Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: LED wavelengths must be selected to provide GCaMP6 excitation, distinguish between oxy- and deoxyhemoglobin absorption for oximetry, and enable correction of GCAMP6 fluorescence emission for correction of confounding hemoglobin absorption. (A) GCaMP6 dynamics are imaged using fluorescence measurements, wherein the fluorophore has a peak excitation at λ = 497nm and peak emission at λ = 512nm. A longpass filter at λ = 514nm is used to block fluorescence excitation light. (B) Spectra from the excitation LED (λ = 454nm, bandpass filtered at 460/60nm). A reference (λ = 523nm) LED is used for correcting for changes in optical properties due to the presence of absorptive hemoglobin. The transmission curve for the 514nm longpass filter at detection is shown for reference. (C) Schematic of the combined GCaMP and OIS imaging system. GCaMP excitation and reference LEDs share a collinear optical path using dichroic mirrors. Green (λ = 523nm), yellow (λ = 595nm), and red (λ = 640nm) are used together to determine differential concentration changes of hemoglobin. Detection is done using a cooled EMCCD with an 85mm f/1.4 lens.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Fluorescence, Blocking Assay, Transmission Assay, Imaging, Concentration Assay

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: Based on previous studies, Thy1 /GCaMP6 fluorescence should provide a signal predominantly driven by cortical neuronal neurons and that is faster than hemodynamic measures. (A) Epifluorescence (i) and confocal (ii) images of 50um coronal slices collected from a representative Thy1 /GCaMP6 mice. GCaMP6 expression (green) is located throughout the cortex (i) and localized to neuronal soma (ii, blue-DAPI stain) and can be seen throughout cortical layers I-VI. (B) Map of stimulus evoked responses to electrical stimulation of left hindpaw (thresholded at >50% peak response) in the anesthetized state. (C) Time series of evoked responses sampled using masks created from the right half of the 50% peak response images in part B. Green reflectance (green trace, top panel) is used to correct raw GCaMP6 emission (teal trace) for changes in absorption due to hemodynamics using a ratiometric approach (see ; the corrected trace is black). The corrected GCaMP6 trace (black) is sensitive to each stimulus presentation (black trace, lower panel), whereas HbO 2 (red) and HbR (blue) follow the slower, canonical hemodynamic response. Error bars are standard error of the mean. Data from 36 blocks across 7 mice are included in all evoked analysis.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Fluorescence, Expressing, Staining

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: Time series of spontaneous GCaMP6 activity filtered over the canonical functional connectivity band: (A) 0.009–0.08H, (B) 0.08–0.4Hz, and (C) the delta band: 0.4–4.0Hz. Corresponding functional connectivity maps constructed using a seed from left motor (Mot.) cortex (black circle) in 0.009–0.08Hz (top right), 0.08–0.4Hz (center right), and 0.4–4.0Hz (bottom right) spontaneous data are also shown. Black and red traces correspond to left and right motor cortices, respectively. The green trace is sampled from right cingulate cortex. Over both frequency bands, left and right motor cortex show highly correlated activity whereas right motor with left cingulate show reduced temporal correlation. At the pixelwise level, correlating the time series from the left motor (black) with every pixel in the brain produces a map showing homotopic functional somatosensory structure across hemispheres. Mot.: Motor, Cing.: Cingulate.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Activity Assay, Functional Assay, Construct

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: (A) Functional connectivity maps were constructed using time windows of spontaneous 0.4–4.0Hz GCaMP6 (awake) data of increasing length from one mouse (Mouse 2). Windows were increased in length by ~2.6s up to ~300s. Spatial similarity (Dice coefficient) was calculated between each of these functional connectivity maps for all networks relative to their corresponding group level map. The functional connectivity maps calculated by using a seed in left motor cortex are highlighted as an example. Across all networks, similarity scores converge at 0.9 with a window length of ~30s. (B) Seed-based functional connectivity maps from Mouse 2 constructed using 30s of spontaneous GCaMP6 data. (C) For each of 7 networks, the same 30s window of spontaneous data for each network was compared to the corresponding group-level network functional connectivity map for each of seven mice (circles). The stability of similarity of 30s of data is present across network and across mice. (D) Using consecutive 30s windows (sampled every ~2.6s) from a single 300s imaging session from each mouse, the spatial similarity between all 30s motor functional connectivity maps and the group level motor functional connectivity map is shown to be relatively stationary. Error bars are standard error of the mean.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Functional Assay, Construct, Imaging

