Author response: Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Understanding circuit-level manipulations that affect the brain's capacity for plasticity will inform the design of targeted interventions that enhance recovery after stroke. Following stroke, increased contralesional activity (e.g. use of the unaffected limb) can negatively influence recovery, but it is unknown which specific neural connections exert this influence, and to what extent increased contralesional activity affects systems- and molecular-level biomarkers of recovery. Here, we combine optogenetic photostimulation with optical intrinsic signal imaging to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice either recovered spontaneously or received chronic optogenetic excitation of right S1FP over the course of 4 weeks. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of stimulus-evoked activity in the unaffected limb. This maneuver also prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited transcriptional changes in several genes relevant for recovery. Our results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of cortical activity and several neuroplasticity-related genes after stroke, and highlight the importance of site selection for targeted therapeutic interventions after focal ischemia. Editor's evaluation Overall, Bice et al., present new work using optogenetics-based stimulation to test how this affects stroke recovery in mice. The study provides interesting evidence that this stimulation may be harmful, and not helpful, and opens new avenues for both basic and therapeutic research. https://doi.org/10.7554/eLife.68852.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Stroke causes direct structural damage to local brain circuits and indirect functional damage to global networks resulting in behavioral deficits spanning multiple domains (Murphy and Corbett, 2009; Baldassarre et al., 2016). In the months following stroke, functional magnetic resonance imaging (fMRI) studies have shown that local circuits lost to infarction remodel in peri-infarct cortex (Cramer and Riley, 2008). This process, termed 'remapping', appears to be tightly focused to perilesional regions in patients exhibiting good recovery, and suggests that peri-infarct cortex may take over the function of brain regions lost to stroke (Brown et al., 2009; Dijkhuizen et al., 2001; Nudo et al., 1996). Similar studies examining brain-wide patterns of synchronized, resting-state, hemodynamic activity after stroke have shown that global patterns of resting-state functional connectivity (RSFC) are also altered (Posner et al., 1988; Felleman and Van Essen, 1991). Shortly after ischemic stroke, disruption of interhemispheric homotopic RSFC predicts poor motor and attentional recovery (Carter et al., 2012; He et al., 2007), and in rats, restoration of homotopic RSFC correlates with improved behavioral performance (van Meer et al., 2010). Similarly, we have shown in mice that functional disruption correlates with infarct size, regional RSFC disruption, the degree of remapping, and behavior (Kraft et al., 2018, Bauer et al., 2014). While functional neuroimaging studies consistently demonstrate local and global changes in functional brain organization after stroke, it is unknown how these processes inter-relate to support recovery of function. Activity in brain regions functionally connected to perilesional tissue can differentially affect recovery potential. In monkeys, the use of the affected limb is required for remapping (Nudo et al., 1996), and conversely, overactivation of the contralateral hemisphere through the use of the 'good limb' is associated with poorer clinical outcome (Turton et al., 1996; Feydy et al., 2002). Additionally, there is substantial indirect evidence that homotopic, interhemispheric connections may directly impact recovery after stroke. Stroke patients with impaired hand movement experience abnormally high interhemispheric inhibition exerted from intact contralesional motor cortex to perilesional motor cortex, with the degree of inhibition increasing with deficit severity (Nudo, 2007). It is thought that an equilibrium between excitation/inhibition across hemispheres may be important for normal unilateral function (Bloom and Hynd, 2005). If the interhemispheric balance is disrupted (e.g. by stroke) local ipsilesional inhibitory influences from distant connections may worsen functional impairment. Prior human studies have primarily used non-invasive brain stimulation techniques (e.g. repetitive transcranial