Small Networks Encode Decision-Making in Primary Auditory Cortex Nikolas A. Francis, Daniel E. Winkowski, Alireza Sheikhattar, Kevin Armengol, Behtash Babadi, Patrick O. Kanold  Neuron  Volume 97, Issue 4, Pages 885-897.e6 (February 2018) DOI: 10.1016/j.neuron.2018.01.019 Copyright © 2018 Elsevier Inc. Terms and Conditions

Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 2P Imaging in Awake Behaving Mice (A) Head-fixed mice were trained to detect a 55 dB SPL, 1 s pure tone presented from a free-field speaker, while neurons in A1 were imaged. Mice responded to tone detection by licking a waterspout. (B) Tone detection behavior. Top: average lick rate during task performance. Horizontal black bar shows when the target tone was presented. Red trace shows the average lick rate for punished hits, i.e., when the first lick occurred before the reward time window (gray shaded region). No water was delivered after the first lick during punished hits, so the mice stopped licking. The blue trace shows the lick rate for rewarded hits, indicating robust licking after receiving water. Bottom: example lick traces for eight sequential trials during task performance. White regions with an X show miss trials, when the mouse did not respond. Blue and red regions show rewarded and punished hit trials, respectively. (C) Head-fixed task performance for roved tone levels to find the level (55 dB SPL) that produced an ∼60%–70% hit rate in the target frequency range (4–32 kHz). (D) Cumulative distribution function (CDF) for response latency (dark, total set of experiments; light, mice). (E) Performance rates for each behavioral response type for experiments (dark) and animals (light). Error bars show two SEMs. Total hit rates are as follows: 65.2% ± 18.8% and 61.5% ± 13.9%, across experiments and animals, respectively. Rewarded hit rates are as follows: 40.2% ± 12.2% and 39.6% ± 6.7% across experiments and animals, respectively. Punished hit rates are as follows: 25% ± 17.7% and 28% ± 15.2% across experiments and animals, respectively. Miss rates are as follows: 35% ± 18.8% and 32.5% ± 15.5% across experiments and animals, respectively. Stars indicate significant differences between pairs of groups indicated by the bars: hits versus misses (bootstrap t test; experiments, p < 0.001; animals, p = 0.0011) and punished hits versus rewarded hits (bootstrap t test; experiments, p < 0.001; animals, p = 0.04). Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Tone-Evoked Neuronal Responses Imaged in Primary Auditory Cortex Layer 2/3 Show Pure-Tone Frequency Selectivity (A) Top left: wide-field imaging used to localize A1, as indicated by a rostro-caudal gradient of high to low frequencies in the 4–48 kHz range (Figure S1). Bottom: mRuby-labeled cell bodies and GCaMP6s-labeled cell membranes. (B) Left: two examples of frequency response areas (FRAs) generated by measuring responses to pure tones presented at 35–75 dB SPL. Cells could show narrow V-shape FRAs monotonic with level or show a wider FRA non-monotonic with level. Right: best frequency (BF) of each cell for tones presented at 55 dB SPL. Gray cells were either too dim (Figure S3) or did not respond to pure tones during passive trials. Color coding is same as in (A). (C) CDFs of the set of pure-tone targets for each experiment (N = 80 experiments) and BFs from all neurons (N = 4,316). (D) Exemplar fluorescence traces from an A1 neuron in response to tones of different frequencies. Left column shows average of ten repetitions of each frequency. Each row shows the data for a single-tone frequency. Right column shows a heatmap for individual trial responses to each tone. Shading indicates two SEMs. (E) Exemplar mean response reliability distributions from two experiments. Arrowheads indicate the mean. Response reliability was defined as the fraction of trials with significant responses in each cell, for the set of tones presented during passive trials (Bandyopadhyay et al., 2010; Rothschild et al., 2010; Kanold et al., 2014; Maor et al., 2016; Winkowski et al., 2013). Reliability was similar for blocks of five or ten trials per frequency. