WaterMaze was written and designed by David Ferster of Actimetrics, Inc., and by Richard Morris and Patrick Spooner of WaterMaze Software.

Transcription

1 WaterMaze Abut WaterMaze... 2 WaterMaze Installatin ) Install the WaterMaze sftware, image acquisitin card and drivers ) Cnnect the camera ) Test the camera ) Cnfigure the cmputer's speakers and sund card ) Munt the camera ) Run WaterMaze ) Install and test the remte cntrl switches fr Atlantis On-Demand Platfrm ) Adjust the lighting ) Test the tracking ) The Atlantis Platfrm... 6 Quick Start: Designing and Running a Prject... 6 Designing a Prject... 6 Running a Prject... 7 Analysis... 7 Setting the Pl... 7 Luminance Prfile Windw... 8 Preferences Windw... 8 Switch Settings and Cnnectins... 9 Trial Cntrl... 9 Atlantis On-Demand Platfrm Cntrl The Prject Windw Menus and Cntrls MENUS The Prject Menu (Handling Prject Files) The Settings Menu (Prgram Settings) The Mde Menu (Run/Design) The Design Menu (Designing a Prject) The Analysis Menu The Help Menu CONTROLS Design Mde Animal Cntrls Trial Cntrls Run Mde Trial Cntrls Animal Cntrls The Basic Prject (Defaults) Setting the Platfrms Cpying platfrm settings frm anther Prject File Setting a Platfrm Markers Save and Exit Cancel Clne Trials Ntes Running a Trial (The WaterMaze Windw) The Trial Viewer Windw The Exprt Windw The Analysis Settings Windw... 23

2 Abut WaterMaze WaterMaze was written and designed by David Ferster f Actimetrics, Inc., and by Richard Mrris and Patrick Spner f WaterMaze Sftware. Abut the prgram WaterMaze is a cmputer prgram fr autmatically tracking and analyzing the path f an animal swimming in a large pl f water. It may als be used fr a variety f ther experimental prcedures that invlve the tracking f animal mvements thrugh space. Flexibility WaterMaze has been designed t prvide individual experimenters with the maximum f flexibility in hw experiments are run and analyzed. Fr example, it can autmatically track the path f either an albin r a hded animal (usually a rat r muse) withut the necessity f attaching either light emitting dides r reflective material t the animal, r altering prgram parameters. Training may be cnducted with the escape platfrm in a fixed psitin, r with it mving t different places frm trial t trial. Once a trial sequence is specified at the start f an experiment, the prgram s unique Prject Manager mdule keeps track f the sequence f animals and trials ver the duratin f the experiment. The prgram can be set such that each animal gets all its daily trials tgether befre the next animal is run, r with all animals being trained n ne trial befre they are mved n t the next. The prgram alerts the experimenter as t the next animal in the sequence t be run, the starting lcatin f the trial, and the platfrm lcatin. Data files are created autmatically and trials stred in the crrect file. The experimenter can easily verride the Experiment Manager where apprpriate and arrange trials in any sequence n demand. Ease f use Fr all its flexibility, WaterMaze is easy t use. Because we are WaterMaze users as well as designers, we have taken extra care t make WaterMaze a natural extensin f the experiment itself. Cntrls and menus are placed in lgical and intuitive arrangements. Anyne familiar with using Micrsft Windws will have n difficulty in learning t use WaterMaze quickly. Help rutines are prvided thrughut the prgram. Vide acquisitin During the trial, the vide capture card installed in the PC grabs images at up t 8 frames per secnd. The prgram can track any mving bject in the scene, identifying the swimming animal whether it swims ver bjects that are lighter r darker than it. A unique algrithm screens ut shadws and lighting changes that might ccur during the trial, making fr rck-slid tracking. An n-line interplatin rutine identifies and smthes ccasinal lss f signal that can ccur if an animal swims briefly underwater r under hanging cue bjects. The path taken, with interplated values, can be displayed in real time as the trial prceeds. At the end f each trial, the experimenter can type in ntes cncerning the trial just cmpleted. After the intertrial interval, whse duratin is specified by the experimenter, the path taken by the animal is autmatically saved fr later analysis (with ther useful infrmatin abut the trial). The prgram then displays the animal and platfrm lcatin f the next trial in the experimental sequence. Multiple data analysis ptins After ne, r a series f trials, r when an entire training sessin is cmpleted, varius types f Data Analysis may be chsen. In the nscreen Trial Viewer, any trial can be easily selected frm trial lists, 2

3 and a cmplete set f analysis parameters will be displayed, alng with a picture f the swim path. Alternatively, the Batch Analysis windw can be used fr analyzing large numbers f trials. Any cmbinatin f analysis prtcls can be applied t almst any cmbinatin f trials and animals. The results are saved in a spreadsheet-cmpatible file fr printing r fr further analysis. Analyses range frm the basic such as latency r path-length t the cmplex such as quadrant analysis, heading directin, and ther parameters taken frm the latest literature. Nt all f these analyses need be cmputed in every experiment; different users may select nly thse analyses mst useful fr their wn applicatin. Finally, the image f the swim path may be exprted t drawing r illustratin prgrams, such as Phtshp, Illustratr, PwerPint r Crel Draw, where the image is fully editable. Advanced Features WaterMaze incrprates a number f advanced features nt fund tgether n ther mre cstly tracking systems. These include the Prject Manager and state-f-the-art frame grabbing technlgy just described. In additin, WaterMaze includes facilities fr cntrl f the n-demand r Atlantis platfrm, which can be triggered t rise t near the surface f the pl if and nly if the animal swims in the apprpriate lcatin fr a pre-defined perid f time. Other features will be added as new techniques develp. (Tell us yur favrite analysis prtcl r suggest a feature that will make yur experiments easier.) Prgram upgrades will be available at n additinal charge. Hardware Requirements WaterMaze runs under the Windws 2000 r Windws XP perating system n all PC-cmpatible cmputers with a Pentium prcessr f at least 800 MHz clck speed. 256MB f RAM is recmmended. WaterMaze Installatin Installatin 1) Install the WaterMaze sftware, image acquisitin card and drivers Befre installing the vide bard, lad the NI-IMAQ sftware frm the CD prvided with the PCI-1407 (P/N ACT-650). Befre installing the digital I/O bard, lad the NI-DAQ sftware frm the CD prvided with the PCI-6503 (P/N ACT-675) which is required nly if using Atlantis On-Demand Platfrm (P/N ACT-203). Shut dwn the cmputer and plug the PCI-1407 (ACT-650) and PCI-6503 (ACT-675) bards int pen PCI-slts. The PCI-1407 shuld be clsest t the prcessr. Run the WaterMaze installer prgram, if yu have nt already. 2) Cnnect the camera Cnnect the camera utput cnnectr t the "Vide" input n the PCI-1407 (ACT-650) using the BNC cable prvided. 3

