Cortical glutamatergic projection neuron types contribute to distinct functional subnetworks

Abstract

The cellular basis of cerebral cortex functional architecture remains not well understood. A major challenge is to monitor and decipher neural network dynamics across broad cortical areas yet with projection-neuron-type resolution in real time during behavior. Combining genetic targeting and wide-field imaging, we monitored activity dynamics of subcortical-projecting (PT^Fezf2) and intratelencephalic-projecting (IT^PlxnD1) types across dorsal cortex of mice during different brain states and behaviors. IT^PlxnD1 and PT^Fezf2 neurons showed distinct activation patterns during wakeful resting, during spontaneous movements and upon sensory stimulation. Distinct IT^PlxnD1 and PT^Fezf2 subnetworks were dynamically tuned to different sensorimotor components of a naturalistic feeding behavior, and optogenetic inhibition of ITs^PlxnD1 and PTs^Fezf2 in subnetwork nodes disrupted distinct components of this behavior. Lastly, IT^PlxnD1 and PT^Fezf2 projection patterns are consistent with their subnetwork activation patterns. Our results show that, in addition to the concept of columnar organization, dynamic areal and projection-neuron-type specific subnetworks are a key feature of cortical functional architecture linking microcircuit components with global brain networks.

Extended Data 1.. IT^PlxnD1 and PT^Fezf2 activation patterns during wakeful resting state across mice.

a. Variance maps for each mouse (in columns) during quiescent and active episodes averaged over two sessions. b. Distribution of variance during quiescent (Q) versus active (A) episodes in IT^PlxnD1 (blue) and PT^Fezf2 (green) (n =12 sessions from 6 mice). c. Difference between IT^PlxnD1 and PT^Fezf2 average variance maps for quiescent and active episodes (n=12 sessions from 6 mice). Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by False Discovery Rate (FDR) = 0.05). Blue pixels indicate values significantly larger in IT^PlxnD1 compared to PT^Fezf2 and vice versa for green pixels. d. Distribution of Pearson’s correlation coefficients between quiescent variance maps within IT^PlxnD1 (blue), PT^Fezf2 (green) and between IT^PlxnD1 & PT^Fezf2 (blue green) (66 pairs within IT^PlxnD1 & PT^Fezf2 and 144 pairs between IT^PlxnD1 & PT^Fezf2 in 12 sessions from 6 mice for each cell type). e. Distribution of Pearson’s correlation coefficients between active variance maps within IT^PlxnD1 (blue), PT^Fezf2 (green) and between IT^PlxnD1 and PT^Fezf2 (blue green) (66 pairs within IT^PlxnD1 & PT^Fezf2 and 144 pairs between IT^PlxnD1 & PT^Fezf2 in 12 sessions from 6 mice for each cell type). f. Distribution of IT^PlxnD1 (blue) and PT^Fezf2 (green) active variance maps projected to the subspace spanned by the top two principal components. g. Distribution of IT^PlxnD1 (blue) and PT^Fezf2 (green) quiescent variance maps projected to the subspace spanned by the top two principal components. h. Average maps of the 75^th (top) and 95^th (bottom) percentile df/f value during active and quiescent episodes for IT^PlxnD1 and PT^Fezf2 (n =12 sessions from 6 mice). *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers (1.5 times more or less than the interquartile range). All statistics in Supplementary table 1.

Extended Data 2.. Spontaneous activity comparison and correlation of sensory response in IT^PlxnD1 and PT^Fezf2 across mice.

