Spontaneous cortical activity alternates between motifs defined by regional axonal projections

Abstract

Using millisecond-timescale voltage-sensitive dye imaging in lightly anesthetized or awake adult mice, we show that a palette of sensory-evoked and hemisphere-wide activity motifs are represented in spontaneous activity. These motifs can reflect multiple modes of sensory processing, including vision, audition and touch. We found similar cortical networks with direct cortical activation using channelrhodopsin-2. Regional analysis of activity spread indicated modality-specific sources, such as primary sensory areas, a common posterior-medial cortical sink where sensory activity was extinguished within the parietal association area and a secondary anterior medial sink within the cingulate and secondary motor cortices for visual stimuli. Correlation analysis between functional circuits and intracortical axonal projections indicated a common framework corresponding to long-range monosynaptic connections between cortical regions. Maps of intracortical monosynaptic structural connections predicted hemisphere-wide patterns of spontaneous and sensory-evoked depolarization. We suggest that an intracortical monosynaptic connectome shapes the ebb and flow of spontaneous cortical activity.

Figure 1. Unique and consensus activation patterns during multiple forms of sensory stimulation

(A) Schematic of unilateral craniotomy showing imaged cortical regions. (B) Photomicrograph of wide unilateral craniotomy, with bregma marked as white circle (top, left). Patterns of cortical activation following (i) stimulation of contralateral C2 whisker, (ii) auditory stimulation, (iii) contralateral forelimb stimulation, (iv) contralateral hindlimb stimulation, and (v) visual stimulation of contralateral eye in isoflurane (0.5%) anesthetized mouse. Note midline activation after all sensory stimulation forms (white arrows), 10–25 ms following primary sensory cortex activation. Responses are mean of 20 trials. Second panel of VSD responses in second row shows anterior (A), posterior (P), medial (M) and lateral (L) directions. (C) Representative montage of VSD response following visual stimulation of left eye in bilateral craniotomy. Responses are mean of 10 trials. (D) (i–iii) Three sequences of spontaneous cortical activity in an anesthetized mouse. See Supplementary Figs. 1, and 4 for group data analysis. (E) Time course of the amplitude of repeated (0.05 Hz), forelimb sensory stimulation-evoked responses measured from an LFP electrode placed within FLS1 before and after RH1692 dye loading. (F) Summary of mean peak amplitude in E. (G) Time course of normalized mean spontaneous LFP power. Recording resumed 30 min following RH1692 washout to encompass periods prior to, during, and after intermittent VSD excitation (denoted as light exposure; total illumination time 300–600 s). (H) Summary of LFP power in G. Error bars represent s.e.m.

Figure 2. Sensory and photostimulation-induced activation exhibit modality specific source locations and termination at a common sink location

(A)(i) Whisker stimulation-evoked VSD activation (average of 20 trials). Black arrows indicate the direction of velocity of VSD signal spread. Relative magnitude of velocity is indicated by arrows size. (ii) For response in (i), measurements of absolute velocity are represented in pseudocolor. Streamlines indicate local direction of velocity flow. (B) Position of maximum velocity (center of mass) of VSD signal spread from sensory stimulation following initial activation on cortex relative to bregma at six 6.67 ms intervals indicated as 1 to 6. Bregma is at coordinate (0,0). (C) Position of sink locations for each sensory modality plotted on cortex relative to bregma. All forms of sensory stimulation shared a common sink location within parietal association area (ptA), dotted box (p>0.05, n=8 mice). Visual stimulation resulted in an additional, sink location within anterior medial cortex (***p<0.001, n=8 mice). (D) Positions of maximum velocity of VSD signal spread following sensory (forelimb and whisker) and photostimulation (FLS1 and BCS1). Maximum velocity positions for each trajectory were normalized to their initial positions. Trajectories from sensory stimulation and photostimulation were similar (p>0.05, for sensory versus photostimulation of the same of modalities, n=7 mice). (E) Positions of sink locations for sensory and photostimulation-induced activation plotted on cortex relative to bregma. Sink locations between sensory-evoked and photostimulation induced activation were similar (p>0.05 for all comparisons, n=7 mice). Error bars represent s.e.m.