4 Cnnect the camera t the 24VAC pwer supply using the tw-stranded wire prvided. Ignre the wire clrs; plarity is unimprtant since this is an AC pwer supply. Plug the pwer supply int any 120V-60Hz utlet. 3) Test the camera Frm Windws's START menu, select Prgrams->Natinal Instruments IMAQ -> Explre IMAQ. A Windw entitled "Explring - Measurement and Autmatin" shuld appear. In the list t the left f the windw, pen Measurement & Autmatin\Devices and Interfaces\IMAQ PIC-1407:img0\Channel 0: rs 170 Frm the tl bar, select Grab. Yu shuld nw see a real-time image frm the camera. If n image appears, check the pwer and signal cnnectins frm the camera and vide bard. If yu still have truble, call Natinal Instruments at and ask fr help with NI-IMAQ. PCI-1411 f PCI-1409: Click the Prperties buttn in the tlbar and set the Bit Depth t 8-Bit r Luminance 8-bit. Once yu have verified that the camera is wrking and prperly cnnected t the image acquisitin bard, yu may quit the camera test prgram by clicking the clse bx at the upper right f the windw 4) Cnfigure the cmputer's speakers and sund card WaterMaze relies n the cmputer's sund card and speakers fr generating tnes that guide the user thrugh a trial. Cnnect the cmputer's speakers as in yur cmputer manual and test the sund system. Yu can test the system by playing a CD (frm the START menu, chse Accessries->Multimedia- >CD Player), r by pening the Sunds cntrl panel, selecting a sund and playing it using the rightward pinting arrw ("Preview:"). If the sunds d nt play, check the speaker cnnectins, whether they are turned n and whether the vlume is set high enugh. Next check whether the drivers fr the sund card that came with the cmputer are prperly installed and functinal. If they are nt, cnsult with the cmputer manufacturer. If yu are able t play a CD r a sund in the Sunds cntrl panel, FreezeFrame shuld be able t play cnditining stimuli as well. 5) Munt the camera Munt the camera securely abve the center f the pl. Be sure that the pwer and signal cables are secured s that they cannt be jstled during a trial. Any mtin f the cable will mve the camera ever s slightly, disrupting tracking. Adjust the zm and aim f the camera s that the water surface fills the center f the image. Fcus carefully! 6) Run WaterMaze When yu first run WaterMaze, it will display a serial number and ask fr a passwrd. Cntact Culburn Instruments (sales@culburn.cm) with yur serial number and a passwrd will be e- mailed t yu. Enter the passwrd. The passwrd is keyed t yur PCI-1407 (ACT-650) image acquisitin bard. Yu may transfer the prgram and bard t any cmputer and the same passwrd will apply. 7) Install and test the remte cntrl switches fr Atlantis On-Demand Platfrm 4

5 Install the PCI-6503 (ACT-675) bard fr use with a switch interface bx Duble-click the Measurement & Autmatin icn. The icn appears n the desktp when the NI-DAQ CD is prperly laded. Select "Devices and Interfaces". Select the PCI If the PCI-6503 is nt listed in the windw, then the bard r the NI-DAQ sftware is nt prperly installed. Duble-click the "Device Number" item in the right-hand windw. In the dialg bx that appears, set Device Number t "1". Click the Test Resurces buttn and make sure that the device passes the test. Cntact Natinal Instruments if it des nt ( ). Cnnect the PCI-6503 t the switch interface bx using the 50-pin ribbn cable prvided. Cnnect a switch t the TRIG input cnnectr n the PCI-1407 r, if installed, cnnect ne r mre switches t the switch input f the interface bx. Open the Switches Windw in WaterMaze (SETTING -> SWITCHES menu). Set which switch cntrl which functins during the WaterMaze trial. Press and hld the switch and verify that the apprpriate Switch indicatr lights. Fr each input, multiple switches can be cnnected in parallel such that any ne switch can be pressed t cntrl a trial. 8) Adjust the lighting Gd lighting is critical fr prper tracking! There are three main cnsideratins fr gd lighting, shadws, reflectins, and evenness. Shadws: Peple mving arund in the experiment rm shuld nt cast strng shadws n the tank. WaterMaze's tracker has a built in algrithm that cmpensates fr changing lighting cnditins during a trial. Nevertheless, a strng shadw cast n the tank during the curse f a trial can ptentially disrupt tracking. Therefre, lights shuld nt be placed in such a way that an experimenter can walk between a light and the tank. The same is true fr brightly lit walls. An experimenter walking in frnt f a brightly lit wall can dramatically change the amunt f light falling n the tank. Reflectins. Light placed abve the tank can be reflected frm the surface f the water directly int the camera lens, especially when there are ripples n the surface caused by the animal's swimming. Check the image t see that reflected images f the lights are nt visible in the image. In additin, ripple the water surface lightly with yur fingers. Even if the lights are nt reflected int the lens when the water surface is flat, ripples made by a swimming animal can smetimes cause reflectins. When lighted ripples are bserved, mve the lights farther away frm the pl. Evenness. It is imprtant that all prtins f the pl be lit evenly. Several light surces placed at pints arund the pl shuld be used. Check the evenness lighting using the Luminance Prfile windw, accessible frm the PREFERENCES -> BRIGHTNESS PROFILE menu. 5