a. Probability distribution of df/f values from IT^PlxnD1 (blue) and PT^Fezf2 (green) during wakeful resting state (average of 12 sessions from 6 mice each, shaded region indicates ±2 s.e.m). b. Mean peak df/f maps of IT^PlxnD1 and PT^Fezf2 during spontaneous behavior (average of 12 sessions from 6 mice for each cell type). c. Difference between IT^PlxnD1 and PT^Fezf2 mean peak df/f maps. Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by FDR = 0.05). Note that no pixels are significantly different. d. Mean temporal dynamics of IT^PlxnD1 and PT^Fezf2 activity with (colored) and without (black) hemodynamic correction from hindlimb sensory area during spontaneous behavior. Activity is aligned to the onset of spontaneous movements (IT^PlxnD1: 367 and PT^Fezf2: 474 trials in 12 sessions from 6 mice each, shaded region indicates ±2 s.e.m). Left image with red dot indicates location used to extract signal. e. Left: Distribution of difference between hemodynamic corrected and uncorrected peak df/f value between 0 to 1 sec after spontaneous movement onset for IT^PlxnD1 (blue) and PT^Fezf2 (green) from panel d. Right: Distribution of Pearson’s correlation coefficient between hemodynamic corrected and uncorrected IT^PlxnD1 and PT^Fezf2 activity from panel d. (IT^PlxnD1: 367 and PT^Fezf2: 474 trials). f. Mean peak normalized activity maps of IT^PlxnD1 (top) and PT^Fezf2 (bottom) in response to corresponding unimodal sensory simulation (n=12 sessions from 6 mice each). g. Distribution of Pearson’s correlation coefficients between sensory activation maps within IT^PlxnD1 (blue), PT^Fezf2 (green) and between IT^PlxnD1 and PT^Fezf2 (blue green) (66 pairs within IT^PlxnD1 & PT^Fezf2 and 144 pairs between IT^PlxnD1 & PT^Fezf2 in 12 sessions from 6 mice each for all stimulations). *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers (1.5 times more or less than the interquartile range). All statistics in Supplementary table 1.

Extended Data 3.. Calcium dynamics of IT^PlxnD1 and PT^Fezf2 at cellular resolution reflect widefield responses

a. Schematic of the whisker stimulation paradigm and the cortical location for two photon imaging (blue circle). b. Left: Example field of view (FOV) of IT^PlxnD1 cell bodies and apical dendrites of PT^Fezf2 in the whisker barrel cortex. Right: Example traces from single IT^PlxnD1 cell bodies and apical dendrites of PT^Fezf2. Numbers indicate the corresponding location on the FOV. Magenta bars indicate whisker stimulation events. c. Heat map of average single neuron responses of IT^PlxnD1 and PT^Fezf2 classified into 3 groups based on their activity during whisker stimulation from the example FOV. d. Average responses across all IT^PlxnD1 and PT^Fezf2 neurons within each group from the example FOV (shaded region indicates ±2 s.e.m). Magenta bars indicate duration of whisker stimulation. e. Average responses across all IT^PlxnD1 and PT^Fezf2 neurons from the example FOV (shaded region indicates ±2 s.e.m). f. Heat map of average single neuron responses of IT^PlxnD1 and PT^Fezf2 classified based on their activity during whisker stimulation across all mice and sessions (IT^PlxnD1 42 FOV’s from n = 4 mice and PT^Fezf2 36 FOV’s from n = 3 mice). g. Average responses across all IT^PlxnD1 and PT^Fezf2 neurons within each group across all mice and sessions (IT^PlxnD1 42 FOV’s from n = 4 mice and PT^Fezf2 36 FOV’s from n = 3 mice, shaded region indicates ±2 s.e.m). h. Average responses across all IT^PlxnD1 and PT^Fezf2 neurons from all mice and sessions combined (IT^PlxnD1 42 FOV’s from n = 4 mice and PT^Fezf2 36 FOV’s from n = 3 mice, shaded region indicates ±2 s.e.m). i. Proportion of neurons in each group from IT^PlxnD1 and PT^Fezf2.

