Knowledge Base

12 - Apply different protocols to different arenas

12 Apply different protocols to different arenas

introduction

This document is intended for EthoVision users who record data in with multiple arenas simultaneously, and want to apply different Trial Control protocols to different arenas. example The camera points at two separate arenas, each with one animal. In arena 1, provide a foot shock 30 seconds from the start of the trial. In arena 2, provide a foot shock at a randomly chosen time between 0 and 90 seconds from the start.

Multiple arenas in your EthoVision experiment

If you work with multiple arenas within an experiment, each arena is treated as an independent replicate. In most cases, you want to apply the same Trial Control protocol to all the arenas. Imagine that you create a Trial Control protocol that stop tracking when the subject has moved a certain distance. You define one Trial Control Settings profile and apply that profile for your trial. Because subjects differ in their behavior, tracking will stop at different times for each of those arenas. That is because EthoVision XT uses multiple copies of the same Trial Control Settings, one for each arena.

Figure 51 Four arenas in the same

camera view. Each arena is an independent replicate. The protocol defined in the Trial Control Settings is applied to each arena independently. 12 - Apply different protocols to different arenas

Same protocol, but independent processes

The Trial Control Settings are applied to each arena independently. Suppose that the protocol is "After starting the trial, switch on a light when the animal enters the zone named Food zone". A light device has been installed on top of both arenas, and a zone named "Food zone" has been defined in the Arena Settings for both arenas. If the animal in Arena 1 enters its Food zone, the corresponding light will be switched on. However, the light on top of Arena 2 won't be activated, unless the animal in Arena 2 enters its own Food zone too (see Figure 53). Although the protocol is the same, it is applied in two copies, one for each arena. This can be represented on a time line: To implement this, select the different physical devices (Light 1 for Arena 1, Light 2 for Arena 2 etc.) on the same line in the Arena - Hardware Mapping window (Arena Settings). This way Figure 52 A simple trial control protocol. When the center point of the subject is detected in the "Food zone", EthoVision sends a command to switch on a light in that arena. Figure 53 Time line of a trial with the same trial control protocol active in two arenas. The circle represents the Food zone. 12 - Apply different protocols to different arenas different arenas refer to the same generic Device name (e.g. "Light"). This "generic" name is then used in the instructions of the Trial Control protocol. Another typical example is the PhenoTyper setup, where the same protocol is applied to a group of 4 or more cages. Conditions and actions are processed independently for each cage. But what if Arena 2 needs a completely different protocol than Arena 1? See the next section.

Different protocols in different arenas

In some cases two or more arenas may need different protocols. For example, your camera view includes three arenas. In Arena 1, a light (Light 1) is switched on at a specific time, for example at one minute after start. In Arena 2, the light (Light 2) is switched on at a moment randomly chosen between 30 s and 2 minutes (because this event is randomly based, it will differ among the animals tested in Arena 2). Arena 3 is then used as a control, with no light stimuli presented. For all arenas, the trial lasts 3 minutes. If we based the Trial Control procedure on an Arena - Hardware Mapping like the one in Figure 54, the two light devices Light 1 and Light 2 would be represented by the same Device name. When using this name in a Trial Control instruction, for example "switch a light using the Device name = Light), that instruction would be executed for both arenas. As a result, the light would be switched on in both arenas twice, one at 1 minute, and a second at a random time between 30 s and 2 minutes. Also if the action is depending on the animal's behavior (for example, "Switch on the light when the animal is in the Food zone"), this command would be executed in both arenas, producing obviously false results. The solution to this problem is to map devices in a different way, by specifying one physical device in each row in the Arena - Hardware Mapping window. Figure 54 In order for the same protocol to be applied to different arenas, the different physical devices specified under Arena 1 and Arena 2 (see the arrows) must refer to the same Device name (e.g. Light). 12 - Apply different protocols to different arenas

simple case: two arenas

To illustrate the concept, we assume that we work with two arenas, and one animal in each arena. The animals are subject to two different treatment protocols.

Experiment Settings

1. Connect all hardware devices to the EthoVision XT computer, via the appropriate port in the Noldus USB-IO box.
2. In the Experiment Settings, under Trial Control Hardware click Settings.
3. Choose Noldus USB-IO box.
4. In the Device Configuration window, assign a hardware device to a port of the USB IO box (or Mini USB IO box), as you normally do. Here Device ID is the name of each physical device. For example, Light 1 will be installed in Arena 1, Light 2 in Arena 2, etc.