6 The best lighting is indirect light reflected ff the ceiling. A few inexpensive halgen flr lamps aimed at a flat, white ceiling abve the pl give fairly even, reflectin-free light. In additin, by lighting the ceiling instead f the walls f the experiment rm, the chance that smene mving in the rm will cast a shadw n the pl is reduced. Light reflected ff f a wall is als effective, as lng as n ne walks between the wall and the tank during a trial. Light reflected ff a single wall, hwever, is likely t leave the edge f the pl nearest the wall prly lit. Make sure a secnd, and even a third light surce is placed ppsite the lighted wall t cmpensate. Nte that the light level need nt be ver bright. Mst cameras autmatically adjust their sensitivity t the ambient light level. 9) Test the tracking Open the Set Pl Windw (SETTING -> POOL menu) and adjust the pl setting t match the pl image. Set up a prject with trials f lng duratin, and run a trial using an artificial animal t test tracking. We use a cardbard cutut the size f the animal's head, munted n a lng piece f very thin, stainless steel tubing. Darken the tubing with a black marker pen s that the prgram tracks the cutut and nt the tubing. A flating bject the size f an animal's head can als be used. Check t see that the prgram can track an bject in every regin f the pl. If nt, adjust the lighting t be mre even, and t minimize shadws and reflectins (see Lighting sectin abve). 10) The Atlantis Platfrm T cntrl the Atlantis platfrms, yu must install a Natinal Instruments PCI-6503 Digital input/utput bard and switch interface bx as described abve. Yu may cntrl 8 different Atlantis platfrms independently frm the first 8 cntrl lines f the bard (see Switches Windw). One platfrm cntrl utput is brught t a phne jack n the frnt f the switch interface bx. The ther 7 utputs are available n the DB-15 cnnectr n the rear f the bx (cntact Actimetrics fr details). Which cntrl line will be used fr each platfrm is specified in the Set Platfrms Windw. Quick Start: Designing and Running a Prject Designing a Prject 1. Fllw the Installatin instructins carefully. Pay clse attentin t lighting f the pl. 2. Open the Pl Windw (SETTINGS -> POOL menu) and adjust the circle defining the pl t match the pl's image. 3. Open the Preferences Windw (SETTINGS -> PREFERENCES menu) and chse apprpriate values fr all the settings. 4. Select the FILE -> NEW menu item t start a new prject. 5. Select the FILE -> SAVE menu item t save and name the prject. 6. Set up the Prject's Platfrms using the PROJECT -> PLATFORMS menu. 7. Set up the basic prject using the PROJECT -> DEFAULTS menu. 8. Specify the animal names individually in the Animal Name cntrl, r specify the name f the first animal and select the PROJECT -> SEQUENCE NAMES menu item. 6

7 9. Edit the trial sequence fr each animal if necessary. Select each animal in turn using the Select buttns, and edit trial sequence (# f days, cnditins per day, and trials per cnditin, platfrm, and trial duratin). 10. Select the FILE -> SAVE menu t save the prject design. Running a Prject 1. Select RUN frm the MODE menu. 2. Use the D Next, D Selected, r Sequence buttns t run trials. Take a reference frame prir t placing the animal in the pl using the nscreen Reference Frame buttn r Remte cntrl buttn. Place the animal in the pl and start the trial using the nscreen Start buttn r the Remte Cntrl buttn. Stp the trial when the animal reaches the platfrm using the nscreen Stp buttn r the Remte cntrl buttn. Save the trial using the Save buttn r wait until the trial is saved autmatically at the end f the intertrial interval. Analysis Use the View r Exprt windws (accessed frm the ANALYSIS menu) t analyze trials. Setting the Pl Settings Windws The Set Pl Windw is used t tell WaterMaze where the pl is lcated within the camera image and hw large it is. Presently, the prgram assumes that the pl is circular. When the windw pens, yu will see a real-time image frm the camera with several superimpsed cursrs (2 vertical and 1 hrizntal line) and the pl circle. Use the cursrs t superimpse the circle n the utline f the pl image. Cursrs. The cursrs cnsist f 2 vertical and 1 hrizntal ne. If these lines are nt visible in the image windw, click the Center cntrl t bring them int view. The clr f the cursrs can be adjusted using the Diagnals clr bx. The psitin f the mst recently selected cursr can be adjusted ne pixel at a time with the Nudge cntrl (fur grey diamnds n a blue backgrund).use the upward and dwnward pinting diamnds fr the hrizntal cursr, and the leftward and rightward pint diamnds fr the vertical cursrs. Diagnals. As a reference pint t the center f the image, the image diagnals can be displayed using the Diagnals buttn. The clr f the diagnal lines is cntrlled by the Diagnals clr bx. Pl Diameter. Enter the diameter f the pl in centimeters int this cntrl. 7