Extended Data 4.. Temporal dynamics of IT^PlxnD1 and PT^Fezf2 within parietofrontal and frontolateral networks centered to lick and hand lift onset.

a. Difference between IT^PlxnD1 and PT^Fezf2 mean activity map from Fig 3a,d. Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by FDR = 0.05, n = 24 maps from IT^PlxnD1 and 23 maps from PT^Fezf2). Blue pixels indicate values significantly larger in IT^PlxnD1 compared to PT^Fezf2 and vice versa for green pixels. b. Distribution of Pearson’s correlation coefficients within IT^PlxnD1 (blue), PT^Fezf2 (green) and between IT^PlxnD1 & PT^Fezf2 (blue green) mean feeding sequence activity maps (n = 253 pairs within IT^PlxnD1, 276 pairs within PT^Fezf2 and 522 pairs between IT^PlxnD1 & PT^Fezf2). c. Distribution of IT^PlxnD1 (blue) and PT^Fezf2 (green) mean feeding sequence activity maps projected to the subspace spanned by the top two principal components (n = 24 maps from IT^PlxnD1 and 23 maps from PT^Fezf2). d. Single trial heatmaps and mean activity of PT^Fezf2 and IT^PlxnD1 from parietal, frontal, FLA and FLP centered to lick (top) and handlift onset (bottom, IT^PlxnD1 - 23 sessions from 6 mice, PT^Fezf2 - 24 sessions from 5 mice, shaded region indicates ±2 s.e.m). ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers (1.5 times more or less than the interquartile range). All statistics in Supplementary table 1.

Extended Data 5.. Temporal dynamics of IT^PlxnD1 in frontolateral and PT^Fezf2 within parietofrontal nodes during feeding with and without hand lift across mice.

a. Single trial heatmaps of PT^Fezf2 activity centered to pellet in mouth onset from parietal and frontal node and IT^PlxnD1 activity in FLP and FLA from eating with (top) and without hand lift (bottom) from all mice and sessions (With handlift : IT^PlxnD1 - 23 sessions from 6 mice, PT^Fezf2 - 24 sessions from 5 mice. Without hand lift: IT^PlxnD1 - 15 sessions from 6 mice, PT^Fezf2 - 13 sessions from 5 mice). b. Left: Mean PT^Fezf2 activity aligned to pellet in mouth onset from parietal node with (light brown) and without (light green) hand lift, frontal node with (dark brown) and without (dark green) hand lift. Right: IT^PlxnD1 activity aligned to PIM onset from FLP with (orange) and without (cyan) hand lift and FLA with (magenta) and without (dark blue) hand lift (shaded region indicates ±2 s.e.m, sample size as in panel a).

Extended Data 6.. Inhibition of parietofrontal and frontolateral regions differentially disrupts sensorimotor components of feeding behavior