magnetic stimulation [rTMS]) to investigate the role of contralateral activity on the brain (Nowak et al., 2010). However, treatment efficacy using these methods is extremely varied (Takeuchi et al., 2005; Nowak et al., 2008; Adeyemo et al., 2012; Hao et al., 2013) and are limited by imprecision and indiscriminate activation or inhibition of all cell types near the stimulated site. Thus, it has been difficult to systematically examine the influence of specific neuronal populations and connection pathways important for recovery in human stroke patients (Nudo, 2007; Takatsuru et al., 2013; Takatsuru et al., 2014). Optogenetics is a powerful approach for increasing the specificity of paradigms designed to modulate local and distant neural activity after experimental stroke (Cheng et al., 2014; Wahl et al., 2017; Song et al., 2017; Tennant et al., 2017; Shah et al., 2017). The aim of this study was to determine if local circuits lost due to stroke in mice remap into perilesional cortex and reconnect with global functional circuits (i.e. resting-state networks), and if this process could be manipulated (spatially and temporally) by contralesional, homotopic excitation. Given that contralateral limb use suppresses recovery, we hypothesized that optogenetically exciting the contralesional cortex would likewise inhibit recovery. To mechanistically examine whether increased contralesional homotopic excitatory activity worsens recovery, we chose to lesion somatosensory forepaw cortex because this modality is amenable to tracking cortical remapping of sensory evoked responses in tandem with local and global network activity. Our results suggest that limited, focal excitation of contralesional cortex impedes perilesional remapping and restoration of RSFC within the somatomotor network. Furthermore, this maneuver profoundly altered more global patterns of cortical activity outside of the targeted circuit as well as recovery of the brain's global functional connectome. These observations were associated with regulation of several molecular pathways relevant for recovery after injury. Results Study design In this study, we used optogenetic targeting in conjunction with optical intrinsic signal imaging (OISI) in CamK2a-ChR2 mice (see Methods) to examine the effects of contralesional excitation on local and global cortical remodeling after focal ischemia. Our experimental design is illustrated in Figure 1. Note that all cortical regions and manipulations are in reference to hemisphere (instead of limb to which a brain region is mapped). We performed photothrombosis targeted to left primary somatosensory forepaw (S1FP) cortex. A randomly selected subset of mice received chronic, intermittent contralesional excitation of right S1FP (+Stim) beginning day 1 after photothrombosis and continued for 5 consecutive days/week over 4 weeks. The remaining mice recovered spontaneously (−Stim). Assessments of behavior (via cylinder rearing) and brain function (via OISI) occurred before, and 1 and 4 weeks after stroke. A sham group controlled for the effects of chronic photostimulation of right S1FP cortex in the absence of stroke and were imaged at the same time points as the stroke groups. Infarct volume and mRNA expression were characterized in a subset of mice from both stroke groups on days 5 and 14 post stroke, respectively. Figure 1 Download asset Open asset Graphical illustration of experiment and timeline. (A) Photothrombosis was delivered to primary somatosensory forepaw cortex (S1FP, shown in green) in the left hemisphere. Beginning on day 1 after focal ischemia, a subset of mice received interventional optogenetic photostimulation of homotopic S1FP in the right hemisphere for 3 min/day. Treatment was given for 5 consecutive days/week for 4 weeks. Optical intrinsic signal imaging of stimulus evoked activity (following electrical stimulation of the forepaws or optogenetic photostimulation of S1FP) and resting-state activity occurred before, and 1 and 4 weeks after stroke. Infarct volume and mRNA expression were characterized in a subset of mice from both groups at 5 and 14 days after photothrombosis. (B) Infarct volume characterized 5 days post photothrombosis for −Stim (n=5) and +Stim (n=4) groups were statistically equivalent. (C) Limb use as measured by cylinder rearing test in –Stim (n=15) and +Stim (n=20) groups. Symmetrical limb use was observed at baseline time points in all mice. Photothrombosis resulted in use asymmetry due to decreased use of right forelimb within the first week of both