Experience-Dependent Plasticity of Odor Representations in the Telencephalon of Zebrafish  Gilad A. Jacobson, Peter Rupprecht, Rainer W. Friedrich  Current Biology  Volume 28, Issue 1, Pages 1-14.e3 (January 2018) DOI: 10.1016/j.cub.2017.11.007 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Responses of Mitral Cells to Repeated Odor Stimulation (A) Calcium signals (ΔF/F0) evoked in the glomerular/mitral cell layer of the OB by repeated odor stimulation (His, 10−4 M; ISI = 2 min; trials 1, 2, and 5). Grayscale image shows raw fluorescence. See also Figure S1. (B) Top: time course of somatic calcium signals of three MCs during trials 1, 2, and 5. Black bar depicts odor stimulus. Bottom: time courses of somatic calcium signals of 64 randomly selected MCs, sorted by response magnitude in trial 1. F0 was calculated by the Favg method. (C) Mean response time course of all MCs in trials 1–8 (n = 281 MCs from 7 experiments in 5 fish). Shaded area shows time window used to measure response amplitude. (D) Mean response amplitude as a function of trial number across experiments, normalized in each experiment to the mean response amplitude across trials. Continuous black line shows exponential fit; dashed line shows asymptote. Only the first trial had a significantly larger response amplitude (one-way ANOVA; df = 7, F = 2.98, p = 0.026). See also Figure S2A. (E) Mean correlation between odor-evoked activity patterns (upper triangle) and between pre-stimulus activity in the same trials (lower triangle). (F) Bars indicate mean correlations between activity patterns in the first trial and subsequent trials (right) and correlations between subsequent trials. Gray lines indicate individual experiments (n = 281 MCs from 7 experiments in 5 fish; correlations of trial 1 versus trials 2–8: 〈r〉=0.78±0.13; within trials 2–8; 〈r〉=0.89±0.05; paired t test; n = 7 experiments; ∗p = 0.017). Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Responses of Dp Neurons to Repeated Odor Stimulation (A) Calcium signals (ΔF/F0) evoked in Dp by repeated odor stimulation (Lys, 10−4 M; ISI = 2 min; trials 1, 2, and 5). Grayscale image shows raw fluorescence. See also Figure S1. (B) Top: time course of somatic calcium signals of three Dp neurons during trials 1, 2, and 5. Black bar depicts odor stimulus. Bottom: time courses of somatic calcium signals of 64 randomly selected Dp neurons, sorted by the response magnitude in trial 1. F0 was calculated by the Favg method. (C) Mean response time course of all Dp neurons (n = 1,446 neurons from 14 experiments in 14 fish) in trials 1–8. Shaded area shows time window used for calculating the response amplitude. (D) Mean normalized response amplitude as a function of trial number. Continuous black line shows exponential fit; dashed line shows asymptote. Trial number had a significant effect on response amplitude (one-way ANOVA; df = 7, F = 7.76, p ≪ 10−3). See also Figures S2B and S3. (E) Mean correlation between odor-evoked activity patterns (upper triangle) and between pre-stimulus activities in the same trials (lower triangle). (F) Bars indicate mean correlations between activity patterns in the first and subsequent trials (right) and correlations between subsequent trials (left). Gray lines indicate individual experiments (n = 1,446 neurons from n = 14 experiments; correlations of trial 1 versus trials 2–8: 〈r〉=0.48±0.2; within trials 2–8: 〈r〉=0.67±0.11; paired t test; n = 14 experiments; ∗∗∗p < 10−3). Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Recovery and Stimulus Specificity of Response Adaptation in Dp (A) Response amplitudes of Dp neurons during a series of odor stimuli (ISI = 2 min; 8 trials) and after different recovery times (filled circles). Black curve shows mean ± SD; colored curves show individual experiments (n = 13 experiments in 8 fish; n = 1,560 neurons). Responses were normalized to the mean of the odor series in each experiment; t = 0 is the time of the last odor application in the series (open circles). The mean response amplitude after recovery was significantly different from the first and last responses during the preceding odor series (paired t test; p < 0.05). (B) Correlation matrix showing mean correlations between