Knowledge Base

12 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 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: DVOCOptogeneticsDemo_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 (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.

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 (see the next picture). 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

(see the picture below, 1). When the mirror is in that position,

carefully tighten the screw on the left to fix the mirror (2). 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 to set 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 the steps above from 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 a folder structure like the following: and three shortcuts on your desktop.

The Led Controller

To control the LED manually:

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

To control the LED from EthoVision XT (through TTL)

1. Turn on the main power (see 1 in the figure above).
2. Set the switch near TTL (5) to Ext.
3. Set the switch near Ain (6) to Int.
4. To turn the LED on, flip the switch (2) on.
5. Connect the TTL cable with BNC connector from the pulser (or the USB-IO box, depending on the setup) to the connector TTL on the back panel of the LED controller (4).
6. In EthoVision XT you can now give the "high" command. See page 100 or 101 depending on the setup.

Other controls

Power adjustment dial (3). Analog input (Ain) connector (7). LED control cable connector (8). LED current cable connector (9). DC power jack (10).

Configuration 1 - Without The Pulser

Connection diagram

In the following example, EthoVision XT controls three LED controllers through the TTL ports in the DanioVision Observation Chamber. Note that each port contains two lines, with connectors white (Line 1) or red (Line 2). Each line controls one LED controller.

Define the pulse sequence

To define a pulse sequence, use actions and conditions in the EthoVision XT's Trial Control Settings. For example a 1-s pulse of light in the first LED can be defined as: Action Light On on port 1 > Time Condition (1 second) > Action Light Off on port 1. For information about creating pulse sequences in EthoVision XT, see the Chapter Optogenetics experiments in the Application Manual - EthoVision XT, which you can find on the EthoVision XT computer under Noldus > EthoVision XT Other Documentation.

USB ports

You need one USB port on the EthoVision XT computer, to control DanioVision.

TTL ports

When using one LED, you can use the TTL-port 2 of DanioVision. Use the white connector at the other end of the cable to connect the LED controller. When using two or three LEDs, you can control two LEDs with one TTL port. Use TTL-port 2 for LED 1 and 2, and TTL-port 3 for LED 3. Remember that the white connector at the other end of the cable means Line 1, and the red connector means Line 2.

Configuration 2 - With Pulsers

Connection diagram

USB ports

On the EthoVision XT computer, you need: One USB port, to connect DanioVision (see page 17) Additional USB ports, one for each Pulser (see page 97). Connect the USB port of each Pulser to a USB port of your computer.

TTL ports

When using one Pulser, you can use the TTL-port 2 of DanioVision. Use the white connector at the other end of the cable to connect the Pulser. When using two or three Pulsers, you can control two Pulsers with one TTL port. Use the DanioVision TTL-port 2 for Pulser A and B, and TTL-port 3 for Pulser C. Connect the other end of the cable to the

Trigger In port of each Pulser. Remember that the white connector

at the other end of the cable means Line 1, and the red connector means Line 2.

Experiment Settings

Choose Setup > Experiment Settings > Click Settings next to Use of Trial

Control hardware. Then follow the section below that applies to

choose the port numbers in the Device Configuration window.

tip Open the sample experiment DVOCOptogeneticsDemo_

Recommended.evxt. This is a live tracking experiment where basic settings in Experiment Settings and Trial Control are already selected for when you work with three pulsers.

Configuration 1 - without Pulser

Each LED controller is connected directly to one TTL port of DanioVision. If for example you have three LEDs, choose Port 2 for the first two LEDs, and Port 3 for the third one. See the corresponding scheme on page 95.

Configuration 2 - with Pulser

Each Pulser is connected to one TTL port of DanioVision. If for example you have three Pulsers, choose Port 2 for the first two Pulsers, and Port 3 for the third one. See the corresponding scheme on page 97. Under Device ID, give each port a name that can be easily recognized.

Arena - Hardware Mapping

Choose Setup > Arena Settings. Draw the arenas (see this manual).

When the arenas are ready, click the Arena - Hardware Mapping button

and make sure that the devices are selected under Arena 1 (A1). The figure below refers to a configuration with three Pulsers.