8 Nte: This cntrl must be prperly set, r measurements f swim velcity and distance will be incrrect. Nte als: Changing the pl settings will alter the psitins and diameters f the platfrms used in nging prjects. If yu change the pl settings in the middle f a prject, be sure t check the platfrm settings. The analysis f trials already cmpleted will nt be affected. Pl clr. The clr f the pl circle can be changed with this cntrl. Swim Path. Clicking the Shw/Hide buttn displays a sample swim path. Adjust the clrs f the pints and line with the crrespnding cntrls. The swim path will be displayed in the WaterMaze Windw using these clrs. Save & Exit. Clses the Windw. Changes made are saved int the MWMCnfig file. Cancel. Clses the windw withut saving the changes made. Luminance Prfile Windw The Luminance Prfile windw is used t measure the evenness f lighting acrss the pl. The camera image is shwn, tgether with tw cursrs, ne a hrizntal line and the ther a vertical line. The graph abve the image shws the luminance f the camera image alng the hrizntal cursr; the graph t the left f the image shws the brightness alng the vertical cursr. The luminance scales f the tw graphs run frm 0 t 255 r frm the minimum t the maximum pssible values. Fr the best tracking perfrmance, it is best t adjust the lighting in the rm s that the luminance prfile acrss the pl is as even as pssible. See "Adjusting the Lighting" in Hardware installatin. Preferences Windw The Preferences Windw is used t set varius infrequently changed prgram parameters. Trial Parameters Default Trial Duratin: The Trial Duratin cntrl in the Prject windw (Design mde) is set t this value by default. This is maximum time a trial is allwed t run, usually the time at which the experimenter places the swimming animal is placed n the platfrm. Typical values are 120 sec (rats) and 90 sec (mice). Intertrial Interval: When the Sequence functin is selected frm the Prject windw (Run mde), trials are dne ne after anther autmatically. After a trial is cmpleted (that is, the prgram stps recrding the swim path), the prgram will wait fr the Intertrial Interval, then autmatically save the trial data and prceed t the next trial. The Intertrial Interval may be verridden by clicking the Save r Exit buttns in the WaterMaze windw. The cuntdwn t the autmatic saving f a trial can be interrupted by the Pause buttn that appears in the WaterMaze windw during the intertrial interval. Autstp Trial: When this cntrl is checked, the prgram stps recrding the animal's swim path after a fixed delay (Autstp Time) frm the time that the animal first reaches the platfrm. The animal must stay n the platfrm fr the entire duratin f the Autstp Time in rder fr the trial t be stpped autmatically. If the animal leaves the platfrm, the autstp timer is reset t 0. If the platfrm is an Atlantis Platfrm, then the prgram stps when the platfrm is raised. Tracking parameters: 8

9 Frame Rate: Cntrls the rate at which the WaterMaze windw cllects and analyzes images during a trial. The maximum rate at which data can be sampled is dependent n the speed f the cmputer's prcessr. Fr a 450 MHz cmputer, fr example, the prgram can't g much faster than 6 frames per secnd. If the frame rate is set higher than the cmputer can handle, the WaterMaze prgram will g as fast as it can. That it is nt ging as fast as specified will be indicated by the appearance f a Frame Rate indicatr appearing in the WaterMaze Windw shwing the real rate. Skipped frames are nt a serius prblem. Except fr the reduced sample rate, data cllectin cntinues withut any harm dne. Save Images: WaterMaze can save images frm a trial nt yur hard drive fr later review. Saved images are als useful fr hand-editing trials in which tracking errrs ccurred (thugh the gal is, f curse, t minimize such errrs). It is therefre gd practice t save images. Image files are stred in the prject flder. They take cnsiderable space n the hard drive and s shuld be regularly discarded r archived n CD, DVD r tape. Sunds Sunds: The WaterMaze prgram uses sunds delivered thrugh the cmputer's speakers t indicate the three phases f a trial. Sund 1 is played intermittently t indicate that a Reference Frame must be taken. Sund 2 sunds briefly when a trial is started. Sund 3 sunds briefly when a trial is stpped. Specify the details f each sund using the crrespnding Frequency and Vlume cntrls. Play a sample f the sund using the Test buttns. If n sund can be heard, check the vlume setting n the cmputer's external speakers. If sunds still are nt audible, test the cmputer's sund card using the Windws Sunds Cntrl Panel. The sund card's drivers may be imprperly installed. Frequencies can range between 100 and 5000 Hz. Vlumes can range between 0 and 255. Switch Settings and Cnnectins Trial Cntrl. During each WaterMaze trial, fur specific functins need t be perfrmed: taking a Reference frame, Starting the trial, Stpping the trial, and Saving the trial. On-screen cntrls are prvided in the WaterMaze Windw t perfrm these functins (and tw f these functins can be autmated in the Preferences Windw). In additin, these fur functins may be perfrmed using remte cntrl buttns. Five different inputs may be used fr the remte cntrl buttns: TRIG. The PCI-1407 vide acquisitin bard has a BNC cnnectr labeled TRIG. DIGITAL PB0-PB3. If yu have installed a PCI-6503 digital input/utput bard, fur f its pins are autmatically cnfigured as inputs fr remte cntrl switches. PB0-PB3 crrespnd t pins 33, 31, 29 and 27 n the PCI PB0-PB2 can be accessed frm the 1/8" (3.5 mm) phne plugs n the frnt f the switch interface bx prvided. Cnnect the switch in the nrmally pen cnfiguratin acrss the tw cnnectrs f the phne plug. Alternatively, PB0-PB7 can be accessed n the DB-15 cnnectr (labeled "Switches") n the back f the interface bx at pins 1-8. Cnnect the switch in the nrmally pen cnfiguratin between the selected pin, and any f pins 9-15 (grund). 9

10 Yu may cnfigure the prgram s that any ne f the fur WaterMaze cntrl functins is cntrlled by any f the 5 digital 2nputs. Use the 4 Signal ppup menus t set the crrespnding input. Nte that mre than ne functin can be assigned t each buttn. If n PCI-6503 is installed, fr example, all fur functins are assigned t the TRIG input. In that case, the ne buttn perfrms each f the fur functins in sequence. The sunds played during the WaterMaze trial (set in the Preferences Windw) indicate which functin the buttn will perfrm at each pint during the trial. Each f the 5 inputs is cnfigured t perate when grunded. When the input is left pen the inputs read as FALSE. When grunded the input reads as TRUE. When a prperly cnnected switch is clsed, the indicatr t the right f the Trial Cntrl cluster shuld turn red. Atlantis On-Demand Platfrm Cntrl Yu may use up t 8 different cntrl lines n the PCI-6503 (PA0-PA7) t cntrl Atlantis platfrms. In the Set Platfrms Windw, each Atlantis Platfrm can be set t be cntrlled by a different line. Here, yu may test each utput line f the PCI-6503 using the crrespnding buttn. Set the Release buttn t the UP psitin t drive the crrespnding utput high (+5V). The pin numbers n the ribbn cnnectr f the 6503 are als indicated fr each line in the Switch setting windw. PA0 can als be accessed frm a 1/8" (3.5 mm) phne plug (labeled "Atlantis") n the frnt f the switch interface bx prvided. The center terminal is signal. Alternatively, PA0-PA7 can be accessed n the DB-15 "Atlantis" cnnectr n the back f the interface bx at pins 1-8. Pins 9-15 are cnnected t grund. Prject Windws The Prject Windw Menus and Cntrls The Prject Manager keeps track f the animals and the sequence f trials t be perfrmed by each animal. The Prject Windw has tw mdes f display, which are chsen frm the MODE menu. The Design Mde is used t specify all aspects f the Prject, including the animals in the prject, the lcatins f the platfrms t be used, and the number and sequence f trials, including the number f trials t be perfrmed in each day, which platfrms will be used fr the different trials, the duratin f each trial, and s n. The Run Mde is used t run the trails in the prject in the desired sequence. MENUS The Prject Menu (Handling Prject Files) NEW: T create a new prject, select the NEW menu item. The current prject and all its parameters cleared frm memry. OPEN: T pen an existing prject, select the OPEN... menu item and select a previusly saved prject file. 10

