Knowledge Base

Introduction

EthoVision XT and Calcium Imaging

To understand the working of neural circuits in the brain, it is vital to be able to view neural activity simultaneously with behavioral data.

Calcium Imaging is a microscopy technique to optically measure the calcium (Ca2+) status of a cell, a tissue or a medium. This allows to monitor the electrical activity in single neurons at a spatial scale that is not possible to achieve with other techniques.

The Inscopix nVista miniature microscope system enables researchers to conduct genetically targeted calcium imaging over time in awake, freely behaving rodents, and has provided insights from multiple brain areas, from the prefrontal cortex all the way to the hypothalamus. nVista's compact, sleek plug-and-play interface makes it easy to add powerful new neural circuit insights to any laboratory's behavioral research arsenal.

You can combine EthoVision XT with the Inscopix nVista system in such a way that EthoVision XT takes control over the recording activity of nVista. For this you need the Noldus USB-IO box interface connected between the two systems.

EthoVision XT and Optogenetic Stimulation

In vivo Calcium Imaging is often coupled with Optogenetic stimulation to causally link neural circuit activity and behavior. For this purpose, the Inscopix nVoke system is coupled with EthoVision XT, which can trigger both calcium imaging and the optogenetic LED stimulation. The two triggering systems can be set independent of one another.

Similarly to what is done for the EthoVision XT and the nVista system, you need the Noldus USB-IO box interface connected between EthoVision XT and nVoke.

nVista vs nVoke

• nVista is the Inscopix system for in vivo Calcium imaging in freely behaving animals. It includes a head-mounted miniature microscope that enables one-photon epifluorescence imaging of calcium dynamics, a correlate of neural activity.
• nVoke is the Inscopix system that enables simultaneous in vivo Calcium imaging and optogenetic manipulations.

EthoVision XT License Needed

To enable control of Inscopix devices by EthoVision XT, you need the Trial and Hardware Control module. If you are not sure whether this function is activated, in EthoVision XT select Help > About EthoVision XT > License Info.

If the Trial and Hardware Control checkbox is not selected, contact Noldus to purchase an upgrade with this module.

Application Examples

Place Preference in the PhenoTyper

The nVista microscope is triggered every time the mouse enters a target region of the PhenoTyper, as defined in the EthoVision XT software.

In an experiment that combines Calcium Imaging and Optogenetics, the PhenoTyper was divided in two side zones, and in each session one side was assigned as stimulus (LED)-paired side. Each time the mouse crossed to the stimulation side, stimulation was delivered until the mouse crossed back into the non-stimulation side (Stamatakis et al. 2018).

Contextual Fear Conditioning

Simultaneous Calcium Imaging and Optogenetic stimulation are performed during a fear conditioning experiment. Optogenetic stimulation during the shock phase reduced the percent time freezing during context retrieval (Jimenez et al. 2020).

Motor Behavior

Simultaneous optogenetics and calcium Ca2+ imaging is performed in different populations of cells to look into the functioning of fast-spiking interneurons in the control of motor behavior in an open field (Owen et al. 2018).

References

• Jimenez et al. (2020). Contextual fear memory retrieval by correlated ensembles of ventral CA1 neurons. Nature Communications 11: 3492. doi:10.1038/s41467-020-17270-w
• Owen et al. (2018). Fast-spiking interneurons supply feedforward control of bursting, calcium, and plasticity for efficient learning. Cell 172(4): 683-695. doi: 10.1016/j.cell.2018.01.005
• Stamatakis et al. (2018). Simultaneous optogenetics and cellular resolution calcium imaging during active behavior using a miniaturized microscope. Frontiers in Neuroscience 12: 496. doi: 10.3389/fnins.2018.00496

Physical Setup

Hardware Components

• Noldus USB-IO box PTIO-002x
• Noldus Optical Isolated TTL-IO interface PTISO-00x0 (also known as opto-isolator)
• Inscopix nVoke or nVista DAQ box

Connection Schemes

Calcium Imaging (nVista)

Connect one of the TTL ports of the USB-IO box to the opto-isolator using a network cable. Connect the Out-1 port of the opto-isolator to the TRIG port of the Inscopix device using cables with BNC connector.

