Sensory and decision-related activity propagate in a cortical feedback loop during touch perception

Abstract

The brain transforms physical sensory stimuli into meaningful perceptions. In animals making choices about sensory stimuli, neuronal activity in successive cortical stages reflects a progression from sensation to decision. Feedforward and feedback pathways connecting cortical areas are critical for this transformation. However, the computational functions of these pathways are poorly understood because pathway-specific activity has rarely been monitored during a perceptual task. Using cellular-resolution, pathway-specific imaging, we measured neuronal activity across primary (S1) and secondary (S2) somatosensory cortices of mice performing a tactile detection task. S1 encoded the stimulus better than S2, while S2 activity more strongly reflected perceptual choice. S1 neurons projecting to S2 fed forward activity that predicted choice. Activity encoding touch and choice propagated in an S1-S2 loop along feedforward and feedback axons. Our results suggest that sensory inputs converge into a perceptual outcome as feedforward computations are reinforced in a feedback loop.

Figure 1

Cellular resolution imaging of population activity in S1 and S2 during tactile detection. (a) Schematic of experimental setup. Mice were trained to report detection of single whisker deflections by licking a lick-port for a liquid reward. Activity of L2/3 neurons in the whisker area of S1 or S2 was monitored by two-photon calcium imaging. (b) Trials began with an auditory cue. On 50% of trials (“Go” trials), a single whisker was deflected with a sinusoidal waveform (0.5 s, 20 Hz). The whisker was not deflected on the other 50% of trials (“NoGo” trials). Trial outcome was determined by lick responses occurring during a response window. (c) Four possible trial outcomes based on the stimulus condition (present vs absent) and the animal’s response (lick vs no-lick). (d) Schematic of brain areas monitored by two-photon imaging. S1 (brown) or S2 (orange) were identified using intrinsic signal optical imaging. (e) Left, View through a cranial imaging window implanted over S1 and S2. GCaMP6 expression (white) is evident in S2. Right, Example field of view for two-photon calcium imaging (max projection through time for a trial). (f) Example GCaMP6 fluorescence traces concatenated across trials from S1 and S2 imaging sessions. Vertical colored lines indicate start of each trial (blue: Hit; black: Miss; green: False Alarm; red: Correct Rejection). Dashed lines: onset of whisker stimulus. (g) Example fluorescence traces (thin lines) and means (thick) showing two S1 and two S2 neurons (one neuron per row). Traces are grouped and colored by trial type. Dashed lines: onset of whisker stimulus. (h) Fraction of neurons that were task-responsive in S1 or S2, for individual mice (gray circles) and means (black; ± SEM).

