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 chapter 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).
• Prizmatix Pulser (left) or PulserPlus (right) (with advanced setup).

Important: To control the LED through EthoVision, on the back panel of the LED controller, near/below the TTL connector, select Ext (but if you want to activate the LED manually using the button located on the front panel, you should select Int). The other switch on the LED controller, just near/above the Ain 0-5V connector, should be in the Int position.

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 1: 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. Take note of this when selecting the TTL commands in EthoVision XT.
  • Note 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 um. To connect the LED Controller [front panel] to the PhenoTyper Top Unit [Fiber Coupler adapter].
• Fiber patch cord, core 1000 um. 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 um).

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.

Limit

• The minimum pulse duration that can be generated with the basic setup (without Pulser) is determined by the sample rate of EthoVision XT. For example, at a sample rate of 25 samples/second, the minimum pulse duration is 40 ms.

Optogenetics Experiments in EthoVision XT

Hardware Settings

1. Choose Setup > Hardware Settings > New.
2. Select USB-IO box from the device list and click Add.
3. Select the correct COM port for the USB-IO box.
4. Click Test to verify the connection.
5. If using the Pulser, also add the Prizmatix Pulser from the device list and select the correct COM port.

Trial Control Settings

In the Trial Control Settings, you define when and how the optogenetic stimulus is delivered based on the behavior of the animal.

1. Choose Setup > Trial Control Settings > New.
2. Add a rule that defines the trigger condition (for example, the animal's center point enters a defined zone).
3. Add an action to that rule to send a TTL output signal via the USB-IO box to the LED controller.
4. Define the duration of the TTL pulse to match the desired stimulation duration.
5. Use the Subrule function to repeat stimulation if needed.

Note: 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. Make sure to select the correct output line in the Trial Control Settings to match the physical cable connection.

Selecting the TTL Output

1. In the Trial Control Settings, select the action Set output.
2. Select the USB-IO box as the device.
3. Select the appropriate TTL port and output line (Output 1 or Output 2) that is connected to the LED controller or Pulser.
4. Set the output state to On to activate and Off to deactivate the LED.

Using the Pulser

Overview

The Prizmatix Pulser and PulserPlus allow you to generate precise, high-frequency pulse trains that cannot be produced by EthoVision XT alone. The Pulser is triggered by a TTL signal from the USB-IO box and then independently generates the configured pulse train to the LED controller.

The Pulser is configured using the Pulser software and stores its settings internally. Once configured and triggered, it operates independently of EthoVision XT for the duration of the pulse train.

Installing the Prizmatix Pulser

1. Connect the Pulser to the PC using a USB cable (USB-A to USB-B).
2. Connect the USB-IO box [TTL control] to the Pulser [Trig. In] using a UTP-to-BNC cable.
3. Connect the Pulser [TTL Out] to the LED Controller [TTL] using a BNC-to-BNC cable.
4. Install the Pulser software on the PC.
5. Open the Pulser software and configure the pulse parameters (frequency, pulse width, number of pulses, etc.).
6. Save the configuration to the Pulser device.

Configuring Pulse Parameters in the Pulser Software

• Frequency: The number of pulses per second (Hz).
• Pulse width: The duration of each individual pulse (ms).
• Number of pulses: The total number of pulses to deliver per trigger event.
• Delay: The delay between the trigger signal and the start of the pulse train (ms).

Triggering the Pulser from EthoVision XT

1. In Setup > Hardware Settings, ensure both the USB-IO box and the Pulser are added as devices.
2. In the Trial Control Settings, create a rule with a behavioral trigger condition (for example, animal enters zone).
3. Add an action to send a TTL output via the USB-IO box to the Pulser [Trig. In].
4. The Pulser receives the TTL trigger and delivers the pre-configured pulse train to the LED controller.

Advanced Setup with PulserPlus

The PulserPlus provides additional channels and more advanced pulse configuration options, allowing simultaneous control of multiple LED controllers with independent pulse parameters per channel.

• Connect each PulserPlus output channel to the corresponding LED controller TTL input using BNC-to-BNC cables.
• Configure each channel independently in the Pulser software.
• Trigger the PulserPlus from the USB-IO box in the same manner as the standard Pulser.

Data Analysis

Analysis Profiles

Choose Analysis > Analysis Profile.

For optogenetics experiments, create analysis profiles that include variables relevant to the experimental design. Typical variables include:

• In zone: To calculate the frequency, total time, and latency to first visit for each zone (for example, the stimulation zone and the non-stimulation zone in a place preference test).
• Distance moved: A measure of overall locomotor activity.
• Velocity: Mean velocity during the trial.
• Time in zone: Total time spent in each defined zone, used to calculate place preference scores.

Place Preference Analysis

1. Define zones for the stimulation side and the non-stimulation side of the arena in Setup > Arena Settings.
2. Create an analysis profile that includes the In zone variable for each zone.
3. Choose Analysis > Results > Statistics and Charts.
4. Compare the total time spent in the stimulation zone versus the non-stimulation zone across sessions to assess place preference or avoidance.

Operant Conditioning Analysis

1. Define the relevant zones (for example, the feeder zone or nose poke zone) in Setup > Arena Settings.
2. Create an analysis profile that includes In zone variables to count the frequency of visits to the operant zone.
3. Use external event markers or hardware event inputs to log the timing of stimulation delivery relative to the behavioral response.
4. Analyze visit frequency and latency to first visit to assess learning and response rates across sessions.

Off-On-Off Stimulus Analysis

1. Use the Trial phases or time bins feature in EthoVision XT to divide the trial into the off, on, and off periods.
2. Create an analysis profile that calculates distance moved, velocity, and time in zones for each phase separately.
3. Compare behavioral measures across the three phases to assess the effect of optogenetic stimulation.

Visualizing Data

Choose Analysis > Results > Integrated Visualization to view track plots and event timelines overlaid with zone visits and stimulus delivery events.

• Use track plots to visualize the animal's position over time in relation to the stimulation zone.
• Use timeline plots to compare the timing of behavioral events with the timing of TTL output signals sent to the LED controller.

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Source: EthoVision XT 19 - Application Manual