Article Figures & Data
Figures
- Figure 1.
Setup for dual mouse brain imaging system. a, Cartoon depiction of surgical preparation for transcranial mesoscale imaging, with custom cut coverslip and titanium bar for head fixation. x denotes the location of bregma. b, Close-up view of mouse positioning during the interaction phase of the experiment. c, Larger field of view render of the imaging system. Numbered components are as follows: (1) Raspberry Pi brain imaging camera; (2) GCaMP excitation and hemodynamic reflectance LED light guide; (3) ultrasonic microphone; (4) stationary mouse; (5) moving mouse; (6) Raspberry Pi infrared behavior camera; (7) stage translation knob; 8) stepper motor with belt controlling stage translation. Blue arrows indicate direction of motion of the translatable rail. d, Example image of whisker overlap during a whisking event captured using a high-speed camera. Image is spatially high-pass filtered to accentuate whiskers.
- Figure 2.
Mice coordinate behavior during interaction. a, Example infrared (IR) bottom camera image of both mice during the interaction phase of the experiment. Regions over each mouse’s whiskers and forelimbs are shown in magenta and cyan boxes, respectively, to estimate motion. b, Average percentage of time spent behaving during the first separated phase of the experiment (top) and the interaction phase (bottom). Intersecting regions show concurrent whisker and forelimb movements (∼5% of total time). c, Timeline of experimental paradigm (top) and ethograms for the stationary and moving mouse (bottom). d, Cross-correlation of each mouse’s binary behavior vectors during the interaction phase where whiskers could freely touch open (black) versus during trials where a mesh (red) or opaque (blue) barrier were placed between the animals, preventing physical touch. Behaviors across mice during the interaction phase were significantly correlated near 0 lag when there was no barrier present (open condition). e, Intersection over union (Jaccard similarity index) for the behavior vectors was significantly greater during the interaction phase across mice for trials with no barriers present compared with mesh or opaque barrier trials. n = 33 mouse pairs; one-way ANOVA with post hoc Bonferroni test for multiple comparisons; F(2,42) = 10.1; p = 2.6 × 10−4. See Extended Data Figures 2-1 and 2-2 for additional data. Asterisk indicates significance.
- Figure 3.
Interbrain synchronization during interaction. a, Example images of dorsal cortical windows for stationary mouse (top) and moving mouse (bottom). b, Representative example of median GCaMP6s activity across the cortical mask for each mouse. c, Pearson correlation coefficients of the two global signals were significantly greater during the interaction phase than either of the two separate phases (n = 35 mouse pairs, p < 0.001; repeated measures ANOVA with post hoc Bonferroni correction for multiple comparisons). Interbrain correlations during interaction were significantly greater than trial-shuffled interaction-phase pairings (n = 35 mouse pairs vs n = 595 shuffled mouse pairs, p < 0.001; t test). d, Interbrain signal correlation does not depend on cagemate (CM) versus noncagemate (NCM) experiments (t test, p = 0.66, cagemates n = 20 mouse pairs, noncagemates n = 13 mouse pairs). e, Expanded view of global signals during interaction phase with behavior annotations overlaid. f, Global cortical signal ΔF/F0 is positively correlated with duration of whisking or forelimb movement (Spearman correlation coefficient; r = 0.40; p < 0.001). g, Example image of transcranial mask with putative cortical regions labeled. Abbreviations: ALM, anterior lateral motor cortex; M2, secondary motor cortex; wM1, whisker motor cortex; aBC, anterior barrel cortex; pBC, posterior barrel cortex; HL, hindlimb; FL; forelimb; lPTA; lateral parietal association area; RS, retrosplenial cortex; V1, primary visual cortex. h, Averaged interbrain correlation matrices across all experiments during the period before interaction (left) and the period during interaction (right). i, Change in interbrain correlation for each region of interest against all other regions, averaged across mice (n = 35 mouse pairs, *p < 0.05; two-way ANOVA with post hoc Tukey–Kramer test). Bars show mean ± SE. j, Time-varying interbrain coherence, computed with a 45-s window on the global cortical signals, and averaged across all experiments, shows an increase in coherence from 0 to 0.2 Hz during the interaction phase (white dashed lines). k, l, Pearson correlation coefficients of the two global signals showed no significant difference between trial phases when an opaque partition (k; p = 0.34, repeated measures ANOVA) or copper mesh (l; p = 0.88, repeated measures ANOVA) were placed between the mice, preventing whisker contact. Asterisks indicate significance. NS indicates not significant. See Extended Data Figures 3-1, 3-2, 3-3, and 3-4 for additional data.