groups. The –Stim mice demonstrated significant improvement by week 4 while +Stim mice exhibited sustained use asymmetry at 4 weeks. Linear mixed-effects model (LMM) revealed a main effect of time (F1.77,39=8.7, p=0.0011). Post hoc tests were performed as t-tests assuming unequal group variance and corrected for multiple comparisons using false discovery rate correction. *=p<0.05; **=p<0.01; ***=p<0.001 compared to baseline time point. Statistical analyses were performed in Graph Pad Prism 8. All data reported as mean ± S.E. Contralesional excitation inhibits behavioral recovery and cortical remapping and after stroke We implemented a photostimulation protocol previously reported to positively affect recovery when chronically applied to ipsilesional tissue after stroke (Cheng et al., 2014). To ensure that our stimulation paradigm did not exacerbate the degree of initial injury, average infarct volume (in mm3) was quantified 5 days after photothrombosis. Both groups had statistically equivalent lesion sizes (t(5.7)=0.11, p=0.92, Figure 1B). Using the cylinder rearing test, symmetrical limb use was observed at baseline in all mice (Figure 1C), with no group differences before stroke (t(15.3)=1.4, p=0.9). Photothrombosis resulted in decreased use of right forelimb within the first week of both groups (−Stim mice: t(9)=3.1, p=0.018 and +Stim mice: t(14)=3.5, p=0.001). Similar performance deficits were observed in both groups at week 1 (t(19)=0.68, p=0.5). At week 4, performance in –Stim mice was comparable to baseline (t(7)=1.4, p=0.19), while +Stim mice continued to exhibit sustained use asymmetry compared to baseline (t(12)=2.5, p=0.028). Recovery of sensorimotor function is associated with the formation of new cortical representations of the affected modality (remapping) (Cramer et al., 1997; Kraft et al., 2018). Use of the unaffected limb is thought to be detrimental to recovery, we therefore hypothesized that contralesional excitation would negatively impact remapping (explored in Figures 2 and 3). Because evoked responses at early time points post stroke can be difficult to detect, group-averaged maps of peak responses at baseline were thresholded at 75% of maximum to define a threshold for what constituted a response for all mice at all time points. For Figure 2, the maximum response above this threshold was used for activation magnitude calculations, and the number of pixels above this threshold was used for the area calculations. All magnitude and area values were then normalized by the mean baseline values. For Figure 3, activation incidence images (i.e. the percentage of mice that exhibited an evoked response) were created by binarizing all pixels above the 75% threshold for each mouse and time point. Figure 2 Download asset Open asset Contralesional activity inhibits cortical remapping after stroke. (A) In the affected (right) limb, stimulus evoked responses following electrical forepaw stimulation were significantly reduced 1 week after stroke in both groups. Mice recovering spontaneously (–Stim, n=10, top row) exhibited robust activations at 4 weeks, while responses in stimulated mice (+Stim, n=15) were comparable to week 1 responses. (B) Quantification of evoked response magnitude and area in the ipsilesional hemisphere. The 2-way repeated measures ANOVA (rmANOVA) during recovery revealed a significant group × time interaction (F1,23=5.12; p=0.033) for evoked response magnitude and a trending group × time interaction (F1,23=4.0; p=0.057) in response area. Main effects of group (F1,23=9.5; p=0.005) and time (F1,23=4.5; p=0.045) were observed for response area while magnitude also exhibited a main effect of time (F1,23=8.32; p=0.008). (C) In the unaffected (left) limb, –Stim mice exhibited a trend toward increased activity at week 1 that subsided by week 4. In +Stim mice, evoked responses were significantly elevated at weeks 1 and 4 compared to baseline. (D) Quantification of evoked response magnitude and area in the contralesional hemisphere. Over the duration of the experiment, 2-way rmANOVA revealed a significant group x time interaction (F2,46=6.02; p=0.0048), as well as a main effect of time (F1.7,39=9.34; p=0.0009) and group (F1,23=6.2; p=0.02) for evoked response area. A main effect of time (F1.8,41=6.43; p=0.0051) was observed for response magnitude. Post hoc tests were performed as t-tests assuming unequal group variance and corrected for multiple comparisons using false discovery rate correction. All analysis performed using total hemoglobin as contrast. Statistical analyses were performed in Graph Pad Prism 8. