DOI: 10.5061/dryad.vdncjsz42
title: iGluSnFR wide-field imaging responses to 2-72 Hz flicker

description: Human primary visual cortex (V1) responds more strongly, or resonates,
 when exposed to ~10, ~15-20, ~40-50 Hz rhythmic flickering light.
 Full-field flicker also evokes perception of hallucinatory geometric
 patterns, which mathematical models explain as standing-wave formations
 emerging from periodic forcing at resonant frequencies of the simulated
 neural network. However, empirical evidence for such flicker-induced
 standing waves in the visual cortex was missing. We recorded cortical
 responses to flicker in awake mice using high spatial resolution widefield
 imaging in combination with high temporal resolution glutamate-sensing
 fluorescent reporter (iGluSnFR). The temporal frequency tuning curves in
 the mouse V1 were similar to those observed in humans, showing a banded
 structure with multiple resonance peaks (8 Hz, 15 Hz, 33 Hz). Spatially,
 all flicker frequencies evoked responses in V1 corresponding to
 retinotopic stimulus location, but some evoked additional peaks. These
 flicker-induced cortical patterns displayed standing wave characteristics
 and matched linear wave equation solutions in an area restricted to the
 visual cortex. Taken together, the interaction of periodic traveling waves
 with cortical area boundaries leads to spatiotemporal activity patterns
 that may affect perception.
description: In vivo glutamate imaging was performed in awake head-fixed
 animals using a charge-coupled device (CCD) camera. To capture the fast
 glutamate sensor iGluSnFR kinetics, images of iGluSnFR activity were
 captured at 150 Hz sampling rate. The glutamate fluorescent indicator was
 excited using blue LED light (Luxeon K2, 470 nm), which was band-pass
 filtered using an optical filter (Chroma Technology Corp, 467-499 nm).
 Light emitted from excited fluorescent indicators was passed through a
 band-pass optical filter (Chroma, 510 to 550 nm; Semrock, New York, NY)
 and a macroscope composed of front-to-front optical lenses. The focal
 length of the lenses was adjusted such that the field of view was 8.6 ×
 8.6 mm (128 × 128 pixels, with 67 μm per pixel). To minimize the effect of
 hemodynamic signal originating from large cortical blood vessels, we
 focused the optical lens at ~1 mm depth.
 <em>Steady-state visual evoked potentials. </em>We
 used a custom-built setup of white light-emitting diodes (LEDs; luminous
 intensity 6900 mcd, color temperature 9000K, Model C513A-WSN, Cree Inc.)
 to deliver visual stimulation (the setup previously used in Gulbinaite et
 al. 2019 NeuroImage). The LED array was placed in a cardboard box, with a
 circular aperture subtending 35° of visual angle covered by a sheet of
 tracing paper (6 cm away from LED array). The visual stimulation box was
 placed on the left side of the animal, 8 cm from the left eye, at a 50°
 angle to the animal’s body axis. Flicker was implemented as a sine-wave
 modulation of the power supply to the LEDs (luminance changes from 0 to
 215 cd/m<sup>2</sup>). On each trial, the animals were exposed
 to 10 s of flicker followed by 10 s inter-trial interval of complete
 darkness (Figure 1B). Mice were exposed to flicker frequencies that ranged
 from 2 to 72 Hz (logarithmically spaced 30 different frequencies). For all
 frequencies, the stimulation started and ended with maximal luminance (215
 cd/m<sup>2</sup>, π/2 phase of the sine-wave cycle). Each
 imaging session lasted up to 30 minutes, during which each flicker
 frequency was presented twice in pseudo-random order. Each animal
 underwent 5 experimental sessions (i.e. 5 recordings on separate days).
 Thus in total, each flicker frequency was presented 10 times. 
