Behavioral performance of two-alternative auditory discrimination learning

(A) Schematic diagram of the auditory discrimination task. Each trial started by presentation of a tone instruction cue with the high (10 kHz) or low (2 kHz) frequency. Three seconds later, the room light was illuminated, and two retractable levers were inserted at the same time. The rats were required to press the right and left levers in response to the high and low tones, respectively. (B) Learning curve of the auditory discrimination in intact rats (n = 14). Inset indicates the cumulative curve of number achieved the success rate of more than 90%. (C) Success rate at Days 2, 6, 10, 13, and 24 (one-way repeated ANOVA, F[1.897,24.659] = 106.706, p = 1.1 × 10-12, post hoc Bonferroni test, Day 2 vs. Day 6, p = 0.001, Day 6 vs. Day 10, p = 3.6 × 10-4, Day 10 vs. Day 13, p = 0.040, and Day 13 vs. Day 24, p = 0.080). (D) Response time (one-way repeated ANOVA, F[2.203,28.642] = 2.386, p = 0.105). (E) Omission rate (F[4,52] = 0.699, p = 0.596). (F) Response bias (one-way repeated ANOVA, F[1.129,14.674] = 0.250, p = 0.653). Data are indicated as the mean ± s.e.m., and individual data are overlaid. *p < 0.05, **p < 0.01, and ***p < 0.001.

Dissociable brain activity patterns during auditory discrimination learning among three striatal subregions

(A) Schematic illustration of an awake rat received intravenously 18F-FDG injection through an indwelling catheter attached to the tail. The rat was conducted for the behavioral experiment and then used for microPET imaging. (B) Schedule for the behavioral training and 18F-FDG-PET scan. After 18F-FDG injection, rats (n = 14 rats) were subjected to the behavioral experiment (30 min) and returned to the home cage (15 min). Ten min before the PET scan started, the rats were anesthetized and then the scan was started (30 min). (C and D) Learning curves of the single lever press task (C) and auditory discrimination task (D). Arrowheads indicate the PET scan days. (E) Behavioral performance on the scan days during the auditory discrimination. Success rate (one-way repeated ANOVA, F[3,39] = 125.012, p = 4.8 × 10-20, post hoc Bonferroni test; Day 2 vs. Day 6, p = 5.0 × 10-7; Day 6 vs. Day 10, p = 0.001; Day 10 vs. Day 24, p = 0.003), response time (one-way repeated ANOVA, F[3,39] = 0.156, p = 0.926), omission rate (one-way repeated ANOVA, F[3,39] = 1.559, p = 0.215), and response bias (one-way repeated ANOVA, F[1.779,23.130] = 17.734, p = 3.6 × 10-5) are shown. (F) Representative images of coronal sections that compared the brain activity in the single lever press task with the activity on Day 2, 6, 10, or 24 in the discrimination task. Left panels show schematic illustrations of striatal subregions. (G) Horizontal (top) and coronal (bottom) images of striatal activation areas shown on Day 10 vs. single lever in (F). (H) Representative images of coronal section that compared the brain activity on Day 2 with that on Day 6, 10, or 24 in the discrimination task. Color bars indicate the T-values, and a value of 3.8 was used as the threshold corresponding to the uncorrected threshold (p < 0.001). (I) Schematic pictures showing voxel of interests for the aDLS, pVLS, and DMS (green, left hemisphere; and purple, right hemisphere). (J-L) Regional 18F-FDG uptakes in the aDLS (J, one-way repeated ANOVA, left aDLS, F[4,52] = 10.322, p = 3.0 × 10-6, post hoc Bonferroni test; single lever vs. Day 6, p = 0.016; Day 2 vs. Day 6, p = 6.5 × 10-5; Day 2 vs. Day 10, p = 0.019; Day 2 vs. Day 24, p = 0.017; Day 6 vs. Day 24, p = 0.041; right aDLS, F[4,52] = 7.462, p = 7.9 × 10-5, post hoc Bonferroni test, Day 2 vs. Day 6, p = 2.9 × 10-4; Day 2 vs. Day 10, p = 0.036; Day 6 vs. Day 24, p = 0.005), pVLS (K, one-way repeated ANOVA, left pVLS, F[2.368,30.784] = 4.152, p = 0.020, post hoc Bonferroni test; single lever vs. Day 10, p = 1.8 × 10-5; right pVLS, F[4,52] = 5.995, p = 4.8 × 10-4, post hoc Bonferroni test, single lever vs. Day 10, p = 0.001; Day 2 vs. Day 10, p = 0.011; Day 6 vs. Day 10, p = 0.032), and DMS (L, one-way repeated ANOVA, left DMS, F[4,52] = 12.836, p = 2.4 × 10-7, post hoc Bonferroni test; Day 2 vs. Day 24, p = 1.8 × 10-5; Day 6 vs. Day 24, p = 4.8 × 10-4; Day 10 vs. Day 24, p = 5.5 × 10-5; right DMS, F[4,52] = 10.717, p = 2.0 × 10-6, post hoc Bonferroni test; single lever vs. Day 24, p = 0.036; Day 2 vs. Day 24, p = 0.011; Day 6 vs. Day 24, p = 2.6 × 10-4; Day 10 vs. Day 24, p = 0.011). Arrowheads indicate the day with the most activations throughout the learning process. Data are indicated as the mean ± s.e.m., and individual data are overlaid. The anteroposterior coordinates from bregma (mm) are shown (F–I). Scale bar; 2 mm (I). *p < 0.05, **p < 0.01, and ***p < 0.001.

