Variants
Open field and novel object tests
An empty box, and everything the animal does in it.
The open field is often run before the water maze. Dr Roger Morris's 1999 essay lists it alongside the elevated plus maze and holeboard as a way to check anxiety and willingness to explore before you test spatial memory.
Open field and novel object recognition in eight steps
Make the background contrast with the animal, calibrate the arena corners, set the threshold, run, then choose a grid that puts each object in its own cell.
- Make the whole background white for dark or hooded animals, or black for white animals, and make objects a similar colour to the floor where possible.
- Click ‘Open Field and NOR’; the ‘Maze and Field Control Panel’ opens. Leave ‘2D-tracking mode’ showing unless you have a 3D licence.
- Click ‘Calibrate’ and select the open field maze type. Enter the height and width of the whole tracking area, click the top-left then the bottom-right corner, and click ‘Save (and return)’.
- Click ‘Run’, choose the number of arenas on the ‘Maze Type’ tab (e.g. ‘Single Field’ or ‘2x2 Fields’), then on the ‘Tracking’ tab move ‘Set X-Y Threshold’ until a cross follows each animal.
- On the ‘Configuration’ tab enter ‘Max secs’ and choose ‘Single Trial’ or ‘Series of Trials’.
- On the ‘Run a Trial’ tab click ‘Prepare to Run’, place the animals and click ‘Run’; ‘End Trial’ stops early.
- Click ‘Analyze’, select OF and ZA, enter a grid size that places each object within one cell, and click ‘Analyze Single’ for one trial.
- For many trials use ‘Setup Custom’ and ‘Auto-Analyze’ to make a CST file.
More detail
Zones are numbered from the top left, down the left column first. For several arenas, enter subjects and descriptors in arena order separated by pipes (e.g. mouse1|mouse2|mouse3|mouse4). Set the freezing criteria if freezing or inactivity matter.
Plan the grid before running: place objects in zone centres and keep placement consistent. Body-centre tracking scores time near objects; sniffing and paw contacts are scored manually. Time bin analysis splits each trial into equal parts and shows early interest in a novel object that whole-trial averages would hide.
Open field zone time, entries and latency
Open field results show how the subject used the arena: % time, % path, entries and latency for each grid cell. Pick a grid size that puts each object or area of interest in its own cell.
More detail
Zone results start at the top-left and run down the left-most column, then the second column, and so on. In the export, a latency of -1 means the zone was not visited. For inner versus outer totals, sum the relevant zone columns in Excel.
Behaviour changes within a trial (for example early interest in a novel object), so whole-trial averages can hide effects; time bin analysis splits each trial into equal parts, and you choose the number of bins. Body-centre tracking scores time near objects; sniffing and paw contacts are scored manually.
Freezing and inactivity (open field)
Freezing results give the number of freezing episodes, time frozen and % time frozen. You decide what counts as freezing by setting how long the subject must stay still and within what distance.
More detail
For example, freezing can be set as not moving more than 0.1 cm in 2 s, or inactivity as not moving more than 5 cm in 10 s. If neither is relevant, set a large time window (e.g. 100 s) and a small movement (0.1 cm) and do not include freezing in your results.
The software reports actual total freezing time, % time and number of episodes; there is no setting for fixed 10-s time sampling. A dark spot on the head can help tracking for freezing.
Reading open field and object test results
In the open field, wall-hugging (thigmotaxis) and time in the outer zones are the usual anxiety-related measures; read them with path length and speed so that activity changes are not mistaken for anxiety.
- Typical control pattern
- When mice are introduced into an open field they explore mainly the peripheral zone, and this tendency to remain close to the walls decreases gradually during the first minutes of exploration (Simon et al., 1994).
- Signs of impairment
- Anxiogenic-like profile: reference anxiogenic drugs increased thigmotaxis in mice (Simon et al., 1994). Higher inactivity or freezing may also reflect fatigue, stress or anxiety, pharmacological effects or motor impairment, so check speed and path length too.
