Interactions between motor cortical forelimb regions and their influence on muscles reorganize across behaviors

Abstract

It remains unclear how classical models of motor cortical hierarchy align with emerging evidence of behavioral organization in motor cortex. To address this, we combined optogenetic inactivation, Neuropixels recording, and electromyography to quantify the pattern and influence of activity in the mouse analogs of forelimb premotor and primary motor cortex (RFA and CFA) during a single-forelimb reaching task and an ethologically-inspired, all-limb climbing behavior. Results revealed that RFA's dominant influence on forelimb muscles and on CFA during reaching is replaced by a dominant influence of CFA on muscles and on RFA during climbing, even when forelimb muscle activity during climbing resembles that during reaching. Short-latency influence between regions on putative excitatory and inhibitory populations in different cortical laminae also showed behavioral specificity. Simultaneous recordings in both areas during climbing revealed a loss of activity timing differences seen during reaching previously interpreted as reflective of hierarchy. These findings demonstrate that hierarchical interactions between forelimb motor cortical regions vary between behavioral contexts.

Fig. 1. Asymmetry in influence on muscles reverses across behaviors.

a Schematic depicting the climbing behavior. b Mouse brain schematic depicting the position of the light stimulus on RFA and CFA. Cartoon adapted from Miri, A. et al. Behaviorally Selective Engagement of Short-Latency Effector Pathways by Motor Cortex. Neuron 95, 683-696.e11 (2017) with permission. c, d Example time series segment of distance climbed with the time of light and control trials, and water reward delivery indicated (c) and normalized muscle activity (d) over a climbing bout. e Mean normalized muscle activity (units are st dev) ± SEM across trials without (black) or with inactivation (cyan bar) of RFA (magenta, top; n = 7303 light and 14,289 control trials across 6 animals) or CFA (blue, bottom, n = 9029 light and 18,397 control trials across 8 animals) for each muscle. The dashed vertical line indicates the shortest latency at which CFA output influences forelimb muscle activity (10 ms). f Mean ± SEM absolute difference between inactivation and control trial averages for each muscle for RFA and CFA inactivation (cyan bar) across animals. For baseline correction, the absolute difference was calculated between resampled control trials to estimate the baseline difference expected by chance. g Mean ± SEM absolute difference between inactivation and control trial averages averaged across all muscles and animals for inactivation (cyan bar) of RFA or CFA during climbing (solid lines). Equivalent means for reaching are overlaid (dashed). h Average absolute difference across muscles between inactivation and control trial averages at 25, 50, and 100 ms after trial onset for individual animals (circles) and the mean across animals (bars) for climbing (filled circles/black bars) and reaching (open circles/white bars) after inactivation of RFA or CFA. Source data are provided as a Source Data file.

Fig. 2. Asymmetry in endogenous neural influence reverses across behaviors.

a, b Mean ± SEM firing rate for cells from all four climbing animals after inactivating RFA (cyan bar) and recording in CFA (a, 170 cells/animal) or vice versa (b, 117 cells/animal) and for control trials. The same number of cells was used from each animal here and in the panels that follow so results equally represent animals despite differing cell yields from recording. Brain schematics were adapted from Saiki–Ishikawa, A. et al. Hierarchy between forelimb premotor and primary motor cortices and its manifestation in their firing patterns. eLife 13, (2024) with permission. c, d Mean ± SEM firing rate for cells from all three reaching animals after inactivating RFA (cyan bar) and recording in CFA (a) 219 cells/animal) or vice versa (b) 31 cells/animal) and for control trials. e Cumulative histograms of the fractional change in firing rate between inactivation and control trials 30–40 ms after trial onset for neurons in each area after inactivation of the other during climbing or reaching. f Mean ± SEM fractional change between control and inactivation trials 30–40 ms after trial onset across neurons in each area after inactivation of the other during climbing (RFA inactivation, n = 680 cells; CFA inactivation, n = 468 cells) and reaching (RFA inactivation, n = 657 cells; CFA inactivation, 93 cells), *p < 0.05, Wilcoxon one-tailed rank sum test CFA vs RFA during climbing, p = 3.01 × 10⁻⁶; RFA vs CFA during reaching, p = 0.009; CFA during climbing vs reaching, p = 4.17 × 10⁻⁹. Source data are provided as a Source Data file.