a. Schematic of optogenetic laser scanning setup. b. Single trial (translucent) and averaged (opaque) tongue trajectories centered to inhibition of frontal (dark brown), parietal (light brown) and frontolateral (magenta) nodes. Control (grey). Note that trajectories evolve from top to bottom with the mouth at top and pellet at bottom (Green schematic). Upward change in value indicates decrease in tongue length. c. Distribution of total tongue length 0.5 seconds before and after inhibition onset of frontal (n=76), parietal (n=88) and frontolateral (n=89 trials) nodes. During pellet retrieval, inhibition of frontal and frontolateral but not parietal nodes resulted in a sharp decrease in tongue extension which recovered on average after about 0.5 seconds. d. Distribution of durations to pick pellet after trial start during control versus inhibition of frontal (n=76), parietal (n=88), and frontolateral (n=89) nodes. Inhibition of frontal and frontolateral but not parietal nodes resulted in a significant delay and disruption in retrieving pellet to mouth. e. Probability of hand lift events during inhibition of frontal (n=67), parietal (n=87) frontolateral (n=100) nodes compared to control. During the hand-lift phase after PIM, inhibition of frontal and frontolateral but not parietal nodes prior to hand lift onset led to substantial deficit in the ability to lift hands towards mouth, resulting in a sharp decrease in the number of hand lifts. f. Left: 5 example hand lift trajectories from side view (top) during control (black) and inhibition of parietal node and corresponding absolute velocities (bottom). Right: Mean vertical hand trajectory from side view (top) and absolute velocity (bottom, shading around trace ±2 s.e.m) during control (black) and inhibition of frontal (n=41), parietal (n=83) and frontolateral (n=59) nodes. Insets: zoomed mean signals. Note increase in velocity fluctuation during parietal inhibition. g. Distribution of absolute velocity integral for 1 sec post hand lift during control and inhibition of frontal (n=41), parietal (n=83) and frontolateral (n=59) nodes. While there was no decrease in handlift probability on parietal inhibition, it resulted in substantial deficits in hand lift trajectory, characterized by erratic and jerky movements. This was reflected in the significant modulation of absolute velocity during lift (see methods). h. Mean normalized vertical trajectory of left finger from front view during food handling from control (black) and inhibition of frontal (n=58), parietal (n=89) and frontolateral (n=101) nodes (shaded region indicates ±2 s.e.m). Inhibiting the frontal and frontolateral nodes severely impeded mice’s ability to bring pellet to mouth during food handling, which recovered immediately after the release of inhibition. Inhibiting the parietal node resulted in only a slight disruption. i,j. Distribution of mean normalized finger to mouth distance (i) and duration of hand held close to mouth (j) during control versus inhibition of frontal (n=58), parietal (n=89) and frontolateral (n=101 trials) nodes during food handling. Frontolateral node consisted of data pooled from FLA and FLP since no major difference was observed. All data is pooled from 3 mice across 7 sessions. *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers. All statistics in Supplementary table 1.

Extended Data 7.. Comparison of inhibition effects between IT^PlxnD1 and PT^Fezf2

a. Distribution of the difference in mean tongue length between control and inhibition trials of frontal IT^PlxnD1 (n=173), PT^Fezf2 (n=140) and FLA IT^PlxnD1 (n=165), PT^Fezf2 (n=98) nodes. b. Distribution of the difference in mean normalized hand to mouth distance for 5 seconds between control and inhibition trials of frontal IT^PlxnD1 (n=353), PT^Fezf2 (n=167) and FLA IT^PlxnD1 (n=455) and PT^Fezf2 (n=202) nodes. c. Distribution of the difference in mean absolute hand velocity for 5 seconds between control and inhibition trials of frontal IT^PlxnD1 (n=353), PT^Fezf2 (n=167) and FLA IT^PlxnD1 (n=455) and PT^Fezf2 (n=202) nodes. All data pooled from 4 mice for IT^PlxnD1 and 3 for PT^Fezf2. *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers. All statistics in Supplementary table 1.

Extended Data 8.. Axonal projection of IT^PlxnD1 and PT^Fezf2 in subcortical structures.

a. Three dimensional rendering of axonal projections of IT^PlxnD1 from FLA and FLP and PT^Fezf2 from frontal and parietal node. Yellow circle indicates injection site. b. Spatial distribution of axonal projections of IT^PlxnD1 from FLA and FLP (top) and PT^Fezf2 parietal and frontal nodes (bottom) within the striatum projected onto the coronal and sagittal plane. c. Spatial distribution of axonal projections of PT^Fezf2 from parietal and frontal nodes within the primary and association thalamus projected onto the coronal and sagittal plane. d. Spatial distribution of axonal projections of PT^Fezf2 from parietal and frontal nodes within the motor Superior colliculus (SCm, magenta), sensory superior colliculus (SCs, yellow) and inferior colliculus (IC, brown) projected onto the coronal and sagittal plane. e. Spatial distribution of axonal projections of PT^Fezf2 from parietal and frontal nodes (bottom) within the hindbrain projected onto the coronal and sagittal plane. f. Brain-wide volume and peak normalized projection intensity maps of IT^PlxnD1 from FLA and FLP and PT^Fezf2 from frontal and parietal nodes from two mice. Black font indicates injection site; larger gray font indicates regions with significant projections; smaller gray font indicates regions analyzed.