- Signs of strong memory
- Anxiolytic-like profile: anxiolytic drugs reduced thigmotaxis in mice (Simon et al., 1994), and in rats thigmotaxis was suppressed by anxiolytic agents (Treit and Fundytus, 1988).
Set-up problems that look like results
- Cross jumps in one area: adjust the threshold with the animal in that area.
- Cross follows the tail or droppings: move ‘Set X-Y Size’ to the right for large animals in small boxes.
- Wrong colour of cross: a green cross tracks dark animals on a light background and a red cross white animals on a dark background; recalibrate with the correct background colour setting.
- Objects or gaps between arenas are tracked: the whole background must be one contrasting colour, and objects should match the floor or be masked.
More detail
Whole-trial averages can hide effects such as early interest in a novel object, so use time bins. For object tests, body-centre tracking scores time near objects, while sniffing and paw contacts are scored manually. For inner versus outer totals, sum the relevant zone columns in Excel. The open field is a very popular animal model of anxiety-like behaviour (Prut and Belzung, 2003).
Prut and Belzung (2003) note that some compounds with a different spectrum of therapeutic efficacy were poorly effective as anxiolytics in the open field test, suggesting that this paradigm may not model features of anxiety disorders. Treit and Fundytus (1988) factored out general activity using analysis of covariance. No preference pattern for novel object recognition is given here; interpret object preference with the method and criteria of the protocol you followed.
Questions people ask
How do I calibrate an open field or zone analysis arena?
On the 'Maze and Field Control Panel' click 'Calibrate', choose 'Open Field or Zone Analysis', enter the arena size, then click the top-left and bottom-right corners. 1) Plug the camera in first. 2) Enter your user name and the height and width of the whole tracking area. 3) Tick 'black' only for light animals on a dark floor. 4) Click the two corners, then 'Save (and return)'.
More detail
Plug in the camera (two for 3D) before calibrating, otherwise restart the software after plugging in. Click 'Calibrate' and select 'Open Field or Zone Analysis'. Position the camera so the area nearly fills the height or width of the view. The tracking area is the arena floor; for divided or multiple arenas it is the whole floor including all arenas; for 48 or 96 wells it is the whole well arrangement (top-left corner of the top-left well to bottom-right of the bottom-right well). Enter user name, then height (top to bottom on screen) and width of the entire tracking area. Tick the box for black if subjects are light and the background dark; leave it unticked for dark or hooded animals on a light background. Click the top-left and bottom-right corners and click 'Save (and return)'.
Going deeper
Always click top-left first and bottom-right second: clicking the bottom corner first reverses north and south in the calibration file. In 3D mode also enter the vertical height to track and click the corners of both the side view (top image) and floor (bottom image). In 2D, ignore the height field or enter 0. For a grid of boxes, click the top-left of the top-left box and the bottom-right of the bottom-right box. 'Click to calibrate with X-Y recorded video' is needed only to track a recorded video, which must show the area in the same screen position.
Should I use 2D or 3D tracking in the open field?
Use 2D (one overhead camera) unless you have a 3D licence and want rearing data from a side camera too. 1) Leave the 2D-tracking mode button as it is for 2D. 2) For 3D, click it to switch to 3D and enter your 3D activation code the first time. 3) Set 3D before both calibrating and running.
More detail
After clicking 'Open Field and NOR', leave the toggle showing '2D-tracking mode' unless you have purchased a 3D tracking licence and will use overhead and side-view cameras at the same time. To use 3D, click it to change to '3D-tracking mode'; the first time, the activation window asks for your 3D code. Click it again to return to 2D. In 3D mode you set both z and xy thresholds, and you can run one tracking area (1x1).
Going deeper
3D is available in the 'Open Field and NOR' module, including zone analysis (not water maze). Raw 3D data (x, y, z with time stamps, at up to 60 Hz) are in the .BOND files in …\Data\Open Field Test\BOND\. If the Z column is missing, the trial was run in 2D: set 3D before calibrating and running. 3D tracking needs a light background. If only one camera is connected, click the toggle back to '2D-tracking mode'. Analysis: 'Z-Plot' shows vertical position against time.