Figure 3. Spontaneous, sensory-, and photostimulation-evoked cortical VSD maps share similar regional patterns

(A) Representative correlation maps from a seed-pixel located within the primary representation of forelimb somatosensory cortex (FLS1) generated from VSD signals after sensory stimulation of contralateral forelimb (eFLS1, left), during 300 s of cortical spontaneous activity (sFLS1, center), or after direct photostimulation of FLS1 (pFLS1, right). (B–D). Similar to A but locating the seed-pixel within (B) primary representation of hindlimb cortex (HLS1), (C) primary representation of C2 barrel cortex (BCS1), or (D) secondary representation of C2 barrel cortex (BCS2). Inset images represent magnification of area denoted by dotted red box and were all re-scaled to the upper calibration bar. Position of bregma marked as white circle. Compass arrows indicate anterior (A), posterior (P), medial (M) and lateral (L) directions. Note that the correlation coefficients (CC) are color-coded. See Supplementary Figs. 3, 5 for group data analysis.

Figure 4. Patterns of sensory-evoked and spontaneous activity in quiet awake mice are similar to those observed under anesthesia

(A) Individual example of neck muscle electromyography (EMG) (>1 Hz) from a head-restrained mouse under isoflurane (0.5%) anesthesia (top trace), or 1 h after awakening (bottom trace). The power spectrum of the EMG signals show different spectra between states of anesthesia (blue) and wakefulness (red). (B) Representative cortical VSD signals in response to contralateral (i) visual or (ii) auditory stimulation during anesthesia (top panel) or in quiet awake states (bottom panel). Images represent the average of 10 trials of stimulation. Note that responses are scaled differently. (C) Quantification of VSD signals to (i) auditory stimulation or (ii) visual stimulation in anesthetized (right panels) or quiet awake states (left panels). Plots are average of VSD signals measured from 5 × 5 pixel boxes (~ 0.11 mm²) placed within (i) A1 (blue traces) and ptA (red traces) for auditory stimulation, or (ii) V1 (blue traces) and M2/AC (red traces) for visual stimulation. (D) Statistical quantification of peak amplitude (left panels), time to peak (center panels), and decay (right panels) of the (i) auditory-evoked or (ii) visual-evoked VSD responses during anesthesia or quiet awake states. (E) Representative montages showing two distinct time epochs (each 633.3 ms) of VSD imaging of spontaneous cortical activity during (i) anesthesia, (ii) or in a quiet awake states. Position of bregma marked as white circle. Compass arrows indicate anterior (A), posterior (P), medial (M) and lateral (L) directions. Error bars represent s.e.m.

Figure 5. Correlation maps generated from sensory-evoked or spontaneous activity during quiet wakefulness are similar to maps obtained during anesthesia

(A) Representative correlation maps generated from a seed-pixel from primary auditory cortex (A1) calculated from 300s of cortical spontaneous activity in isoflurane induced anesthesia (1^st column from left), quiet wakefulness (2nd column from left) or after auditory stimulation in anesthetized or quiet awake states (3^rd and 4^th column from left respectively). Inset images represent magnification of area denoted by dashed red box and were all re-scaled to the right calibration bar. (B–D) Similar to A, the correlation maps were calculated within primary visual cortex (V1) (B), primary representation of hindlimb somatosensory cortex (HLS1) or secondary representation of hindlimb somatosensory cortex (HLS2) (C, D). CC denotes correlation coefficient. Position of bregma marked as white circle. Compass arrows indicate anterior (A), posterior (P), medial (M) and lateral (L) directions. See Supplementary Fig. 7 for group data analysis.

