Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Procedure for measuring exploratory behavior using and open field circular arena and SEE
Step Procedure performed Ages (wks) Equipment Time (min) Data collected 1 10-wk-old male mice are randomly selected and then brought to the testing room and acclimated before testing begins 10 transfer cages 30 - 2 each mouse is placed near the wall at 12'clock position within the circular arena and videotaped for the entire duration of the session 10 circular arena with video tracking (VCR) 30 activity and locomotor characteristics 3 using the Strategy for the Exploration of Exploration (SEE) software various behavioral endpoints are defined and analyzed from each mouse videotaped recording 10 computer processor 30
• Testing arena 2.5 m diameter circular arena with a non-porous, primer-gray painted floor and 50 cm high, primer-gray painted, continuous walls. A number of visual landmarks of various shapes and sizes are attached in different locations to the arena wall and to the walls of the room outside the arena. Fluorescent lights mounted on the ceiling illuminate the arena.
A video camera is mounted on the ceiling and centered above the arena. Camera output is fed into a Panasonic 7200 VCR.
• Data analysis
Raw video data from each session were translated into quantifiable endpoints in the following steps:
video tracking performed by an automatic tracking system with a spatial resolution of ~1 cm and a time resolution of ~0.04 s (Ethovision 2.0, Noldus Information Technology)
data smoothing using the Lowess algorithm with improvements to reduce tracking noise and outliers (Hen et al., 2004)
segmention of the smoothed path and endpoint computation using the investigators' Strategy for the Exploration of Exploration (SEE) software, to define progression segments separated by lingering episodes (stops). SEE can be used to query, visualize, and quantify complex properties of behavioral data accumulated over time and to easily design new endpoints for improved discrimination and replicability (Drai et al., 2001; Kafkafi, Lipkind, et al. 2003).
separation of the smoothed and segmented path into units of behavior along the wall and in the center and computation of endpoints concerning wall vs. center behavior using the Wall Center Separation Procedure, which is part of the SEE software (Lipkind et al. 2004).
- Disinfectant for cleaning circular arena in between testing
- Mop for cleaning
Acclimation to test conditions
Mice are allowed to acclimate to procedure room before testing begins.
Procedure for measuring exploratory behavior using and open field circular arena and SEE
a. Mice are shipped in two batches.
b. Mice are housed in the home colony 2 wks before testing.
c. Each mouse is transported from the animal facility to the testing room and placed in the arena near the wall at the 12 o'clock position and videotaped for a 30 min period.
d. Following taping, fecal boli were collected, and the arena floor is cleaned and swept with a wet mop between sessions.
e. Sessions are run during the dark cycle, starting 2 hrs after onset and continued for 8 hrs on each experimental day.
f. Mice are tested in random order, and the order of testing within a strain is recorded and incorporated in the mouse identification number, along with batch number and laboratory code (labcode-batch-order).
See Kafkafi, Lipkind, et al., 2003, Kafkafi, Pagis, et al., 2003, and Lipkind et al., 2004 for more details.
Coordinated experiments were conducted in three laboratories in 3 different locations
1- Department of Zoology
Tel Aviv University (Labcode TAU)
Ilan Golani (PI), Yoav Benjamini.
2- Maryland Psychiatric Research Center (Labcode MPRC)
University of Maryland
Baltimore Maryland, USA
3- National Institute on Drug Abuse (Labcode NIDA)
Baltimore Maryland, USA
Neri Kafkafi (see new address)
g. Eight strains are tested in each laboratory.
h. Thirty-three endpoints are calculated and submitted as measurements for selected strains.
i. The first 17 measurements listed are submitted for all strains; the remaining 16 measurements are submitted for 5 strains only.
Arena center: more than 15 cm away from arena wall.
Arena-Crossing incursions: incursions in which the maximal distance from wall is higher than the intermediate incursions cutoff.
Center lingering: lingering episodes that are performed in the center, defined by the wall center separation procedure.
Center segments: segments of smoothed path in which radial speed or distance from wall are higher than intrinsic cutoffs, determined separately for each animal using the wall center separation procedure. [Center segments include all the movement that is not performed along the wall]
Curvature: the change in direction between data points. [Data points are located 5 cm from each other; all curvature data are normalized to 5 cm, e.g., MCW units are degrees per 5 cm]
Diversity: average distance between any two stops, taking into account spatial and temporal scattering of stops, thereby weighing the distribution and durations of the stops.
End point: behavioral measure obtained using an open field test and the application of Software for the Exploration of Exploration (SEE)
Excursion: a round trip starting and ending at the homebase
Homebase: the place with the highest occupancy in the arena
Incursions: trips to the center, which start and end near the wall. [An incursion includes one or more center segments and may include one or more center lingering episodes]
Intermediate incursions: incursions in which the maximal distance from wall is higher than the near-wall incursions cutoff and lower than an intrinsic cutoff, determined separately for each animal using the wall center separation procedure.