How should I arrange objects and the background for open field, NOR or social tests?
Make the whole background one contrasting colour and keep objects close to the floor colour, so only the animal is tracked. 1) White background for dark or hooded animals, black for white animals, including gaps between arenas. 2) Colour or mask objects to match the floor. 3) Place objects so they fall inside analysis grid cells.
More detail
Arrange the camera and arena so the analysis zone grid lines match borders of interest; for object recognition, set it up so each object sits within one grid cell. Zone grids (overlaid during analysis, set by the square root of the number of zones) are different from grids of multiple arenas, and you can re-analyse with different zone sizes. Objects form part of the background, so where possible make them a similar colour to the floor; for social tests with an animal under an up-turned cup, colour the cup base like the floor; mask other contrasting objects. The background throughout the tracking area, including unvisited areas and spaces between arenas, must be white for dark or hooded animals or black for white animals.
Going deeper
Plan the grid before running: place objects in zone centres and keep placement consistent (mark the grid temporarily). Body-centre tracking scores time near objects; sniffing and paw contacts are scored manually, or use the Precision Tracker for head position in small arenas. In large arenas where the animal is small relative to the cell, standard tracking works well for NOR. Objects can also be masked in software via the Morris Water Maze module (contact HVS).
How do I set the tracking threshold and size in the open field?
With lighting and objects as for real trials, put an animal in and move the threshold slider until a green or red cross follows it everywhere. 1) Keep the Size slider left for small animals in big arenas. 2) Move it right for large animals in small boxes, to ignore the tail and droppings. 3) If the cross jumps in one area, adjust with the animal there. 4) Then run a practice trial.
More detail
On the 'Maze and Field Control Panel' click 'Run' (enter the tracking activation code if asked). On the 'Maze Type' tab choose 'Open Field or Zone Analysis' and the number of arenas ('Single Field', '2x2 Fields' or '3x3 Fields'), then open the 'Tracking' tab. Set the lighting exactly as for trials, place any objects, put an animal in each area and use 'Set X-Y Threshold' (and 'Set Z Threshold' in 3D) until a green or red cross tracks the animal wherever it goes. Use 'Set X-Y Size': left for small animals in large areas, right for relatively large animals in small boxes so the body, not the tail, is tracked and small marks such as faeces are ignored. If no slider position works, improve lighting and camera settings for even colour, good contrast and no glare or shadow; then adjust the sliders with the animal in any problem area.
Going deeper
A green cross tracks dark animals on a light background; a red cross tracks white animals on a dark background. If the wrong one appears, recalibrate with the correct background colour setting. If no cross appears, check neither the threshold nor the size slider is at 0; start with size just above zero, sweep the threshold slowly to find where the cross appears, then set size to body width and fine-tune. Blurring the video is not needed to ignore tails; use the size slider. For recorded videos, brightness and contrast may need adjusting if conditions were not set up before recording.
How do I run open field trials, including several arenas at once?
Choose the number of fields, set 'Max secs', then enter details and click 'Prepare to Run' and 'Run' as you release the animal. 1) For one arena choose 'Single Field' and enter one subject. 2) For several arenas enter each subject ID separated by pipes (|), and the same for descriptors. 3) Order is top-left, top-right, bottom-left, bottom-right for '2x2 Fields'. 4) Click 'Run' just after your hand leaves the view.
More detail
You can run one tracking area (1x1) in 3D, or one or several (for example 2x2 or 3x3) in 2D, as single trials or a series. Select the number of fields on the 'Maze Type' tab, open the 'Configuration' tab, enter 'Max secs' and choose 'Single Trial' or 'Series of Trials'. On the 'Run a Trial' tab enter Trial Name (included in each .hvs file name; with multiple arenas, the arena number is added), User Name, Subject and Descriptor (usually group) and an optional Remark; Start Point can be left blank. For multiple arenas enter each subject ID in turn separated by pipes (|), and the same for descriptors; typing them in a text file and pasting is easiest. Click 'Prepare to Run', place the subjects and click 'Run' when the trial should start; 'End Trial' stops early; 'Return' goes back to the menu.