11 SAVE: T save the current prject, select the SAVE menu item. If the prject has already been named, any changes will be saved int the existing file. If the current prject is a new ne and has nt yet been saved, yu will be prmpted fr the filename. SAVE AS: T save the existing prject with a new name, select the SAVE AS... item and yu will be prmpted fr a new prject file name. The Prject Windw title bar: The title bar displays the name f the currently pen file, such as Prject Manager: Mutants When changes are made t a Prject, an "*" is appended t the file name t indicate that unsaved changes have been made. The "*" is cleared when the changes are saved. The Settings Menu (Prgram Settings) The Settings menu items are used t specify settings that are nt assciated with a Prject, and are rarely changed. POOL: The POOL menu item pens the Set Pl windw in which yu tell the prgram where the pl is in the vide image. Be sure t update the POOL setting whenever the camera r pl is repsitined. PREFERENCES: The Preferences windw is used t specify varius default parameters, such as the frequency f the sunds used t indicate the switch mde during a trial, the intertrial interval, etc... SWITCH TEST: The Switch windw is used t set and test the peratin f the switch used t start and stp trials and t test the peratin f the Atlantis Platfrm cntrls (if these are installed). The Mde Menu (Run/Design) The Prject Windw has tw mdes f display. The single item in this menu is used t switch the Prject Windw between Run mde and Design mde. The name f the item changes accrding t which mde the prgram is currently in. DESIGN MODE: This is used t specify all aspects f the Prject, including the number and names f animals in the prject, the lcatins f the platfrms t be used, and the number and sequence f trials, including the number f trials t be perfrmed in each day, which platfrms will be used fr the different trials, the duratin f each trial, and s n. RUN MODE: This is used t run the trials in the prject in the desired sequence. The Design Menu (Designing a Prject) DEFAULTS: Opens the Basic Prject windw, which is used t specify the default parameters used fr the trial sequence in each animal, such as the platfrm t be used and the default trial duratin. PLATFORMS: Opens the Set Platfrms windw. Used t specify the number, names, lcatins, and prperties f the platfrms t be used in the prject. Platfrm settings are stred in the Prject file. Therefre, if the PLATFORM item is selected and n Prject file has been pened, a warning is displayed. START LOCATIONS: The user may specify up t 30 different starting lcatins fr each prject. Depending n which lcatin is specified fr a trial, a small dt is placed in the WaterMaze Windw image t indicate where the animal shuld be placed in the pl. Selecting this menu items pens the Set 11

12 Start Lcatins windw, which is similar in layut t the Set Platfrms windw. Lcatins are used nly as a guide fr the user during a trial. SEQUENCE NAMES: This feature is useful if the animals t be studied in the prject are numbered in sequence. Fr example, imagine that the 12 animals in a prject are named "mutant045", "mutant046", "mutant047",... "mutant056". Enter the "mutant045" int the Animal Name text bx fr animal 1, set the # f animals t 12, and select the SEQUENCE NAMES menu item. WaterMaze will fill in the names f the remaining 12 animals by adding 1 t each name. CLONE TRIALS: Opens the Clne Trials windw, which is used t cpy the trial settings fr ne animal t the settings fr any number f ther animals. NOTES: Opens the Ntes windw. A descriptive nte can be entered fr each animal. The ntes are stred in the Prject file. INTERLEAVE ANIMALS: Specifies the rder in which a day's trials are perfrmed. With this menu item checked, trials are perfrmed in the rder animal 1 - trial 1, animal 2- trial 1, animal 3- trial 1,...animal 1 -trial 2, animal 2 - trial 2... With the INTERLEAVE ANIMAL item unchecked, the day s trials fr animal 1 are cmpleted befre prceeding t animal 2, etc. In either case, the specified rder f trials can be verridden using the Select and D Selected cntrls when running trials. The Analysis Menu VIEW: Opens the Analysis View windw, which is used fr viewing the swim path and analysis parameters fr individual trials. EXPORT: Opens the Analysis Exprt windw, which is used fr exprting analysis parameters fr grups f animals int spreadsheet cmpatible files. SETTINGS: Certain analysis functins can be mdifies by setting assciated cntrl values in the Analysis Settings windw. The Help Menu Shw/Hide Tl tips: Many cntrls have tl tips assciated with them, brief explanatry messages that pp up when the cursr is held ver the cntrl fr a few secnds. Use this menu t turn n (default) r ff these messages (which can get annying nce yu've learned the prgram). Help: Opens this help system in Windws Explrer. The help file fr a given prgram windw can be reached directly by typing F1 at any time in the prgram. CONTROLS Design Mde Animal Cntrls The cntrls t the left f the screen specify the number f animals in the prject and their names. # f animals: Specifies the number f animals in the prject. A number may be typed in r the arrws used t change the number. Animal Name: Type the name f each animal int the crrespnding rw. Or type in the first name and use the DESIGN -> SEQUENCE NAMES menu item. 12