 <em>Visual evoked-potentials. </em>In addition to
 sine-wave stimulation, we recorded responses to single light pulses (20
 ms) using 1500 cd/m<sup>2</sup> luminance stimuli to
 characterize spatiotemporal dynamics in response to single stimuli and
 on/off responses.
description: # iGluSnFR wide-field imaging responses to 2-72 Hz flicker
 [https://doi.org/10.5061/dryad.vdncjsz42](https://doi.org/10.5061/dryad.vdncjsz42) ## Description of the data and file structure There are two ZIP files containing widefield imaging DATA from two visual stimulation protocols used in the experiment: (1) sine-wave modulated luminance changes (flicker; Spatiotemporal_resonance_DATA_Flicker.zip) and (2) pulse stimulation (Spatiotemporal_resonance_DATA_Pulse.zip). Data is organized in folders. Each folder contains single-animal data, e.g. Glu1, Glu2 etc. Organization of the folders: #### (1) **Spatiotemporal\_resonance\_DATA\_Flicker.zip** In each animal's data folder you will find a subfolder named "Ready4preprocessing" with the following file types: * datafiles (*.raw); scripts to load this datafile type can be found in Spatiotemporal_resonance_CODE.zip, file named *imreadallraw.m*, for an example how to use it, see *1_Preprocessing_singleTrial_SSVER.m* line 154 * stimulus onset/offset timestamp files (*.abf); scripts to load this datafile type can be found in Spatiotemporal_resonance_CODE.zip file named *abfload.m*, for an example how to use it, see *1_Preprocessing_singleTrial_SSVER.m* line 78 * stimulus files (*Rec.mat); variable *frex* contains flicker frequency on each trial (Hz), variable *trialDuration* indicates trial duration (10 sec), *flickeringLEDsAmplitude* variable indicates light intensity setting of a custom-built LED stimulation device (arbitrary units) and it corresponds to maximum luminance of 215 cd/m2 - transformation matrix used to align across recordings (*adjusted.mat); generated using *0_Coregistration_across_recordings.m* scrip * matrix marking pixels containing the brain data (Mask*.tif; brain pixels = 1; non-brain = 0) Recordings are organized by day in alphabetic order, such that a1 and a2 are the files for recording day 1; b1 and b2 are for recording day 2, etc. **File naming**. Both *.raw and *.abf files contain information about the recording (e.g. a1_06_20180412_ssvep_92000fr_150Hz_intensity_15_2_74Hz_animal_1): * recording: e.g. a1 * file-order during data acquisition: e.g. 06 * recording date: e.g. 20180412 * number of camera frames in the recording: e.g. 92000fr * sampling rate: e.g. 150Hz * stimulation intensity (LED stimulation device): e.g. 15 * range of frequencies used in the experiment: e.g. 2-74Hz * which animal: animal 1 Each animal's data folder also contains vasculature images from each recording. These images were used to align data across recordings. Use the code *0_Coregistration_across_recordings.m* if you want to redo the realignment, or use the *adjusted.mat files instead. #### (2) **Spatiotemporal\_resonance\_DATA\_Pulse.zip** In each animal's data folder you will find the following file types: * datafiles (*.raw); scripts to load this datafile type can be found in Spatiotemporal_resonance_CODE.zip, file named *imreadallraw.m*, for an example how to use it, see *1_Preprocessing_singleTrial_Pulse.m* line 154 - stimulus onset/offset timestamp files (*.abf); scripts to load this datafile type can be found in Spatiotemporal_resonance_CODE.zip file named abfload.m, for an example how to use it, see *1_Preprocessing_singleTrial_SSVER.m* line 79 - transformation matrix used to align across recordings (*adjusted.mat; obtained using *0_Coregistration_across_recordings.m* script); - matrix marking pixels containing the brain data (Mask*.tif; brain pixels = 1; non-brain = 0) ## Code The main scripts are *1_Preprocessing_singleTrial_PULSE.m* and *1_Preprocessing_singleTrial_SSVER.m* (preprocessing is done separately for two stimulation paradigms). The other scripts will be called by this main script. The scripts in the folder allow to preprocess the data and prepare it for the analysis. The full analysis scripts are provided on: [https://osf.io/u37ar/](https://osf.io/u37ar/) **Requirements:** MATLAB Image Processing Toolbox and Signal Processing Toolbox. The code was run on MATLAB 2019b.