Knowledge Base

Introduction

Optogenetics and EthoVision XT

In the last decade, a method was developed to specifically activate or even inhibit small groups of neurons with light. Today, scientists can insert light-sensitive receptor proteins (originally found in algae) into single mammalian neurons in vivo, making these neurons sensitive to activation by light of specific wavelengths. This allows scientists to control the activity of these neurons and study their downstream influence on a variety of biological processes.

Optogenetics is an important development for behavioral research. Not only can the actual influence of specific neurons on behavior more specifically be determined, but also, with the help of an automatic video tracking system such as EthoVision XT, behavior can be manipulated via optogenetic methods in real time. With the Trial & Hardware Control module, EthoVision XT can control light pulses given by a third party device, based on the behavior of the animal (for instance, presence in a trigger zone).

This manual focuses on the optogenetics solution for rodents that combines EthoVision XT, PhenoTyper (as test environment) and the Prizmatix optogenetics devices. For how to conduct optogenetics experiments with zebrafish, see the DanioVision DVOC-0041 Reference Manual.

Examples

Place Preference

The animal is placed in an arena with two distinct sides, one of which is paired with optogenetic stimulation. Depending on whether this stimulation is activating or deactivating, and which neurons are affected, this can be a rewarding or aversive stimulus for the animal. If in further sessions the animal spends more time on the stimulated sides, it is fair to conclude the stimulus had a rewarding effect. If the animal avoids this side, the stimulus could then be considered aversive.

Operant Conditioning

In operant conditioning tests, the animal learns to perform an action in order to get a reward or avoid an aversive stimulus. The behavioral response can be recorded with EthoVision XT, either by detecting the animal (or its nose point) in a certain zone, such as the feeder, or by analyzing the external signal initiated by the nose poke or lever press. Following this action, EthoVision XT sends out the command for optogenetic stimulation.

Off-On-Off Stimulus Test

In an off-on-off stimulus test, you can program EthoVision XT to turn optogenetic stimulation on for a period of time during the test. For example, a 15 minute test during which continuous optogenetic stimulation takes place in the middle 5 minutes.

References

1. Guru, A., Post, R.J., Ho, Y., Warden, M. (2015). Making Sense of Optogenetics. International Journal of Neuropsychopharmacology, Aug 2015, 1-8.
2. Zalocusky, K., Deisseroth, K. (2013). Optogenetics in the behaving rat: integration of diverse new technologies in a vital animal model. Optogenetics 2013: 1-17.
3. Stamatakis, A.M., Stuber, G.D. (2012). Activation of lateral habenula inputs to the ventral midbrain promotes behavioral avoidance. Nature Neuroscience, 15(8), 1105-1107.
4. Kravitz, A.V., Tye, L.D., Kreitzer, A.C. (2012). Distinct roles for direct and indirect pathway striatal neurons in reinforcement. Nature Neuroscience, 15, 816-818.

Physical Setup

Components (Example)

Devices

• PC with EthoVision XT installed.
• Noldus USB-IO Box.
• PhenoTyper with adapted Top Unit (optional).
• Prizmatix LED controller (for different light wavelengths). To control the LED through EthoVision, on the back panel of the LED controller, near/below the TTL connector, select Ext. If you want to activate the LED manually using the button located on the front panel, select Int. The other switch on the LED controller, just near/above the Ain 0-5V connector, should be in the Int position.
• Prizmatix Pulser or PulserPlus (with advanced setup).

Main Cables and Fiber Cords

• USB cable (USB-A to USB-B). Use this cable to connect the EthoVision XT PC to the USB-IO box [USB, type-B]. Also use this cable to connect the Pulser to the PC.
• UTP to BNC cable. To connect the USB-IO box [TTL control] to the LED Controller [TTL] or Pulser [Trig. In]. Note that each TTL port of the USB-IO box has two output lines. The white RCA jack corresponds to Output 1 and the red RCA jack to Output 2. If you received UTP-to-BNC cables with four BNC connectors, only use those marked with Output to connect the LED controller or Pulser.
• BNC to BNC cable. From Pulser [TTL Out] to LED Controller [TTL].
• Fiber patch cord, core 1000 micron. To connect the LED Controller [front panel] to the PhenoTyper Top Unit [Fiber Coupler adaptor].
• Fiber patch cord, core 1000 micron. This is located inside the PhenoTyper Top Unit. It connects the FC adaptor to the Rotary joint.
• Single fiber. To connect the Rotary joint fixed on the bottom plate of the PhenoTyper Top Unit to the implantable cannula. Available in various core diameters (e.g. 500 micron).

Other Components

• Zirconia ferrules (sleeves) for 2.5 mm and 1.25 mm cannulae.
• Optogenetics implantable cannulae (2.5 mm, 1.25 mm).

Basic Setup

Aim

With a basic setup, without the Pulser, EthoVision XT controls activation of the LED directly via the Noldus USB-IO box. With this setup you can, for example, activate the LED for one second.

Notes

• Activation of the LED can be repeated during a trial using the Subrule function in the Trial Control Settings.
• For generating two pulses of different length, you must create separate Trial Control commands.

Limitations

• The pulse cannot be shorter than 0.1 s.
• Accuracy of onset, offset and duration of a pulse is approximately the time taken by three samples. When tracking at 30 fps, this time is 0.1 s. The higher the sample rate, the higher the accuracy.