13 Select: T view the trial sequence fr any ne animal, click the crrespnding Select buttn. Trial Cntrls The cntrls t the right f the screen specify the trial sequence fr the animal that is selected by the Select buttns. These cntrls are the heart f the Prject Manager and are used t specify the exact sequence f trials t be perfrmed fr the selected animal. Make sure yu specify the starting psitins and platfrm lcatins befre specifying the trial sequence. # f days: Specifies the number f days that the animal will be run. Cnditins/day: Specifies the maximum number f different cnditins that will be used in a single day. A cnditin is cmprised f a platfrm and default trial duratin. Nte that a cnditin, which includes a platfrm, a start psitin, a maximum trial duratin, is distinct frm a trial perfrmed using that cnditin. That is, an animal may be trained fr several trials per day t a single cnditin. If each day, the same platfrm, start psitin and maximum duratin will be used, then cnditins/day can be set t 1. Cnditins: The cluster f cntrls labeled "Cnditins" takes n the number f rws specified in the # f days cntrl, and the number f clumns specified in the Cnditins/day cntrl. Thus, an experiment lasting 5 days with 2 cnditins wuld have 5 rws and 2 clumns. Fr each cnditin, yu may specify the Number f trials t be perfrmed, the Start lcatin t by used, which Platfrm t use fr the trials and the default Duratin fr the trials. Fr example, cnsider an animal that is t be trained fr 4 trials/day with the platfrm mving lcatin between trial 1 and trial 2 and nt thereafter. There are 2 cnditins and thus 2 clumns. The first cnditin shuld be set at 1 trial and the secnd set at 3 trials. Befre setting up a trial sequence, Start lcatins and Platfrms shuld first be defined using the crrespnding ptins in the Design Menu. Discard: Depress this buttn t eliminate an animal frm the prject fr any reasn after the prject trials have started. Lcked: This buttn is depressed whenever the trials fr the selected animal have begun. When it is depressed, the trial parameter cntrls (# f days, Cnditins/day, etc.) are disabled and cannt be changed. T re-enable these cntrls, click again n the Lcked buttn t turn ff the lcked cnditin. Yu may nw change the trial parameters fr the selected animal. Ding s is dangerus, hwever, and may crrupt the data that have been already cllected. CAUTION! Changing the number f trials that are t be dne n a day fr which all trials have been cmpleted will have disastrus cnsequences. Ding s will crrupt data that have already been cllected. Run Mde Trial Cntrls Shw Next: Indicates which animal is next in the Prject sequence by highlighting the crrespnding Select buttn fr that animal. D Next: Determines which animal is next in the specified Prject sequence and pens the WaterMaze windw with the crrespnding platfrm, trial duratin and animal name. Sequence: Same as D Next, but instead f returning t the Prject windw when the trial is dne, the WaterMaze windw is pened repeatedly accrding t the specified trial sequence. The Sequence mde is canceled when the Cancel buttn in the WaterMaze Windw is pressed (instead f the Save buttn). 13

14 D Selected: Opens the WaterMaze windw with the name, platfrm and trial duratin fr the animal specified by the Select buttns. Thus, the D Selected buttn verrides the sequence specified fr the Prject. Animal Cntrls Animal Name: Indicates each animal's name. This cntrl is disabled in RUN mde, and enabled in DESIGN mde. Yu may nt change the name f an animal nce a trial has been perfrmed fr this animal. Animal names may nt cntain any f a set f reserved characters, including < >,.?! \ and /. Select: Displays the Trial List fr the selected animal. T perfrm a trial n ne animal, click the crrespnding Select buttn and then the D Selected buttn in the Trial Cntrls cluster (abve). Day/Trial: (Indicatr nly). They indicate the day and trial number f the last perfrmed trial fr the crrespnding animal. If fr example, the secnd trial n the 4th day f trials has been perfrmed, and 5 trials are t be run n the 4th day, the Day indicatr will read "4" and the Trial indicatr will read "2/5". Trial List: The trial list shws a cmplete list f the trials perfrmed and t be perfrmed fr the selected animal. The clumns indicate fr each trial: the trial number, the day n which the trial is t be run, the trial number fr that day, the name f the platfrm t be used, and -- if the trial has been run -- the trial duratin. Discard: Same as in Design Mde. Click n the buttn t eliminate an animal frm a prject nce the prject's trials have begun. When the Discard buttn is depressed, the animal will be skipped by the prgram when Shw Next, D Next r Sequence are selected. An animal may be temprarily discarded. If fr sme reasn, yu wish t defer perfrming an animal's trials until the end f the day, fr example, press the Discard buttn, perfrm the trials fr the remaining animals using the D Next r Sequence buttns, then press the Discard buttn again and use D Next r Sequence (r D Selected) t return t the deferred animal. The Basic Prject (Defaults) This windw allws yu t specify sme basic default aspect f a Prject. When setting up a new prject frm scratch, this windw shuld be used first, especially when the trial sequence fr the animals in the prject are t be very similar. The windw settings are adpted fr all animals. Settings may be changed in the Prject Manager windw fr individual animals and individual days as desired. # f animals. Sets the number f animals in the Prject. # f days. Sets the starting # f days fr all animals in the prject. These may be changed later fr individual animals within the Prject Manager windw. Trials per day. Sets the starting Trials per day fr all days and all animals in the prject. These may be changed later fr individual animals within the Prject Manager windw. Platfrm. Sets the starting Platfrm fr all days and all animals in the prject. These may be changed later fr individual animals within the Prject Manager windw. Trial duratin. Sets the starting Trial Duratin fr all days and all animals in the prject. These may be changed later fr individual animals within the Prject Manager windw. 14

