Knowledge Base

Experiments with Calcium Imaging

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 is 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 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 Experiments with Calcium Imaging 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 Experiments with Calcium Imaging

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)

Figure 15.1 Basic connections for triggering video recording from EthoVision XT in nVista/nVoke systems. Connect one of the TTL ports of the USB-IO box to the Opto-isolator using a network cable (here in orange). Connect the Out-1 port of the Opto-isolator to the TRIG port of the Inscopix device using cables with BNC connector (in gray). Experiments with Calcium Imaging

Calcium Imaging + LED stimulation (nVoke)

Figure 15.2 Basic connections for triggering video recording and OG-LED

stimulation from EthoVision XT in nVoke systems. 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 (in gray). 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. See also page 331. Experiments with Calcium Imaging

notes

Why use a 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) at one end and a BNC connector at the other end. However, we recommend to use a Noldus Opto-isolator also to prevent ground loops to occur and preserve the TTL signals. Use one opto- isolator per TTL port of the USB-IO box. Experiments with Calcium Imaging

Basic settings

ethovision xt: 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 (see page 321), 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 the steps 3-4 above 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. See Figure 15.2 and page 331. 6. Click OK. Next, open the Arena Settings (see below).

ethovision xt: arena settings

Choose Setup > Arena Settings. Calibrate and define the outline of the

of 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. Experiments with Calcium Imaging 3. Optionally, edit the Device name (default: Device A, B, etc.). You will see this name back in the Trial Control Settings and in the Analysis profile. 4. Repeat the steps 2-3 to map the remaining devices.

inscopix devices

1. Connect the microscope to the subject and pass the cord through the tracking chamber/PhenoTyper and connect it to the Inscopix device (see page 321).
2. On the Inscopix computer, launch the data acquisition software and adjust the imaging settings, like the frame rate and the gain.
3. In the data acquisition software, select to trigger from external hardware. If you do 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.

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

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, the 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

See the scheme on page 321. Experiments with Calcium Imaging

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 (see page 324).

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 at the right of the 10-minutes time box. Experiments with Calcium Imaging

tip In the Hardware Action box you can test whether Ethovision XT

triggers the nVista. To do so, click the Test button.

note 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. Experiments with Calcium Imaging

Hardware connections

See the scheme on page 322. 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 (see page 324).

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 at the right of the Condition box just created. Experiments with Calcium Imaging 8. When that applies, 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. Experiments with Calcium Imaging

Hardware connections

See the scheme on page 322. 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/lines to the arena (see page 324).

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 action 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. Experiments with Calcium Imaging

Detection settings

general

Ensure your subjects are detected well by Ethovision XT. Choose Setup > Detection Settings.

See also detection settings on page 17. For more details, see the

EthoVision XT Help.

Sample rate

In the Video section, specify the sample rate. Some commonly-used

values are: For tracking rats: 5 samples/second. For tracking mice: 12.5 samples/second. When tracking the nose- and tail-base points of rodents: 25-30 samples/second.

detect immobility

There are two ways to detect immobility of the subject during the trial.

Activity measures the number of pixels that change their intensity,

so whenever the subject sits still, the Activity value will be low. However, when the subject is connected to a microscope cable, the cable can move continuously and keep Activity high. Experiments with Calcium Imaging As a result, Activity measurements may not detect immobility with sufficient accuracy. Instead, use Mobility.

Mobility is based on the change of the detected shape (i.e., the

yellow blob). You can define Mobility in the Analysis profile, so you do not need to specify anything in the Detection Settings. However, make sure that the yellow blob covers the entire body of the subject and is not affected by the cable (see below).

remove the effect of the microscope cable

When the microscope cable is detected as the subject, it changes both the Mobility measure and the position of the center point. The latter affects distance moved and other readouts.