Impacts of excitotoxic lesion of striatal subregions on the acquisition of auditory discrimination

Rats were given intracranial injection of PBS or IBO solution into the aDLS (n = 8 for each injection), pVLS (n = 8 for each injection), or DMS (n = 7 for PBS injection, and n = 6 for IBO injection) before start of the single lever press task. (A-C) Representative images of NeuN immunostaining and individual schematic illustrations showing lesioned area in the aDLS (A), pVLS (B), or DMS (C). Dotted lines in the images indicate the range of the lesioned area. ac, anterior commissure; cc, corpus callosum; and LV, lateral ventricle. (D-F) Learning curves during the single lever press task in the groups injected into the aDLS (D, two-way repeated ANOVA, group, F[1,14] = 1.275, p = 0.278, day, F[1.512,21.171] = 26.281, p = 7.0 × 10-6, group × day, F[1.512,21.171] = 0.834, p = 0.418), pVLS (E, two-way repeated ANOVA, group, F[1,14] = 2.542, p = 0.133, day, F[1.547,21.658] = 40.183, p = 2.2 × 10-7, group × day, F[1.547,21.658] = 0.953, p = 0.380), or DMS (F, two-way repeated ANOVA, group, F[1,11] = 0.025, p = 0.876, day, F[1.205,13.252] = 14.818, p = 0.001, group × day, F[1.205,13.252] = 0.610, p = 0.478). (G-I) Learning curves during the auditory discrimination task in the groups injected into the aDLS (G, two-way repeated ANOVA, group, F[1,14] = 11.578, p = 0.004, day, F[2.805,39.274] = 60.733, p = 1.8 × 10-14, group × day, F[2.805,39.274] = 3.863, p = 0.018), pVLS (H, two-way repeated ANOVA, group, F[1,14] = 7.221, p = 0.018, day, F[3.646,51.041] = 51.794, p = 9.2 × 10-17, group × day, F[3.646,51.041] = 1.612, p = 0.190), or DMS (I, two-way repeated ANOVA, group, F[1,11] = 0.469, p = 0.507, day, F[2.677,29.452] = 48.860, p = 3.9 × 10-11, group × day, F[2.677,29.452] = 1.094, p = 0.362). (J-L) Changes in the proportion of the WSW or LSL strategy through the discrimination learning in the groups treated into the aDLS (J, two-way repeated ANOVA, group, F[1,14] = 13.451, p = 0.003, day, F[3.048,42.673] = 54.777, p = 9.1 × 10-15, group × day, F[3.048,42.673] = 3.288, p = 0.029 for the WSW; group, F[1,14] = 5.039, p = 0.041, day, F[3.866,54.119] = 14.578, p = 5.2 × 10-8, group × day, F[3.866,54.119] = 2.471, p = 0.057 for the LSL), pVLS (K, two-way repeated ANOVA, group, F[1,14] = 9.251, p = 0.009, day, F[3.206,44.881] = 47.168, p = 2.7 × 10-14, group × day, F[3.206,44.881] = 1.862, p = 0.146 for the WSW; group, F[1,14] = 0.762, p = 0.397, day, F[4.773,66.815] = 24.018, p = 1.9 × 10-13, group × day, F[4.773,66.815] = 1.338, p = 0.260 for the LSL), or DMS (L, two-way repeated ANOVA, group, F[1,11] = 0.703, p = 0.420, day, F[3.052,33.568] = 48.424, p = 2.3 × 10-12, group × day, F[3.052,33.568] = 0.846, p = 0.480 for the WSW; group, F[1,11] = 0.002, p = 0.965, day, F[3.442,37.866] = 13.787, p = 1.0 × 10-6, group × day, F[3.442, 37.866] = 0.581, p = 0.654 for the LSL). Data are indicated as the mean ± s.e.m., and individual data are overlaid (except for panels J–L). The anteroposterior coordinates from bregma (mm) are shown (A–C). Scale bars; 2 mm (A–C). *p < 0.05 and **p < 0.01.