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p< 0.0001. Figure 3 with 1 supplement see all Download asset Open asset Activation incidence and effect size of forepaw remapping maps of activation incidence for evoked responses reported in Figure 2 in the (A) affected and (B) unaffected limb of each group (–Stim [n=10] and +Stim [n=15]). 100% of mice produced robust responses at baseline in both limbs. (A) About 1 week after stroke, both groups exhibited significantly reduced activations in the affected limb, with at most 50% of mice having detectible responses in limited regions of cortex. By 4 weeks after stroke, 80% of –Stim mice exhibited reproducible responses in large regions of somatosensory cortex, posterior to the original forepaw region. Conversely, only 67% of +Stim mice had detectible responses that were smaller in total area. (B) In the unaffected limb, responses in the –Stim mice were largely similar across time points. However, responses in +Stim mice were generally larger (in line with Figure 2) and exhibited increased activity in forepaw motor cortex compared to the baseline time point. (C) Map of effect size showing main topographical differences in remapping between groups at 4 weeks following electrical stimulation of the affected forepaw. Cohen's D was calculated using mouse-level T-maps (see main text) as –Stim minus +Stim so that an effect would manifest as a positive value. Contours of original and remapped forepaw response shown for reference. Statistical analyses were performed in MATLAB (2018a). At baseline, electrical stimulation of the right forepaw (affected limb) resulted in consistent activation of left S1FP cortex in 100% of mice in both groups (Figures 2A and 3A). Photothrombosis targeted to left S1FP resulted in >70% loss of evoked response magnitude in the first week following injury in both groups (–Stim: t(9)=4.6, p=0.001; +Stim: t(14)=4.4, p<0.001), and at least an 80% reduction in response area in both groups (−Stim: t(9)=8.6, p<0.0001; +Stim: t(14)=6.5, p<0.0001, Figure 2B). Responses from the affected limb reappeared in 80% of the mice in the –Stim group (Figure 3A) by 4 weeks, and exhibited magnitudes statistically indistinguishable (t(9)=1.3, p=0.22) from baseline responses (Figure 2B). These new cortical representations in –Stim mice were focused to regions lateral and posterior to the infarct (near barrel cortex) (Figure 2A, Figure 3A), significantly larger than those at week 1 (t(9)=2.3, p=0.045), but remained smaller than baseline representations (t(9)=6.0, p=0.0002, Figure 2B). In +Stim mice, only 67% of mice had detectible responses at week 4 (Figure 3A). Week 4 responses in +Stim mice were 60% smaller in magnitude (t(14)=4.6, p=0.0004) and 87% smaller in area (t(14)=7.57, p<0.0001) than those at baseline and were indistinguishable from response area (t(14)=0.1, p=0.92) and magnitude (t(14)=0.5, p=0.63) observed at week 1 (Figure 2B). Left forepaw (unaffected limb) stimulation resulted in consistent activation of right S1FP cortex in 100% of the mice in both groups prior to photothrombosis (Figure 2C, Figure 3B). In +Stim mice, evoked responses from the unaffected limb at week 1 were 81% larger than baseline responses (t(14)=3.5, p=0.003, Figure 2D). Activation magnitude in –Stim mice trended (t(9)=1.7, p=0.1) toward larger responses at week 1, but returned to baseline levels by week 4 (t(9)=1.4, p=0.2). In +Stim mice, activation area was 80% larger at week 1 (t(14)=6.48, p<0.0001) and 60% larger at week 4 (t(14)=4.12, p=0.001) than those at baseline. Furthermore, stimulation of the unaffected limb in +Stim mice activated forepaw representations in contralesional motor cortex in 80% of mice that were not observed at baseline (Figure 3B). Post stroke activation area in –Stim mice did not significantly differ from baseline at either time point (week 1: t(9)=0.6, p=0.66; week 4: t(9)=1.3, p=0.21, Figure 2D), nor elicit responses in contralesional motor cortex (Figure 3B). To further examine the effect of contralesional stimulation on group-wise cortical remapping, T-maps were calculated for each mouse for all imaging sessions of evoked activity at 4 weeks following electrical stimulation of the affected forepaw. From these T-maps, a map of Cohen's D was calculated as –Stim minus +Stim so that an effect would manifest as a positive value (Figure 3C). Computed differences between groups included the region of forepaw remapping in the ipsilesional hemisphere dominated by –Stim mice. Peak Cohen's