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.

Experiments with Calcium Imaging

nVista vs nVoke

• nVista: Inscopix system for in vivo Calcium imaging in free behaving animals. It includes a head-mounted miniature microscope that enables one-photon epifluorescence imaging of calcium dynamics, a correlate of neural activity.
• nVoke: 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: 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 (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: 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)

Basic connections for triggering video recording from EthoVision XT in nVista/nVoke systems:

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

Calcium Imaging + LED Stimulation (nVoke)

Basic connections for triggering video recording and OG-LED stimulation from EthoVision XT in nVoke systems:

1. 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 a BNC connector.
2. Note that a TTL port on the USB-IO box can send out signals through two independent lines (Output 1 and Output 2).
3. 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.

Source: EthoVision XT 18 - Application Manual, Noldus Information Technology