Article Figures & Data
Figures
- Figure 1.
Design of the behavioral task and rats' performance. A , Open field, reward cylinders (food site and water site), and a tone cylinder (tone site) for the task. Windows for nose pokes (reward locations) are indicated. The tone cylinder also has a small window at the same position as the reward cylinders. B , Schematic of the movements required of rats to receive rewards at the food and water sites. To start the task, rats have to visit, insert, and keep their noses in the tone site until the tone (8 kHz, 0.4 s) is presented (tone delay). Green, red, and blue areas indicate tone delay, food reward delay, and water reward delay, respectively. For details regarding the tone and reward delay periods, see Materials and Methods. C–E , Numbers of reward successes, choice errors, and wait errors under the ERD condition (food, C ; water, D ) and ETD condition (both rewards, E ). Under the ERD condition, the number of tone wait errors is negligible, and therefore these are not counted. For similar reasons, the number of reward wait errors under the ETD condition is not counted. The number of successes for food and water are merged. The number of choice errors for food and water and the number of tone wait errors for food and water are also considered together. F–H , Reward success rate ( F ), choice error rate ( G ), and wait error rate ( H ) during the ERD condition (red, food; blue, water) and the ETD condition (green, both rewards). **p < 0.001, significant differences compared with the ERD condition (Mann–Whitney U test). # p < 0.01, significant differences between food and water under the ERD condition (Mann–Whitney U test). Data from three rats are plotted as mean ± SEM.
- Figure 2.
Locations and classification of putative serotonin neurons. A , Location of the electrode track in each rat; boxes are reconstructed from the final electrode position. B , Results of classification of putative serotonin neurons based on spike duration (d in waveform inset, x-axis, in milliseconds) and baseline firing rate (y-axis, spikes−1). Filled red circles show neurons with decreased firing rates after systemic injection of 8-OH-DPAT. Filled black circles show putative non-5-HT neurons on the basis of no response to 8-OH-DPAT. The boundary (solid green line) was obtained with a logistic regression model from the results of neural responses to 8-OH-DPAT (see Materials and Methods). Open red and black circles, which were not tested by 8-OH-DPAT, are classified into putative 5-HT and non-5-HT neurons, respectively. Inset classification plot shows the range of firing rates. Inset waveforms show examples of putative serotonin (red) and non-serotonin (black) neurons. Gray shadings indicate SD.
- Figure 3.
Activity of serotonin neurons during tone delay and reward delay periods. A , B , Activity of two example neurons recorded in the dorsal raphe nucleus shown separately for food (left) and water (right) during the CD condition (tone delay and reward delay are 2 s). For each reward, raster plots of neural activity (top) and perievent time histograms smoothed with a Gaussian filter (SD of 50 ms) (bottom) are aligned at the time of tone site entry (left) and at the time of reward site entry (right). Raster plots represent neural activity in the order of occurrence of trials for each reward site from bottom to top. Each dot represents a spike. The tone for food and water site is food tone and water tone, respectively. Reward choice error and reward wait error trials are excluded. Green, red, and blue areas indicate tone delay, food delay, and water delay periods, respectively. Light blue areas indicate water spout presenting period. C , D , Comparison between baseline activity and reward delay activity ( C ) and tone delay activity ( D ) in DRN 5-HT neurons (n = 63). Open circles indicate food delay activity ( C ) and tone delay activity preceding food ( D ). Open triangles indicate water delay activity ( C ) and tone delay activity preceding water ( D ). Red and blue indicate statistically significant delay activity (Mann–Whitney U test, p < 0.01). Black, No significant difference from baseline. E , Averaged activity of the 63 neurons during the CD condition. F , Average firing rate during tone and reward delay periods. Averaged firing rates during the baseline (B), food tone delay (FTD), water tone delay (WTD), food delay (FD), and water delay (WD) are shown. ***p < 0.0001, significant differences compared with baseline activity (Wilcoxon's signed-rank test). # p < 0.0001, significant differences compared with tone delay activity (Wilcoxon's signed-rank test). In A , B , and E , gray shadings indicate SEM.
- Figure 4.