Calcium Imaging and LED Stimulation (nVoke)

Connect the Out1 and Out2 ports of the opto-isolator to the TRIG port and the GPIO port of the Inscopix device using cables with BNC connector. Note that one TTL port on the USB-IO box can send signals through two independent output lines (Out 1 and Out 2). Make sure that the Trial Control procedure sends the correct commands to the Inscopix DAQ box through the two lines Out 1 and Out 2, one for TRIG and the other for GPIO.

Notes

Why Use an Opto-isolator?

If you do not have the Noldus opto-isolator, you can connect the USB-IO box and the Inscopix directly. For this you need a cable with an Ethernet (RJ45) connector at one end and a BNC connector at the other end. However, we recommend using a Noldus opto-isolator to prevent ground loops and preserve the TTL signals. Use one opto-isolator per TTL port of the USB-IO box.

EthoVision XT Settings

Experiment Settings

After creating a new experiment, choose Setup > Experiment Settings. Define the number of arenas, the features that you want to track, and the camera properties.

To define the Inscopix devices:

1. Select Use of Trial Control hardware, and click the Settings button.
2. Depending on the type of USB-IO box you use, choose either Noldus USB-IO box or Noldus Mini USB-IO box.
3. Locate the TTL port that you use to connect the USB-IO box, and from the Device Type list choose Custom Hardware.
4. Under Device ID, enter a name for the device. For example, nVista Video, or nVoke Video, nVoke LED.
5. Repeat steps 3 and 4 to declare more devices. If you want to trigger Calcium Imaging and LED stimulation, you must use two TTL ports from the USB-IO box, one for the TRIG input and one for the GPIO[n] input of the nVoke device.
6. Click OK. Next, open the Arena Settings.

Arena Settings

Choose Setup > Arena Settings. Calibrate and define the outline of the arena(s) and the zones.

To map the Inscopix devices to the arena(s):

1. Click the Arena - Hardware Mapping button at the bottom-right corner of the screen.
2. Check that the device appears under the arena name. In the case of multiple devices, assign each of them to one arena and make sure that they do not occur in the cells for other arenas.
3. Optionally, edit the Device name (default: Device A, B, etc.). You will see this name in the Trial Control Settings and in the Analysis profile.
4. Repeat steps 2 and 3 to map the remaining devices.

Inscopix Devices

1. Connect the microscope to the subject and pass the cord through the tracking chamber or PhenoTyper and connect it to the Inscopix device.
2. On the Inscopix computer, launch the data acquisition software and adjust the imaging settings, such as the frame rate and the gain.
3. In the data acquisition software, select to trigger from external hardware. If you are doing Calcium Imaging only, you can use the Basic Recording mode in the Inscopix software. Make sure that the Recording Start mode is set to Triggered Recording.
4. When using optogenetics stimulation, in most cases you must use the Advanced Recording mode. Specify the type of stimulation, power, number of repeats, etc. See also the nVista and nVoke User Manual for examples.

Acquiring Data

Program Control of Devices in EthoVision XT

Trial Control Settings

The Trial Control Settings in EthoVision XT allows you to program activation of calcium imaging and optogenetic stimulation by Inscopix devices at specific times or when specific events occur, for example when the subject enters a target zone of the arena.

In addition, in Trial Control Settings you can define the conditions for the start and stop of data acquisition.

In EthoVision XT, choose Setup > Trial Control Settings > New, and enter a name for the new settings profile.

For more information on how to work with Conditions and Actions in Trial Control Settings, see the EthoVision XT Help (press F1 in EthoVision XT) and the EthoVision XT 17 - Trial and Hardware Control - Reference Manual, which you can find in the Apps screen under Noldus > EthoVision XT 17 Other Documentation.

Example 1 - Image Recording (nVista)

Use Case

EthoVision XT starts tracking after locating the subject's center point in the arena. Next, EthoVision XT sends out a high TTL pulse to the TRIG port of nVista. After a time delay of 10 minutes, EthoVision XT stops tracking the subject.

Hardware Connections

Use the basic connections for triggering video recording from EthoVision XT in nVista systems.

Prerequisites in EthoVision XT

You specified the TTL port and output line (either 1 or 2) for TRIG in the Experiment Settings and mapped the port to the arena.

Procedure

1. In the Trial Control Settings, under Actions, click the button next to Custom hardware.
2. Specify the device (e.g. Device A) and the type of signal (Output 1 High).
3. Optionally, edit the name in the Action name field.
4. Click OK and insert the Action box between the Condition - In zone box and the Action - Start track box.
5. Click Settings in the Condition - Time box and select 10 minutes. This way tracking stops after 10 minutes.
6. If you want to stop recording in nVista when you stop behavior tracking, bring a new Action box in the flow line which instructs the system to send out a low TTL pulse to nVista. Make sure that you place the Action box to the right of the 10-minute time box.