Going deeper
No automatic start is used: click 'Run' as you release the animal, just after your hand leaves the camera view, because auto-start can trigger on hands. Grids such as 3x3 are for one animal per sub-arena; for a single arena use 'Single Field'. Using a 3x3 grid for one animal produces nine .hvs files and needs a subject and descriptor for every sub-arena. If a subject name is too long for the trial list, 'Look at HVS' shows the full file name. Several animals together in one arena need a different (multi-animal) system; contact HVS.
How do I run a series of open field trials from a TXP file?
List each trial's subject and descriptor in a .txp file, then choose it on the 'Configuration' tab with 'Series of Trials'. 1) In Excel leave columns A and B blank; put subject in C and descriptor in D. 2) Use pipes (|) for several arenas. 3) Save as name.txp in Master Experiment Files. 4) 'Configuration' tab > 'Series of Trials' > 'Choose TXP/MEF', then 'Prepare to Run' and 'Run'.
More detail
Create the list of subjects and descriptors in run order and save it with a .txp extension in C:\HVS Image 2027\Users\[username]\Config\Master Experiment Files\ (it opens there automatically, though you can browse elsewhere). In Excel leave the first two columns blank and use the third and fourth for subject and descriptor, one line per trial; for multiple arenas separate subjects and descriptors with pipes, for example mouse1|mouse2|mouse3|mouse4 and groupA|groupA|groupB|groupB. Save as [filename].txp, choosing CSV as the type if 'All files' is not available. In Notepad each line reads ,,mouse1,groupA. Then open the 'Configuration' tab, select 'Series of Trials', click 'Choose TXP/MEF' and pick the file; the first subject and group appear on the 'Run a Trial' tab.
Going deeper
If the software reports the .txp is not found, it was probably saved as .txt, .csv or .xls: save again with 'All files' type and the .txp extension. Missing commas shift fields (subject appears as descriptor). Lines beginning with a double quote are remarks. Tick 'Record' to save video frames (needs its own activation); untick it when tracking from recorded videos. If you cannot find 'Record' in your version, contact HVS via the Support button or contact page.
How do I analyse open field trials and set the grid size?
In 'Analyze' choose Open Field (OF and ZA), type a Grid Size and press Enter, then click a trial and 'Analyze Single' for its summary. 1) Grid Size 3 gives a 3x3 grid. 2) Pick a size that puts each object in its own cell. 3) Cells are numbered down the left column first, then the next column. 4) Re-analyse with other grid sizes at any time.
More detail
Click 'Analyze' and select Open Field (OF and ZA). Keep Video Height and Width at 480 and 640 for live trials; for recorded videos set them to the video's size. Click a trial name to see its path; to see simultaneous arenas together, tick 'Retain Previous Plots' and click each. Enter the Grid Size (rows and columns) and press Enter. Zone results start at the top-left and run down the left-most column, then the second column, and so on. Choose a grid size that places objects or special areas within one cell or block of cells; results then give % time, % path, entries and latency for those cells. Ignore the Detection Area buttons in open field. 'Analyze Single' shows on-screen results (total path, duration, average speed, % zones visited, zone entries, % time per zone, freezing/inactivity); Print or Export saves a full text file, including latency to each zone (-1 = not visited) and the zone entry sequence.
Going deeper
For 5x5, cell 5 is bottom-left and cell 6 the top of the second column. For inner versus outer totals, sum the relevant zone columns in Excel. The 3x3 multi-arena layout is separate from the analysis grid (for example, set the analysis grid to 3 to split a three-chamber box).
How do I set freezing or inactivity criteria in the open field?
Set Time Window (seconds still) and Movement (distance) under Freezing in the analysis screen. 1) For example, freezing = less than 0.1 cm in 2 s; inactivity = less than 5 cm in 10 s. 2) If you do not need it, enter a large window (e.g. 100 s) and 0.1 cm and leave freezing out of your results.