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: Based on the temporal delay between evoked GCaMP6 and HbO 2 /HbR responses combined with similar observed 0.08–0.4Hz functional connectivity structure, delay-shifted GCaMP6 likely shares functional network information with HbO 2 and HbR. (A) GCaMP6 (black), HbO 2 (red), and HbR (blue) evoked responses to 2Hz 0.5mA electrical stimulation of left hind paw (see ). Data from a sampling window spanning from approximately t = 0s to t = 7.5s were included in subsequent cross-correlational delay analysis. (B) Cross-correlation from the GCaMP6 and HbO 2 time courses in (A), and the GCaMP6 and HbR time courses in (A), both show a delay of 0.65s between responses. (C) An example of spontaneous GCaMP6, HbO 2 , and HbR time traces from the right motor cortex seed over the 0.08–0.4Hz band. HbO 2 and HbR data was shifted based on the lag with the maximum correlation calculated between the spontaneous hemoglobin data and spontaneous GCaMP6 data per seed (as shown by arrows). (D) Top and Bottom: Seed-based group-averaged functional connectivity mapping in filtered (0.08–0.4Hz) anesthetized data using HbO 2 (top) or HBr (bottom) and cross-correlation curves (with mean delays of maximum correlation in black text with standard errors of the mean in parenthesis) between each HbO 2 network seed trace and corresponding GCaMP6 seed trace. Middle: Seed-based GCaMP6 functional connectivity maps and corresponding intercontrast seed-based functional connectivity maps for GCaMP6 vs temporally-shifted HbO 2 (top) and GCaMP6 vs temporally-shifted HbR (bottom). Correlation between spontaneous HbO 2 and GCaMP6 contrasts (filtered between 0.08–0.4Hz) are better optimized by removing the intercontrast time shift between these contrasts compared to the analogous correlation between spontaneous HbR and GCaMP6 contrasts.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Functional Assay, Sampling

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: (A) Seed-based functional connectivity maps for seven canonical functional networks in anesthetized and awake GCaMP6 mice (N = 7) at delta-band frequencies. Under anesthesia (top), delta activity (0.4–4.0Hz) drives a strongly correlated/anticorrelated network structure between anterior and posterior brain regions that is not observed in awake animals (second row). This high magnitude correlation feature is preserved in the difference images (third row) and after performing pixelwise t-tests and thresholding the t-statistic maps at a Bonferroni adjusted α = 3.1e-6 (fourth row). Together, these results show that the topography of functional connectivity maps in the anesthetized state is less sensitive to seed-based network-specific features compared to functional connectivity topography during wakefulness. Finally, horizontal line profiles through the center of each seed show higher ipsilateral and contralateral focality in the awake (red) functional connectivity maps compared to anesthetized (blue) maps (fifth row and table in ). (B) Seed-based pairwise functional connectivity matrices. Regional correlation coefficients between each seed reveal heterogeneous connectivity structure within each contrast across different states. Clusters of networks are present in the anesthetized matrix (top), including between cingulate, motor, and somatosensory networks that are diminished in the awake mouse (bottom).

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Functional Assay, Activity Assay

Journal: PLoS ONE

Article Title: Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice

doi: 10.1371/journal.pone.0185759

Figure Lengend Snippet: (A) Power spectra from the whole brain from a representative mouse. The presence of a peak at ~1.5Hz in both the anesthetized GCaMP6 (black) and EEG (dark green) corresponds to increased delta band (0.4–4.0Hz) activity observed in the brain under anesthesia and deep, slow-wave sleep (see ). Note that this peak is absent when the mouse is awake (GCaMP6, transparent black, EEG, transparent green). Maximum cross-correlation between the whole brain signal and each pixel’s timeseries were calculated across the FOV. Cortex-wide delay topography may underlie the striking correlated/anti-correlated structure observed in the anesthetized functional connectivity maps due to the effect of phase shifts on the magnitude and sign of zero-lag correlations. Asterisks represent significant (p<0.0001) comparisons across state following two-sample Welch’s t-tests. Error bars are standard error of the mean. (B) Group-wise delay maps in anesthetized (top) and awake (bottom) mice (N = 7). The map from anesthetized mice show a temporal separation along the anterior-posterior axis of the brain, with anterior somatomotor regions leading the whole brain signal by < = 0.06s and posterior visuoparietal areas lagging the whole brain signal by < = 0.06s. This time shift is in agreement with the striking correlated/anti-correlated structure in the delta band functional connectivity maps (see ). This structure is not present in awake mice. Group-average maps are masked using pixels identified to be significant (p<0.001) following one-sample t-testing. (C) Top: Delay maps from consecutive 300s epochs from one mouse as it transitions out of anesthesia to awake reveals a loss of anterior-posterior delay structure across time. Middle section: functional connectivity maps for a left visual seed from each epoch (top row), difference images between each successive epoch and E1 (middle row), and pixelwise t-statistic maps after performing paired t-tests between E1 and each other epoch (bottom row). The boundary of the area of maximum delay in the anterior, cingulate network in the delay map in E1 (top section of C) is overlaid on the E6 t-statistic map for reference. These data show that the correlation magnitude of the parietal region differs across state and that there is agreement across delay and functional connectivity analyses in the implicated cingulate network. All analysis was performed after dividing each 300s epoch into ten 30s windows. Bottom: Spectrogram over the delta frequency band (0.4–4.0Hz) from each epoch showing the loss of delta-band activity (peak at ~1.5Hz) with time across epochs.

Article Snippet: Transgenic mice expressing GCaMP6 driven by the thymus cell antigen 1 ( Thy1 ) promotor were acquired from Jackson Laboratories (JAX Strain: C57BL/6J- Tg(Thy1-GCaMP6f)GP5 .

Techniques: Activity Assay, Functional Assay