- Figure 4.
Ridge regression model in open interaction trials confirms somatotopic representation of whisker and forelimb behaviors across animals. a, Binary event vectors (colored lines) considered in the ridge regression model. Model included nine separate interaction trials which had been concatenated together (dotted lines; partner limb and whisker behavioral variables were only assessed during the together phase). b, Explained variance for the full model after 10-fold cross-validation, projected back onto the cortical map. Scale bar: 2 mm. c–e, Unique contribution for each stationary mouse behavioral model variable; taken as the difference in explained variance between the full model and the reduced model with the specified variable randomly permuted. f, g, Same as c–e, except for the partner mouse behaviors. h–j, Same as c–e, except for trial-associated events. See Extended Data Figures 4-1, 4-2, 4-3, and 4-4 for additional data.
Tables
Description # Manufacturer Part number Aluminum Breadboard 18 × 24 × 1/2”, 1/4”−20 Taps 1 Thorlabs MB1824 Ø1” Pillar Posts with 1/4”−20 Taps, 2” 4 Thorlabs RS2 Ø1” Pillar Posts with 1/4”−20 Taps, 3” 8 Thorlabs RS3 Ø1” Pillar Posts with 1/4”−20 Taps, 6” 8 Thorlabs RS6 Clamping Fork, 1.24” Counterbored Slot, Universal 4 Thorlabs CF125 Ø1/2” Pedestal Post Holder 3 Thorlabs PH2E Ø1/2” Optical Post, SS, 8–32 Setscrew, 1/4”−20 Tap, L = 8” 3 Thorlabs TR8 Ø1/2” Optical Post, SS, 8-32 Setscrew, 1/4”−20 Tap, L = 12” 1 Thorlabs TR12 Right-Angle Clamp for Ø1/2” Posts, 3/16” Hex 2 Thorlabs RA90 Ø25 mm Post Spacer, Thickness = 3 mm 1 Thorlabs RS3M RPi Camera (F), Supports Night Vision, Adjustable-Focus 2 Waveshare 10299 Flex Cable for Raspberry Pi Camera or Display - 2 m 2 Adafruit 2144 Triple light guide and imaging parts Triple Bandpass Filter (camera) 1 Chroma 69013m Liquid Light Guide 1 Thorlabs LLG0338-4 SM1 Adapter for Liquid Light Guide 1 Thorlabs AD3LLG SM1 Lens Tube, 3.00” Thread Depth 3 Thorlabs SM1L30 SM1 Lens Tube, 1.00” Thread Depth 3 Thorlabs SM1L10 SM1 Lens Tube, 2.00” Thread Depth 1 Thorlabs SM1L20 SM1 Retaining Rings 2 Thorlabs SM1RR-P10 Dichroic Cage Cube 2 Thorlabs CM1-DC Cage Cube Connector 1 Thorlabs CM1-CC Compact Clamp with Variable Height 1 Thorlabs CL3 Bi-Convex Lens 4 Thorlabs LB1761 AT455DC size: 26 × 38 mm 1 Chroma AT455DC 25 × 36 mm Longpass Dichroic Mirror, 550 nm Cutoff 1 Thorlabs DMLP550R Ø1” Bandpass Filter, CWL = 620 ± 2 nm, FWHM = 10 ± 2 nm 1 Thorlabs FB620-10 ET480/30× size: 25 mmR R=Mounted in Ring 1 Chroma ET480/30x Ø1” Bandpass Filter, CWL = 440 ± 2 nm, FWHM = 10 ± 2 nm 1 Thorlabs FB440-10 Royal-Blue (448 nm) Rebel LED 1 Luxeon Star SP-01-V4 Blue (470 nm) Rebel LED 1 Luxeon Star SP-01-B6 Red-Orange (617 nm) Rebel LED 1 Luxeon Star SP-01-E6 Machined parts (stainless steel) Milled as-1.50_2_v2.SLDPRT 3 Spacer_with_wire_hole_as-.500_v2.SLDPRT 3 LED_mount_as-1.50_v2.SLDPRT 3 3D-printed parts (black PLA) TripleLEDLightGuide_Base.stl 1 Light_Guide_Mount_V2.stl 1 Data structure Type of test Test values and power Figure 2d All groups normally distributed One-way ANOVA with post hoc Bonferroni multiple comparisons test F(2,42) = 10.2; p = 2.4 × 10–4 Figure 2e All groups normally distributed One-way ANOVA with post hoc Bonferroni multiple comparisons test F(2,42) = 10.1; p = 2.6 × 10–4 Figure 3c All groups normally distributed Repeated measures ANOVA with Bonferroni’s multiple comparisons test F(2,68) = 11.2; p = 0.002 Figure 3d All groups normally distributed Two sample t test t(33) = −0.44; p = 0.66 Figure 3f Groups not normally distributed Spearman correlation