TITLE: ANY-maze Protocol (Elevated Plus Maze) v6.2
AUTHORS: Cristina Corral, Lieselot Carrette, Olivier    George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for running rats in the Elevated Plus Maze with ANY-maze software analysis of the experimental data.</div><div class = "text-block"><span style = "font-weight:bold;">TECH SUPPORT OPTIONS</span></div><div class = "text-block"><span>Under the tab titled </span><span style = "font-style:italic;">Support</span><span> in ANYmaze project files can be sent to ask software support questions</span></div><div class = "text-block">Email contact from training:</div><div class = "text-block">Emmanuel G. Vlamakis (Software Engineer)</div><div class = "text-block">Email: Emmanuel@StoeltingCo.com</div><div class = "text-block">Public link for tech support</div><div class = "text-block"><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.anymaze.co.</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">uk/suppo</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">r</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">t</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">.</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">h</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;:;">t</span></a><a href="http://www.anymaze.co.uk/support.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;">m</span></a><span>  </span></div></div>

[STEPS]
?. Open a "New Experiment" from the file tab and name the protocol and modify settings as described below(Or open EPM CC, which has all the settings as recommended below)From the dropdown menu below the protocol title select Video tracking mode to record video tracking From the dropdown menu below the Video tracking mode select Elevated Plus Maze as the optional image(If manuel scoring with key inputs is needed instead of ANY-maze tracking, under the take note dropdown in the same dropdown selecting the TakeNote video observation mode option is analogous to Video tracking mode without ANY-maze tracking)
?. To add a Video Source click the plus button titled Add item (located in the light gray ribbon bar above the protocol page) and then click New video sourceOr right click the tab on the left titled Video SourcesFor each video added there should only be for one rat in the video and it should be primarily only footage of the experiment (so around 5-6 minutes long total)Title the video source (corresponding rat information is recommended, i.e. Video Source r3 for a rat with ID 3 and change the title when the video source is changed)From the dropdown menu select the last option of video file to select a file from the computer A live camera feed can be used as well if necessary, usually it not needed for EPMTo select the live feed option, from the dropdown menu select the option WEB CAMERA (Port X) with X being the corresponding port for the live camera feed
?. To add an Apparatus (a general region of interest) click the plus button Add item and then click New apparatus Title the apparatus Arms (corresponding rat information is recommended, i.e. Arms r3 for rat 3)Click a point in the middle of the video and then draw the apparatus map using the multiline tool (double click the mouse to end the multiline shape) to draw the borders for the analysis of the arms Verify that there are no stray lines or shapes in the frame so that incorrect regions are not trackedIt is recommended that the checkmark at the bottom of the page is checked to save results without requesting confirmationSet up the ruler line length using a known length (measured before testing)
?. To add a Zone (location for analysis,open and closed arms distinction) click the plus button Add item and then click New zoneTitle the zone Open armsFrom the dropdown menu select This is not a hidden zoneFrom the dropdown menu below the previous menu select Position of the zone remains the same in all testsRight click the zones of interest to highlight zones when animal is in themRepeat above steps for the Closed Arms  zone
?. To set Zone entry settings, click on any generated zone (i.e. Open Arms) and select Zone entry settingsZone entry follows the 4 paw rule so that 85% of the rat needs to be in a zone for it to be counted under the automatic Use the center of the animal setting, but these settings can be adjusted from the tab Zone entry settings It can be set to track from the position of the animal's head or the animal's entire area with a manual setting of the minimum and maximum percentage of the animal that must be in a zoneBased on testing using in lab videos the Center of Area tracking setting is the most accurate for tracking in the open arm and closed arm regions, but not for tracking events such as entering and exiting the arms
?. OPTIONAL: To add Points (i.e. the ends of the plus maze) click the plus button Add item and then click New PointOr right click the tab on the left titled PointsIt is a measure of the time spent in proximity to a point, so it can be used to note when the rats reach an end of the maze (1 point created for every end, so a total of 4 points)
?. Select the animal color  by right clicking the tab on the left titled Animal Color  to select if the animal color will be lighter or darker than the apparatus background
?. Select to track the animal's head and tail by clicking the tab on the left titled Tracking the animal's head & tail and select Yes, I want the animal's head and tail to be trackedBy selecting this option the file is larger, but the test will not have to be rerun if you would like to change any track parameters
?. To add Stages (set the time of the experiment) click the plus button Add item and then click New StageSet the test duration for 5 min Select to Perform each trial for all the animals before starting the next trial
?. Set the automatic starting of tests by clicking the tab on the left titled Automatic starting of tests and select Yes, automatically start tests
?. Set what to record in the test by clicking the tab on the left titled What to record while testing and from the dropdown menu select Record a maximum of 10 positions/second and in the 2nd drop down menu about video quality select Medium quality