Trial Control (no Pulser)

important Before you follow this section, make sure that (1) the

dichroic mirror is installed in the DanioVision chamber (page 90), and (2) all components are connected (page 95) and powered up, and (3) that the LED controller is set to TTL control (page 93). When the LED is controlled directly from EthoVision XT, activation occurs in Trial Control, using the Action boxes. The duration of the light pulse is determined by a Time condition placed between the Action Output 1 High and Output 1 Low. In the example below, LED 1 (Device D in the Arena - Hardware mapping

window; see above) is activated with a TTL signal Output 1 High and

after 0.5 it is deactivated with a TTL signal Output 1 Low. The LED

Controller must be connected to DanioVision through TTL Port 1 - Line 1 (see page 95).

Use Output 2 High/Low commands to control the other LED controller

connected to the same DanioVision TTL port. Make sure that the Int-Ext switch near TTL on the LED controller is set

to Ext!

Trial Control (with Pulsers)

important Before you follow this section, make sure that (1) the

dichroic mirror is installed in the DanioVision chamber (page 90), (2) all components are connected (page 97) and powered up, and (3) that the LED controller is set to TTL control (page 93).

Procedure

1. Open all the instances of the Pulser software by clicking on the

desktop shortcuts Pulser A, Pulser B, and Pulser C. Each instance

controls one pulser. 2. For each instance of the software, next to Select COM port, choose the correct COM port for the corresponding Pulser. These can be found on the pulser or on the connection diagram. 3. Next to Select Operation Mode, choose one of the following:

When testing the Pulser, choose (0) Execute pulse sequence

after START. By pressing the Start Sequence button, the LED

flashes the configured sequence.

When running the actual trials, choose (3) Execute pulse

sequence after trigger HIGH, then stop when LOW. If you now

click the Start Sequence button, the Pulser waits for a TTL trigger

to start the sequence. 4. Enter the other settings in the dialog to control the pulse duration and the time between pulses. For details, see the Prizmatix Pulser/ PulserPlus User Manual. 5. Start EthoVision and open the example experiment: DVOCOptogeneticsdemo_recommended.evxt In the Trial Control Settings you find predefined routines (called subrules) to control three Pulsers. The main procedure (top row) has been configured to consecutively trigger the three Pulsers using the corresponding subrules. Make sure the pulsers all have the correct

COM port, are all in mode 3 and the start sequence button has been

pressed. See also configure the com port for each pulser on page

103. Here below you find the basic actions to control the triggering of the Pulsers. For more information, see also the Chapter Optogenetics experiments in the Application Manual - EthoVision

XT, which you can find in Start > Noldus > EthoVision XT Other

Documentation.

Basic subrule

A basic set of instructions in a subrule activates the Pulser a number of times, as specified in the Subrule Reference box. The boxes within the subrule specify an action to trigger the pulse

sequence (Send a signal High to a device through line 1 or 2), a Time

(wait a number of seconds) and a second action which stops the pulse sequence (Send signal Low to a device through line 1 or 2). In the following example, the subrule triggers the sequence in the Pulser A through Output Line 1, for 5 seconds. The name of the device

selected next to Custom Hardware is 570 +520 nm, because it control

two Pulsers, one for a LED of 570 nm (through Output Line 1) and one

for a LED of 520 nm (through Output Line 2). You find the name 570

+520 nm in the Arena - Hardware Mapping window of the Arena

Settings in the demo experiment. In practice, the subrule would look like the following: Make sure that the Int-Ext switch near TTL on the LED controller is set

to Ext!

Configure The Com Port For Each Pulser

Aim

To configure the COM port in such a way the Pulser/PulserPlus accepts commands from EthoVision XT.

Procedure

1. Connect the Pulser/PulserPlus to the PC using the USB cable with type-A to type-B connectors.
2. In the Control Panel, open the Device Manager.
3. Under Ports (COM & LPT), right-click USB Serial Port and select Properties.
4. Click the Port Settings tab.
5. Make sure that the parameters are as below, then click OK.
6. Take note of the port number (in the example above it is COM3).
7. Repeat the procedure for each Pulser/PulserPlus.

Notes

The COM port number is retained in the Pulser, so if you disconnect the Pulsers and reconnect them, you do not have to assign the COM ports again.

tip To know which COM port is assigned to which Pulser/PulserPlus

device, disconnect the USB cable from one Pulser/Pulser Plus. Then,

re-connect it. The item that appears under Ports (COM & LPT)

indicates the COM port.

tip To change the COM port for a specific Pulser, in the Control

Panel, open the Device Manager. Under Ports (COM & LPT), right-

click the COM port, select Properties, Port Settings, then Advanced.

From the COM Port Number list select the COM port among those

available.

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Source: Reference Manual - DanioVision DVOC-0041