coefficient r = 0.40; p = 6.0 × 10−81 Figure 3i All groups normally distributed Two-way ANOVA with post hoc Bonferroni test Trial phase: F(1,680) = 136; p = 1.0 × 10−28 Brain region: F(9,680) = 0.81; p = 0.61 Interaction: F(9,680) = 1.2; p = 0.30 Figure 3k All groups normally distributed Repeated measures ANOVA F(2,28) = 1.0; p = 0.34 Figure 3l All groups normally distributed Repeated measures ANOVA F(2,30) = 0.22; p = 0.88 Extended Data Figure 2-2a All groups normally distributed One-way ANOVA Whisker movements: F(2,51) = 0.68; p = 0.51 Forelimb movements: F(2,51) = 1.02; p = 0.37 Extended Data Figure 2-2b All groups normally distributed One-way ANOVA Whisker movements: F(2,45) = 0.28; p = 0.76 Forelimb movements: F(2,45) = 0.41; p = 0.67 Extended Data Figure 2-2c All groups normally distributed One-way ANOVA Whisker movements: F(2,30) = 0.85; p = 0.44 Forelimb movements: F(2,30) = 0.05; p = 0.95 Extended Data Figure 3-1b All groups normally distributed One-way ANOVA F(2,9) = 0.91; p = 0.44 Extended Data Figure 3-3b All groups normally distributed Two-way ANOVA with post hoc Bonferroni test Trial phase: F(1,680) = 44.1, p = 6.3 × 10–11 Brain region: F(9,680) = 0.30, p = 0.98 Interaction: F(9,680) = 0.01, p = 1.0
Movies
- Movie 1.
Cortical signal responses for interacting mice. Data from two head-restrained tTA-GCaMP6s mice at the onset of the interaction phase. The two pseudo-colored images show the processed calcium images from the left and right mouse. The color bar indicates the ΔF/F0 of the signals. Behavior video is shown below.
Extended Data Figure 2-1
Protocol for estimating behaviors. a, Raw motion energy within the whisker region of interest. b, Smoothed whisker motion energy. Motion energy exceeding a threshold of the mean + 1 SD (red line) is classified as binary movement behavior (shaded areas). c, Example montage of whisking behavior. Images are taken from beneath the mouse (see Figs. 1 and 2). Individual frames are cropped and displayed with saturated pixels, and a Canny edge detection algorithm was run over the whisker region to enhance visualization of whiskers. A whisker protraction event can be seen at 0.14 s. d–f, Same as a–c for forelimb movements. Individual frames are cropped to aid with visualization. Left and right paws are labelled with red and cyan markers, respectively. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
Number of whisker or forelimb movements do not change between trial phases. Number of whisker movements (left) and forelimb movements (right) for all trial phases during open social interaction experiments (a) and barrier controls (b, c). During: during interaction period while mice are stationary and together (face to face); before/after: before/after interaction period while mice are stationary and apart. No significance between trial phases for all conditions (open n = 33 trials, mesh n = 16 trials, opaque n = 11 trials). Download Figure 2-2, TIF file.
Extended Data Figure 3-1
No interanimal correlation observed in Thy1-GFP mice. a, Representative example of GFP activity global signals over the entire cortical mask for the stationary mouse (top) and moving mouse (bottom). Green shading indicates period when mice were together. b, Pearson correlation coefficients computed at each phase of the experiment. Dashed line shows median correlation coefficient between global signals for the GCaMP mice during the interaction phase of the experiment from Figure 3c. No significant difference was observed between phases. n = 4 trials, p = 0.4, one-way ANOVA. Download Figure 3-1, TIF file.
