After opening the program, the interface can look as follows:
Files should be loaded into the program one condition at a time (i.e. files of a single sucrose concentration and genotype).
First, enter the genotype. To enable the loading of files, enter the sucrose concentration of the to-be-loaded files and press enter. Multiple files can be loaded at once by selecting the relevant files using the Shift key to select ranges or the Control key to select individual files. Note that it is possible to load files of the same concentration in multiple actions; the list of these newly loaded files will be appended to the existing one. After pressing OK, the user is prompted with the following screen:
This window asks the user for a set of parameters that holds for all data files that were selected to be loaded. First, the length of the sucrose pulse(s) has to be entered. All pulses in a loaded data set are assumed to be of the same length. Currently, the maximum number of pulses in a single file is limited to 2. Next, the user has to enter the starting time for each pulse. If there is only a one pulse per file, the second value has to be blank. After pressing OK, all selected data will be loaded and a progressbar will show the progress of loading. Once loading has finished, the interface could look as follows (depending on the program version):
If the loaded group was non-existent, its label will now have been added to the 'Group' popup menu (1). All files contained in the selected group are shown as a list under 'Files' (2). Selecting one of these files will display the data in the plot window (3).
Back to topOnce all files have been loaded, click the checkbox ´Files linked´ (4) as in the above figure to associate submaximal sucrose stimulations (e.g. 0.25M) with RRP-depleting sucrose stimulations. For WT neurons, 0.5M sucrose is typically sufficient to deplete the RRP. However, for some genotypes the fusion energy barrier can be increased, requiring a stronger stimulus (e.g. 1.0M) to deplete the RRP. Therefore, every submaximal stimulus is linked in a pairwise manner to an RRP-depleting stimulus. If more than two concentrations were applied to a single cell, all these recordings are fitted simultaneously.
This process of linking recordings is necessary, as fitting ´non-linked´ submaximal sucrose stimulations will result in wrong estimates of the RRP size and the release rate. It is important to note that this association is achieved via a particular comparison of filenames, namely according to the following format:
[prefix]0001.abfThe matching is performed by stripping off the extension (.abf) and the recording number (here: 0001), and subsequently matching the remaining [prefix] of the RRP-depleting data set with the [prefix] of the 'submaximal' data set. For instance, in the above figure, the to-be-matched [prefix] would be '130821_WT_c09_'.
The user should determine which concentration to designate as 'RRP-depleting'. All recordings with lower concentrations than the maximal one will be considered 'submaximal'.
Back to topFor future reference, it is possible to save the current session via File->Save session. This will store the current data structure, containing among others the raw data and fit results (see the Theory section for a description of the data structure), in a Matlab .mat-file.
Previously saved session files can be loaded via File->Load session. Note that the software version with which the session file was saved is key here - if the data structure employed by the program has changed between time of loading and time of saving, the session file cannot be loaded.
In order to load and analyse recordings in a format different from .abf-files, the user should write a script which processes these data and returns a data structure as specified in the theory section.
Back to topThe preferences menu (Edit->Preferences) enables the user to modify parameters related to the model, the way the data is loaded, and the procedure used to fit data to the model. The preferences menu can look as follows:
The 'Model parameters' shown on the left control particular properties of the model and the way data is handled by the program:
The user fix certain model parameters during a fit by ticking the 'Use fixed parameters' box. Doing so will prompt the user, when starting the fit, with a window asking which parameter(s) to keep fixed. Subsequently, this/these parameter(s) can be given a particular value, or (by leaving the field empty) the parameter(s) for the second pulse in a trace can be kept at the same value(s) as fitted from the first pulse. To start a fit using these settings, use Fit->Fit all - fitting a single pulse via 'Refit data' will not work with fixed parameters.
Control over the 'Experimental features' is switched off by default:The 'Fitting parameters' shown on the right control the fitting procedure:
Pushing the 'Default' button will return the model or fitting parameters to their original values.
Back to topOne can choose to either fit all data in one go (via Fit->Fit all), or (re)fit traces one by one by right-clicking on a trace in the 'Files' list and selecting 'Refit data'. See also the section about the fitting procedure on the Theory page.
Back to topThe software keeps track of the actions performed by the user via a log, which can be accessed via the Help menu (Help->Show log). The log keeps track of a number of actions/events, including the loading of raw data files; performance during the fitting procedure (one can for instance monitor the duration of a fit); manually changed parameters when reviewing fit results; possible errors/problems encountered during the fitting procedure; etc. The severity of a logged action is colour-coded, ranging from green to red. The log can look as follows:
Users can enter debug mode via the Help menu (Help->Debug). Note: recommend only for experienced Matlab users!
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