Lingering episodes: segment of smoothed path where tracking coordinates are those in which speed is lower than an intrinsic cutoff, determined separately for each animal using SEE segmentation procedure; lingering episodes (or stops) alternate with progression segments.
Lingering diversity: average distance between any two stops, weighted by the distribution of the duration of these stops; lingering diversity captures the spatial and temporal scatter of stops; lingering diversity increases as a greater area is covered, as an area is covered more homogeneously, and as the duration of stopping is distributed more homogeneously over the arena
Lingering mean speed: an approximate measure of local movements, which probably incorporate scanning movements, some sideways and forward steps, rearing, and stretch-attend behavior.
Maximal speed of progression segment: maximum speed obtained while in motion necessary for the discrimination of progression from lingering behavior
Near-Wall incursions: incursions in which the maximal distance from wall is lower than an intrinsic cutoff, determined separately for each animal using the wall center separation procedure (see Investigator Notes).
Number of progression segments: number of stops made in 30 min session
Occupancy: total time spent in a given location multiplied by the number of visits to that location.
Progression rate of turn: measures the change of direction (in degrees) during one second of progression (using absolute values, i.e., no discrimination between left and right turns). The 'time for turn' (TTRN) is the inverse of this rate, multiplied by 360, i.e., the time in seconds it would take to complete a 360° turn at the momentary rate of turn
Progression radius of turn: measures the momentary radius of path (using absolute values, i.e., no discrimination between left and right turns)
Progression segments: segments of smoothed path in which speed is higher than an intrinsic cutoff, determined separately for each animal using SEE segmentation procedure; by definition, number of progression segments equals number of lingering episodes
Segment acceleration: equals maximum speed divided by duration of motion
Session: 30 min of testing
Spatial spread of lingering episode: maximal distance between any two points during a stopping or lingering episode.
Wall segments: segments of smoothed path in which radial speed and distance from wall are lower than intrinsic cutoffs, determined separately for each animal using the wall center separation procedure. [During wall segments the animal runs along the arena wall, i.e. in parallel to the wall and in close proximity to it]
Wall lingering: lingering episodes that are performed near the wall, defined by the wall center separation procedure.
NA: (not applicable) assigned if the number of roundtrips from homebase (NEXC) is less than 4 or if the number of progression segments (NP) is less than 5. NA is informative with regard to strain performance, indicating the option of not moving out of the spectrum of possible responses. However, strain means do not reflect this characteristic (NA is omitted from strain mean calculations). The following table is included to show which strains contain NA data, by measurement:
Measurement short name/ Total number tested
129S1/SvImJ A/J BALB/cByJ DBA/2J Total 35 34 35 36 140 CNTRT 1 2 2 0 5 DST 1 2 2 0 5 DSTRD 1 2 2 0 5 DVRS 1 2 2 0 5 HBO 1 2 2 0 5 LMS 1 2 2 0 5 LMXHS 1 2 2 0 5 LNGP 1 2 2 0 5 MSDR 1 2 2 0 5 NEXC 1 2 2 0 5 NLEXC 1 12 7 1 21 PMXS 1 2 2 0 5 SPTL 1 2 2 0 5 TL 1 2 2 0 5 TRADA 1 2 2 0 5 TTRN 1 2 2 0 5
Investigator Notes: The algorithm for separating incursions into types was slightly modified from the algorithm described in Lipkind et al. (2004). As before, we defined two ranges of possible cutoff values -- one for the 1st (small) cutoff value in each mouse, and another for the 2nd (large) cutoff value in each mouse (so that each mouse has 2 cutoff values, and, consequently, three incursion types, as described in Lipkind et al. 2004). However, instead of using the entire range of the non-transformed values for each of these two groups, we used the range of the log-transformed values, not including far-out outliers (i.e., values above the upper quartile plus 3 times the inter quartile range, and below the lower quartile minus 3 times the inter quartile range).
Additional information for selected measurements
Median of incursions maximal wall distance (MIMWD): the median of the maximal distance from wall attained in incursions.
Proportion of incursions beginning with a stop (PIBS): the proportion of incursions that are directly preceded with a stop (lingering episode) out of all the incursions.
Average segment length in incursions (ASLI): the median of the average segments lengths in incursions.
Outbound/inbound speed ratio in incursions (OISRI): the median of the ratio between the mean speed in the outbound (directed towards the center) and the inbound (directed towards the wall) portions in each incursion.
Data collected by investigator
Complete panel of behavioral endpoints using OFT and SEE: CNTRT, DST, DSTRD, DVRS, HBO, LMS, LMXHS, LNGP, MSDR, NEXC, NLEXC, PMXS, SPTL, TL, TRADA, TTRN.
Kafkafi N, Mayo C, Drai D, Golani I, Elmer G. Natural segmentation of the locomotor behavior of drug-induced rats in a photobeam cage. J Neurosci Methods. 2001 Aug 30;109(2):111-21.