Comparison between tone delay activity and reward delay activity of DRN serotonin neurons under the constant delay condition. A , Comparison between tone delay activity and reward delay activity in DRN 5-HT neurons (n = 63). Open circles indicate the comparison between tone delay activity that precedes food and food delay activity. Open triangles indicate the comparison between tone delay activity that precedes water and water delay activity. Red and blue represent activity in which the difference in firing rate was statistically significant (Mann–Whitney U test, p < 0.01). B , Comparison between food delay activity and water delay activity in DRN 5-HT neurons (n = 63). Red and blue indicate neurons with statistically significant higher food and water delay responses, respectively (Mann–Whitney U test, p < 0.01). C , Comparison between the tone delay activity that precedes food and the tone delay activity that precedes water in DRN 5-HT neurons (n = 63). Red and blue indicate neurons with a significantly higher tone delay response preceding food and a higher tone delay response preceding water, respectively (Mann–Whitney U test, p < 0.01).
- Figure 5.
Modulation of serotonin neural activity to extended reward delay and extended tone delay. A , Example of a 5-HT neuron during the extended reward delay test (the reward delay was gradually increased every 5 min; tone delay, 1.5 s). B , Activity of an example neuron (same neuron as A ) during the extended tone delay test (the tone delay was gradually increased every 5 min; reward delay, 2 s). C , Average firing rate of serotonin neurons during reward delay periods of different lengths (mean ± SEM). The average firing rates of 3, 5, 7, and 9 s reward delay periods are merged with those of 2, 4, 6, and 8 s reward delay periods, respectively. Red indicates the food delay firing rate. Blue indicates the water delay firing rate. Food site: 2 s delay (n = 46), 4 s delay (n = 45), 6 s delay (n = 47), 8 s delay (n = 43), 12 s delay (n = 37), and 20 s delay (n = 15). Water site: 2 s delay (n = 46), 4 s delay (n = 45), 6 s delay (n = 47), 8 s delay (n = 46), 12 s delay (n = 39), and 20 s delay (n = 13). D , Average firing rate during the first 2 s and the last 2 s of the long reward delay period (6–20 s) (mean ± SEM). *p < 0.01, Wilcoxon's signed-rank test.
- Figure 6.
Activity of serotonin neurons during reward wait error and reward choice error in the extended reward delay test. A , Population activity aligned to the onset of the reward presentation (red, food; blue, water) and to the reward wait error (pink, food wait error; cyan, water wait error) (left, food site, n = 26; right, water site, n = 24). Gray shadings represent SEM. Light yellow areas indicate the periods that were used to analyze average firing rate. B , Average firing rate during the first and last 2 s of the waiting period after entry into the reward site in a case of successful entry (red, food; blue, water) and in the case of wait error entry (pink, food wait error; cyan, water wait error) (left, food site, n = 26; right, water site, n = 24; ± SEM). C , Population activity aligned to reward site success entry (red, food; blue, water) and to reward choice error entry (orange, water choice error; green, food choice error) (left, food site, n = 43; right, water site, n = 23). D , Average firing rate during 2 s after reward site entry in a case of successful entry (red, food; blue, water) and in a case of choice error entry (orange, water choice error; green, food choice error) (left, food site, n = 43; right, water site, n = 23; ±SEM). **p < 0.001, ***p < 0.0001, Wilcoxon's signed-rank test. n.s., Not significant.
- Figure 7.
Effects on expected reward omission on serotonin neural activity. A , Example of a 5-HT neuron aligned to water site entry during the water omission test. Light yellow areas indicate the water omission period. Red dots indicate water site exit. During the water omission period, food site success entry was rewarded. Right, The raster (top) and the histogram (bottom, 100 ms bins) during water omission period is aligned to the onset of water site exit. B , Population activity aligned to water site rewarded entry (blue) and to water omission entry (cyan, left; n = 24). Population activity aligned to water site exit after water omission entry (right; n = 24). Gray shadings represent SEM. Light yellow areas indicate the periods that were used to analyze average firing rate. C , Average firing rates during a 2 s period after water site rewarded entry, after water omission entry, and before water site exit (n = 24; ±SEM). D , Population activity of the first 3 trials (left) and all trials (right) after changing to the water omission condition (n = 24). E , Average firing rate for 1 s before and 1 s after the expected water reward was omitted (left, first 3 trials; right, all trials; n = 24; ±SEM). *p < 0.01, Wilcoxon's signed-rank test. n.s., Not significant.
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