Figure 2

Coding of stimulus and choice in S1 and S2. (a) Activity (mean ± SEM ΔF/F₀ across mice) averaged across Hit (blue), Miss (black), False Alarm (green) and Correct Rejection (red) trials from S1 imaging sessions (12 sessions, 5 mice, 274 ± 52 neurons per mouse; mean ± SEM). Evoked ΔF/F₀ responses on Hits were larger than on Misses (t_(1,369) = 16.79, P = 1.17 × 10⁻⁵⁷; 1,370 neurons). Cyan shading: first 0.25 s after stimulus onset, which preceded 96.5% of first licks. Dashed line: last time point before median first-lick time. (b) Same as a for S2 (11 sessions, 5 mice,103 ± 26 neurons per mouse). Evoked ΔF/F₀ responses on Hits were larger than on Misses (t₍₆₀₆₎ = 20.32, P = 2.22 × 10⁻⁷⁰; 607 neurons; paired t-tests). In S2, evoked ΔF/F₀ responses on False Alarms were larger than on Correct Rejections (t₍₆₀₆₎ = 4.07, P = 5.33 × 10⁻⁵; paired t-tests). (c) Mean evoked ΔF/F₀ responses normalized to Hits across individual neurons in S1 and S2 (mean ± SEM across mice; circles show individual mice). For both S1 and S2 neurons, responses on Misses were smaller than on Hits (Miss/Hit ratio: S1: 0.57 ± 0.04; z = 22.3, **P = 1.73 × 10⁻¹¹⁰; 1,370 neurons; S2: 0.32 ± 0.05; z = 18.84, **P = 3.76 × 10⁻⁷⁹; 607 neurons; Wilcoxon sign rank tests), with stronger modulation for S2 compared with S1 (z = 7.46, **P = 8.38 × 10⁻¹⁴; Wilcoxon rank sum test). (d) Cumulative histograms (means across mice) of “stimulus probability” (SP, left), “detect probability” (DP, middle) and DP/SP ratio (right) for all (responsive and non-responsive) neurons in S1 (brown) and S2 (orange). DP/SP ratio was higher in S2 (medians: 1.01 vs 0.97; D = 0.093, **P = 1.96 × 10⁻⁷; 2,490 S1 and 1,471 S2 neurons; Kolmogorov-Smirnov test); SP was higher in S1 compared with S2 (medians: 0.55 vs 0.52; D = 0.063, **P = 0.0013; Kolmogorov-Smirnov test) while DP was similar (D = 0.044, P = 0.056; Kolmogorov-Smirnov test). S1: 12 sessions, 5 mice, 498 ± 74 neurons per mouse; S2: 11 sessions, 5 mice, 294 ± 47 neurons per mouse). (e) Maps from one mouse showing distributions of stimulus- and decision-encoding neurons in S1. White neurons are those whose SP or DP 95% confidence intervals included 0.5. Responsive and non-responsive neurons are included. Black “+” marks: center of the somatotopic column of the stimulated whisker. (f) Same as e for S2. (g) Cumulative histograms of pairwise distances among SP neurons (black), DP neurons (gray), and all neurons (dashed blue; responsive and non-responsive) in S1. Both SP and DP neurons had smaller pairwise distances among themselves compared with all neurons (both **P < 5 × 10⁻⁵; 26,898 SP, 12,387 DP, and 431,962 all-neuron pairs; permutation tests). (h) Same as g for S2. (i) Mean SP (black) and DP (gray) values averaged across all neurons (8 bins of 50 µm; ± SEM) as a function of distance from the center of the somatotopic column representing the stimulated whisker in S1. SP and DP both decreased with distance from the column center (SP: −0.028 per 100 µm; DP: −0.017 per 100 µm; test of zero slope: F_(1,74) = 54.84, P = 1.711 × 10⁻¹⁰; difference in slopes for SP and DP: F_(1,74) = 3.58, P = 0.062; 78 binned values total from 5 mice; ANCOVA). (j) Same as i for S2 (test of zero slope: F_(1,54) = 3.11, P = 0.083; difference in slopes for SP and DP: F_(1,54) = 0.92, P = 0.342; 58 values from 5 mice). (k) Performance of a classifier (mean ± SEM across mice) in decoding the stimulus condition from population activity at each time point reached higher levels for S1 (84 ± 7% correct by 0.25 s after stimulus onset, and 89 ± 5% by the median reaction time of 0.52 s) compared with S2 (68 ± 8% correct by 0.25 s, and 82 ± 4% by 0.52 s; performance diverged by 0.32 s: U = 76, P = 0.045; 7 S1 and 9 S2 sessions; one-tailed Wilcoxon rank sum test). Cyan shading and vertical dashed line as in a. (l) Same as in k for decoding choice. Performance was higher for S2 compared with S1 (79 ± 3% vs 72 ± 1% at 0.35 s; U = 40, P = 0.021; one-tailed Wilcoxon rank sum test). (m) Mean pairwise noise correlations between each responsive neuron and other responsive neurons (top), each DP neuron and other DP neurons (middle), or each SP neuron and other SP neurons (bottom). Noise correlations were higher in S2 (all **P < 5 × 10⁻⁵; S1: 1,370 responsive, 536 SP and 338 DP neurons; S2: 607 responsive, 287 SP and 259 DP neurons; permutation tests).

Figure 3

Feedforward propagation of activity from S1 to S2 predicts choice. (a) Schematic of retrograde labeling of S2-projecting (S2p) neurons in S1. (b) Coronal section showing CTB-Alexa555 (red) at the S2 injection site and GCaMP6 (green) in S1. (c) In vivo identification of S2p neurons. Dashed circles indicate GCaMP6-expressing neurons labeled with CTB-Alexa555. (d) Activity (mean ± SEM ΔF/F₀ across 3 mice, 8 sessions total) of S2p neurons averaged across trial types. Responses on Hits were larger than on Misses (0.040 ± 0.003 ΔF/F₀ vs 0.020 ± 0.006 ΔF/F₀; t₍₈₇₎ = 5.28, P = 9.24 × 10⁻⁷; 88 neurons; paired t-test). Conventions as in Fig. 2a. (e) Same as d for unlabeled neurons (Hit: 0.027 ± 0.003 ΔF/F₀; Miss: 0.019 ± 0.007 ΔF/F₀; t₍₆₄₇₎ = 13.19, P = 2.43 × 10⁻³⁵; 648 neurons; paired t-test). (f) Mean evoked ΔF/F₀ responses normalized to Hits across individual neurons (mean ± SEM across mice; circles show individual mice). For both S2p and unlabeled neurons, responses on Misses were smaller than on Hits (Miss/Hit ratio: S2p: 0.21 ± 0.14; z = 6.51, **P = 7.43 × 10⁻¹¹; 88 neurons; unlabeled: 0.43 ± 0.12; z = 13.81, **P = 2.11 × 10⁻⁴³; 648 neurons; Wilcoxon sign rank tests), with slightly stronger modulation for S2p compared with unlabeled neurons (*P = 0.031; permutation test). (g) S2p neurons showed higher SP compared with unlabeled neurons (medians: 0.57 vs 0.54; **P < 5 × 10⁻⁵; 133 S2p and 1,134 unlabeled neurons; permutation test). Includes responsive and non-responsive neurons. (h) S2p neurons showed higher DP compared with unlabeled neurons (medians: 0.58 vs 0.54; **P < 5 × 10⁻⁵; permutation test). (i) Performance of a classifier (mean ± SEM across mice) in decoding the stimulus condition from population activity reached a higher level for S2p compared with unlabeled neurons (73 ± 2% vs 63 ± 5% at 0.24 s after stimulus onset; R = 34, P = 0.012; 8 sessions; one-tailed Wilcoxon sign rank test). Conventions as in Fig. 2k. (j) Same as i for decoding choice (68 ± 7% vs 62 ± 5% at 0.24 s; R = 30, P = 0.055; one-tailed Wilcoxon sign rank test). (k) Cumulative histograms of mean pairwise noise correlations between each S2p neuron and other S2p neurons (orange), or each unlabeled neuron and other unlabeled neurons (brown). Noise correlations were higher among S2p neurons (**P < 5 × 10⁻⁵; 88 S2p and 648 unlabeled neurons; permutation test).

