the top left well of the plate. Select top-right mark and outline the viewing area of the top right well. Next select bottom mark to outline the bottom right well.

After defining the well tracking areas click build to prompt the software to delineate the viewing areas of the remaining wells. In the calibration area, click draw scale and draw a horizontal line across the plate. Once the line is drawn, a dialogue box labeled calibration measurement will appear.

Enter the well plate length in this box and click OK.Click apply to group in calibration area. To exit the drawing manager, click the draw areas icon. Next click the tiles icon.

Using the cursor highlight all of the boxes that appear on the viewing screen so that each box is green. Click view and full screen. Then in the detection threshold box, click bkg and use the threshold adjustment bar to set the pixel detection threshold.

Once the appropriate threshold is selected, click apply to group. In the box labeled movement threshold, enter the desired movement speed tracking parameters. Once speed parameters are set, click apply to group.

Next, click protocol parameters from the drop down menu. In the dialogue box, select the time tab and enter the observation and integration times. Click ok and open the light driver settings dialogue box by selecting light driving from the parameters drop down menu.

Set the light dark photo period times and light intensity for each photo period. Then click ok. Next save the observation protocol.

First place the well plate containing the experimental fish in the behavioral recording chamber. Then open the previously developed tracking protocol. In the video tracking viewer, ensure that all of the larvae are visible, that only one larvae is present in each well, and that the wells are aligned with the defined observation areas.

Next click on experiment and execute. Specify the name and save location of the data. And click on the several live images icon to highlight all of the predefined viewing areas.

Finally, close the panel of the recording chamber and click background followed by start on the computer monitor. Following 96 hours of exposure to caffeine fathead minnow larvae photomotor response was altered by caffeine at lower levels than zebrafish. However, a markedly larger number of photomotor end points were affected in zebrafish.

Additionally, light and dark locomotor activity was analyzed across three speed thresholds for distance moved, number of movements, and duration of movements. In both species, caffeine inhibited activity at all significantly affected locomotor end points. While performing this procedure it's important to remember to connect behavioral assays with a narrow specific window of time.

Because time of day can influence larval fish behaviors. Following this procedure, methods can be applied to standardized guidelines in other chemicals to answer additional questions related to fish behaviors. After watching this video, you should have a good understanding of how to observe behaviors and photomotor responses of larval fish models when performing toxicity bioassays with standardized guidelines of relevance to the environmental and biomedical sciences.

Don't forget that working with certain chemicals can be hazardous, so be sure to wear appropriate personal protective equipment when performing this protocol.