Influence of transient inhibition of striatal subregions at different timings on the performance of auditory discrimination

Rats received intracranial injection of SAL or MUS solution into the aDLS (n = 8 for each injection) or pVLS (n = 6 for each injection). (A and B) Representative images of cresyl violet staining and schematic illustrations showing placement sites of the tip of guide cannula in the aDLS (A) or pVLS (B). Dotted lines in the images indicate the position of the cannula placement. Ac, anterior commissure; cc, corpus callosum; and LV, lateral ventricle. (C and B) Effects of transient striatal inhibition on the performance. For aDLS inhibition (C), success rate (left, two-way repeated ANOVA, stage, F[2,28] = 28.708, p = 1.7 × 10-7, group, F[1,14] = 8.840, p = 0.010, stage × group, F[2,28] = 0.757, p = 0.478; unpaired Student’s t-test, early, t[14] = 0.558, p = 0.586; middle, t[14] = 2.764, p = 0.015; Welch’s t-test, late, t[12.010] = 1.617, p = 0.132, p = 0.016 after a Bonferroni correction method) and subtracted success rate on day N-1 from that on day N (right, two-way repeated ANOVA, stage, F[2,28] = 1.231, p = 0.307, group, F[1,14] = 10.797, p = 0.005, stage × group, F[2,28] = 2.360, p = 0.113; unpaired Student’s t-test, early, t[14] = 0.285, p = 0.780, late, t[14] = 1.271, p = 0.225, Welch’s t-test, middle, t[9.603] = 3.331, p = 0.008, p = 0.016 after a Bonferroni correction method). For pVLS inhibition (D), success rate (left, two-way repeated ANOVA, stage, F[2,20] = 17.642, p = 3.8 × 10-5, group, F[1,10] = 43.942, p = 5.9 × 10-5, stage × group, F[2,20] = 4.729, p = 0.012; simple main effect, early, p = 0.055, middle, p = 0.109, late, p = 4.2 × 10-5) and subtracted success rate on day N-1 from that on day N (right, two-way repeated ANOVA, stage, F[2,20] = 12.105, p = 3.6 × 10-4, group, F[1,10] = 16.310, p = 0.002, stage × group, F[2,20] = 8.500, p = 0.002; simple main effect, early, p = 0.595, middle, p = 0.085, late, p = 3.0 × 10-6). (E and F) Subtracted proportion of the WSW or LSL strategy in the groups injected into the aDLS at the middle stage (E, unpaired Student’s t-test, t[14] = 2.038, p = 0.061 for the WSW, and t[14] = 2.714, p = 0.017 for the LSL) and the late stage (F, unpaired Student’s t-test, t[14] = 0.898, p = 0.384 for the WSW, and t[14] = 0.226, p = 0.824 for the LSL). (G and H) Subtracted proportion of the WSW or LSL strategy in the groups injected into the pVLS at the middle stage (G, unpaired Student’s t-test, t(10) = 1.924, p = 0.083 for the WSW, and Welch’s t-test, t[6.095] = 1.364, p = 0.221 for the LSL) and the late stage (H, unpaired Student’s t-test, t[10] = 3.629, p = 0.005 for the WSW, and t[10] = 1.577, p = 0.146 for the LSL). Data are indicated as the mean ± s.e.m., and individual data are overlaid. The anteroposterior coordinates from bregma (mm) are shown (A and B). Scale bars; 2 mm (A and B). *p < 0.05, **p < 0.01, and ***p < 0.001.