Advanced Setup (With Pulser)

Aim

With this setup, EthoVision XT controls activation of the LED via the Prizmatix Pulser and PulserPlus devices. With this setup you can send out very specific sequences of pulses, at a frequency higher (max 0.5 kHz) than with the basic setup. The sequence is predefined in the Pulser or PulserPlus.

PulserPlus is the device with three BNC connectors.

Connection Notes

• On the LED controller, make sure that the switch near/below the TTL connector is in the Ext position. Keep the switch near/above the Ain 0-5V connector in the Int position.
• Connect the Trigger In / Trig. In port of the Pulser or PulserPlus to the USB-IO box with the white RCA jack if you use Output 1, or with the red jack if you use Output 2.
• Connect the Pulser or PulserPlus to the PC with a USB cable.
• If you received UTP-to-BNC cables with four BNC connectors, only use those marked with Output to connect the Trigger In/Trig. In port on the Pulser or PulserPlus.
• If you have the PulserPlus, set the Operation mode switch to PC.

Measuring the LED Light Output

In some circumstances you may want to measure the LED light intensity at the tip of the cannulae. For this you need a sensor and a power meter. Note that a standard power meter may not give accurate measurements.

An example is the Thorlabs S140C or Thorlabs S142C coupled with the Thorlabs PM100USB interface, which shows the measurements on a computer screen.

EthoVision XT Settings

Create an experiment. If you use PhenoTyper, you can create an experiment from a PhenoTyper template. Choose File > New from Template and in the guided wizard, from the Arena template list, choose PhenoTyper (for one cage) or PhenoTyper quad (for two to four cages).

Experiment Settings

Choose Setup > Experiment Settings.

General Settings

• Set Video Source to Live Tracking and select the camera image.
• Choose the Tracked Features that apply to your experiment.

Use of Trial Control Hardware

1. Under Trial Control Hardware, select Use of Trial Control Hardware.
2. Click Settings and choose Noldus USB-IO box.
3. In the window that appears, locate the TTL port of the USB-IO box that is connected to the LED controller, and from the Device type list choose Custom Hardware. Under Device ID, give this device a unique name (for example, LED controller 1, or Pulser if you use the Prizmatix Pulser or PulserPlus).
4. Repeat the previous step if you have more LED controllers or Pulsers.

Make sure that Custom Hardware is selected in the Device Configuration window, not TTL Tester or others.

Arena Settings

Choose Setup > Arena Settings > Open Arena Settings 1.

Arena-Hardware Mapping

1. Make sure that you have drawn all the arenas.
2. At the bottom of the Arena Settings window, click the Arena-Hardware Mapping button.
3. In the Arena-Hardware Mapping window, click Add device one or more times depending on how many physical devices (LED controllers or Pulsers) are connected.
4. Assign the devices to the arenas. For setups with one cage (and one device), the device is automatically assigned to Arena 1. For setups with multiple cages (and devices), assign each device to the corresponding arena.
5. For each row, rename the Device name (for example, rename Device A to LED controller 1 or Pulser 1). Give devices unique names, which is also helpful when analyzing the data and events of specific devices.

To Test the LEDs

This procedure is only valid for the Basic setup (without Pulser).

1. In the Arena-Hardware Mapping window, click the row that corresponds to the device you want to test and click the Test button.
2. Choose Output 1 High and click Test. Check that the LED is activated.
3. Choose Output 1 Low and click Test. Check that the LED is deactivated.

Trial Control Settings

In the Trial Control Settings you can program activation and deactivation of the LED controller during the trial. Choose Setup > Trial Control Settings.

The following example shows how to switch on a LED one minute after the start of the trial, keep the LED active for one second, and then switch it off.

To Switch On the LED

1. Under Actions, click the button next to Custom Hardware.
2. From the Custom Hardware list, select the LED controller device (this is the name given under Device name in the Arena-Hardware Mapping window).
3. From the Action to perform list, select Output 1 High (or Output 2 High if you connected the USB-IO box to the LED controller using the cable with the red RCA connector).
4. Optionally, rename the Action name (for example, rename Hardware act (1) to Start Pulse).
5. Click OK and insert the new Action box in the Trial Control sequence, usually after a condition. In this example, the stimulation is given one minute after the start. Therefore, a Time Condition box is inserted between the Start track box and the Action box.

With this Trial Control procedure, the stimulus does not stop unless you insert another Action box that specifies Output 1 Low.

To Switch Off the LED After Some Time

1. Follow the instructions above to program activation of the pulse.
2. Under Conditions, click the button next to Time. Select After a time of and enter the duration of the pulse (minimum possible: 0.1 s). Insert the Condition box immediately after the Action box.
3. Under Actions, click the button next to Custom Hardware.
4. From the Custom Hardware list, select the LED controller device (this is the name given under Device name in the Arena-Hardware Mapping window).
5. From the Action to perform list, select Output 1 Low (or Output 2 Low if you connected the USB-IO box to the LED controller using the cable with the red connector).
6. Optionally, rename the Action name (for example, from Hardware act (2) to Pulse Off).
7. Click OK and insert the new Action box after the second Time Condition box.

The sequence can be read as follows: one minute after the start of tracking, activate the LED controller, then wait one second and deactivate the LED controller.

To Activate the LED When the Animal Is in a Zone

It is often required to activate the LED multiple times, for example, every time the subject enters a trigger zone, or every time the subject presses a lever. In such situations you need to create a Subrule that can be repeated indefinitely. The Subrule contains the following instructions:

• Check that the subject is in the zone (or that the lever has been pressed).

Source: EthoVision XT 17.5 Application Manual - Optogenetics Experiments