SEE Workshop:

SEE Workshop is a software-based Strategy for Exploring Exploration behavior by high-quality ethologically-relevant measures (end-points). This is accomplished by following a special multi-stage analysis procedure consisting of several stages and carried out by five sub-programs (modules):

1) SEE Path Smoother : Careful smoothing of the raw data, so as to preserve the fine fabric of the mouse’s stop-and-go behavior and calculation of momentary speeds (Hen et al., 2004). The raw data for this module are the time series of the animal's coordinates in space, sampled at a high rate (25 Hz in the European PAL system and 30 Hz in the US NTSC system) that allows a meaningful computation of stops, velocities and accelerations, and although not obligatory for running the next stages, is highly recommended. The input data file/s for this module is the time series of X,Y coordinates which are the output of a computerized tracking system such as “.csv” files (for example files created by EthoVision™) or “.dxy” files (for example files created by PhotoBeam AccuScan).

2) SEE Path Segmentor : Parsing of the data into intrinsic ethologically-relevant building blocks of locomotion, consisting of slow local movements (“lingering episodes”) and faster progression segments (“movesegments”, or progression segments) - (Drai et al., 1999).

3) SEE File Creator: This program creates SEE files, which are output files that are necessary for the calculation of behavioral measures (end-points) by the Endpoint Calculator module (see below) and for using the advanced Mathematica™ based program SEE Professional package (or any other software, such as mathlab you might want to use for developing your own functions). The input files for this program are the output files of both SEE Path Smoother module (“.txt” files) and SEE Path Segmentor module (”.inf” files) programs.

4) Arena Builder: This module is software designed for applying robust separation of the path of the animal into Wall or Center segments based on intrinsic geometric and statistical criteria for finding the threshold. This module also estimates the real contour shape of the arena in order to get an accurate threshold even when the arena is not a perfectly circular. The input files for this module are the files from the SEE File Creator module (Lipkind et al., 2004). For now the algorithm is applicable only to an empty (including no objects), circular arena.

· Both the segmentation (SEE Path Segmentor) and separation (Arena Builder) procedures yield distinct components.

5) Endpoint Calculator: In this module behavioral measurements (end-points) are finally performed both over all data and on each of the components separately thus creating a table of end-points in a standard and common format (.csv file) on which further analysis and statistics can be applied.

Instructions for program use:

1. Open the SEE Workshop.

2. Select the path of the folder containing the input files by clicking the ‘Add..’ button. Only .csv files (may be generated in Microsoft Excel) and .dxy files can be placed in this folder. Workshop analyses all the input files found in this folder as a batch. You may select several folders at a time.

3. Files with a csv extension should be of the following format:

a) Header: If you used Ethovision for tracking proceed to (d).

If you used any other tracking system download the file named "header for csv file.csv" copy its contents, and paste at the beginning of each file you intend to analyze.

b) Columns: The first column should be a running time index. Note that it must be continuous! The second column can show running time index in seconds, but in the absence of such data it also can contain any constant value throughout the file. The third column should contain the x – coordinates, and the 4^th column should contain the y-coordinates. The fifth column can show the area of the animal's image, but, if such data are not available, it also can contain any constant value throughout the file. Any other columns after the 5^th will be ignored by workshop. An example of a proper csv file is available in the SEE website.

c) All values should have no more than 2 digits after the decimal point.

d) Rows with missing data should have a running time index and all the cells should be filled with a dash, or hyphen (-).

e) The first row must always contain data (and not dashes) in all cells.

4. Use the checkboxes to select the modules you wish to process. You can select more than one module, but upon selecting a module one must ascertain that all the previous modules have been run on these files. Remember that the program processes each module at a time over all the data files in the selected folders.

Note:

· The output files will be created in the same folders containing the input files.

· When applied, the program rewrites over existing output files that were already calculated in the past and located in the selected folders. However, this will only cause a loss of computational time due to the unnecessary recalculation.

· The modules depend on having the right input files (which are basically the output files of their previous modules. It is strongly recommended for new users to use the modules as ordered. Skipping any one of the modules will damage further processing and will prevent end-points calculation.

5. Set the parameters in the selected modules and click the ‘Process’ button.

Visualization of Smoothed data:

SEE workshop enables you to visualize the time series of locations (x or y) from the raw data of a selected file even before any process was applied. After applying the SEE Path Smoother module you will be able to view simultaneously both the raw (in red) and smoothed coordinates (in blue), as well as the speed time series attained from the smoothed locations. Use the ‘Visualization of Smoothing’ button to open a data file. Enter range of frames and press the ‘Update’ button to view the data.

Note:

· The visualization module is independent of the other modules and can be used separately.