Extended data 9.. IT^PlxnD1 and PT^Fezf2 show distinct spatiotemporal dynamics and spectral properties under ketamine anesthesia.

a. Example single trial traces of IT^PlxnD1 (blue) and PT^Fezf2 (green) activities from 6 different cortical areas during ketamine/xylazine anesthesia. Colors represent cortical areas as indicated (VC – primary visual Cortex, RSp – medial retrosplenial cortex, HL – primary hindlimb sensory cortex, MOs – secondary motor Cortex, MOp – primary motor cortex, BC – barrel cortex). b. Example spectrogram of IT^PlxnD1 and PT^Fezf2 activity from MOp and RSp of one mouse. c. Mean relative power spectral density of IT^PlxnD1 (blue) and PT^Fezf2 (green) activity from MOp and RSp (18 sessions across 6 mice each, shaded region indicates ±2 s.e.m). While IT^PlxnD1 exhibited oscillations at approximately 1–1.4 Hz, PT^Fezf2 fluctuated predominantly at 0.6–0.9 Hz. d. Distribution of average relative power within MOp and RSp of IT^PlxnD1 and PT^Fezf2 between 0.6–0.9 Hz and 1–1.4 Hz (n = 18 sessions across 6 mice each). e. Spatial map of the average relative power between 0.6–0.9 Hz and 1–1.4 Hz from IT^PlxnD1 (top) and PT^Fezf2 at each pixel (bottom, 18 sessions across 6 mice each). While IT^PlxnD1 was strongly active within the frontolateral at both frequency bands, PT^Fezf2 was predominantly active in the retrosplenial regions at 0.6–0.9 Hz. f. Example space-time plots of the neural activity across a slice of the dorsal cortex (red dashed line) from IT^PlxnD1 (top) and PT^Fezf2 (bottom). Middle, zoomed-in activity from indicated top and bottom panels visualizing the distinct spatial dynamics across dorsal cortex. g. Example activation sequence of the most dominant pattern (1^st dimension) identified by seqNMF from IT^PlxnD1 (top) and PT^Fezf2 (bottom, suppl. methods). The top dimension accounted for more than 80 % of the variance in IT^PlxnD1 and over 90% in PT^Fezf2. *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers. All statistics in Supplementary table 1.

Extended Data 10.. Spatiotemporal dynamics of IT^PlxnD1 and PT^Fezf2 under ketamine anesthesia across mice.

a. Mean spectrogram of IT^PlxnD1 and PT^Fezf2 activity from MOp and RSp (18 sessions across 6 mice for each cell type). b. Difference between IT^PlxnD1 and PT^Fezf2 average relative power maps for each frequency bands (18 sessions from 6 mice for each cell type). Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by FDR = 0.05). Blue pixels indicate values significantly larger in IT^PlxnD1 compared to PT^Fezf2 and vice versa for green pixels. c. Distribution of IT^PlxnD1 (blue) and PT^Fezf2 (green) spatial power maps for each frequency band projected to the subspace spanned by the top two principal components (n = 18 maps in each group). IT^PlxnD1 and PT^Fezf2 both clustered independently with further segregation between IT^PlxnD1 0.6–0.9 Hz and 1–1.4 Hz frequency bands, substantiating the distinct activation patterns between the two populations. d. Activation sequence of the most dominant pattern (1^st dimension) identified by seqNMF from IT^PlxnD1 (top) and PT^Fezf2 (bottom) activity combined across mice and sessions.