Figure 6. Spontaneous cortical activity can be decomposed into unique repeating sensory motifs

(A) Representative montages show examples of VSD response to five different forms of sensory stimulation. Each image of spontaneous activity was compared with the sensory evoked templates (pattern at initial cortical response, highlighted by solid red box). VSD responses represent the average of 20–45 trials of stimulation. (B) Top, plot of concurrent correlation of instantaneous patterns of spontaneous activity (2.6 s of spontaneous VSD data as denoted by red dashed square in C with multiple templates of sensory evoked responses. Spontaneous activity was considered to be a “match” to the evoked template if the correlation value was greater than a given threshold (see Supplementary Fig. 8A). Bottom, representative montages (20 ms intervals) showing cortical spontaneous activity that resembles sensory stimulation derived templates, corresponding to shaded gray regions highlighted in the top panel. (C) Pseudocolor image representing a 30 s time course of concurrent correlation of different templates of sensory evoked responses (rows representing whisker (WK), auditory (AD), visual (VL), forelimb (FL), and hindlimb (HL)) with a 30 s epoch of spontaneous activity. The color of each column of pixels represents the correlation value at different times. (D) Concurrent correlation as in C, but using templates of sensory-evoked responses containing the first three frames of sensory-evoked activity following the initial response (highlighted by dashed red box in A).

Figure 7. Comparison of cortical axonal projection patterns to correlation maps of spontaneous cortical activity

(A)(i). Schematic showing injection of AAV tracer to map axonal projections of different cortical regions within the adult mouse brain (data obtained from the Allen Institute for Brain Science). Color-coded cortical locations are as described in Fig. 1. To quantitatively compare the axonal projection maps with functional correlation maps, 2D representations of Allen Mouse Brain Connectivity datasets were created (ii–vi, See Methods). (vii) 3D reconstruction, using Brain Explorer software from Allen Institute for Brain Science, of primary motor cortex axonal projections. (B) Left, example of primary somatosensory trunk (S1-Trunk) and primary motor (M1) cortex axonal projection maps are shown. Pixel intensities scaled logarithmically. Seed-pixel correlation maps obtained from VSD imaging of spontaneous activity within S1-Trunk or M1 cortex are shown in center. Spatial co-localization of axonal projection (left) and correlation (center) maps are shown in right. White dashed circles indicate position of secondary somatosensory cortex. Blue and white stars indicate positions of tracer injection and the seed-pixel, respectively. Position of bregma marked as white circle. Compass arrows indicate anterior (A), posterior (P), medial (M) and lateral (L) directions. See Fig. 8 for group data analysis.

Figure 8. Axonal projection maps are most similar to their corresponding functional maps

(A)(i) Axonal projection maps (left) of primary barrel somatosensory cortex (BCS1). Blue asterisk denotes AAV injection site. Pixel intensities scaled logarithmically. Note relatively weak pixel intensity in the area outside the injection site (dashed circle). Seed-pixel correlation map (center; presumed similar location to AAV injection site) of BCS1 obtained from spontaneous activity. White asterisk denotes location of seed-pixel. Right, overlay of anatomical and correlation maps. Inset images represent the areas denoted by dashed red boxes and were all re-scaled to the upper right calibration bars. Similar to (i), axonal projection and correlation maps shown for (ii) retrosplenial cortex (RS) and (iii) parietal association cortex (ptA). (B) Matrix showing similarity between VSD correlation (n=7 mice) and axonal projection maps for nine cortical regions. (C) Ordered strength of ptA axonal projections (black) to eleven regions of interest (ROIs) compared to normalized correlation maps obtained from spontaneous activity (red; n=10 mice) or ChR2-evoked VSD responses (blue; n=6 mice). ROIs are ordered based on strength of ptA axonal projection density to other ROIs. The rank order of axonal projection strength was similar to the rank order of functional connectivity strength measured from both spontaneous activity and ChR2-evoked responses (r_{projection(ptA)&spontaneous(ptA)} = 0.92, r_{projection (ptA)&ChR2(ptA)} = 0.89, p<0.001 both comparisons). Tests of significance were made against a null hypothesis of axonal projection strength and HL functional connectivity strength (r_{projection(ptA)&spontaneous(HLS1)}=−0.37, r_{projection(ptA)&ChR2(HLS1)} = −0.38, p>0.20 both comparisons).