15 Setting the Platfrms The Set Platfrms windw is used t specify the lcatin and prperties f the multiple platfrms t be used in a Prject. The Windw cntains a real-time view f the camera image, and several cntrls and cursrs fr changing platfrm settings. Cpying platfrm settings frm anther Prject File The platfrm settings are stred in the Prject File. Yu may find that yu wuld like t start a new prject using the same platfrm settings that were established in a previus prject file. Instead f setting up the platfrms again frm scratch in the new prject, pen the ld prject file, select FILE -> SAVE AS... and save the ld prject file with a new name. Nw edit the animal and trial settings in the new file, leaving the platfrm settings intact. Setting a Platfrm 1. Select the number f the platfrm yu wuld like t edit using the Selectr ppupmenu. 2. Enter the name f the platfrm int the Name text edit bx. 3. Indicate whether the platfrm is t be physically present r nt using the Present checkbx. Unless an Atlantis platfrm is t be used, this setting has n practical effect n the running f an actual trial. It can be used as an aid in setting up a trial r in later analysis. 4. Indicate whether an Atlantis Platfrm is t be used. If s, a set f cntrls becmes visible at the bttm right f the windw fr setting the Atlantis Platfrm parameters. Trigger diam and Dwell Time. The Atlantis platfrm will be raised when the animal remains within a trigger zne defined by the Trigger diameter (centimeters) fr a perid defined by the dwell time. Raise after. If, after the perid defined by this cntrl (secnds) the animal has nt triggered the raising f the platfrm by swimming in the Trigger zne, it will be raised anyway. Clr. Defines the clr f the circle that is t indicate Atlantis trigger zne in bth this windw and in the WaterMaze windw. Trigger line. The Atlantis Platfrm is cntrlled frm the PCI-6503 Digital I/O bard. With this cntrl yu may specify which f the 24 digital lines f the 6503 will be used fr each Atlantis platfrm. Each platfrm may be cntrlled by a different line. Trigger lines 1-8 cntrl pins 47, 45, 43, etc. n the Set the diameter f the platfrm (centimeters) using the Diameter cntrl. 6. Mve the platfrm int the desired psitin using the cursr at the center f the platfrm circle. If the platfrm circle and cursr are nt visible n the screen, click the Center Cursr buttn t bring them int view. Use the clr bx t set the clr f the platfrm circle in this windw and in the WaterMaze windw. 15

16 The Nudge cntrl (grey diamnd n blue backgrund) can be used fr fine cntrl ver the psitin f the platfrm circle. The X and Y indicatrs shw the psitin f the platfrm center with respect t the center f the pl. They are updated by mving the cursr f the platfrm, but cannt be entered directly. Markers Varius markers can be displayed in the image t guide the placement f the platfrms. Diagnals. These define the center f the pl (nt the center f the image). Diagnal clrs are set in the Set Pl windw. Marker Circle 1 and 2. These are cncentric with the pl and their diameters expressed as a percentage f the pl diameter. Circle clrs are the same as fr diagnals (set in the Set Pl windw). Pl. Shws the utline f the pl. Clr is set in the Set Pl windw. Save and Exit Clses the windw, n ne case Saving the platfrm setting t the currently pen prject file. Cancel Clses the windw withut saving any f the changes made. Clne Trials The Clne Trials windw is used t cpy the experimental trial sequence frm ne animal t anther. Fr example, after setting up a cmplex series f trials fr ne animal in a prject, yu can cpy the series t all ther animals in the prject. In the Frm listbx, select ne animal whse trial sequence yu wish t cpy. In the T listbx, select multiple animals whse trial sequences will be replaced with that f the animal selected in the Frm list. (Shift-Click t select mre than ne animal.) Click the Clne--> buttn t cpy the trial sequences accrding t the listbx selectins and return t the Prject Manager windw. Click Cancel t return t the Prject Manager windw withut making any changes. Ntes This windw allws yu t save ntes in the Prject file, ne nte per animal. Simply select an animal in the Animal listbx and type any desired text int the Nte text edit bx. Ntes may be added and amended at any time during an experiment. Save & Exit and Cancel clse the windw either with r withut saving the changes made. 16

17 WaterMaze Windw Running a Trial (The WaterMaze Windw) The WaterMaze windw is reached frm the Prject Manager Windw. In the Run mde, click the D Next, D Selected r Sequence buttn. The WaterMaze Windw pens shwing The name f the animal t be run The trial number fr the animal The next animal in the sequence t be run The camera image The name f the platfrm t be used The utline f the platfrm (and the Atlantis platfrm trigger zne) superimpsed n the image A small spt t indicate the starting lcatin specified by the user The Reference Frame. Yu must capture a reference frame befre the animal is intrduced int the tank. WaterMaze finds the animal in an image by cmparing that image t a reference frame, that is, an image f the tank taken withut the animal in it. Prir t each trial, therefre, WaterMaze plays an intermittent tne t remind yu t take the reference frame. Press the Reference Frame cntrl in the WaterMaze Windw, r press the Reference remte cntrl switch until the tne ceases. Yu may set the pitch and vlume f the tne in the Preferences Windw (Sund #1). Starting the trial. Once the reference frame has been taken, the trial can be started. Intrduce the animal int the tank and immediately press the Start cntrl in the WaterMaze Windw, r press the remte switch nce. This starts the trial, as indicated by a brief tne, the frequency and vlume f which are set in the Preferences Windw (Sund #2). Make sure yu have intrduced the animal int the pl and mved ut f the image befre pressing the start buttn, r the animal will nt be tracked prperly during the first few frames f the trial. Running the trial. The trial prceeds autmatically. An asterisk-shaped cursr indicates where the prgram has lcated the animal. If the Shw Path buttn is n, the swim path is pltted as the animal swims. The clr f the swim path pints is set in the Set Pl Windw. Stpping the trial. A trial can be halted in ne f fur ways. Press the Cancel buttn. This halts the trial and exits the WaterMaze Windw withut saving the trial. Press the Stp buttn r the Stp remte switch. This halts the trial and plays Sund #3. The trial can then be saved (with the Save buttn r the remte cntrl switch) r discarded (with the Cancel buttn). Select AutStp Trial in the Preferences Windw. In this case, the trial is autmatically halted when the animal reaches the platfrm and stays in it fr the time specified in the Preferences windw (Autstp time). 17

