Knowledge Base

1. Introduction

Larval stage zebrafish display a robust startle response, which is mediated by neural pathways similar to that in higher vertebrates. Noldus Information Technology has developed the DanioVision Tapping Device DVTD-0010 that works as a startle stimulus for zebrafish larvae, to be used in combination with the DanioVision Observation Chamber and the EthoVision XT software. In this system, a single mechanical impulse is given to the well plate holder which startles the zebrafish larvae. Here we demonstrate the capability of this solution by listing a number of test results.

Questions Addressed

1. Can the DanioVision Tapping Device trigger a startle response in zebrafish larvae?
2. Does the startle response differ between larvae in light and dark conditions?
3. At what age of the larvae can the DanioVision Tapping Device evoke a startle response?
4. Does the response differ between larvae located in different areas of the well plate?

2. Methods

DanioVision and the Tapping Device

We used a DanioVision Observation Chamber (DVOC-0040/T) connected to a Temperature Control Unit (DVTCU) that kept the water at 28°. The Tapping Device is located inside the DanioVision chamber, in such a way that when activated it would hit a metal plate connected to the DanioVision well plate holder. The larvae were filmed using a digital camera with a video resolution of 640 x 480 pixels, and a frame rate of 60 fps.

How the DanioVision Tapping Device Works

The DanioVision Tapping Device is based on a solenoid, an electromagnetically inductive coil wound around a metal core. The core is made of two parts, a fixed one and a plunger connected to a push pin. When a current flows in the solenoid, a magnetic force is created between the plunger and the core, causing the plunger and the push pin to quickly move forward. The pushpin hits a metal plate attached to the basin in which the well plate is kept. When deactivated, a spring makes the plunger resume its position.

EthoVision XT and Test Protocols

We used EthoVision XT 10.1 on a Dell Precision workstation. The Trial Control procedures of EthoVision XT controlled the timing of the stimulus. In most cases the stimulus was given repeatedly at regular intervals of 1 minute, with a rest period of 5–10 minutes between stimuli of different intensity. In other tests, the intensity of the stimulus was constant in each trial.

The stimulus intensity can be adjusted in the Trial Control procedures of EthoVision XT, on a scale from 1 to 8. Additional tests using an accelerometer placed on an empty well plate showed that higher stimulus intensities resulted in higher accelerations.

Tests were carried out on 2 to 9 dpf larvae, with different genotypes (wild type and genetically modified) and subject to different experimental treatments. In the Trial Control procedures, the Tapping Device and the White Light stimuli could be combined and programmed. In general, tests were carried out in two different lighting conditions: either with full light (with the intensity of the DanioVision White Light set to 100%) or in darkness.

Data Analysis

To analyze the startle response we used the distance moved by the center point of each subject. We compared the 1-second periods immediately before and after administering the stimuli, respectively. Most of the results reported here are from control, wild-type animals.

3. Results

Startle Response

In all test conditions, zebrafish larvae reacted to the DanioVision Tapping Device. We found a significant increase in the subject's distance moved in the 1-second period after stimulus administration compared with the previous 1-second period (paired one-tailed t-tests, n=96; always P<0.001). Although significant variation in the data was due to between-subject differences, the response was consistent across systems and laboratories. A startle response could be detected independent of the animals' baseline.

Effect of Lighting Conditions

Zebrafish larvae were generally less active in dark conditions, but showed similar responses as when they were exposed to the DanioVision white light. The difference in response was not significant (ANOVA, P >0.5).

Effect of Subject Age

Tests conducted in different laboratories on larvae of age 2, 3, 6 and 9 dpf showed that the startle response can be detected from the age of 3 to 9 dpf, and for all stimulus intensities. Tests carried out on 5 and 6 dpf larvae showed no significant difference in the degree of response between day 5 and 6 (ANOVA, P >0.5).

Effect of Position in the Well Plate

We tested whether the degree of the startle response differed between animals in different positions of a 96- or 48-well plate. We analyzed the distance moved before and after stimulus administration between animals in the top vs. bottom rows, and between animals in the left vs. right columns. The degree of response did not differ between groups in any of the tests (P >0.05). We conclude that there is no evidence that the Tapping Device stimulus results in differential responses at various distances from the stimulus source.

4. Conclusions

• For all Tapping Device stimulus intensities, in both light and dark conditions, the DanioVision Tapping Device is capable of inducing a startle response in animals of 3 dpf and older.
• It is in principle possible to design experiments where the animals are subject to stimuli of different intensity.
• There is no evidence that the startle response measured by EthoVision XT depends on light conditions or the position of the subject in the well plate.

5. Acknowledgments

We acknowledge the kind collaboration of the following researchers and their groups:

• Dr. Stefan Spulber, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden
• Dr. Edor Kabashi, Institut du Cerveau et de la Moelle, Paris, France
• Dr. Hiroko Nakatani, URM INSERM 788, Le Kremlin-Bicêtre, France

Sjors Coenders is Senior Solution Engineer and Fabrizio Grieco is Behavioral Research Consultant at Noldus Information Technology.

Source: EthoVision XT 18 - THC - Trial and Hardware Control, Noldus Information Technology