How to run the protocols

Pre-flight checklist

Before running either protocol, ensure:

Protocol 1 — GUI-based

Protocol 1 is run through the G4_experiment_conductor GUI.

Tip

A detailed explanation of Protocol 1 can be found here.

Step-by-step

  1. Open the experiment conductor: Type G4_experiment_conductor in the MATLAB Command Window.

  2. Load the protocol: Click File → Load, then navigate to the appropriate .g4p file within the protocols/ folder. For the most recent version, use:

    • Left hemisphere recording: protocols/bkg4/LHS/protocol1_10kHz_4reps_12px_6px_LHS_2sbkg_200msfl_150msint_07-10-25_15-45-60/
    • Right hemisphere recording: protocols/bkg4/RHS/

  3. Verify settings: Check that the processing_settings.mat file is present in the protocol folder and that settings.is_ephys_grid = 1.

  4. Run the protocol: Click the Run button. The protocol will take approximately 6 to 7.5 minutes to complete.

  5. Automatic processing: After the protocol finishes, the processing functions should run automatically (provided the processing_settings.mat is configured correctly). This generates 4 grid plots showing the cell’s responses.

Between Protocol 1 and Protocol 2

After P1 completes, you need to determine two things before running P2:

1. Identify the peak_frame

Examine the 6 pixel flash grid plots (both bright and dark contrasts). Each subplot shows the cell’s voltage response to a flash at that location on the screen. The number above each subplot is the frame number of that flash within the pattern.

  • Find the subplot with the largest response (biggest deflection from baseline).
  • Note the frame number above that subplot — this is your peak_frame.
  • The peak_frame will be used to generate the Protocol 2 stimulus at the same location but with higher spatial resolution. The preferred contrast of the cell is determined automatically based on the peak_frame value.
ImportantCheck RF position

If the peak_frame corresponds to a flash near the edge of the stimulus area, the 30 x 30 pixel Protocol 2 area may extend beyond the arena boundary. The code will warn you if this is the case. Consider whether Protocol 2 is still worthwhile for edge cases.

Protocol 2 — Script-based

Protocol 2 is generated and run entirely through a single MATLAB script. Unlike Protocol 1, it does not use the G4_experiment_conductor GUI.

Tip

A detailed explanation of Protocol 2 can be found here.

Step-by-step

  1. Run the script: Type generate_protocol2 in the MATLAB Command Window, or run the script directly.

  2. Fill in the pop-up dialog: A GUI dialog will appear with the following fields:

    Field Description Example
    Frame The peak_frame number from the P1 6px grid plot with the largest response 42
    Side Which side of the arena was used in P1 (L for left, R for right) L
    Age Age of the fly in days (1-8, or NaN if unknown) 3
    Strain Fly genotype — select from the dropdown list 42F06_T4T5_control
Important

If a new strain is being run that is not in the dropdown list, add it to the field Items for the variable strainDropdown within the script get_input_parameters.m.

  1. Wait for generation: The script will:

    • Convert the peak_frame to screen coordinates using patt_frame_to_coord.m
    • Determine the ON/OFF contrast from the peak_frame value
    • Generate all flash patterns, bar patterns, and position functions
    • Assemble the experiment into currentExp.mat
    • Create a new timestamped folder (format YYYY_MM_DD_HH_MM)

  2. The protocol runs automatically: After generation, the script calls run_protocol2() which connects to the PanelsController and runs all stimuli with 3 repetitions. The protocol takes approximately 20 to 25 minutes.

  3. During the experiment: The arena will cycle through the stimulus sequence (flashes → bar sweeps at different speeds → bar flashes) three times. You should see the arena LEDs changing as each stimulus is presented.

  4. Post-experiment: After completion:

    • TDMS log files are automatically converted to .mat format
    • Metadata is saved to the experiment folder
    • Experiment details are exported to the Google Sheets log (unless strain is set to test)
    • The script returns the peak_frame value for the inverse contrast, so you can run P2 again with the opposite contrast if desired
NoteRunning both contrasts

Protocol 2 only presents one contrast (ON or OFF) per run. After the first run completes, the script returns the peak_frame value that would generate the protocol at the same location with the inverse contrast. You can use this to run P2 a second time to collect data for both contrasts.

After Protocol 2

Once Protocol 2 is complete, you can analyse the data using process_protocol2(). See How to analyse P2 for instructions.