A distributed circuit for associating environmental context with motor choice in retrosplenial cortex

Abstract

During navigation, animals often use recognition of familiar environmental contexts to guide motor action selection. The retrosplenial cortex (RSC) receives inputs from both visual cortex and subcortical regions required for spatial memory and projects to motor planning regions. However, it is not known whether RSC is important for associating familiar environmental contexts with specific motor actions. We test this possibility by developing a task in which motor trajectories are chosen based on the context. We find that mice exhibit differential predecision activity in RSC and that optogenetic suppression of RSC activity impairs task performance. Individual RSC neurons encode a range of task variables, often multiplexed with distinct temporal profiles. However, the responses are spatiotemporally organized, with task variables represented along a posterior-to-anterior gradient along RSC during the behavioral performance, consistent with histological characterization. These results reveal an anatomically organized retrosplenial cortical circuit for associating environmental contexts with appropriate motor outputs.

Fig. 1. A task for association of context with trajectory in the absence of locomotion.

(A) Schematic of behavioral setup. Mice use a rotary joystick to choose trajectories in a virtual T-maze presented on monitors spanning the horizontal visual field. (B) Schematic of the T-maze task for head-fixed mice in the absence of locomotion. Left turns are rewarded in context 1, whereas right turns are rewarded in context 2. (C) Example behavioral session. Dots indicate decisions (green = correct and red = incorrect); gray tick marks indicate licking; cyan shading indicates the decision window. Columns on the right show correct (C; yellow/blue) and incorrect (I; orange/cyan) responses in the two contexts. Trials in which no choice was made within the decision window are left blank. (D) Summary of the behavioral responses for the session shown in C (yellow = correct left, orange = incorrect right, blue = correct right, and cyan = incorrect left). (E) Fraction of response types during behavioral training (n = 18 mice). Mice are habituated to the training rig for 5 days (gray shaded area), during which the joystick can only rotate the correct direction to establish context-reward associations. Following habituation, the fraction of free decisions is progressively increased (10 to 80% of trials); only free decision trials are analyzed. Lines indicate mean ± SEM; colors as in (D). (F) Performance during behavioral training (n = 18 mice). Performance of individual mice is shown in gray. Average performance is shown in black. (G) Plots displaying performance during the first 5 days of training (S_1–5), last 5 days of training (S_46–50), widefield imaging sessions (WF), and two-photon imaging sessions (2P). Mouse performance was significantly above chance during late training and imaging sessions [S_1–5 = 50.0%; S_46–50 = 86.4%; WF = 78.9%; 2P = 75.5%; asterisks indicate lower 95% confidence interval (CI) > 50%]. Gray dots, individual mice; Black overlay, median ± 95% CI.

Fig. 2. Differential mesoscale activity in RSC during context-trajectory associations.

(A) Schematic of widefield imaging of calcium activity across dorsal cortex during task performance. (B) The skull of transgenic mice expressing GCaMP6s was polished and overlaid with cyanoacrylate and a coverslip to allow imaging of an 8- to 10-mm-diameter region of dorsal cortex. Scale bar, 1 mm. (C) Regions of dorsal cortex imaged during performance of the task. Region parcellation based on the Allen Mouse Brain Common Coordinate Framework (80). (D) Average registered activity maps obtained in 1.5-s epochs during traversal of the T-maze for all trial types (n = 20 sessions in four mice). Arrowheads and dashed lines indicate trial start (black), decision point (gray), and trial end (cyan). (E and F) Maps obtained by subtracting correct trials minus incorrect trials. Note that activity is higher in RSC in correct trials before the decision point (pooled across both contexts). After decisions, activity is higher in SMC in correct trials. Arrowheads and dashed lines indicate trial start (black), decision point (gray), and trial end (cyan). (G) Bar plots indicating significantly higher RSC activity in correct trials before the decision point. After decisions, SMC and SSC exhibit higher activity (3 to 6 s), whereas RSC and PPC have both higher (3 to 4.5 s) and lower (RSC, 6 to 7.5 s; PPC, 4.5 to 7.5 s) activity in correct trials. VC activity, by contrast, is lower by the end of correct trials (4.5 to 7.5 s). All values are means ± SEM, asterisks indicated statistical significance, *P < 0.01; Wilcoxon sign-rank test with Bonferroni correction (α = 0.01).