Extended Data Figure 3-2
Seed pixel correlation maps for putative cortical ROIs from an example mouse. Pearson correlation coefficients over the entire trial (spanning both separate and together phases) were calculated between every pixel within the cortical mask and the averaged signal obtained from a five by five-pixel neighborhood chosen from the specified region in each panel. Scale bar: 2 mm. Download Figure 3-2, TIF file.
Extended Data Figure 3-3
Intrabrain correlations increase during interaction phase of the trial. a, Averaged intrabrain correlation matrices across all experiments during the period before interaction (left) and the period during interaction (right). b, Change in intrabrain correlation for each region of interest against all other regions, averaged across mice (n = 35 mouse pairs, *p < 0.05; two-way ANOVA with post hoc Tukey–Kramer test). Bars show mean ± SE; y-axis is scaled similarly to interbrain correlation changes from Figure 3i. Download Figure 3-3, TIF file.
Extended Data Figure 3-4
No ultrasonic vocalizations detected during social-interaction tests. Example data from the social interaction experiment (top), compared to a control experiment taken from a breeder mouse introduced to a female (bottom). Ultrasonic vocalizations are clearly observed in the female stimulus control experiment, but not in the two-mouse imaging experiments. Download Figure 3-4, TIF file.
Extended Data Figure 4-1
Ridge regression model on an additional mouse in open interaction trials. a–j, Binary event vectors (colored lines) considered in the ridge regression model. Model included three separate interaction trials which had been concatenated together (dotted lines; partner limb and whisker behavioral variables were only assessed during the together phase). b, Explained variance for the full model after 10-fold cross-validation, projected back onto the cortical map. Scale bar: 2 mm. c–e, Unique contribution for each stationary mouse behavioral model variable; taken as the difference in explained variance between the full model and the reduced model with the specified variable randomly permuted. f, g, Same as c–e, except for the partner mouse behaviors. h–j, Same as c–e, except for trial-associated events. Download Figure 4-1, TIF file.
Extended Data Figure 4-2
Ridge regression model on an additional mouse in open interaction trials. a–j, Binary event vectors (colored lines) considered in the ridge regression model. Model included two separate interaction trials which had been concatenated together (dotted lines; partner limb and whisker behavioral variables were only assessed during the together phase). b, Explained variance for the full model after 10-fold cross-validation, projected back onto the cortical map. Scale bar: 2 mm. c–e, Unique contribution for each stationary mouse behavioral model variable; taken as the difference in explained variance between the full model and the reduced model with the specified variable randomly permuted. f, g, Same as c–e, except for the partner mouse behaviors. h–j, Same as c–e, except for trial-associated events. Download Figure 4-2, TIF file.
Extended Data Figure 4-3
Ridge regression model in mesh barrier trials. a, Binary event vectors (colored lines) considered in the ridge regression model. Model included four separate interaction trials which had been concatenated together (dotted lines; partner limb and whisker behavioral variables were only assessed during the together phase). b, Explained variance for the full model after 10-fold cross-validation, projected back onto the cortical map. Scale bar: 2 mm. c–e, Unique contribution for each stationary mouse behavioral model variable; taken as the difference in explained variance between the full model and the reduced model with the specified variable randomly permuted. f, g, Same as c–e, except for the partner mouse behaviors. h–j, Same as c–e, except for trial-associated events. Download Figure 4-3, TIF file.
Extended Data Figure 4-4
Ridge regression model on an additional mouse in mesh barrier trials. a–j, Binary event vectors (colored lines) considered in the ridge regression model. Model included four separate interaction trials which had been concatenated together (dotted lines; partner limb and whisker behavioral variables were only assessed during the together phase). b, Explained variance for the full model after 10-fold cross-validation, projected back onto the cortical map. Scale bar: 2 mm. c–e, Unique contribution for each stationary mouse behavioral model variable; taken as the difference in explained variance between the full model and the reduced model with the specified variable randomly permuted. f, g, Same as c–e, except for the partner mouse behaviors. h–j, Same as c–e, except for trial-associated events. Download Figure 4-4, TIF file.
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