Task design and behavioral trajectories in a delayed cue-guided navigation task.

(a) Left, mice initiated each trial by nose-poking at a clear door at the home base. The nose-poke triggered a 1-second visual cue where an LED strip on either the right (blue) or left (green) wall turned on. This was followed by a 1-second delay period with no cues. At the end of the delay period, the door opened, and the mice must run the length of the track (1 m) and turn toward the side of the arm where the cue was presented. Trials were reset when the mice returned to the water port at the home base. Incorrect trials were not rewarded. Right, top view of the track. (b) Average duration of the Run epoch of a trial (n = 1517 trials from 8 mice). (c) Example session showing left-ward (green) and right-ward (blue) trajectories. Thick lines show the mean trajectory. Only correct trials were included. Dashed horizontal line, position in the central arm where left-ward and right-ward trajectories diverged significantly. (d) Distribution of the position of significant divergence between left-bound and right-bound trials across all sessions (n = 27 sessions from 2 mice). The absence of early divergence indicates that trajectories did not differ at trial onset.

Strategy to optogenetically silence dorsal hippocampus (CA1) and prelimbic mPFC.

(a) Experimental animals (n = 8, including one wild-type control). Five mice were Parvalbumin (PV)-cre x Ai32 mice, two mice were injected with an AAV expressing Dlx-ChR2 in the dorsal hippocampus (CA1) and prelimbic mPFC. (b) Stereotaxic coordinates targeting mPFC and dorsal hippocampus. (c) Histological verification of viral expression and fiber placement for each mouse. T7, T6, and T8 had bilateral hippocampal fibers only; the remaining mice had bilateral fibers in both the hippocampus and mPFC. Green, ChR2-GFP expression in PV+ cells. Red, ChR2-mCherry expression in Dlx+ cells. Horizontal white lines correspond to optic fiber tips. Scale bar, 1 mm.

Example sessions illustrating epoch-specific effects of optogenetic silencing.

(a) Example session from a PV-Cre x ChR2 mouse (T9) with hippocampal silencing during the early run phase (first 2s). Top-left, trial-by-trial choices (green, correct; magenta, incorrect, with shaded blocks indicating stimulation trials). Bottom-left, average performance in blocks of 10 trials. Top-right, schematic of the stimulated task-epoch. Bottom-right, average performance during baseline and stimulation trials. (b) Example session from a Dlx-ChR2 mouse (T22) with silencing during the delay period (∼30% of trials, randomly interleaved). Format as in (a). Block-averaged performance is not shown as each block contains a mixture of stimulation and baseline trials. (c) Same mouse as in (b) (T22), with silencing during the early run phase (∼30% of trials). (d) Example session from a control mouse (T7) with light delivery during the early run phase. No impairment is observed.

Optogenetic silencing of dorsal hippocampus reveals epoch-specific effects on memory-guided behavior.

(a) Task epochs targeted for optogenetic stimulation. To control for time since cue delivery, a subset of mice was tested with a longer delay, with stimulation applied to the final 2 s of the delay. Unless otherwise noted, all manipulations were bilateral. (b) Position of the animal along the central arm at the onset of stimulation for the three run manipulations. Stimulation windows were aligned to distinct portions of the run epoch based on median trial duration (∼2.4 s): immediately after door opening (t = 0 s), after the stop-to-run transition had occurred (t = 0.8 s), and near the choice point (t = 1.6 s). Each dot represents the position from a single trial. Horizontal lines indicate the median position at those timepoints. (c, d) Performance during baseline (gray) and hippocampal stimulation (blue) trials across task epochs. Each line represents the average performance of one mouse. Red lines, PV-Cre × Ai32 mice; blue lines, Dlx-ChR2 mice. Perturbations during early central arm traversal produced large and consistent impairments across animals, whereas manipulations during other task epochs resulted in minimal effects. (Paired t-test; Cue: n = 7 mice, t(6) = 0.8177, p = 0.4448; Delay: n = 7 mice, t(6) = -0.0218, p = 0.9833; Cue+Delay: n = 7 mice, t(6) = -1.6816, p = 0.1436; Run (first 2s): n = 7 mice, t(6) = 6.6353, p = 5.653 × 10−4; Run (after 800 ms): n = 5 mice, t(4) = 19.797, p = 3.841 × 10−5; Run (after 1.6s) : n = 3 mice, t(2) = 1.1407, p = 0.3722; Delay + Run: n = 7 mice, t(6) = 8.2827, p = 1.677 × 10−4; Longer delay: n = 4 mice, t(3) = -0.6665, p = 0.5527; Unilateral hippocampus run (first 2s): n = 9 mice, t(8) = 2.4801, p = 0.0381). (e) Summary of the average effect across stimulation conditions. Colors correspond to stimulation windows shown in (a). The largest impairments were observed for manipulations targeting early central arm traversal, whereas other conditions showed smaller or inconsistent effects (Comparison between Run (first 2s), bilateral and unilateral hippocampus, t-test of difference from baseline, n = 9 sessions from 7 mice, t(14) = 2.7326, p = 0.0162). (f) Trial duration during baseline and stimulation trials for the bilateral hippocampal Run (first 2 s) manipulation. Hippocampal silencing increased trial duration. Each dot is a trial. Gray lines connect the mean trial duration for individual mice across conditions (Wilcoxon rank sum test, n = 414 & 338 trials, Z = -5.64, p = 1.68 × 10−8). (g) Relationship between trial duration and performance during baseline and hippocampal stimulation trials. During baseline trials (black), slower trials were associated with reduced performance (Spearman correlation, R = -0.21, p = 1.37 × 10−5). In contrast, during hippocampal silencing (blue), this effect of trial duration was lost with uniform impairment (Spearman correlation, R = - 0.01, p = 0.787). Numbers indicate the number of trials contributing to each bin. *p<0.05, ***p<0.001