Step 1 - Define the contrast

The first thing to do is to make sure that the entire body of the subject is well detected throughout the arena. Use for example Dynamic subtraction and choose the smallest range of contrast that enables detection of the subject's body. In this example the cable is still detected. This will be removed in th next step. Make sure that the contour of the subject is not too much indented and noise (i.e. the orange areas) is limited. Experiments with Calcium Imaging

Step 2 - Erode and then dilate the contour

In the Detection Settings, locate the Subject Contour options. Select 1

or a higher value for the first Erosion and a similar value for Dilation

until the cable is no longer highlighted in yellow. The Erosion filter removes the pixels from the contour (and therefore the cable too); the Dilation filter restores the original size of the yellow blob but excludes the cable. Experiments with Calcium Imaging

Aligning data

sync signal

To align the data streams from EthoVision XT and Inscopix nVista/ nVoke, there are two solutions: Have EthoVision XT send a time code signal (TCAP). Have EthoVision XT send a sequence of TTL pulses. Either way, the Inscopix system records the signal in one of its channels. The difference between the two is that with TCAP you need to export the TCAP signal that is stored in Inscopix back to EthoVision XT, together with the Inscopix data, so that EthoVision XT reads the time code and aligns the Inscopix data with the tracks.

Note

The nVista/nVoke system can also send a sync pulse to EthoVision XT through the USB-IO box every time a video frame is recorded. However, the pulse generated from the SYNC port of nVista/nVoke has a duration of 10 ms. That is too little for EthoVision XT to detect and record the signal. Therefore, this solution is not optimal to sync the two data streams.

using the tcap signal

Prerequisites

For this solution you need: The External Data add-on module for EthoVision XT. Contact Noldus if you need to purchase it. An interface cable between EthoVision XT and the BNC connector of the nVista/nVoke DAQ box. Contact Noldus to have one made for you. Experiments with Calcium Imaging A way to convert the Inscopix data to a format compatible with EthoVision XT: fixed sample rate, without gaps.

Procedure

1. Set the TCAP signal in the Experiment Settings of EthoVision XT, and specify the sample rate of the Inscopix channel that will receive that signal.
2. Start acquisition in Inscopix and then start the trial in EthoVision XT. nVoke/nVista records the TCAP signal, which should be visualized on the system's screen.
3. Export the Inscopix data to text files. Note that here the sample rate must be fixed and all samples must be written in the file, otherwise import won't work.
4. After import EthoVision shows the TCAP signal and other Inscopix data together with the track data. For more information, see External Data in the EthoVision XT Help.

using a ttl signal

Connections

Connect a TTL port of the USB/IO box to one of the GPIO ports of the Inscopix DAQ box. For this you need a cable with a RJ45 connector at one end and a BNC connector at the other end.

Procedure

1. In the Experiment Settings in EthoVision XT, define a TTL port of the

USB-IO box. Select Custom Hardware from the list. In the Arena

Settings, map that TTL port to one of the arenas. 2. In the Trial Control Settings, add a subrule that activates the TTL port

every few seconds. For example: TTL Output 1 High for 1 second, then

TTL Output 1 Low for 4 seconds. Let the subrule repeat a number of

times or when a Trial Control variable reaches a specific value. In general, make sure that the ON-OFF sequence runs through the duration of the trial. Experiments with Calcium Imaging Example:

tip To stop the subrule at the end of the trial, add an Action box

immediately before the Stop Trial box, where a Trial Control Variable, for example END, gets the value 1. In the Subrule reference, specify to end the subrule when END =1. 3. Run a test trial and make sure that Inscopix records the signal coming to the GPIO port. 4. Visualize the data in both systems. In EthoVision XT, you can

visualize the TTL signal by selecting, for example, Trial Control state

in the Analysis profile and defining the state from the action Output

1 High to the action Output 1 Low (here the numbering depends on

which TTL line you use). 5. If the data are well aligned, the time between the first and the last pulse should be the same in EthoVision XT and Inscopix, or differ by a few milliseconds.

For more information

The nVista/nVoke User Manual. The EthoVision XT 17 - Trial and Hardware Control - Reference Manual. Experiments with Calcium Imaging

mark ethovision events on the calcium imaging timeline

You can also use the GPIO ports on the nVoke/nVista box to send specific events to the Calcium Imaging software. In the following example, a setup that includes a fear conditioning system, all GPIO ports are set to digital and work as inputs.

GPIO-1 receives a signal from a TTL port of the USB-IO box that is set

to high when tracking starts.

GPIO-2 receives a signal from a TTL port of the USB-IO box that is

set to high when a 5-kHz tone is given.

GPIO-3 receives a signal from a TTL port of the USB-IO box that is

set to high when a 15-kHz tone is given.