Figure 55 Schematic representation of

the treatments in two arenas. 12 - Apply different protocols to different arenas

Arena Settings

1. In the Arena Settings, define and calibrate the arenas as usual. Next, click the Arena - Hardware Mapping button in the Arena Settings window.
2. In the Arena - Hardware Mapping window, click Add device two or more times to create multiple rows, one row for each physical device that has to work independently.
3. For each arena, assign a device in a different row. Make sure that the other cells of the row show .

Figure 56 Adding devices to the Arena - Hardware Mapping window,

Figure 57 First row: The physical device Light 1 is assigned to Arena 1 with the generic name "Light Arena 1". Second row: The physical device Light 2 is assigned to Arena 2 with the generic name "Light Arena 2". 12 - Apply different protocols to different arenas

Define the protocols

Make a sketch of the events in each arena. Draw a time line for each arena. On the time line, write down the events that should occur, and their duration. This will help creating the Trial Control protocols.

Trial Control Settings

There may be parts of the protocol shared between arenas. In the example of Figure 59, the first 30 seconds after the start of the trial are the same for the two arenas. Therefore, one condition box after the Start track box will suffice. Figure 58 Make a sketch of your protocols on different time lines.

Figure 59 A condition box that

waits 30 seconds for both arenas. 12 - Apply different protocols to different arenas The sequence of instructions for the two treatments must originate from the same point (so they will start at the same time) and proceed in parallel. Note that the two branches do not have to end back into the main start-stop sequence. However, you must make sure that the trial does not stop before the two branches have been processed. In this example, we have set the trial to stop after 2 minutes. Therefore, a Time box has been added to the Start-stop sequence that waits 90 s to complete the trial (30 s of the first box + 90s = 120 s) Sequence for Arena 1 - According to the protocol, for Arena 1 the light must be switched on immediately after the 30 s period, then 2 s later it must be switched off. This can be programmed with two hardware actions (ON and OFF) and a Time condition in between. The actions must specify the device Light Arena 1. Figure 60 After a 30-s period, two procedures are activated, one for each arena. Figure 61 Instruction sequence for switching on a light in Arena 1 for two seconds. 12 - Apply different protocols to different arenas Sequence for Arena 2 - According to the protocol, for Arena 2 the light must be switched on at a time randomly chosen between 0 and 90 s. This can be programmed with a time condition that determines the random time, followed by a sequence similar to that for Arena 1. The actions must specify the device Light Arena 2. The Random time condition defines a time between 0 and 88 seconds, to make sure that the 2-seconds light flash is always given in its entire duration within 90 seconds.

Repeat a sequence within the trial

In most cases, a sequence of actions is repeated a number of times, or until a condition is met. If you want the two sequences described above to be repeated, you must use the Sub- rule function in Trial Control. Figure 62 Instruction sequence for switching on a light in Arena 2 for two seconds. The first box defines a random time chosen between 0 and 90 s. Figure 63 Setting two protocols to be repeated a number of times. Note that the two branching sequences are connected at their end to the stop box of the sub-rule through an operator. Only when the two sequences are processed, control is passed for a new start of the sub-rule. How repeats start is specified in the Sub-rule Reference settings (click Settings in the Reference box). 12 - Apply different protocols to different arenas The Sub-rule settings ensure that the sub-rule is repeated 10 times, and the time between the start of 2 successive repeats is 2 minutes. For more information on how to use Sub-rules, see Sub-rules on page 55.

advanced case: six arenas

In this use case, we plan to record data in six arenas, with one animal per arena. This use case is considered advanced for several reasons: Each treatment (odor stimulus and a subsequent foot shock) is duplicated in two arenas. This example shows that even when two of a number of arenas share the same protocol, each arena must have its own trial control procedure with unique Device names defined. Together with two treatments that require different protocols, a third group (control) is added that requires no foot shock. Control animals are recorded simultaneously with the other animals. All animals share part of the protocol. They all receive an odor stimulus by means of an olfactometer. Therefore, part of the Trial Control procedure must work for all arenas.

