Knowledge Base

Introduction

Optogenetics allow scientists to control the activity of specific neurons and study their downstream influence on a variety of biological processes, including behavior. In rodent studies, this technique requires optical fiber implantation. When working with zebrafish larvae, mostly transparent, fiber optics are not needed. Light simply needs to shine in the right direction, making it easy to assess the effect of stimulation of specific, light-sensitive neurons on the behavior of the fish.

By inserting light-sensitive receptor proteins into neurons in vivo, it is possible to make those neurons sensitive to activation by light of specific wavelengths. In zebrafish studies, the light-sensitive receptor protein halorhodopsin (NpHR) has successfully been used to inhibit swimming behavior in zebrafish larvae (Arrenberg et al. 2009). Other research showed that channelrhodopsin-2 (ChR2) activation induced backward swimming in a sparse transgene expression line (Zhu et al. 2009).

Optogenetics Add-On

Application of optogenetic stimulation in zebrafish larvae requires the correct wavelength (i.e., color) of light. When using the DanioVision Observation Chamber, the Optogenetics Add-on provides a way to accurately control and precisely time the application of optogenetic stimulation to up to 96 individuals simultaneously (working with 96 well plates).

The Optogenetics Add-on for DanioVision is an optogenetic LED light source based on the Prizmatix Modular LED system. It consists of one, two or three LED wavelengths. The Optogenetics Add-on can be programmed and controlled using the EthoVision XT software included with your DanioVision system. You can set user-defined time conditions for the optogenetic stimulation. In comparison to manual control, this offers far better temporal precision and adds efficiency to longitudinal studies.

References

• Arrenberg, A.B., Del Bene, F., Baier, H. 2009. Optical control of zebrafish behavior with halorhodopsin. PNAS: 17968–17973.
• Zhu, P., Narita, Y., Bundschuh, S.T., Fajardo, O., Scharer, Y.P., Chattopadhyaya, B., Bouldoires, E.A., Stepien, A.E., Desseroth, K., Arber, S., Sprengel, R., Rijli, F.M., Friedrich, R.W. 2009. Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system. Frontiers in Neural Circuits: 21.

Sample Experiment

Your DanioVision system comes with an EthoVision XT sample experiment: DVOC_Optogenetics_Demo_Recommended.evxt. It contains examples of Trial Control rules for triggering pulse sequences in one up to three Pulsers.

Additional Manuals

• For connecting hardware: Prizmatix UHP-T LED Illuminator User Manual.pdf
• For programming the Pulser: Prizmatix Pulser User Manual.pdf
• For connecting the DanioVision Temperature Control Unit: Reference Manual - DanioVision Temperature Control Unit DV-TCU.pdf

Install the Dichroic Mirror in DanioVision

When you receive the DanioVision Observation Chamber DVOC-004x with the Optogenetics Add-on, the LEDs are already mounted so you only have to connect the cables. However, the dichroic mirror comes in a separate box to prevent damage during shipping. This section explains how to install it.

Note: The function of the dichroic mirror is to direct the color light beams to the well plate, and at the same time allow light to reach the camera, so you can film and track the fish during the test.

1. Locate the box containing the mirror.
   Important: Please wear the supplied gloves to avoid damaging or dirtying the mirror. Avoid touching the mirror surface and only hold its edges.
2. Locate the arrow on one side of the mirror. Once the mirror is mounted, this arrow should be at the top of the mirror and point towards the light source, that is, the backside of the DanioVision chamber.
3. Attach the mirror. Slide the mirror upwards along the brackets as indicated by the arrows.
4. Position the mirror just above the mark on the right-hand bracket. When the mirror is in that position, carefully tighten the screw on the left to fix the mirror. Do not apply any force as this may damage the mirror.
5. The mirror is now ready to use.

Important: The LED is extremely powerful and may cause eye injury when looking directly into the light source. For testing, we recommend setting the LED intensity to low.

The Pulser Software

Please skip this section if you do not use the Pulser/PulserPlus devices. Follow the procedure below if you use one or more Pulser/PulserPlus devices. To control multiple devices, multiple instances of the Pulser software must be running on the EthoVision XT computer.

Important: Do not connect the Pulser/PulserPlus device to the computer until the software setup process is complete.

Install the Pulser Software Once

1. Copy the installation files and folders to your computer's hard drive.
2. Run the setup.exe file and follow the instructions as they appear on the screen. Next, restart the computer to complete the software installation.
3. Open the folder where the software has been installed (default: C:\Program Files (x86)\Prizmatix Pulser Plus 3.1) and make a shortcut to the EXE file. Give the shortcut a name that is easily recognized, like Pulser A or the name of the first LED.
4. To install or update the Pulser/PulserPlus drivers, see the Pulser/PulserPlus User Manual.

Install Multiple Instances of the Pulser Software

To control two or three LEDs independently using the dedicated Pulser/PulserPlus devices, install one instance of the Pulser software for each Pulser/PulserPlus.

1. Install the first instance of the Pulser software (see above).
2. Browse to the folder C:\Program Files (x86) and copy the entire folder Prizmatix Pulser Plus 3.1.
3. Paste the folder in the same location C:\Program Files (x86). Rename the folder to Prizmatix Pulser Plus 3.1 PulserB.
4. Open the new folder and make a shortcut for the EXE file that you find there. Place the shortcut on your desktop. Name it like Pulser B or the name of the second LED.
5. If you have a third LED, repeat steps 2 to 4 above to create a new folder which will contain a third instance of the Pulser software.

Source: DanioVision DVOC-0041 Reference Manual, Noldus Information Technology