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 Tone-Evoked Activity in A1 Is Modulated by Behavioral Choice (A) Heatmap of individual neuronal responses. Each row shows a neuron’s average response across repetitions of the same pure-tone frequency during passive (left), hit (middle), and miss (right) trials. Facilitative and suppressive responses are shown as positive (warm colors) and negative (cool colors) values, respectively. The vertical black line shows the tone onset time. Neurons in all three panels were sorted by peak response latency during hit trials. (B) Population average response time courses for both facilitative and suppressive hit trials (red), miss trials (blue), and passive trials (black). Shading shows two SEMs. (C) Boxplots for the distribution of population responses, after collapsing the time course for each cell into an average value across time after the tone onset. Conditions are color coded as in (B). ∗p < 0.001, KW test. The average facilitative ΔF/F responses for passive, hit, and miss trials were 14.8% ± 0.62%, 24% ± 0.71%, and 10% ± 0.5%, respectively. The average suppressive ΔF/F responses for passive, hit, and miss trials were −2.9% ± 1.4%, −22.1% ± 1.4%, and −5.4% ± 1.04%, respectively. Error bars show 95% confidence intervals. (D) CDFs for attentional gain, measured as the differences in ΔF/F between hit and passive conditions. CDFs were computed by taking the cumulative sum of the histogram of attentional gain values. Left, middle, and right panels show the CDFs for the total population of cells, experiments, and mice, respectively. CDFs for facilitative and suppressive responses in black and gray, respectively. The mean attentional gain for facilitative responses was 9.9% ± 0.93%, 9.9% ± 2.0%, and 9.9% ± 3.7%, across cells, experiments, and mice, respectively. The mean attentional gain for suppressive responses was 19.9% ± 2%, 20.7% ± 3.5%, and 23.8% ± 5.0%, across cells, experiments, and mice, respectively. Arrowheads indicate mean values. ∗Significant difference from 0 (bootstrap t test, p < 0.001). The magnitude of attentional gain in suppressive responses across individual cells, experiments, and mice was significantly greater than the attentional gain for enhanced responses (bootstrap t test, p < 0.001). (E) Attentional gain for groups of cells according to the octave difference between BF and the target frequency (ΔTAR). Attentional gain was positive for both groups (ΔTAR ≤ 0.5, 12.8% ± 0.6%; ΔTAR > 0.5, 11.4% ± 0.6%; bootstrap t test, p < 0.001) but similar (bootstrap t test, p = 0.11). Error bars show two SEMs. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Neurons in A1 Encode Decision Timing and Cost (A) Left: time-averaged activity aligned to licks during the silent period is similar to 0 (bootstrap t test, p = 0.5). Right: the time-averaged difference in activity of lick-aligned hit-miss trials (i.e., attentional gain, or “choice”) is positive (bootstrap t test, p < 0.001). Error bars show two SEMs. (B) Population average time course of neural responses to tones during rewarded (blue) and punished (red) hits. Mice were trained to withhold licking for 0.5 s after the tone onset. Shading indicates two SEMs. (C) Left: average behavioral versus neural response latencies for each experiment. Vertical and horizontal lines mark 0.5 s wait period. Right: average neural response latency for punished and rewarded hits. ∗p < 0.001 (bootstrap t test). Error bars show two SEMs. (D) The time course of the difference trace from (B) shows the dynamics of decision cost (green; rewarded hit – punished hit). Shading indicates two SEMs. (E) CDFs for decision-cost signaling for all cells (left), experiments (middle), and mice (right). Arrowheads indicate the means (9.5% ± 0.78%, 9.1% ± 1.7%, and 10.2% ± 3.1% ΔF/F). ∗Means different from 0 (bootstrap t test, p < 0.001). (F) Model of A1 activity that is modulated by attentional gain, the cost of behavioral choice, and sensory input. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 5 Auditory Attention Modulates Pairwise Noise Correlations in A1 L2/3 (A) CDF of average pairwise neuronal noise correlations (r) for each neuron in the study (N = 4,316) during passive (black) and hit (blue) trials. The vertical dotted line shows the inflection point (r = 0.09) where the sign of rHIT-rPASSIVE (i.e., Δr) reverses. Δr values indicated by the gray arrows pointing below (Δr+) and above (Δr−) the inflection point were separated for subsequent panels. (B) Probability density functions (PDFs) for Δr+ and Δr−. ∗Means different from 0 (bootstrap t test, p < 0.001). (C) r values for cells grouped by the octave distance between best frequencies (ΔBF). The mean values for Δr+ for hit versus passive were as follows: ΔBF ≤ 0.5, 0.12 ± 0.007 versus −0.02 ± 0.007; ΔBF > 0.5, 0.1 ± 0.007 versus −0.05 ± 0.007. The mean values for Δr− for hit versus passive were as follows: ΔBF ≤ 0.5, 0.16 ± 0.005 versus 0.27 ± 0.006; ΔBF > 0.5, 0.14 ± 0.005 versus 0.24 ± 0.006. ∗Differences between BF groups in Δr− group for