Figure 4

Activity in a feedback loop between S2 and S1. (a) Schematic of S1→S2 axon imaging experiment. AAV-GCaMP6 was injected in S1. A glass window was placed over S2. (b) Left, Image of a coronal section showing GCaMP6 fluorescence in the injection (S1) and imaging (S2) areas. Right, Example field of view from L2/3 of S2. Dashed circles indicate example regions of interest (ROIs). (c) Schematic of S2→S1 axon imaging experiment. AAV-GCaMP6 was injected in S2. A glass window was placed over S1. (d) Left, Image of a coronal section showing GCaMP6 fluorescence in the injection (S2) and imaging (S1) areas. Right, Example field of view and ROIs from L1 of S1. (e) Activity (mean ± SEM ΔF/F₀ across 4 mice, 7 sessions total, 160 axons total) of S1→S2 axons for each trial type. Responses on Hits were larger than on Misses (0.11 ± 0.027 ΔF/F₀ vs 0.079 ± 0.029 ΔF/F₀; t₍₁₅₉₎ = 3.25, P = 0.001; 160 axons; paired t-test). Conventions as in Fig. 2a. (f) Same as e for S2→S1 axons (10 sessions, 4 mice, 440 axons) of S2→S1 axons. Responses on Hits were larger than on Misses (0.031 ± 0.004 ΔF/F₀ vs 0.016 ± 0.002 ΔF/F₀; t₍₄₃₉₎ = 3.13, P = 0.002; paired t-test). A subset of S2→S1 data in panels f, g–i are reanalyzed from ref. . (g) Mean evoked ΔF/F₀ responses normalized to Hits across individual axons (mean ± SEM across mice; circles show individual mice). For both S1→S2 and S2→S1 axons, responses on Misses were smaller than on Hits (Miss/Hit ratio: S1→S2: 0.61 ± 0.10; S = 99, *P = 0.003; 160 axons; S2→S1: 0.29 ± 0.11; S = 313, **P = 1.15 × 10⁻¹⁸; 440 axons; sign test), with stronger modulation for S2→S1 axons (z = 3.82, **P = 1.33 × 10⁻⁴; Wilcoxon rank sum test). (h) Performance of a classifier (mean ± SEM across mice) in decoding the stimulus condition from population activity reached a higher level for S1→S2 compared with S2→S1 axons (82 ± 2% vs 66 ± 6% at 0.28 s after stimulus onset; z = 2.05, P = 0.044; 7 S1→S2 and 10 S2→S1 sessions; Wilcoxon rank sum test). Conventions as in Fig. 2k. (i) Same as h for decoding choice. (j) Schematic of feedforward and feedback propagation of task-related activity (dashed: hypothetical functional pathways). (k) Top, schematic of optogenetic silencing experiment. In mice expressing channelrhodopsin-2 in GABAergic neurons, a 473 nm laser was directed over the C2 column (in S1), whisker S2 or a control area (within whisker S1 but ~1 mm from the C2 column). Photostimulation was randomly delivered on 30–40% of all behavioral trials. Bottom, example electrophysiology traces showing responses of an S1 neuron to whisker stimulation with and without laser illumination. (l) Cell-attached electrophysiology recordings were targeted to the C2 column in awake mice (16 neurons in 2 mice). The C2 whisker was stimulated in the presence or absence of laser illumination directed to the recording site (centered over the C2 column in S1), S2 or the control area. Silencing was quantified as the ratio of whisker-evoked spike count in the presence vs absence of illumination. S1 (C2 area) illumination produced stronger silencing of whisker-evoked responses compared with illumination of either S2 or the control area. Silencing was similar for S2 and the control area. Vertical axis is broken to accommodate one outlier. (m) Task performance (mean ± SEM across 4 mice) was reduced by illuminating either S1 (from 74 ± 1.7% to 58 ± 1.5% correct) or S2 (from 77 ± 2.0% to 57 ± 4.6% correct), to a similar degree (S1 vs S2 reductions: z = −0.81, P = 0.421; 12 S1 and 15 S2 sessions; Wilcoxon rank sum test). Illuminating the control area caused a much smaller drop in performance (from 75 ± 2.6% to 70 ± 1.8% correct; S2 vs control area reductions: z = −3.27, P = 0.001; 12 control area sessions; Wilcoxon rank sum test). Gray lines: performance of individual mice averaged across sessions for interleaved trials with (bolts) and without (no bolts) illumination. Black symbols: mean ± SEM across mice.