Fig. 3. Suppression of RSC activity impairs context-trajectory associations.

(A) Transgenic mice expressing GCaMP6s were injected with AAV-CAG-Flex-ArchT-tdTomato (red dots: approximate injection sites) along the rostrocaudal extent of RSC in both hemispheres to drive expression of the inhibitory opsin ArchT. (B) Example mouse showing expression of ArchT (red) and GCaMP6s (green). Scale bar, 500 μm. (C) Activity maps showing the suppression of spontaneous activity in RSC immediately following (1-s window) randomly presented light pulses (550 nm, 0.5 to 50 mW) compared to immediately before the pulse (1-s window) in an example ArchT-expressing mouse at different power intensities. (D) Change in RSC spontaneous activity in the region exhibiting expression of ArchT (red) and in a control region outside of RSC only exhibiting expression of GCaMP6s [green; see (B)]. Increasing light-emitting diode (LED) power progressively decreased activity in RSC (mean ± SEM; n = 12 sessions in four mice; asterisks indicate statistical significance, Wilcoxon sign-rank test). (E) Behavioral performance of ArchT⁺ mice (left) was impaired in randomly interleaved trials (25%) by optogenetic inhibition of RSC activity during initial traversal of the stem of the T-maze and the decision window (n = 12 sessions in four mice). Asterisks indicate significant effect of condition in linear mixed-effects model. No effect was seen in ArchT⁻ mice (right; n = 6 sessions in two mice). Lines indicate mean ± SD. n.s., not significant. (F) No change in the fraction of behavioral responses between trials with and without optogenetic stimulation in ArchT⁺ mice (Wilcoxon sign-rank test; mean ± SD). (G) Fraction of correct and incorrect responses for both contexts in ArchT⁺ mice during light off trials and light on trials. Asterisks indicate significant effect of condition in linear mixed-effects model, *P < 0.001. Lines indicate mean ± SD.

Fig. 4. Populations of RSC neurons exhibit selective responses spanning trial duration.

(A and B) Imaging of excitatory neuron populations using two-photon microscopy (A) through a cranial window over bilateral RSC (B) during task performance. (C) Example 4-mm cranial window (left; scale bar, 500 μm) and two-photon imaging field (right); scale bar, 50 μm. (D) Example cells exhibiting selective responses for particular contexts (cells 1 to 2) or for both contexts with different dynamics (cell 3). For each cell: top: ΔF/F trace across concatenated correct trials for (context 1, yellow; context 2, blue). Middle: normalized responses across correct trials for context 1 (left) and context 2 (right). Bottom: average activity across correct context 1 (left) and context 2 (right) trials. (E) Average normalized activity of all neurons (n = 10 mice) preferring correct trials in context 1 (top, n = 2634 neurons) and context 2 (bottom, n = 2560 neurons). Averages include only even trials, sorted by peak latency on odd trials. (F) Average normalized activity for all responsive neurons (n = 5194 neurons from 10 mice) for the preferred (left) and nonpreferred (right) context. Averages include only even trials, sorted by peak latency on odd trials. (G) Fraction of neurons with peak activity at each trial time (mean ± 97.5% CIs). (H) Average peak response magnitude of all neurons as a function of trial time (mean ± SEM). (I) Neuronal activity trajectories for correct trials in both contexts obtained by targeted dimensionality reduction. Trajectories are initially similar but diverge as a result of the context and motor decision, followed by parallel displacement in the outcome dimension for both trajectories (s, trial start; d, decision point; e, trial end). a.u., arbitrary units. (J) Distance between trajectories described by neuronal activity in correct trials for both contexts (mean ± bootstrap-estimated SEM). Arrowheads indicate trial start (black), decision point (gray), and trial end (cyan).

Fig. 5. RSC neurons encode task variables during context-trajectory associations.