both active and passive (bootstrap t test, p < 0.001). Error bars show two SEMs. Inset shows differences in Δr between BF groups (Δr (ΔBF ≤ 0.5) − Δr (ΔBF > 0.5)) and indicates that Δr− cells with ΔBF < 0.5 showed the largest reduction in r. (D) Noise correlations as function of BF relative to the target frequency (ΔTAR). The average noise correlations for Δr− in hit versus passive trials were as follows: ΔTAR ≤ 0.5, 0.14 ± 0.008 versus 0.26 ± 0.008; ΔTAR > 0.5, 0.15 ± 0.006 versus 0.27 ± 0.006. The average noise correlations for Δr+ in hit versus passive trials were as follows: ΔTAR ≤ 0.5, 0.11 ± 0.01 versus −0.04 ± 0.009; ΔTAR > 0.5, 0.11 ± 0.008 versus −0.04 ± 0.006. (E) Effect of behavioral choice on noise correlations across all neurons. ∗p < 0.001, KW test. Error bars show two SEMs (D and E). Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 6 GC Subnetwork Structure Was Modulated by Task Performance (A) GC analysis was used to find directional links (i.e., effective connections) between neurons. Example GC networks during passive (top), hit (middle), and miss (bottom) trials. Positive (+) and negative (−) links are shown in orange and black, respectively. Magnification shows example of directionality for individual GC links. (B) Example traces of pairs of positive or negative GC-linked neurons. (C) CDF of GC link weight. ∗Difference between the passive and hit trials (bootstrap t test, p < 0.001). Trends were similar across mice (Table S1). (D) GC link angle. ∗The significant rostro-caudal directionality of links during hit trials (Rayleigh test, p = 0.03). (E) Pairwise noise correlations for cells inside a GC subnetwork (GCIN) and cells outside of a subnetwork (GCOUT). Average Δr− noise correlations for GCIN cells for hit versus passive trials were as follows: 0.098 ± 0.006 versus 0.135 ± 0.008. Average Δr− noise correlations for GCOUT cells were as follows: 0.103 ± 0.005 versus 0.11 ± 0.011. GC average Δr+ noise correlations for GCIN cells for hit versus passive trials were as follows: 0.08 ± 0.007 versus 0.13 ± 0.01. Average Δr+ noise correlations for GCOUT cells for hit versus passive trials were as follows: 0.09 ± 0.006 versus 0.09 ± 0.02. For both Δr− and Δr+ groups, GCIN cells had larger noise correlations during passive trials compared to GCOUT cells, and significantly decreased noise correlations during hit trials (green and purple ∗ bootstrap t test, p < 0.001). Noise correlations in GCOUT cells were similar for hit and passive trials (bootstrap t test; Δr−, p = 0.62; Δr+, p = 0.66). Error bars show two SEMs. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 7 Tone Detection Drives GC Subnetworks into Small and Localized Clusters of Four to Five Neurons with Diverse Frequency Tuning (A) CDF for number of +links. Asterisk (∗) indicates differences between passive and hit trials (bootstrap t test, p < 0.001). (B) CDF for number of −links. (C) CDF for GC subnetwork size. Blue asterisk (∗), significant difference between the hit and miss trials (bootstrap t test, p = 0.019). (D) CDF for GC link length. (E) BF differences (ΔBF) between pairs of cells for GCIN and GCOUT cells. Populations were similarly distributed and means were similar to 0 (bootstrap t test, p > 0.05). Error bars show 95% confidence intervals. (F) Illustration of passive and active (i.e., hit) GC networks. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 8 GC Subnetworks Have Enhanced Encoding of Behavioral Choice (A) Prediction accuracy (PA) of decoded neural activity plotted as a function of time. The black curve shows the PA averaged across experiments. The green line shows the PA for shuffled data. Shading shows two SEMs. (B) PA CDF is shown for all 80 experiments. (C) Both facilitative and suppressive cells (Figures 3A and 3B) had PA greater than chance (bootstrap t test, p < 0.001) but were not different (bootstrap t test, p = 0.74). (D) Cells with BF near and far from the target frequency had PA greater than chance (bootstrap t test, p < 0.001), but were not different (bootstrap t test, p = 0.1). (E) GC subnetwork membership enhances PA. Both GCIN and GCOUT cells had PA greater than chance (bootstrap t test; GCIN, p < 0.001; GCOUT, p = 0.008). ∗PA of GCIN cells was larger than GCOUT cells (bootstrap t test, p = 0.012). Error bars show two SEMs (C–E). (F) PA during each experiment was tested against the number of neurons used for prediction. Arrowhead: four neurons are sufficient to predict behavior above chance. Neuron 2018 97, 885-897.e6DOI: (10.1016/j.neuron.2018.01.019) Copyright © 2018 Elsevier Inc. Terms and Conditions