Figure 1.. Distinct activity patterns of IT^PlxnD1 and PT^Fezf2 during wakeful resting and upon sensory input

a. STP images of GCaMP6f labeled PT^Fezf2 and IT^PlxnD1 neurons across dorsal cortex. Arrow indicates anterior – posterior axis. Yellow text indicates approximate location of layer 2/3 (L2/3), layer 5a (L5a), layer 5b (L5b) and layer 6 (L6). Sagittal schematic depicts major projection patterns of IT and PT. b. mRNA in situ images of Fezf2⁺ (left), PlexinD1⁺ (middle) cells. Double in situ overlaid (right) shows Satb2⁺ (red) and PlexinD1⁺ (green). PlexinD1⁺ cells represent subset of Satb2⁺ IT cells. c. Example z-scored variance of behavior from video recordings (black trace) and corresponding variance of neural activity from IT^PlxnD1 (blue) and PT^Fezf2 (green). Gray blocks indicate active episodes. d. Average variance maps of spontaneous activity during active (right) and quiescent (left) episodes (n=12 sessions from 6 mice). e-f. Distribution of percentage of cross-validated IT^PlxnD1 and PT^Fezf2 activity variance explained by full frame behavior variance (e) and specific body part (f) from encoding model (n=12 sessions from 6 mice). g. Illustration of unimodal sensory stimulation paradigm. h. Mean activity maps of IT^PlxnD1 and PT^Fezf2 in response to corresponding sensory simulation (average of 12 sessions from 6 mice). i. Single trial IT^PlxnD1 and PT^Fezf2 activity within orofacial (yellow), whisker (red) and visual (purple) areas during orofacial (os), whisker (ws) and visual (vs) stimulation. j. Single trial heat maps of IT^PlxnD1 and PT^Fezf2 activity from whisker (bc), orofacial (oc) and visual cortex (vc) in response to corresponding sensory stimulus from 1 example mouse for each cell type. k. Mean activity of IT^PlxnD1 and PT^Fezf2 in whisker, orofacial and visual cortex during corresponding sensory stimulus (n=240 trials in 12 sessions from 6 mice, shaded region indicates ±2 s.e.m). l. Distribution of IT^PlxnD1 and PT^Fezf2 activity intensity in whisker, orofacial and visual cortex during corresponding sensory stimulus (n=240 trials in 12 sessions from 6 mice); *p<0.05, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and whiskers extend to extreme points excluding outliers (1.5 times above or below interquartile range). All statistics in Supplementary table 1.

Figure 2.. Distinct PT^Fezf2 and IT^PlxnD1 subnetworks tuned to different sensorimotor components of a feeding behavior

a. Schematic of the head-fixed feeding behavior showing the sequential sensorimotor components. b. Example traces of tracked body parts and episodes of classified behavior events. Colored lines represent different body parts as indicated (light green shade: handle-and-eat episodes; orange shade: chewing episode). c. Mean IT^PlxnD1 and PT^Fezf2 sequential activity maps (200 ms steps) during the feeding sequence before and after pellet-in-mouth (PIM) onset (IT^PlxnD1 – 23 sessions from 6 mice, PT^Fezf2 – 24 sessions from 5 mice). Note the largely sequential activation of areas and cell types indicated by arrows and numbers: 1) left barrel cortex (IT^PlxnD1) when right whiskers sensed approaching pellet; 2) parietal node (PT^Fezf2) while making postural adjustments as pellet arrives; 3) forelimb sensory area (IT^PlxnD1) with limb movements that adjusted grips of support bar as pellet approaches closer; 4) frontal node (PT^Fezf2) during lick; 5) orofacial sensory areas (FLP (Frontolateral Posterior), IT^PlxnD1) when pellet-in-mouth; 6) parietal node again during hand lift; 7) FLA (Frontolateral Anterior)-FLP (IT^PlxnD1) on handling and eating the pellet. d. Difference between IT^PlxnD1 and PT^Fezf2 average activity maps at each time step as in panel c. Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by False Discovery Rate = 0.05). Blue pixels indicate values significantly larger in IT^PlxnD1 compared to PT^Fezf2 and vice versa for green pixels. e. Spatial maps of IT^PlxnD1 (top) and PT^Fezf2 (bottom) regression weights from an encoding model associated with lick, PIM, hand lift, eating and handling, and chewing (IT^PlxnD1 - 23 sessions from 6 mice, PT^Fezf2 - 24 sessions from 5 mice).