Fig. 3. Only the dominant area exerts a larger effect on narrow-waveform neurons.

a, b Mean ± SEM firing rate for all wide- (a, n = 1299) or narrow-waveform (b, n = 230) neurons in CFA after RFA inactivation and for control trials. c Cumulative histogram of the fractional change between inactivation and control trials 30-40 ms after trial onset for wide- (n = 850) and narrow-waveform (n = 194) neurons in CFA after RFA inactivation during climbing or reaching. d Mean ± SEM fractional firing rate change between inactivation and control trials 30–40 ms after trial onset for all wide- and narrow-waveform neurons in CFA after RFA inactivation during climbing and reaching (n = 631 wide, n = 205 narrow), *p < 0.05, Wilcoxon one-tailed rank sum test; wide vs narrow during reaching, p = 0.035. e, f Same as (a, b), but for RFA neurons (n = 1583 wide, 187 narrow) recorded during CFA inactivation. g, h Same as (c, d), but for RFA neurons (n = 900 wide, 127 narrow during climbing; n = 331 wide, 88 narrow during reaching) during CFA inactivation. *p < 0.05, Wilcoxon one-tailed rank sum test; wide vs narrow during reaching, p = 0.035; wides during climbing vs. reaching, p = 0.006; narrow during climbing vs reaching, p = 6.57 ×10⁻⁹; wide vs narrow during climbing, p = 2.15 × 10⁻⁷. Source data are provided as a Source Data file.

Fig. 4. Effects of inactivation on cell types vary by cortical layer.

a, b Mean ± SEM firing rate for wide- (a) 324 in L2/3, 167 in L5, 130 in L6) and narrow-waveform (b) 60 in L2/3, 52 in L5, 38 in L6) neurons in CFA assigned to each laminar cell group after RFA inactivation, and for control trials. c Mean ± SEM fractional firing rate change between inactivation and control trials 30–40 ms after trial onset for all neurons in different laminar cell groups (wide L2/3, n = 324; narrow L2/3, n = 60; wide L5, n = 167; narrow L5, n = 52; wide L6, n = 130; narrow L6, n = 38) in CFA after RFA inactivation during climbing. *p < 0.05, Wilcoxon one-tailed rank sum test; narrow L5, p = 0.030. d Mean ± SEM difference between the fractional firing rate change of neurons in each laminar cell group 30–40 ms after trial onset and the mean fractional change of the equivalent group during reaching (wide L2/3, n = 117; narrow L2/3, n = 80; wide L5, n = 95; narrow L5, n = 41; wide L6, n = 201; narrow L6, n = 34). *p < 0.05, Wilcoxon Signed Rank Test; wide L2/3, p = 2.06 × 10⁻²⁸; narrow L2/3, p = 0.032; wide L5, p = 2.74 × 10⁻⁶. e–g Same as a–c but for RFA neurons (wide L2/3, n = 103; narrow L2/3, n = 12; wide L5, n = 156; narrow L5, n = 34; wide L6, n = 132; narrow L6, n = 12) recorded during CFA inactivation, instead of vice versa. *p < 0.05, Wilcoxon one-tailed rank sum test; wide L2/3, p = 1.41 × 10⁻⁶_; narrow L2/3, p = 0.015. h Same as (d), but for RFA neurons recorded during CFA inactivation (during reaching: wide L2/3, n = 73; narrow L2/3, n = 5; wide L5, n = 79; narrow L5, n = 22; wide L6, n = 52; narrow L6, n = 30). *p < 0.05, Wilcoxon Signed Rank Test; narrow L2/3, p = 0.016; wide L5, p = 3.20 × 10⁻³; narrow L5, p = 6.40 × 10⁻³. Source data are provided as a Source Data file.