Optogenetic silencing of prelimbic mPFC leads to epoch-specific effects on memory-guided behavior.

(a) As in Fig. 4a, schematic showing the task epochs targeted for mPFC silencing. (b) Average performance of baseline (gray) versus mPFC stimulation (blue) trials across mice. Each line corresponds to the average performance of a single mouse. Red lines, PV-Cre x Ai32 mice. Blue lines, Dlx-ChR2 virus-injected mice. Perturbations targeting early central arm traversal produced impairments, whereas manipulations during other task epochs resulted in smaller or inconsistent effects. (Paired t-Test; Cue: n = 4 mice, t(3) = 1.098, p = 0.3524; Delay: n = 4 mice, t(3) = -0.5466, p = 0.6227; Cue+Delay: n = 4 mice, t(3) = 0.2722, p = 0.8031; Run (first 2s): n = 4 mice, t(3) = 3.4740, p = 0.0402; Run (after 1.6s): n = 2 mice, t(1) = 1.8526, p = 0.3151; Delay + Run: n = 4 mice, t(3) = 4.09, p = 0.0264; Unilateral mPFC run (first 2s): n = 6 mice, t(5) = 0.8203, p = 0.4494). (c) Summary of the average effect from (b), comparing across stimulation conditions. Colors correspond to stimulation windows shown in (a). As with hippocampal silencing, only stimulations targeted to the central arm traversal led to an impairment. Unilateral manipulations had very little effect. (d) Trial duration during baseline and stimulation trials for the bilateral mPFC Run (first 2 s) manipulation. Gray lines connect the mean trial duration for individual mice across conditions (Wilcoxon rank sum test, n = 257 & 188 trials, Z = -6.2, p = 5.42 × 10−10). (e) Relationship between trial duration and performance during baseline and mPFC stimulation trials. Same as Fig. 4g. Baseline trials (black) (Spearman correlation, R = -0.17, p = 0.0067). The effect of trial duration persisted during mPFC silencing (Spearman correlation, R = -0.32, p = 1.02 × 10−5). Numbers indicate the number of trials contributing to each bin. *p<0.05, **p<0.01, ***p<0.001

Optogenetic silencing during early central arm traversal increases perseveration and alters behavioral trajectories.

(a) Example session where optogenetic stimulation targeted hippocampal fibers during RUN (first 2s). Trial-by-trial behavior is shown with leftward and rightward choices. Black, correct trials; magenta, incorrect trials; blue boxes, blocks of 10 stimulation trials. The mouse chose the left arm on 73.33% of stimulation trials compared to 53.33% during baseline, consistent with a leftward bias during stimulation. (b) Percentage of leftward cues during baseline vs. stimulation trials. Across sessions, cue distribution remained near 50% for both hippocampus (blue) and mPFC (red) targeting. (c) Same as (b), but for the chosen arm rather than the cue. While baseline choices were near 50%, stimulation induced a directional bias (left bias, above horizontal line; right bias, below horizontal line). (d) Example trajectories from a hippocampal stimulation session targeting the RUN phase. Gray, correct no-stimulation trials; magenta, incorrect no-stimulation trials; teal, stimulation trials (correct and incorrect). Circles mark leftward turns; squares mark rightward turns, defined as the point of maximal horizontal motion into the side arms. (e) Position along the central arm at the point of maximal horizontal motion toward a side arm. Gray, correct no-stimulation trials; magenta, incorrect no-stimulation trials; teal, stimulation trials (correct and incorrect). Each dot is a trial. During stimulation, but not during no-stimulation error trials, the point of maximal horizontal motion occurred farther along the central arm (Kruskal Wallis test followed by Tukey-Kramer post-hoc tests, n = 98,17,110 trials respectively, Chi-sq (2) = 56.601, p = 5.12 × 10−13). All box plots show median ± interquartile; whiskers show range excluding outliers.***p<0.001.