Figure 3.. IT^PlxnD1 and PT^Fezf2 within frontolateral and parietofrontal nodes show distinct temporal dynamics during feeding behavior

a,d. Mean activity map of PT^Fezf2 (a, 24 sessions from 5 mice) and IT^PlxnD1 (d, 23 sessions from 6 mice) during feeding from 1 second before to 2 seconds after PIM. b,e. Example PT^Fezf2 (b) and IT^PlxnD1 (e) activity from FLA (magenta), FLP (orange), frontal (dark brown) and parietal (light brown) nodes during feeding behavior; vertical bars indicate behavior events. c,f. Single trial heatmaps of PT^Fezf2 activity from frontal and parietal (c) and IT^PlxnD1 from FLA and FLP nodes (f) centered to lick, PIM, and handlift onset (5 sessions from one example mouse). g. Mean PT^Fezf2 activity within frontal and parietal node centered to lick, PIM, and handlift onset. Grey dashed lines indicate median onset times of other events relative to centered event (5 sessions from one example mouse, shaded region indicates ±2 s.e.m). Grey shade indicates eating-handling episode. h. Mean IT^PlxnD1 activity within FLA and FLP centered to lick, PIM, and handlift onset (5 sessions from one example mouse, shaded region indicates ±2 s.e.m). i. Single trial heatmaps of IT^PlxnD1 and PT^Fezf2 activities within parietal, frontal, FLP and FLA centered to PIM (IT^PlxnD1 - 23 sessions from 6 mice, PT^Fezf2 - 24 sessions from 5 mice). j. Mean IT^PlxnD1 and PT^Fezf2 activity within parietal (light brown), frontal (dark brown), FLP (orange) and FLA (magenta) centered to PIM (sample size as in panel i, shaded region indicates ±2 s.e.m). Left inset: Overlaid activity maps of IT^PlxnD1 (blue) and PT^Fezf2 (green) after thresholding indicating distinct nodes preferentially active during the feeding sequence. k. Distribution of IT^PlxnD1 and PT^Fezf2 activities centered at PIM onset from parietal, frontal, FLP and FLA projected to the subspace spanned by first two linear discriminant analysis dimensions (IT^PlxnD1 - 23 sessions from 6 mice, PT^Fezf2 - 24 sessions from 5 mice).

Figure 4.. Feeding without hand lift selectively occludes PT^Fezf2 activity in parietal node.

a. Schematic of feeding without hand lift trials. b. Single trial heatmaps of PT^Fezf2 within frontal and parietal nodes centered to lick and PIM onset during feeding without handlift (2 sessions from one example mouse). c. Mean PT^Fezf2 activity within frontal and parietal nodes centered to PIM during feeding with (dashed lines, 5 sessions from one example mouse) and without handlift (solid lines, 2 sessions from the same example mouse, shaded region indicates ±2 s.e.m). Grey box indicates eating-handling episode during handlift sessions. d. Single trial heatmaps of IT^PlxnD1 within FLA and FLP centered to lick and PIM onset during feeding without handlift (3 sessions from one example mouse). e. Mean IT^PlxnD1 activity within FLA and FLP centered to PIM during feeding with (dashed, 5 sessions from one example mouse) and without handlift (solid, 3 sessions from the same example mouse, shaded region indicates ±2 s.e.m). f. Distribution of PT^Fezf2 activity intensity during 1 sec post PIM onset from parietal and frontal nodes during with and without handlift (no lift: 440 trials in 13 sessions from 5 mice, lift: 647 trials in 24 sessions from the same 5 mice). g. Distribution of IT^PlxnD1 activity intensity during 1 sec post PIM onset from FLA and FLP during with and without hand lift (no lift: 544 trials in 15 sessions from 6 mice, lift: 781 trials in 23 sessions from the same 6 mice). h. Mean PT^Fezf2 (top) and IT^PlxnD1 (bottom) activity maps during 1 sec post PIM onset with (left) and without (right) handlift. i. Difference in PT^Fezf2 and IT^PlxnD1 mean spatial activity maps between feeding with and without handlift. Only significantly different pixels are displayed (two-sided Wilcoxon rank sum test with p-value adjusted by FDR = 0.05, sample size as in panel f,g). Note that parietal areas in PT^Fezf2 and no pixels in IT^PlxnD1 are significantly different.***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers. All statistics in Supplementary table 1.