(A) Example cells exhibiting significant decoding of a single task variable (cell 1, context; cell 2, motor; cell 3, outcome) or multiple task variables (cell 4) using a support vector machine decoder (chance = 0.5; mean ± bootstrap-estimated SEM). (B) Top: proportion of cells (n = 5194 from 10 mice) significantly encoding one, two, three, or no task variables (see Materials and Methods). Middle: cells encoding a single task variable. Bottom: cells encoding a combination of two task variables. (C) Decoding of context (top), motor (middle), or outcome (bottom) by the entire population of recorded cells (n = 5194 from 10 mice) combined across sessions using a support vector machine classifier (chance = 0.5; mean ± bootstrap-estimated SEM). (D) Average trajectory described by population encoding of context, motor, and outcome mapped to each axis (s, trial start; d, decision point; e, trial end). Note that encoding of context precedes encoding of the eventual motor action, followed by encoding of outcome. (E) Bar plots showing significant encoding of the different task variables in different epochs (n = 5194 neurons from 10 mice; lower 5% CI > 0.5; mean ± bootstrap-estimated SEM). Arrowheads indicate trial start (black), decision point (gray), and trial end (cyan).

Fig. 6. Encoding of task variables varies along the anterior-posterior axis of RSC.

(A) Maps displaying the contribution of individual RSC neurons to the encoding of context, motor, and outcome variables across different epochs throughout the trial using a support vector machine classifier (n = 5194 from 10 mice). Some cells are saturated to display dynamic range. (B) Encoding of context (top row), motor (middle row), and outcome (bottom row) by populations of neurons in posterior (n = 1726 neurons), medial (n = 2493 neurons), and anterior (n = 975 neurons) RSC (mean ± bootstrap-estimated SEM; shuffled trials in gray). Note that context is preferentially encoded in posterior RSC, whereas motor and outcome decoding are distributed across RSC with differing spatial and temporal dynamics. (C to E) Bar plots showing significant decoding in posterior (C; n = 1726 neurons), medial (D; n = 2493 neurons), and anterior (E; n = 975 neurons) RSC of context, motor, and outcome in different task epochs (asterisks, lower 5% CI > 0.5; mean ± bootstrap-estimated SEM). (F) Spatiotemporal encoding maps of context, motor, and outcome variables by RSC neurons (n = 5194). Context is mainly encoded in posterior RSC, with higher performance after trial start. By contrast, motor encoding is increased before trial start, revealing pretrial bias in motor output. Moreover, motor decoding transitions from posterior-to-anterior RSC before the decision point. Outcome is encoded throughout RSC, with a sharp increase in performance close to the reward time. (G) Proposed circuit architecture. Environmental context information from visual cortex enters posterior RSC, which then triggers motor planning activity in anterior RSC, including neurons projecting to CFA, the motor region controlling forelimb motor planning and movement. Outcome information is distributed across all RSC. Arrowheads indicate trial start (black), decision point (gray), and trial end (cyan).

Fig. 7. An anatomical gradient of visual inputs and motor outputs in RSC.

(A) Schematic of viral tracing experiments. An anterograde tracer (AAV.Syn.flex.GCaMP7b) was injected bilaterally into regions PM and AM of visual cortex in Emx1-Cre mice to achieve expression in excitatory projections neurons from visual cortex to RSC. A retrograde tracer (AAVrg.CAG.tdTomato) was injected bilaterally into CFA. Last, a cranial window was implanted over RSC for visualization under the widefield microscope. Cross indicates bregma. (B) Widefield image of RSC taken 21 days after virus injection. Scale bar, 500 μm. (C) Example coronal sections taken from anterior (−1.3 mm posterior to bregma) to posterior (−3.9 mm posterior to bregma) of RSC. Axonal expression of anterograde viral tracer (green) is present primarily in posterior RSC, while retrogradely labeled cell bodies (red) are almost exclusively located in anterior RSC. Nuclear stain [4′,6-diamidino-2-phenylindole (DAPI), blue] was used for structural visualization. Scale bar, 200 μm. (D) Normalized expression density (mean ± SD) for the anterograde tracer (visual cortical axons, green) in superficial layers (0 to 200 μm from pial surface) and the retrograde tracer (CFA-projecting cell bodies, red) in deep layers (200 to 1000 μm from pial surface) in dRSC. Distributions of expression density are significantly different (n = 4 hemispheres from two mice; P = 0.0046, Kolmogorov-Smirnov test).