?. Set what to display while testing by clicking the tab on the left titled What to display while testing and select List any active zone
?. Set the treatment groups and number of animals by clicking the tab on the right titled Treatment groupsSelect the Use Treatment groups checkmarkSelect the button The user will manually assign the animals to their groupsRECOMMENDED: To use established Animal ID codes to identify the animal, click the tab on the right titled Animal ID and click the checkmark Use my IDs to refer to Animals Next click the tab on the top gray bar titled Experiments (to the right of the Protocol tab)Click the orange pill icon titled View treatments on the top Add each treatment under the Name column and under the No. animals column add the number of animals being tested with the corresponding treatmentClick the rat icon titled View animals (next to View Treatments) on the top to assign the status of the rats and the treatment group from the drop down menus To add or delete animals use the corresponding icons on the top bar next to the View animals icon, however a deleted animal will still be counted in the total Animal number, so it is recommended to edit an existing animal instead of deleting a ratFor example, Rat Animal number "3" was deleted (Animal ID number 722), but adding new animals (n=2 added) adds them to the end of the available animals, so unassigned rats animal numbers "11" and "12" were added with rat "3" still being deleted, so the total amount of rats is 11, but the animal number max is 12
?. To run multiple tests at once set up a new video source and new corresponding apparatus (as the plus may be shifted) and in the zone section select the zones to be tracked for the new apparatus (but do not add new open and closed arm zones)A maximum of 4 tests at once is recommended as more tests cannot be easily viewed on one laptop screen at onceChange the title of the video source and apparatus of each rat when the video source is changed to avoid confusion
?. OPTIONAL: To make changes to software terminology click the tab on the top titled Options and on the left side click Terminology
?. Start the experiment by clicking the tab on the top titled Tests (If running multiple video sources at once see notes at the end of this card first)For any test ensure that the apparatus is in the correct location (select a time in the middle to see if it was moved in the beginning before the test was run) and that the zones are properly designated, if they need to be adjusted the test must be stopped and run after the adjustments To each video source click the green play button (cyan arrow in image) once to change the testing status from Ready  (green highlight over text) to Waiting to Start (orange highlight over text)Click the blue play button (Red arrow in image) to begin the video sourceThe test should not automatically begin if the experimenter is still in frame (a person icon in the upper left hand side should be present until the experimenter has left), if it does then click the red square stop button to redo the testOnce the experimenter has left the frame the test should begin (indicated by the red highlight over text, the tracking of the animal, and testing status listed as Testing...)If the test does not automatically begin, scrub the video playback to where the test begins and click the green play button a second time to manually begin the testIf the test needs to be stopped, click the red square stop icon and if the test needs to be rerun click from the Testing status drop down menu Rerun testOnce the test is completed it will say Completed under the Testing status dropdown menu and it will be in the Ready status for the next rat in the list Multiple video sources at once:If there are multiple video sources, select the green plus icon next to the green play button icon to select the corresponding rat (via their animal ID) to the video source and set up each test individually to ensure that they are working for around 30 seconds before setting up and starting the next one (Select scale to fit to view the entire video source on the screen)
?. Results and Data are available by clicking the tab on the top titled Results Results can generate reports of data, such as data significance and preliminary graphsSelect the results needed and then on the top gray bar select the icon View the reportThis section has optional data tools such as  Text (shows data collated by groups to show amount of subjects, SD, mean, etc), Graph (shows line, column, or scatter graphs), or statistical (shows statistical analysis of results)The tab on the top titled Data can generate spreadsheets to copy and transfer data to other data processing software,To generate a spreadsheet from the Data tab click Select data (if greyed out then already on the page) on the top gray bar and select the data then select the View spreadsheet icon next to the Select data icon to verify that the data that was selected in the results tab is available to save as a spreadsheet If data is missing or not needed go back to the results tab on the top to edit the results to include by selecting the checkmark boxes The tab on the top titled Data can generate spreadsheets to copy and transfer data to other data processing software,To generate a spreadsheet from the Data tab click Select data (if greyed out then already on the page) on the top gray bar and select the data then select the View spreadsheet icon next to the Select data icon to verify that the data that was selected in the results tab is available to save as a spreadsheet To save the spreadsheet select the Save icon on the top gray banner and the file can be saved as CSV file to be opened in excel or PRISM for further statistical analysis
?. For miscellaneous  potential issues see the Help tab in the rightmost top corner for an ANY-maze reference guide and the Support tab to contact technical support, see what drivers are installed, and to check for software updates (also see this protocol description) The Options tab is used for sound, appearance terminology, User management, administrative options, and printing options