More detail
Freezing is complete immobility apart from breathing, and inactivity is a looser criterion of little movement over a longer window; HVS Image detects both with a time window and a movement distance. Use the Time Window and Movement boxes under Freezing to set how many seconds the subject must stay still, and within what distance; for example freezing as not moving more than 0.1 cm in 2 s, or inactivity as not moving more than 5 cm in 10 s. If neither is relevant, set a large Time Window (e.g. 100 s) and a small Movement (0.1 cm) and do not include freezing in your results. Results include number of freezing episodes, time frozen and % time frozen.
Going deeper
The software reports actual total freezing time, % time and number of episodes; there is no setting for fixed 10-s time sampling. For side-view fear-conditioning chambers, use 2D with one camera mounted high in front, mask light areas inside the chamber with black, and adjust lighting so bars do not reflect. A dark spot on the head can help tracking for freezing.
How do I export open field results for many trials to Excel?
Use 'Setup Custom' to choose your measures, then Auto-Analyze the trials into a CST file for Excel. 1) Select a trial and click 'Setup Custom'. 2) Name the CST file, tick the items you want (ignore 'Maze Variables'), click 'Save Spec'. 3) Select the CST file (top right) and your trials. 4) Click 'Auto-Analyze', then open the CST in Excel (or click 'View CST').
More detail
Select a trial and click 'Setup Custom'; enter the CST file name; tick the items you want in the top section and in 'Open Field and Zone Analysis Variables' (ignore 'Maze Variables'); click 'Save Spec'. In the top-right panel of the analysis screen select that CST file, select the trials (Ctrl-click or Shift with arrow keys) and click 'Auto-Analyze'; traces may flash past. Click 'View CST' (or double-click the CST name) to view it. CST files are comma-separated, in C:\HVS Image 2027\Users\[username]\Data\Open Field Test\CST. In Excel each trial is a row, with a column per item, and % time per zone has one column per cell in the standard order.
Going deeper
Excel import: Data > From Text/CSV, choose All Files, pick the .cst, delimiter Comma, Load (older Excel: Data > Get External Data > From Text, delimited, comma). You can make several CST files for the same trials with different grid sizes or items. Multi-arena grid cells appear top-left first to bottom-right last (in 3x3, the ninth). Path coordinates from exported files can be plotted in Excel or other plotting software.
Where are recorded video frames saved and how do I turn them into a video?
Frames are saved per trial in …\Data\[experiment type]\Videos\, and you can turn them into a video with the FFmpeg tool supplied. 1) Tick 'Record' before the trial (if your version shows it). 2) Find the trial's folder under Videos. 3) Use FFmpeg from C:\HVS Image 2027\Helper Apps\ffmpeg\ (it comes with its own tutorial).
More detail
If 'Record' is ticked, each frame is saved in C:\HVS Image 2027\Users\[username]\Data\[experiment type]\Videos\. FFmpeg, the leading open-source converter, is supplied with its own documentation in C:\HVS Image 2027\Helper Apps\ffmpeg\. Saving frames rather than video avoids compression running while the next trial is tracked, keeps every frame at original resolution, and lets you choose compression and frame rate yourself.
Going deeper
Video frame recording needs its own activation code, separate from tracking and analysis. The frame-rate slider runs from 0 (no recording) to 1000 ms between frames; 100 ms gives about 10 frames/s; very short intervals may give fewer frames depending on PC speed (maximum about one frame per 0.03 s). Frames take a lot of disc space: move old Videos folders off the PC; HVS Image warns when less than 10% of the disc is free.
How should I set up an open field test?
Use a plain, uniformly lit arena, clean between animals and let animals settle in the room first. Your local ethics committee, licence conditions and vet always take precedence.
More detail
Mouse protocol: 50 × 50 × 38 cm, 500–1000 lux, at least 30 min acclimation, 10 min test, 95% ethanol cleaning [Seibenhener & Wooten 2015]. Centre time and thigmotaxis reflect anxiety [Prut & Belzung 2003].
Going deeper
Light level strongly affects open field anxiety measures: keep it constant.
See HVS Image for the open field and related tests.
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