Multi-unit recording of neurons in striatal subregions and firing activity related to the behavioral outcome of RS-HR neurons in the aDLS

(A) Schematic illustration of a freely moving rat used for simultaneous multi-unit recordings in the aDLS and pVLS. Six rats were used for the following analysis. (B) Representative images showing positions of electrode tips (arrows) in the aDLS and pVLS (left rows). The recording sites estimated by electrode tracks and electrical marks in individual rats are shown (center and right rows). ac, anterior commissure; LV, lateral ventricle. (C) Sequence of some events in correct and error trials. The delay periods were pseudorandomly added between a correct lever press and the reward sound (0.5 ± 0.2 s). The room light turned off the extended time (4 s) after the correct response or immediately after the error response. (D) Mean firing rate (top rows) and auROC values (bottom rows) of RS-HR neurons in the aDLS during the period when the RS is presented (green shadows, 500 ± 200 ms after the lever press) at the early (n = 43 neurons), middle (n = 37 neurons), and late (n = 44 neurons) stages. Time bins with significant differences between the mean firing rate in the HR or LR trial and any of the rates in other three trials (Wilcoxon signed rank test) or the distribution of auROC values and 0.5 (Wilcoxon rank-sum test) are represented by the circles at the top. (E) Averaged firing rate during the RS period in the HR and LR trials at the early, middle, and late stages (Kruskal-Wallis test: HR trial, χ2 = 3.028, p = 0.220; and LR trial, χ2 = 1.249, p = 0.536). (F) Cumulative probability of the proportion of RS-HR neuron number at the three stages (Kruskal-Wallis test, χ2 = 45.378, p = 1.4 × 10-10; post hoc Tukey-Kramer test, p = 7.1 × 10-5 for early vs. middle, p = 1.0 × 10-9 for early vs. late, and p = 0.040 for middle vs. late). Data are indicated as the mean ± s.e.m. (D) or the median and quartiles with the maximal and minimal values (E). The anteroposterior coordinates from bregma (mm) are shown (B). Scale bars; 2 mm (B). *p < 0.05 and ***p < 0.001.