activity patterns evoked by odors A and B (n = 646 neurons from 7 experiments in 5 fish). Correlation coefficients between patterns evoked by the same odor (mean correlation ± SD: r = 0.54 ± 0.22) were higher than correlation coefficients between patterns evoked by different odors (r = 0.24 ± 0.19; paired t test; n = 7 experiments; p ≪ 10−3). (C) Mean response of Dp neurons to successive stimulation with two odors A and B, normalized to the mean response amplitude across all trials (n = 448 cells from n = 4 experiments in 4 fish). Gray lines show responses from individual experiments. Continuous black lines show independent exponential fits for responses to odors A and B. Dashed black line is an extrapolation of the fit to odor A responses. Red lines illustrate relative amplitude of the first B trial relative to the first and last A trials. The first response to odor B was significantly smaller than the first response to odor A but significantly larger than the last three responses to odor A and significantly larger than responses 3–8 to odor B. ∗p < 0.05; ∗∗∗p < 0.001. See also Figure S2C. Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Variability Results from Ongoing Activity and Stimulus-Dependent Representation Shift (A) Mean correlation between activity patterns evoked by the same odor in different trials as a function of inter-trial distance. Inset shows correlation coefficients along different diagonals that were averaged to represent different inter-trial distances. Line shows linear fit (r = −0.98, p < 10−3). Mean decrease across experiments: −0.028 per interval; n = 14 experiments; one-sample t test; p = 0.007. (B) Pattern correlation as a function of inter-trial distance, averaged after normalizing correlations in each experiment to their mean. The slope of the linear fit (dashed line) is defined as the representation shift index (RSI). Linear regression (6 time intervals × 14 experiments = 84 data points): r = −0.57, p ≪ 10−3. Error bars indicate the SEM. (C) Correlation between adjacent trials as a function of position along trial sequence (mean ± SD). Results from each experiment (n = 14) are represented by a light gray line. Correlation did not change significantly (two-way ANOVA; df = 5, F = 0.5, p = 0.78). (D) Mean trial-to-trial variability (SD) of response amplitudes of Dp neurons with low (SD = 10.1% ± 5.2% ΔF/F0) and high (SD = 13.5% ± 6.8% ΔF/F0) spontaneous activity. Gray lines show results from individual experiments. Paired t test (n = 14 experiments): ∗∗∗p < 0.001. See also Figure S4. (E) Pattern correlations calculated separately for neurons with low and high spontaneous activity. (F) Mean correlations between all trial pairs for neurons with low and high spontaneous activity. Gray lines show results from individual experiments. Low spontaneous activity: 〈r〉=+0.66; high spontaneous activity: 〈r〉=0.59; paired t test; n = 14 experiments; ∗∗p = 0.002. (G) Control, mean correlation of activity patterns across all neurons and trial pairs; corrected, mean correlations of activity patterns after subtracting estimates of ongoing activity in each trial. Correlations were increased significantly by 〈Δr〉=+0.05 (9.7% ± 8.8%; paired t test; n = 10 experiments; ∗∗p = 0.003). Shuffled, same after subtracting estimates of ongoing activity from mismatched trials. Correlations were significantly decreased by 〈Δr〉=−0.13 (−34% ± 49%; paired t test; n = 10 experiments; ∗∗p = 0.005). Gray lines show results from individual experiments. Error bars indicate the SEM. Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Representation Shift Is Driven by Sensory Input (A) A series of ≥5 odor applications (ISI = 2 min) was followed by application of the same odor after 10–37 min. The fourth-from-last trial in the initial series was defined as the reference trial (t = 0). Data points show the mean pattern correlation between the reference trial and subsequent trials as a function of time. The rightmost data point (black) shows the mean time difference and the mean pattern correlation between the delayed trial and the reference trial. All error bars show SD. The dashed line is a linear fit to the first three data points. The solid line is the correlation expected for an odor series extended by one additional stimulus. Data are from n = 10 experiments from 6 fish; n = 1,322 neurons. (B) Results of individual experiments (n = 10) shown in different colors. Dashed line is a linear fit to the first three data points (open circles, correlations between trials in the series). Solid circle shows correlation between reference trial and the delayed trial. Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Effect of NMDA Receptor Antagonist on Odor Responses in Dp (A) Example of calcium signals (ΔF/F0) evoked in Dp by repeated odor stimulation (His, 10−4 M; ISI = 2 min; repetitions 1, 2, and 5 are shown) in the presence of D-AP5. Grayscale image shows raw fluorescence. (B) Top: time course of somatic calcium signals of three Dp neurons in the presence of D-AP5 during trials 1, 2, and 5. Black bar depicts odor stimulus. Bottom: time courses of somatic calcium signals of 64 randomly selected Dp neurons, sorted by the response magnitude in trial 1. F0 was calculated by the Favg method. (C) Mean response time course of all Dp neurons in the presence of D-AP5 in trials 1–8. Shaded area shows time window used for calculating the response amplitude. (D) Mean response amplitude averaged over all cells and trials under control condition and during application of D-AP5 (Wilcoxon’s rank sum test, p = 0.66; Kolmogorov-Smirnov test, p = 0.20). Error bars indicate the SD. (E) Percentage of responses exceeding different % ΔF/F0 thresholds under control conditions and in D-AP5. (F) Mean normalized response amplitude as a function of trial number in the presence of D-AP5. Continuous black line shows exponential fit; dashed line shows asymptote. Amplitude was not significantly dependent on trial number (n = 479 neurons from 6 experiments in 4 fish; one-way ANOVA; df = 7, F = 2, p = 0.08). See also Figure S2D. (G) Mean correlations between odor-evoked activity patterns (upper triangle) and between pre-stimulus activities in the same trials (lower triangle) in the presence of D-AP5. (H) Bars indicate mean correlations between activity patterns in the first and subsequent trials (right) and correlations between subsequent trials (left) in the presence of D-AP5. Gray lines indicate individual experiments. Correlations of trial 1 versus trials 2–8: 〈r〉=0.82±0.08; between trials 2–8: 〈r〉=0.86±0.08. Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Effects of NMDA Receptor Blockade on Response Variability (A) Top: time course of somatic calcium signals of three Dp neurons in the absence of odor stimulation (spontaneous activity) before (left) and during application of D-AP5 (right). Bottom: spontaneous activity of 64 randomly selected Dp neurons before and during application of D-AP5. F0 was calculated by the Fmin method. (B) Mean percent of time that neurons were active (>20% ΔF/F0) under control conditions and in the presence of D-AP5. Gray lines correspond to means across cells from individual experiments. Decrease in spontaneous activity was significant across experiments (n = 5; paired t test; p = 0.006) and within each experiment (n = 90, 81, 70, 78, and 119 neurons; Wilcoxon signed-rank test; ∗∗∗p ≪ 10−3 in each experiment). See also Figure S4. (C) Pattern correlation in Dp as a function of inter-trial distance in the presence of D-AP5 (dark gray). Linear regression (continuous line) was not significant (6 experiments × 6 intervals = 36 data points; 〈r〉=−0.09, F = 0.28, p = 0.6). Results obtained under control conditions are shown for comparison (light gray; same as Figure 4A). (D) RNI (representation noise index) in Dp under control conditions (left; 0.26 ± 0.09; n = 14 experiments) and in the presence of D-AP5 (right; 0.13 ± 0.1; n = 6 experiments). Difference was statistically significant (two-sample t test; ∗p = 0.01). Error bars indicate the SD. (E) Comparison of RSI (representation shift index) under control conditions (left; RSI = 5.4 ± 6.7) and in the presence of D-AP5 (right; RSI = −0.5 ± 1.2). Boxes, 95% confidence intervals from linear regression analysis; red lines, RSI. p < 0.05. Sample sizes are the same as (D). Current Biology 2018 28, 1-14.e3DOI: (10.1016/j.cub.2017.11.007) Copyright © 2017 Elsevier Ltd Terms and Conditions