18 The trial times ut because the animal des nt reach the platfrm in the time specified fr this trial by the Trial Duratin in the Prject Windw. Swim Path. At the end f the trial, the swim path pints (which are displayed during the trial) are cnnected by a line, the clr f which is set in the Set Pl Windw. The line skips thse pints in the path fr which the calculated swim speed exceeds the Velcity Threshld set in the Preferences Windw. The prgram assumes that these pint represent errrs by the tracking algrithm and s it edits this pints ut f the path and instead extraplates the path between valid pints recrded prir t and fllwing the errrs. Saving the trial. Press the Save buttn r the Save remte cntrl switch t save the trial. Press the Cancel buttn t exit the WaterMaze windw withut saving the trial. If the WaterMaze windw has been pened using the Sequence buttn in the main windw, at the end f the trial a timer appears and begins t cunt dwn the secnds starting frm the Intertrial Interval that has been set in the Preferences Windw. The Pause buttn temprarily halts the cuntdwn. The trial is saved when the timer reaches 0. T verride the timer and save the trial immediately, press the Save cntrl, r the Save remte cntrl switch. The trial is appended t a file named fr the animal, and the WaterMaze windw clses. If the trial has been started using the Sequence buttn in the Prject Windw, then the WaterMaze Windw will autmatically pen again fr the next trial in the Prject. Nte: Occasinally, the animal yu have actually run may nt be the animal that is indicated in the windw (under "Animal" at the upper right f the windw). Smetimes the peratr can mistakenly chse a different animal frm the ne requested by the prgram. Instead f thrwing ut the trial, yu may use the Animal ppup menu t crrect the errr, by selecting the animal that was actually run. Of curse, this feature will nt be very helpful if the platfrm fr the requested animal and the platfrm fr the animal that was run are different. Atlantis Platfrm cntrl. If the platfrm specified fr the trial has been set up as an Atlantis platfrm, several indicatrs will appear at the lwer left f the image windw. The Dwell Time and the Raise After time (bth set in the Set Platfrm Windw) are shwn in digital indicatrs and each has an assciated slide indicatr. The Trigger Zne (als set in the Set Platfrm Windw) is indicated as an extra circle surrunds the psitin f the platfrm in the image windw. During the trial, the Raise After slide indicatr will gradually turn red. When the duratin f the trial reaches the Raise After time, the indicatr becmes cmpletely red and the platfrm will be released. That is, the digital line n the PCI-6503 that has been selected fr the current Atlantis platfrm (set in the Set Platfrm and Switches Windws) will be driven high (+5V). Once the animal enters the trigger zne (selected in the Set Platfrm Windw), the Dwell Time slide indicatr will start t turn red. If after the Dwell Time, the animal has remain in the trigger zne cntinuusly, the indicatr turns cmpletely red and the platfrm is released. The indicatr is reset t zer if the animal exits the trigger zne befre the indicatr turns cmpletely red. Frame rate. This indicatr will appear nly if the cmputer cannt sample as fast as the Frame Rate specified in the Preferences Windw. It shws the actual frame rate at which the data are being recrded. This indicatr is merely a warning, and nt a serius errr. Except fr the lwered sample rate, data cllectin is unaffected. T avid this warning, lwer the Frame Rate specified in the Preferences Windw. Analysis Windws 18

19 The Trial Viewer Windw The trial viewer can be used t view and analyze any cmpleted trial in the Prject. Open the Trial Viewer windw by selecting the Analysis -> View menu f the Prject Manager windw. T view a trial: 1. Select the animal fr which yu wuld like t view a trial using the Animal listbx. 2. Select the trial that yu wuld like t view using the Trial listbx. The trials that have been cmpleted have the Time entry in the Trial listbx filled in. 3. Select the prtin f the trial yu wish t analyze using the Trial Subset cntrls. The Start and End cntrls determine the start and end f the prtin t be analyzed in either secnds r percent, depending n the setting f the accmpanying switch. The All buttn sets the switch t "%" and the start and end times t 0 and 100. Fr the selected trial, several types f infrmatin are displayed. Sme analysis variables are assciated with a cntrl parameter as listed belw. Platfrm Parameters. Psitin, diameter, present? Atlantis Parameters. Atlantis?, trigger diameter, dwell time, raise after. Trial Duratin. The ttal recrding time. Distance Traveled (Path length). Path-length is calculated using Pythagras therem n separated pairs f crdinates. The separatin (in image frames) is determined by the Smthing cntrl. This smthing acts as lw-pass filter that reduces jitter, i.e., the number f ccasins that the distance calculatin is artifactually increased when the image-analyzer cursr jumps frm the rats black head t black marks n its bdy, r between pints reflecting small head-mvements. Average Speed. Distance/Time (cm/sec). In ur experience in Edinburgh, speed is remarkably unifrm at circa 25 t 30 m/sec fr animals swimming in water at 25 C. Speed declines slightly as animals get tired (e.g. after several successive lng trials). % time near walls. Percentage (%) f time spent within a specified distance frm the side walls f the pl. The bundary distance is specified in centimeters using the Width cntrl. The analyses is useful fr assessing thigmtaxic tendencies in animals, which can be increased in animals given high dses f antichlinergic drugs, r certain kinds f brain lesins. The Shw buttn makes visible the regin near the walls used in the analysis. Directinality. Directinality measures the animal's heading in degrees f angle relative t the platfrm at a selected distance alng the path frm the start. The pint at which directinality is t be measured is indicated in centimeters using the Distance cntrl. The animal's directin is calculated as a line thrugh tw pints in the path, ne 3 frames back frm the selected distance, and ne 3 frames ahead. When the animal is heading in a directin clckwise frm the platfrm, angles are reprted as psitive. The Shw buttn makes visible the tw vectrs used in the calculatin. Gallagher's Prximity. Derived frm Gallagher, Burwell & Burchinal, 1993, Behaviral Neurscience, 107: The animal's distance t the platfrm is first averaged ver each 1-secnd interval f the trial. 19