Sustained activity after the reward of FL-HR neurons in the aDLS and pVLS

(A) Firing rate (top rows), auROC (middle rows), and licking rate (bottom rows) after the reward of FL-HR neurons in the aDLS at the early (n = 27 neurons), middle (n = 31 neurons), and late (n = 49 neurons) stages. Time bins with significant differences between the mean firing rate in the HR trial and any of the rate in other three trials (Wilcoxon signed rank test) or the distribution of auROC values and 0.5 (Wilcoxon rank-sum test) are represented by the circles at the top. Timings of the RS and FL are shown. (B) Averaged firing rate for 5 s after the FL in the HR trial (Kruskal-Wallis test, χ = 16.396, p = 2.8 × 10, post hoc Tukey-Kramer test, early vs. middle, p = 0.726, early vs. late, p = 7.2 × 10-4, middle vs. late, p = 0.009). (C) Averaged total number of time bins above 3 S.D. of the baseline firing of aDLS neurons for 5 s after the FL in the HR trial (Kruskal-Wallis test, χ2 = 12.803, p = 0.002, post hoc Tukey-Kramer test, early vs. middle, p = 0.173, early vs. late, p = 0.001, middle vs. late, p = 0.219). (D) Cumulative probability of the proportion of FL-HR neuron number (Kruskal-Wallis test, χ2 = 39.280, p = 3.0[×[10-9, post hoc Tukey-Kramer test, early vs. middle, p = 1.3 × 10-7, early vs. late, p = 0.995, middle vs. late, p = 2.3 × 10-7). (E) Distribution of the correlation coefficient between the numbers of spikes of FL-HR neurons and licking. Closed column indicates the number of neurons showing significant correlations between the two parameters (5 out of 107 neurons). (F) Firing rate (top rows), auROC (middle rows), and licking rate (bottom rows) after the reward of FL-HR neurons in the pVLS at the early (n = 17 neurons), middle (n = 11 neurons), and late (n = 64 neurons) stages. Time bins with significant differences between the rate in the HR trial and any of the rate in other trials (Wilcoxon signed rank test) or the distribution of auROC values and 0.5 (Wilcoxon rank-sum test) are represented by the circles at the top. The timings of the RS and FL are shown. (G) Averaged firing rate of pVLS neurons after the FL in the HR trial (Kruskal-Wallis test, χ2 = 7.652, p = 0.022; post hoc Tukey-Kramer test, early vs. middle, p = 0.975, early vs. late, p = 0.076, and middle vs. late, p = 0.096). (H) Averaged total number of time bins above 3 S.D. of the baseline firing after the FL in the HR trial (Kruskal-Wallis test, χ2 = 0.023, p = 0.989). (I) Cumulative probability of the proportion of FL-HR neuron number (Kruskal-Wallis test, χ2 = 47.922, p = 3.9 × 10-11; post hoc Tukey-Kramer test, early vs. middle, p = 0.011, early vs. late, p = 1.7 × 10-4, and middle vs. late, p = 9.7 × 10-10). (J) Distribution of the correlation coefficient between the number of spikes in the pVLS and the licking. Closed column indicates the number of neurons showing significant correlations between the two parameters (5 out of 92 neurons). Data are indicated as the mean ± s.e.m. (A and F) or the median and quartiles with the maximal and minimal values (B, C, G, and H). **p < 0.01 and ***p < 0.001.

Transient activity related to the beginning and ending of a behavior of CO-HR and CR-HR neurons in the pVLS

(A) Firing rate (top rows) and auROC values (bottom rows) of CO-HR neurons in the pVLS at the early (n = 4 neurons), middle (n = 21 neurons), and late (n = 24 neurons) stages. Time bins with significant differences between the mean firing rate in the HR trial and either of the rate in other three trials (Wilcoxon signed rank test) or the distribution of auROC values and 0.5 (Wilcoxon rank-sum test) are represented by the circles at the top. (B) Averaged firing rate of CO-HR neurons in HR and LL trials (0-600 ms after the CO) (Wilcoxon signed rank test; early, p = 0.875, middle, p = 0.006, and late, p = 0.004). (C) Cumulative probability of the proportion of CO-HR neuron number (Kruskal-Wallis test, χ2 = 51.114, p = 8.0 × 10-12; post hoc Tukey-Kramer test, early vs. middle, p = 9.6 × 10-10, early vs. late, p = 0.002, and middle vs. late, p = 6.4 × 10-4). (D) Firing rate (top rows) and auROC values (bottom rows) of CR-HR neurons in the pVLS at the early (n = 11 neurons), middle (n = 22 neurons), and late (n = 41 neurons) stages. Time bins with significant differences between the mean rate in the HR trial and any of the rate in other trials (Wilcoxon signed rank test) or the distribution of auROC values and 0.5 (Wilcoxon rank-sum test) are represented by the circles at the top. The timing of the RS is presented. (E) Averaged firing rate of CR-HR neurons in HR and LL trials (0-600 ms after the CR) (Wilcoxon signed rank test; early, p = 0.278, middle, p = 0.758, and late, p = 0.279). (F) Cumulative probability of the proportion of CR-HR neuron number (Kruskal-Wallis test, χ2 = 13.786, p = 0.001; post hoc Tukey-Kramer test, early vs. middle, p = 0.003, early vs. late, p = 0.005, and middle vs. late, p = 0.976). Data are indicated as the mean ± s.e.m. (A and B) or the median and quartiles with the maximal and minimal values (B and E). **p < 0.01 and ***p < 0.001.