Experiment Settings

1. Connect all hardware devices to the EthoVision XT computer, via the appropriate port in the Noldus USB-IO box.
2. In the Experiment Settings, select the Number of arenas.
3. Under Trial Control Hardware click Settings.
4. Select Noldus USB-IO box.

Figure 64 Schematic representation of the

treatments in six arenas. Arenas 1-2, 2-3 and 4- 5 share the same treatment. 12 - Apply different protocols to different arenas 5. In the Device Configuration window, assign a hardware device to a port of the USB IO box (or Mini USB IO box). Here Device ID represents a name for each physical device. For example, Shocker 1 will be installed in Arena 1, Shocker 2 in Arena 2, etc. Since the olfactometers work the same way in all arenas, they can be connected to the same port of the USB IO box. Therefore, you need 4 ports for the shock generators (Arenas 1 to 4) and one port for the olfactometer.

Arena Settings

1. In the Arena Settings, define and calibrate the arenas as usual. Next, click the Arena - Hardware Mapping button in the Arena Settings window.
2. In the Arena - Hardware Mapping window, click Add device two or more times to create multiple rows, one row for each physical device that has to work independently. In this case, 5 rows.
3. For each arena, assign a device in a different row. Make sure that the other cells of the row show . Note that: Although Arena 1 and 2 and Arena 3 and 4 share the same treatment, they must be

linked to different Device names. This to avoid that any command given to for

Figure 65 The Arena - Hardware Mapping window. In the first four rows, a physical device is assigned to one arena; for example Shocker 2 in Arena 2. The name "Shocker 2" in the Device name column will be used in the Trial Control instructions. In the last row, the olfactometer is selected for all arenas, using the Device name "Olfactometer". This name will be used in Trial Control. 12 - Apply different protocols to different arenas example the shocker in Arena 3 does not go to Arena 4. In principle we could use the

same command (and thereby the same Device name) for Arena 1 and 2 because the

shock is given at the same time, but to keep this case more general we leave them separate. The arenas with no shock treatment (Arenas 5 and 6) have no shocker device selected. The olfactometer is used in all arenas, and at the same time. This means that the command to activate/deactivate the olfactometer can be represented by one Device name in Trial Control (see the last row in the table above). Therefore, the same Device name is assigned to all arenas.

Define the protocols

Make a sketch of the events in each arena. Draw a time line for each arena. On the time line, write down the events that should occur, and their duration. This will help creating the Trial Control protocols. Figure 66 Make a sketch of your protocols on different time lines. 12 - Apply different protocols to different arenas

Trial Control Settings

Sequence for Arena 1 and 2 -

Note that only the sequences for Arena 1 and 2 are connected to the Stop trial box. There is no need to connect the sequences for Arena 3 and 4 and the sequence for the olfactometers to the Stop Trial box, because these instructions proceed in parallel with those for Arena 1 and 2. The track duration is the same for all arenas. Figure 67 Overview of the Trial Control Settings. Indicated are the parts of the procedure applied to specific arenas. The total length of the trial is 4 minutes and 30 seconds. Figure 68 The trial control sequence for controlling a foot shocker at 28 s from the start, in both Arena 1 and Arena 2. The shock ends at 28+2 = 30 s from the start. 12 - Apply different protocols to different arenas

Sequence for Arena 3 and 4 -

Figure 69 The trial control sequence for controlling a foot shocker at a time randomly chosen within a 90-s period. In order to center the 90-s period for the random shock in the 4-min period after the offset of the odor (see Figure 66), a 75-s condition has been added (75+90+75 = 240 s = 4 minutes). Note that the 30-s Condition box and the two 75-s conditions could be replaced by one 105-s Condition box, connected to both Random time conditions. In this picture the 105-s time has been kept split in two parts (30 s +75 s) to visually match the sequences for the other arenas. 12 - Apply different protocols to different arenas

Repeat the sequence within the trial

If you want the sequences described above to be repeated a number of times within a trial, you must use the Sub-rule function in Trial Control. Here, the sub-rule is repeated 10 times. Note that the end box of the sub-rule is connected to the sequence for Arena 1 and 2. This sets the duration of the repeats (30 min. with odor stimulus + 4 minutes), and the trial as a whole. For more information on how to use Sub-rules, see Sub-rules on page 55. Figure 70 A sub-rule has been added to repeat the protocol a number of times in the same trial.

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Source: EthoVision XT 17 - Trial and Hardware Control Reference Manual