Figure 5.. Inhibiting IT^PlxnD1 and PT^Fezf2 disrupts distinct components of feeding

a. Top: Optogenetic setup layout (left) with inhibition location (right). Bottom: Example GtACR1 expression in IT^PlxnD1 and PT^Fezf2 left cortex. b. Left: 10 example tongue trajectories centered to inhibition of IT^PlxnD1 and PT^Fezf2 frontal (brown) node. Control (grey). Trajectories proceed from mouth (top) to belt (bottom, green schematic). Right: Mean tongue trajectories following inhibition of IT^PlxnD1 frontal (brown, n=173), FLA (magenta, n=165) and PT^Fezf2 frontal (n=140) and FLA (n=98 trials) nodes. Black (control). c. Distribution of mean tongue length for 1.5 seconds during control and inhibition of IT^PlxnD1 and PT^Fezf2 frontal and FLA nodes. Sample size as in b. d. Handlift probability during control and inhibition of IT^PlxnD1 frontal (n=201), FLA (n=200) and PT^Fezf2 frontal (n=82) and FLA (n=38) nodes. e. Top two rows: example hand trajectory during inhibition (brown) of IT^PlxnD1 and PT^Fezf2 frontal node. Control (grey). Trajectories proceed from belt (bottom) to mouth (top). Bottom: Mean single hand trajectories during inhibition of IT^PlxnD1 frontal (inh (n=353), control (n=363)), FLA (inh (n=455), control (n=461)) and PT^Fezf2 frontal (inh (n=167), control (n=176)) and FLA (inh (n=202), control (n=204)) nodes. Control (Black). f,g. Distribution of mean normalized hand-mouth distance (f) and mean absolute hand velocity (g) for 5 seconds during control and inhibition of IT^PlxnD1 and PT^Fezf2 frontal and FLA nodes. h. Distribution of mean hand near mouth duration for 5 seconds during control and inhibition of IT^PlxnD1 frontal nodes. i. Mean trace of inter-finger 1–2 distance during inhibition of IT^PlxnD1 frontal (brown) and FLA (magenta) nodes. Control (black). Red line over finger 1 and 2 illustrates the variable measured. j. Distribution of mean inter finger 1–2 distance for 5 seconds during control and inhibition of IT^PlxnD1 frontal and FLA nodes. Sample size for panels f-j as in e. All data pooled from 4 mice for IT^PlxnD1 and 3 for PT^Fezf2. *p<0.05, **p<0.005, ***p<0.0005. For box plots, central mark indicates median, bottom and top edges indicate 25^th and 75^th percentiles and the whiskers extend to extreme points excluding outliers. Shaded region indicates ±2 s.e.m. All statistics in supplementary table 1.

Figure 6.. Brain wide projections of IT^PlxnD1 and PT^Fezf2 from frontolateral and parietofrontal networks.

a. Anterograde projections of IT^PlxnD1 from FLA and FLP and PT^Fezf2 from frontal and parietal nodes within isocortex projected to the dorsal cortical surface from an example mouse. b. Brain-wide volume and peak normalized projection intensity maps of IT^PlxnD1 from FLA and FLP and PT^Fezf2 from frontal and parietal nodes from an example mouse. Black font indicates injection site; larger gray font indicates regions with significant projections; smaller gray font indicates regions analyzed. c. Schematic of the projection of IT^PlxnD1 from FLA and FLP and PT^Fezf2 from frontal and parietal nodes. Circle indicates the site of injection.