GPIO-4 receives a signal from a TTL port of the USB-IO box that is

set to high or low when the optogenetic stimulation is given/ stopped, respectively, or when a shock is given/stopped, respectively. Experiments with Calcium Imaging

Data analysis in EthoVision XT

Once you acquire your trials, you want to pair the behavioral tracking with the calcium imaging/optogenetic stimulation events. To view and analyze those events, you must first define them in the Analysis profile. Here we report a few examples. Choose Analysis > Analysis Profile > New.

define a simple event

With the variable Hardware command you can visualize the time that

the USB-IO box sends a trigger command to nVista/nVoke. 1. In the Dependent Variables pane, under Hardware, choose Hardware command. Experiments with Calcium Imaging 2. Choose the device and the output state, for example Output 1 High. Note that Output represents one of the two communication lines present in each TTL port of the USB-IO box. Choose the line that you used in the Action box in the Trial Control Settings to trigger the device (see the examples on page 327). 3. Visualize the event (Analysis > Results > Integrated Visualization).

analyze intervals (global)

You can analyze the behavior of the subject within an interval based on events. For example, calculate the average velocity of the subject from the time that stimulation starts (i.e., when the TTL Output 1 is High) to the time that stimulation end (i.e., nVoke is triggered with Output 1 Low. To analyze the behavior within intervals, you must use the Nesting function in the Data profile. Experiments with Calcium Imaging 1. Choose Analysis > Data Profile > New. 2. In the Components pane, under Nesting, choose Hardware state. 3. Select the device and the status of the device. For example, Device A - Is Output 1 High - true. This selects the time that the TTL Output 1 signal remains high. 4. Insert the box in the flow line. 5. Visualize the interval (Analysis > Results > Integrated Visualization).

note If Output 1 stays high multiple times during one trial, EthoVision

XT considers the cumulative time that Output 1 stays High, and that is used as the analysis interval. When you want to analyze the time defined by a specific triggering event, see below for a different solution.

analyze specific intervals

You can use Free Intervals to specify a single interval based on one of the occurrences of a triggering event. Suppose that nVoke is triggered in ten intervals, and you want to analyze the second interval. Experiments with Calcium Imaging 1. Choose Analysis > Data Profile > New. 2. In the Components pane, under Nesting, choose Free interval. 3. For both Start criterion and Stop criterion, select Hardware. Define

the start and stop event. For example, Start criterion: Output 1 High;

Stop criterion: Output 1 Low. 4. Select Calculate Result for interval and choose the occurrence. In this example, 2. 5. Insert the box in the flow line. 6. Visualize the interval (Analysis > Results > Integrated Visualization). 7. In the Analysis profile, define the variable that you want to calculate within the interval (e.g. velocity).

note You can also use the commands of Trial Control to define

intervals. For example, define an interval that starts when the tone cue is given (i.e. when the Action Tone cue becomes active). You can also use Conditions (e.g. the interval starts when the Condition Wait 30 seconds becomes active). If an Action follows immediately a Condition, then the time that the condition becomes true is essentially the same as the time that the following action becomes active, because both boxes are processed in the same sample time. In that case it does not matter which box you use for defining the interval.

export the raw data

You can export the raw data (behavior and hardware events) with their time stamps, for example to sync EthoVision XT data with calcium imaging in a third-party application. Choose Analysis > Export > Raw data.

Note that Raw data also include the Trial Control events and states

(e.g. a condition being true) once you include them in the Analysis profile.

Select the Hardware log option if you want to export hardware

events. Experiments with Calcium Imaging

align ethovision xt and inscopix data

Computer clocks

Ensure that the two computers used for data acquisition (EthoVision XT and Inscopix IDAS) have their clocks synchronized, for example using Network Time Protocol. Point all PCs at a time server or point one and set it up to act as a time server to the other.

Unix time

Inscopix software often exports time stamps to Unix (Epoch) time. Unix time is also known as Epoch Time or Unix timestamp. It's a system that counts the number of seconds that have elapsed since the Unix Epoch, i.e. January 1st, 1970. To put in simple words, Unix time is the total number of seconds that have elapsed since 00:00:00 UTC Thursday, 1 January 1970.

Convert EthoVision XT time to Unix time

You can use an Excel formula to convert the trial time in EthoVision XT to Unix time. In general, the formula goes like this: Unix time = (EthoVision Time - DATE(1970,1,1))*86400 Because the trial (or recording) time in EthoVision XT is relative time (e.g. 0.200), you need to convert it to absolute time using the Reference time cell in the raw data (A in the next image). Then use a second formula (B) that adds the trial (or recording) time to the reference time A.

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