Knowledge Base

The DanioVision Optogenetics Add-On

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 behavioral 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 106: 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 3: 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 the 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: This manual, page 80.

Install the Dichroic Mirror in DanioVision

If you purchase the DanioVision Observation Chamber DVOC-0050 with the Optogenetics Add-on, the LEDs are already mounted so you only have to connect the cables (see the next section). However, the dichroic mirror comes in a separate box to prevent damage during shipping. This section explains how to install it. Please follow the instructions below.

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. 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. See the next sections.

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 3.2.1) 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 3.2.1.
3. Paste the folder in the same location C:\Program Files (x86). Rename the folder to Prizmatix Pulser Plus 3.2.1 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 to create a new folder which will contain a third instance of the Pulser software. Make a shortcut to the new EXE file and rename it to e.g. Pulser C.
6. You should at this point have the appropriate folder structure and three shortcuts on your desktop.

LED Controller UHPTLCC

This device has a switch in the middle of the front panel.

Basic Connections

1. Make sure the power switch (1) is set to Off.
2. Connect the 12VDC connector (10) to the power outlet through its power cable and adapter.
3. Connect the LED cables (LED Control cable and LED Current cable) that come from the DanioVision system to the two LED connectors (8 and 9, respectively).

To Control the LED Manually

1. Set the switch near TTL (5) to Int.
2. Set the switch near Ain (6) to Int.
3. Turn on the main power (see 1 in the figure above).
4. To turn the LED on, flip the switch (2) on.

To Control the LED from EthoVision XT Through TTL

1. Set the switch near TTL (5) to Ext.
2. Set the switch near Ain (6) to Int.
3. Turn on the main power (see 1 in the figure above).
4. To turn the LED on, flip the switch (2) on.
5. Connect the TTL cable with BNC connector from the pulser (or the back of the DanioVision chamber, depending on the setup) to the TTL connector on the back panel of the LED controller (4).
6. In EthoVision XT you can now give the "high" command using the Trial Control actions. See page 157 or 159 depending on the setup.

Other Controls

• Power adjustment dial (3).
• Analog input (Ain) connector (7).

LED Controller UHPTLCC-02

This device does not have a switch in the middle of the front panel.

Basic Connections

1. Make sure the power switch (9) is set to Off (O).
2. Connect the 12VDC connector to the power outlet through the power cable and adapter.
3. Connect the LED cables (LED Current cable and LED Control cable) that come from the DanioVision system to the two LED connectors (7 and 8, respectively).

To Control the LED Manually

1. Set the switch near TTL (4) to Int.
2. Set the switch near Ain (5) to Int.
3. Turn on the main power (9).
4. To turn the LED on and off, press the button (1).

To Control the LED from EthoVision XT Through TTL

1. Set the switch near TTL (4) to Ext.
2. Set the switch near Ain (5) to Int.
3. Turn on the main power (9).
4. Do one of the following, depending on whether you work with Pulsers or not:
   • If you do not use the Pulsers, connect the TTL-In of each LED controller to the connectors at the back of DanioVision: Trigger Out 1 for LED Controller 1, Trigger Out 2 for LED Controller 2, and TTL A (or B) for LED Controller 3. For this latter connection you need a special cable with an 8-pin connector at one end and a BNC connector at the other end. See Configuration 2 - With the Pulsers on page 149.
   • If you use the Pulsers, connect the Trigger In of each Pulser to the connectors at the back of DanioVision: Trigger Out 1 for Pulser A, Trigger Out 2 for Pulser 2, and TTL A (or B) for Pulser C. For this latter connection you need a special cable with an 8-pin connector at one end and a BNC connector at the other end. See Configuration 2 - With the Pulsers on page 151.
5. In EthoVision XT you can now trigger the LED controllers or the Pulsers using the Trial Control actions. See page 157 or 159 depending on the setup.

Other Controls

• Power adjustment dial (2).
• Analog input (Ain) connector (6).

Configuration 1 - Without the Pulsers

In the following example, EthoVision XT controls three LED controllers through the TTL ports in the DanioVision Observation Chamber.

EthoVision XT Connections

• One Ethernet connection to control the DanioVision and Optogenetics hardware devices.
• One Ethernet connection for the DanioVision camera. See page 14.

Next Steps

• Experiment settings on page 153.
• Arena settings on page 154.
• Trial control (without the Pulsers) on page 157.

For information about creating pulse sequences in EthoVision XT, see the chapter Optogenetics Experiments in the EthoVision XT 18 Application Manual. To open this manual, from the Windows Start menu choose All Apps > Noldus > EthoVision XT 18 Other Documentation.

Configuration 2 - With the Pulsers