Pavlovian conditioning dopamine ramps depend on ITI.

a. Top, fiber photometry approach schematic for nucleus accumbens core (NAcC) dLight recordings. Bottom, head-fixed mouse. b. Pavlovian conditioning task setup. Trials consisted of an 8 s auditory cue followed by sucrose reward delivery 1 s later. c. Cumulative Distribution Function (CDF) of ITI duration for long (solid line, mean 55 s) and short ITI (dashed line, mean 8 s) conditions. d. Experimental timeline. Mice were divided into groups receiving either a 3 kHz fixed and dynamic up↑ tone or a 12 kHz fixed and dynamic down↓ tone. e. Tone frequency over time. f. Peri-stimulus time histogram (PSTH) showing average licking behaviors for the last 3 days of each condition (n = 9 mice). g. Average anticipatory lick rate (baseline subtracted) for 1 s preceding reward delivery (two-way ANOVA: long ITI vs short ITI F(1) = 9.3, **p = 0.0045). h. ANCCR simulation results from an 8 s dynamic cue followed by reward 1 s later for long ITI (teal) and short ITI (pink) conditions. Bold lines show the average of 20 iterations. i. Left, average dLight dopamine signals. Vertical dashed lines represent the ramp window from 3 to 8 s after cue onset, thereby excluding the influence of the cue onset and offset responses. Solid black lines show linear regression fit during window. Right, closeup of dopamine signal during window. j. Average peak dLight response to cue onset for LD and SD conditions (paired t-test: t(8) = 6.3, ***p = 2.3×10−4). k. dLight dopamine signal with linear regression fit during ramp window for example SD trials. Reported m is slope. l. Session average per-trial slope during ramp window for the first day and last 3 days of each condition (one-sided [last day LD < first day SD] paired t-test: t(8) = -2.1, *p = 0.036; one-sided [last day SD > first day SF] paired t-test: t(8) = 2.4, *p = 0.023). m. Average per-trial slope for last 3 days of each condition (Tukey HSD test: q = 3.8, LD vs SD **p = 0.0027, SD vs SF *p = 0.011). All data presented as mean ± SEM. See Supplementary Table 1 for full statistical details.

Statistical Details.

Per-trial dopamine ramps correlate with previous ITI.

a. Scatter plots showing the relationship between dopamine response slope within a trial and previous ITI for all trials in the last 3 days of SD condition. Each animal plotted individually with linear regression fit (black line). b. Linear regression β coefficients for previous ITI vs. trial slope calculated per animal (one-sided [< 0] one sample t-test: t(8) = -2.2, *p = 0.031). c. Scatter plot of Z-scored trial slope vs. previous ITI pooled across mice for all trials in the last 3 days of LD condition (linear regression: t(2671) = -4.1, R2 = 0.0063, ***p = 3.8 ×10−5). The Z-scoring per animal removes the effect of variable means across animals on the slope of the pooled data. All data presented as mean ± SEM. See Supplementary Table 1 for full statistical details.

VR navigation dopamine ramps depend on ITI.

a. Head-fixed VR approach schematic. b. VR navigation task setup. Trials consisted of running down a patterned virtual hallway to receive sucrose reward. VR monitor remained black during the ITI. c. Experimental timeline. Following training, mice were assigned to either long or short ITI conditions for 8 days before switching. d. Velocity PSTH aligned to trial onset for long (teal) and short (pink) ITI conditions (n = 9 mice). e Average change in velocity at trial onset. Bottom asterisks indicate both conditions significantly differ from zero (one-sided [> 0] one sample t-test: long ITI t(8) = 6.4, ***p = 1.0 ×10−4 ; short ITI t(8) = 7.9, ***p = 2.3 ×10−5). Top asterisks indicate significant difference between conditions (paired t-test: t(8) = 4.3, **p = 0.0028). f. Velocity PSTH aligned to reward delivery. g. Average velocity during 1 s preceding reward (paired t-test: t(8) = 0.71, p = 0.50). h. PSTH showing average dLight dopamine signal aligned to trial onset. i. Comparison of peak dLight onset response (paired t-test: t(8) = 7.6, ***p = 6.3 ×10−5). j. Left, average dLight dopamine signal across distances spanning the entire virtual corridor. Vertical dashed lines represent the ramp window from 20 to 57 cm (10 cm before end of track). Solid black lines show linear regression fit during window. Right, closeup of dopamine signal during window. k. Session average per-trial slope during ramp window for all days of each condition. l. Scatter plot showing relationship between average per-session slope and inter-reward interval for the last 3 days in long (teal) and short (pink) ITI conditions. Black line indicates linear regression fit (linear regression: t(53) = -2.6, R2 = 0.12, *p = 0.012). m. Comparison of average per-trial slope during ramp window for last 3 days of both conditions (one-sided [long < short] paired t-test: t(8) = -2.1, *p = 0.035). All data presented as mean ± SEM. See Supplementary Table 1 for full statistical details.