D in the region of remapping was 0.44, an intermediate effect, indicating that –Stim mice exhibit a larger, more active portion of remapped cortex compared to +Stim mice. Focal excitatory stimulation in control mice results in increased evoked responses of right and left S1FP To determine if the effects of contralesional stimulation were conditional on focal ischemia, stimulus evoked responses were characterized in mice experiencing chronic excitatory stimulation of right S1FP in the absence of photothrombosis (Figure 3—figure supplement 1). Robust evoked activity was observed in all control mice and time points (Figure 3—figure supplement 1A). Interestingly, by 4 weeks, this group also exhibited increased evoked activity in forepaw representations of motor cortex, similar to contralesional responses observed in +Stim mice after stroke. In control mice, stimulus evoked responses in the right hemisphere exhibited larger response area at 1 week (t(9)=4.4, p=0.002) and 4 weeks (t(8)=6.1, p=0.0003) compared to baseline, and between weeks 1 and 4 (t(8)=3.96, p=0.004) (Figure 3—figure supplement 1B top). Response magnitude in the right hemisphere was larger at week 4 compared to week 1 (t(8)=3.33, p=0.01) (Figure 3—figure supplement 1B top). In the left hemisphere, response area was significantly increased at 4 weeks compared to baseline (t(6)=4.43, p=0.004) and at 4 weeks compared to 1 week (t(7)=3.02, p=0.02) (Figure 3—figure supplement 1B bottom). Response magnitude did not significantly change over time, but trended toward a larger response between weeks 1 and 4 (t(7)=2.57, uncorrected p=0.037). Contralesional stimulation inhibits recovery of somatomotor forepaw circuitry Both remapping (Brown et al., 2009; Kraft et al., 2018) and the formation of new homotopic functional connections (van Meer et al., 2010; Carter et al., 2012) are associated with better recovery following stroke. Having observed differential remapping in +Stim and –Stim groups, we examined how contralesional excitation affected reintegration of local S1FP circuitry into the somatomotor resting-state network. The regions of interest (ROIs) for RSFC analysis were defined by stimulus evoked responses at baseline and week 4 (see Methods): original S1FP representations in both hemispheres, new (remapped) S1FP representations in the lesioned hemisphere, and forepaw representations in contralesional motor cortex, primary motor forepaw cortex (M1FP), (Figure 4A). Group-averaged S1FP RSFC maps at baseline reveal positive ipsilateral RSFC with sensory and motor regions, as well as bilateral RSFC with original S1FP in both groups (Figure 4B). About 1 week after photothrombosis, bilateral RSFC between original ipsilesional and contralesional S1FP in both groups is significantly reduced (–Stim: t(9)=6.2, p=0.0002; +Stim: t(14)=3.1, p=0.008; Figure 4B and C). Furthermore, at week 1, RSFC between contralesional S1FP and the region of future ipsilesional remapping has not been established in either group (–Stim: t(9)=4.6, p=0.0014; +Stim: t(14)=3.5, p=0.0036) (Figure 4B and D, Figure 4—figure supplement 1A). However, in –Stim mice 4 weeks after stroke, new 'homotopic' functional connections between contralesional S1FP and remapped ipsilesional S1FP were observed having RSFC strength equivalent to baseline values (t(9)=0.29, p=0.78). Conversely, RSFC between contralesional S1FP and remapped S1FP at 4 weeks in the +Stim group was significantly reduced from baseline (t(14)=2.2; p=0.042) and indistinguishable from week 1 (t(14)=0.28, p=0.79; Figure 4B and D, Figure 4—figure supplement 1). Furthermore, significant disruption of intrahemispheric, ipsilesional RSFC between remapped S1FP and motor persisted at 1 and 4 weeks in +Stim mice (1 week: t(14)=2.9, p=0.011; 4 weeks: t(14)=3.8, p=0.002) (Figure 4—figure supplement 1B). Figure 4 with 2 supplements see all Download asset Open asset Contralesional stimulation inhibits recovery of somatomotor forepaw circuitry (A) regions of interest (ROIs) overlaid on a representative white light image of the dorsal mouse skull. ROIs were determined from evoked responses at baseline (original primary somatosensory forepaw [S1FP] in the left or right hemispheres) or at 4 weeks after stroke (remapped left S1FP). Responses at 4 weeks contained increased activity in right M1FP and included as an ROI for S1FP resting-state functional connectivity (RSFC) analysis. (B) Group-averaged maps of RSFC for the original right S1FP ROI in both –Stim and +Stim (n=15) groups reveal ipsilateral connectivity with sensory