Fig. 5. Relations between firing in RFA and CFA change across behaviors.

a, b Scatter plots of the firing rates for cells recorded in CFA (a) and RFA (b) during periods of active climbing versus all other time periods for wide- and narrow-waveform neurons. Brain schematic was adapted from Saiki-Ishikawa, A. et al. Hierarchy between forelimb premotor and primary motor cortices and its manifestation in their firing patterns. eLife 13, (2024) with permission. c Cumulative histogram of the mean firing rates of wide- and narrow-waveform neurons recorded in CFA and RFA during climbing. d The fraction of neurons recorded in CFA and RFA for each animal during climbing (circles; n = 10 mice) and the mean across animals (black bars) whose firing rate time series was significantly correlated with that of at least one muscle (p-value threshold <0.05). The mean fraction in each area across animals during reaching (n = 6 mice) is overlaid (white bar). *p < 0.05, Wilcoxon one-tailed rank sum test; fraction in CFA vs RFA during climbing, p = 0.012. e The difference between the fraction of neurons in CFA and RFA whose activity was significantly correlated with that of at least one muscle for each animal (circles) and the mean across animals (bars) during climbing (n = 10 mice) and reaching (n = 6 mice). *p < 0.05, Wilcoxon one-tailed rank sum test; climbing vs reaching, p = 0.008. f For an example mouse, the normalized activity change from baseline summed across the top three principal components (PCs) for all recorded CFA or RFA neurons, and the top PC for muscle activity surrounding muscle activity onset. Circles indicate the time of estimated activity onset in each area. g Time from muscle activity onset at which the neural activity change from baseline exceeded a low threshold for each animal during climbing (circles; n = 10 mice) and the mean onset time across animals (black bars). The mean onset times across animals during reaching (n = 6 mice) are overlaid (white bars). h The difference between activity onset times in CFA and RFA for each animal (circles) and the mean across animals (bars) during climbing and reaching. *p < 0.05, Wilcoxon one-tailed rank sum test; climbing vs reaching, p = 0.044. i The activity variance in the 150 ms before muscle activity onset, defined as a fraction of the total activity variance from 150 ms before to 150 ms after muscle activity onset for each animal during climbing (circles; n = 10 mice) and the mean onset time across animals (black bars). The mean onset times across animals during reaching (n = 6 mice) are overlaid (white bars). j The difference between the fraction of variance captured before muscle activity onset in CFA and RFA for each animal (circles) and the mean difference across animals (bars) during climbing and reaching. Source data are provided as a Source Data file.

Fig. 6. Variance captured by activity patterns shared at a lag changes across behaviors.

a Scatter plot illustrating the delays of all across-region components detected by DLAG versus the fractional variance captured in each region for one mouse (2 sessions). Open circles indicate that the time delay was not significantly different from zero. Lines connect variance capture for individual components. b Fractional variance capture of CFA activity (left) and RFA activity (right) by across-region components in which CFA activity led RFA (CFA➤RFA), or RFA led CFA (RFA➤CFA), and by within-region components, in DLAG models fit with four across-area and four within-area components. Connected dots indicate individual mice (n = 14). c Cumulative histogram of all significant component delays from all mice during climbing and reaching from DLAG models fit with four across-area and four within-area dimensions. Dashed lines indicate the median delay for each behavior. (d,e) The median delay across all significant component delays when varying within- (d) or across-region (e) dimensionality during reaching (open circles) and climbing (filled circles) with the other number of dimensions set to four. Positive values indicate CFA led more components, whereas negative values indicate RFA led more components. f, g Mean ± SEM difference between the variance captured in RFA activity by CFA-leading components and the variance captured in CFA activity by RFA-leading components across animals, for reaching (open circles) and climbing (filled circles) when varying the within-region (f) or the across-region (g) dimensionality. Values below zero indicate RFA-leading components captured more variance in CFA activity than vice versa. Source data are provided as a Source Data file.