Statistical Details.

Dependence of ANCCR on eligibility trace time constant.

a. Schematic showing exponential decay of cue eligibility traces for a two-cue sequential conditioning task (left) and a multi-cue conditioning task (right) with a long inter-trial interval (ITI). In this case, a long ITI results in a proportionally large eligibility trace time constant, T, producing slow eligibility trace decay (Supplementary Note 1). Reward delivery time indicated by vertical dashed line. b. Schematized ANCCR magnitudes (arbitrary units) for cues in the two-cue (left) and multi-cue (right) conditioning tasks with a long ITI. Since the eligibility trace for the first cue is still high at reward time, there is a large ANCCR at this cue. The remaining cues are preceded consistently by earlier cues associated with the reward, thereby reducing their ANCCR. c. Same conditioning task trial structure as in a, but with a short ITI and smaller T, producing rapid eligibility trace decay. d. Schematized ANCCR magnitudes for cues in both conditioning tasks with a short ITI. Since the eligibility trace for the first cue is low at reward time, there is a small ANCCR at this cue. Though the remaining cues are preceded consistently by earlier cues associated with the reward, the eligibility traces of these earlier cues decay quickly, thereby resulting in a higher ANCCR for the later cues.

Pavlovian conditioning histology and responses.

a. Mouse coronal brain sections showing reconstructed locations of optic fiber tips (red circles) in NAcC for Pavlovian conditioning task. b. Example average dLight traces for the last three days of all conditions. Vertical dashed lines at 3 and 8 s represent the ramp window period. Black lines display the linear regression fit during this period. c. Same as in b but for the average dLight traces across all animals. d. Comparison of average baseline subtracted lick rate during the ramp window across all conditions (one-way ANOVA: F(3) = 0.81, p = 0.50). e. Individual plots for each mouse displaying the cumulative distribution of per-trial slopes for the last three days in all conditions. Vertical dashed lines indicate the average trial slope for LF (grey), LD (teal), SD (pink), and SF (purple) conditions.

Trial-by-trial correlations for long ITI/dynamic tone condition.

a. Scatter plots showing the relationship between dopamine response slope within a trial and previous ITI for all trials in the last 3 days of LD condition. Each animal plotted individually with linear regression fit (black line). b. Linear regression β coefficients for previous ITI predicting trial slope (one-sided [< 0] one sample t-test: t(8) = 0.36, p = 0.64). c. Scatter plot of Z-scored trial slope vs. previous ITI pooled across mice for all trials in the last 3 days of LD condition (linear regression: t(1050) = 0.64, R2 = 3.9 ×10−4, p = 0.53).

VR task histology and responses.

a. Mouse coronal brain sections showing reconstructed locations of optic fiber tips (red circles) in NAcC for VR navigation task. b. Left, CDF of ITI duration for long (teal), medium (grey), and short (pink) ITI conditions. Middle, CDF plot of inter-reward interval (IRI) durations for each condition. Right, CDF plot of trial durations for each condition. c. Comparison of average trial duration for long and short ITI conditions (paired t-test: t(8) = 1.0, p = 0.34). d. Lick rate PSTH aligned to reward delivery indicates minimal anticipatory licking behavior. e. Quantification of average anticipatory lick rate 1 s before reward delivery. Bottom asterisks indicate a positive anticipatory lick rate for both ITI conditions (one-sided [> 0] one sample t-test: long ITI t(8) = 2.9, *p = 0.010; short ITI t(8) = 4.4, **p = 0.0012), though there was no significant difference between conditions (paired t-test: t(8) = 1.1, p = 0.31) f. CDF plots for each mouse separately showing the distribution of per-trial slopes for the last three days in both conditions. Vertical dashed lines indicate the average trial slope for long (teal) and short (pink) ITI conditions.

Trial-by-trial correlation of dopamine response slope vs previous inter-reward interval (IRI) in the VR task.

a. Scatter plot of dopamine response slope on a trial and the previous IRI for individual animals in the VR task during the short ITI condition. Here, we are measuring the environmental timescale using IRI instead of ITI because the trial duration in this task (see Supplementary Note 1) depends on the running speed of the animals, which varies trial to trial. Thus, IRI measures the net time interval between successive trial onsets. In the Pavlovian conditioning task, IRI and ITI differ by a constant since the trial duration is fixed. b. Linear regression β coefficients for previous IRI vs trial slope calculated per animal (one-sided [< 0] one sample t-test: t(8) = -0.48, p = 0.32). c. Scatter plot of Z-scored trial slope vs. previous IRI pooled across mice for all trials in the last 3 days of the Short ITI condition (linear regression: t(1301) = -2.11, R2 = 0.0034, *p = 0.035).