and motor regions, as well as bilateral RSFC with left forepaw cortex. Similar deficits are observed in both groups 1 week after stroke. RSFC in both groups is while ipsilateral connectivity By 4 RSFC in right S1FP in the –Stim group new functional connections in perilesional somatosensory regions, remapped left S1FP as well as more motor RSFC of right S1FP in the +Stim group largely by 4 weeks. Quantification of RSFC over time in ROIs in (C) RSFC in original S1FP is significantly reduced in both groups after and only in –Stim mice: 2-way repeated measures ANOVA (rmANOVA) revealed a significant group × time interaction and a main effect of time (D) RSFC with remapped forepaw regions is by 4 weeks after stroke in –Stim mice during recovery, 2-way rmANOVA revealed a significant group × time interaction and a main effect of time RSFC between right S1FP and right exhibiting increased activity in right M1FP during left forepaw +Stim mice not exhibit increased ipsilateral RSFC between right M1FP and right 2-way rmANOVA revealed a significant group × time interaction All post hoc tests were performed as t-tests assuming unequal group variance and corrected for multiple comparisons using false discovery rate correction. Statistical analyses were performed in Graph Pad Prism 8. *=p<0.05; **=p<0.01; ***=p<0.001; reported in Figure and D, electrical stimulation of the unaffected forepaw resulted in a trend toward or significant in (+Stim) stimulus evoked activity in contralesional S1FP and M1FP following stroke. We examined if increased contralesional somatomotor activity was associated with increased RSFC between these regions (Figure In +Stim mice, RSFC between sensory and motor did not change over time from baseline exhibiting increased area and magnitude in these regions after photothrombosis. At week 4, –Stim mice exhibited RSFC between sensory and motor regions compared to +Stim mice the of the contralesional hemisphere during recovery. At week 1, +Stim mice demonstrated a but significant in RSFC compared to –Stim mice. However, this did not in +Stim mice over In control mice, photostimulation did not significantly homotopic S1FP RSFC (Figure 4—figure supplement or between right S1FP and right M1FP (Figure 4—figure supplement these results suggest that more cortical remapping is associated with a to more normalized patterns of homotopic RSFC in the affected Furthermore, larger patterns of activation in the unaffected hemisphere are not of increased RSFC within that hemisphere. network after stroke are suppressed by homotopic contralesional stimulation network disruption following focal outside of the lesioned to brain regions from the infarct et al., networks toward more normal patterns of intrinsic organization after stroke (i.e. homotopic to support better behavioral performance (Carter et al., 2012; He et al., 2007). Because +Stim mice exhibited sustained behavioral deficits and S1FP RSFC at week 4 compared to –Stim mice, we hypothesized that contralesional focal excitatory stimulation also altered the degree to which global RSFC in +Stim mice. cortex were for all comparisons within our for both groups at week 4 (Figure and are by functional then by hemisphere right –Stim mice (Figure exhibit homotopic RSFC compared to +Stim mice (Figure Furthermore, ipsilateral between brain regions are also more for between ipsilesional motor and regions, in –Stim mice compared to +Stim mice. differences at week 4 were calculated as –Stim minus +Stim (Figure to +Stim mice, –Stim mice exhibit increased interhemispheric RSFC between ipsilesional sensory and motor cortex (e.g. 1, Figure homotopic RSFC within the somatosensory and motor networks (e.g. 2, Figure as well as increased and RSFC within and cortex (e.g. 3, Figure Additionally, mice recovering spontaneously exhibit increased between (e.g. motor and and posterior 4, Figure Figure 5 with 2 supplements see all Download asset Open asset network after stroke are suppressed by increased contralesional forepaw activity cortex for (A) –Stim and (B) +Stim (n=15) groups 4 weeks after photothrombosis. are by functional then by hemisphere (C) functional connectivity (RSFC) (–Stim minus the group-averaged differences between experimental groups. increased and (C) RSFC within the forepaw somatosensory and motor as well as in distant and regions in the –Stim group Additionally, mice recovering spontaneously exhibit increased between somatosensory and motor regions (D) RSFC differences examined using analysis of the in 2 of the variance between group differences