In the Hardware Action box you can test whether EthoVision XT triggers the nVista by clicking the Test button.

Make sure to instruct nVista/nVoke to record video based on the TRIG signal.

Example 2 - Manual Video Recording, Triggered OG-LED Stimulation

Use Case

EthoVision XT starts tracking after locating the subject's center point in the arena. You start video recording in nVoke manually by clicking the Record button. At the planned time, for example at 10 minutes after start, EthoVision XT sends out a high TTL pulse to the GPIO[n] port of nVoke.

In the Inscopix DAQ software, under Video Recording configuration, make sure that Trigger is set to NONE and Control is set to Video Recording ON.

Hardware Connections

• Use the basic connections for triggering video recording and OG-LED stimulation from EthoVision XT in nVoke systems. You do not need to use the TRIG port of the nVoke device.
• Set the GPIO[n] to Input in the Inscopix DAQ software.

Prerequisites in EthoVision XT

In the Experiment Settings, you defined the TTL port and output line (either 1 or 2) connected to the GPIO[n] of nVoke. In Arena Settings, you mapped that port to the arena.

Procedure

Because calcium imaging is started manually in this example, you only have to program the activation of the OG-LED. In the following example, the Time condition is set to wait 10 minutes before the triggering action.

1. In the Trial Control Settings, under Conditions, click the button next to Time.
2. Specify the time (e.g. 10 minutes). Optionally, edit the name in the Condition name field.
3. Click OK and insert the Condition box after the Action - Start track box (or wherever it applies in your protocol, for example in a sub-rule).
4. In the Trial Control Settings, under Actions, click the button next to Custom hardware.
5. Specify the device (e.g. Device A) and the type of signal (Output 1 High).
6. Optionally, edit the name in the Action name field.
7. Click OK and insert the Action box to the right of the Condition box just created.
8. When applicable, define a Condition and an Action (Output Low) for stopping the trigger action and insert them in the trial control flow line.

Notes

• In the Inscopix DAQ software, make sure to set the Trigger mode for the GPIO[n] port to Follow.
• When the Trigger is set to low, LED stimulation is stopped. To set the Trigger to Low, add an Action to the same device/TTL port and output line as above, and select the signal Output Low.
• You can also let the DAQ software run its own pulse protocol while the Trigger is ON. In that case the Control for the GPIO[n] port is set to Pulse protocol.
• For more information, see the nVista and nVoke User Manual.

Example 3 - Triggered Video Recording and LED Stimulation

Use Case

EthoVision XT starts tracking after locating the subject's center point in the arena. Both video recording and the OG-LED are triggered by EthoVision XT when the corresponding conditions (time or event) are met.

Hardware Connections

• Use the basic connections for triggering video recording and OG-LED stimulation from EthoVision XT in nVoke systems.
• Set the GPIO[n] to Input in the Inscopix DAQ software.

Prerequisites in EthoVision XT

In the Experiment Settings, you defined two TTL lines from the USB-IO box: one connected to the TRIG and the other to the GPIO[n] port of nVoke. In Arena Settings, you mapped the ports and lines to the arena.

Procedure

Define actions and conditions as explained for the previous examples.

• Condition to trigger video recording.
• Action to trigger video recording through the TRIG port.
• Condition to trigger OG-LED stimulation.
• Action to trigger the OG-LED stimulation through the GPIO[n] port.

When applicable, define additional conditions and actions to stop video recording and OG-LED stimulation.

Notes

• In the Inscopix DAQ software, make sure to set the Trigger mode for the TRIG port to Follow, and the Control is set to Video Recording ON. In an alternative configuration, you can set Control to Recording schedule to record video for a specific time.
• In the Inscopix DAQ software, make sure to set the Trigger mode for the GPIO[n] port to Follow. Select the type of Control you require (e.g. Pulse protocol).
• See the Example Configuration #3 in the nVista and nVoke User Manual.

Data Analysis

Detection Settings

Ensure your subjects are detected well by EthoVision XT. Choose Setup > Detection Settings. For more details, see the EthoVision XT Help.

Sample Rate

In the Video section, specify the sample rate.

Source: EthoVision XT 17.5 Application Manual - Experiments with Calcium Imaging