Role of Posterior Medial Thalamus in the Modulation of Striatal Circuitry and Choice Behavior

Abstract

The posterior medial (POm) thalamus is heavily interconnected with sensory and motor circuitry and is likely involved in behavioral modulation and sensorimotor integration. POm provides axonal projections to the dorsal striatum, a hotspot of sensorimotor processing, yet the role of POm-striatal projections has remained undetermined. Using optogenetics with slice electrophysiology, we found that POm provides robust synaptic input to direct and indirect pathway striatal spiny projection neurons (D1- and D2-SPNs, respectively) and parvalbumin-expressing fast spiking interneurons (PVs). During the performance of a whisker-based tactile discrimination task, POm-striatal projections displayed learning-related activation correlating with anticipatory, but not reward-related, pupil dilation. Inhibition of POm-striatal axons across learning caused slower reaction times and an increase in the number of training sessions for expert performance. Our data indicate that POm-striatal inputs provide a behaviorally relevant arousal-related signal, which may prime striatal circuitry for efficient integration of subsequent choice-related inputs.

Keywords: Behavioral Choice; Optogenetics; PV Interneurons; Photometry; Posteromedial Thalamus; Sensorimotor Integration; Spiny Projection Neurons; Striatum; Synaptic Physiology; Thalamostriatal Signaling.

Figure 1.. POm Equally Innervates Striatal Cell Types with Faster Latency In PV Interneurons

(A) Schematic detailing pAAV-ChR2-EYFP injection unilaterally into POm (Right), and optogenetic stimulation of POm-striatal afferents whilst recording from identified and unidentified neurons via ex vivo slice of posterior DLS (AP range: −0.34 to −1.22 relative to Bregma; Left). See Figure S1. Illumination (2.5ms pulses of 470nm light, ~0.6mW intensity) was delivered through the 40x objective. (B) Representative injection site (orange) in POm (Left), and viral spread of all electrophysiology injections within highlighted POm (purple; Right). S1BF = S1 Barrel Field. Scale = 1mm. (C) Red box inset from panel (B) highlighting stereotypical POm-cortical projection pattern to S1BF L1 and L5a.^,, Right: POm-striatal axons within posterior DLS. CC = corpus callosum. Scale = 200μm. (D) Representative cell type-specific PSPs to SP stimulation. Colored lines = average PSP of 20 sweeps. Gray lines = 20 individual traces. Solid vertical and dashed horizontal lines = latency and amplitude, respectively. Red dashed line = 0mV. Blue tick = photostimulation (PS). Time scale = 10ms. Voltage scale = 4mV. (E) Amplitudes evoked by each cell type were similar (D1-SPNs = 20 cells from 6 mice, D2-SPNs = 11 cells from 5 mice, PVs = 17 cells from 7 mice, unidentified SPNs = 7 cells from 4 mice). Inset shows grand average PSPs. Time scale = 10ms. Voltage scale = 2mV. (F) Latency to maximum PSP amplitude is significantly quicker in PVs than all other cell types. (G-H) Representative responses of (G) D1-SPN (Top) and D2-SPN (Bottom), and (H) PV (Top) and putative SPN (Bottom) to PPR stimulation. PPR is defined as the ratio of PSP amplitude of pulse 2 over the ratio of PSP amplitude of pulse 1. PPR PS parameters = five 2.5ms pulses with 50ms interpulse intervals (20Hz). Time scale = 100ms. Voltage scale = 2mV. See Figure S2I) Stimulation of POm-striatal afferents evokes similar PPR responses. (J-K) Representative responses of (J) D1-SPN (Top) and D2-SPN (Bottom), and (K) PV (Top) and putative SPN (Bottom) to train stimulation. Colored lines = average of 5 individual gray traces. Train PS parameters = thirty 2.5ms pulses with 64.2ms interpulse intervals (15Hz). Time scale = 1000ms. Voltage scale = 2mV. (L) Relative PSP amplitude (average of pulses 5–15 compared to pulse 1) is significantly larger than both SPNs. Data are mean ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2.. Mice Rapidly Learn to Discriminate Between Two Textures, and All Three Activity Parameters Markedly Increase Across Learning

(A) Schematic detailing pAAV-hSynapsin1-axon-jGCaMP8s-P2A-mRuby3 injection unilaterally into POm (Right), and a 400μm cannula implanted in the left posterior DLS (Left). (B) Representative injection site in POm (Left), and cannula placement in the posterior DLS along with ascending POm axons (Right). Scale = 1mm. (C) Top: Stimulating timing and texture movement representation during a trial. Note = both LEDs (isosbestic = 405nm; axon-jGCaMP8s = 470nm) were constantly on for every session. Bottom: Outcomes for each stimulus-response pair. (D) Schematic representing texture movement and potential outcomes during a single trial of the Go/NoGo whisker discrimination paradigm. (E) Changes in Hit Rate, FA Rate, Sensitivity (d’) and Bias of the FP cohort (n = 5 mice) as they transition from the Learning to the Expert phase. Note that mice are classified as Expert when they achieve a Hit Rate ≥ 0.80 and a FA Rate ≤ 0.30 for two consecutive sessions. Red line = 0. See Figure S3. (F) Average number of sessions required for expert discrimination of the FP cohort. (G-K) Three activity parameters (licking, axonal calcium, and pupil activity) from a representative (G) Shaping (session 1), (H) Early Learning (first two sessions after Shaping), (I) Late Learning (last two sessions before Expert), (J) Expert, and (K) Reward sessions from the same mouse (FPOm-18). Top: licking activity within a session (150 trials). Colored ticks = lick. Vertical black line = sound cue representing trial start as the texture moves towards the whisker field. Vertical red lines = start (texture arrival at endpoint in whisker field) and end (texture departure towards starting point) of the PT window (time where mice can respond by licking). Vertical brown line = average reaction time (RT; time of first lick that triggers an outcome) across all trials in each session. Colored boxes = 500ms grace period (licking does not trigger any outcomes). Note = no response line is present in the Reward session (K) as licking does not trigger any outcomes, and water was automatically delivered at PT end. Top Middle: Lick histogram. Middle: Heatmap sorted by trial outcome (to the Right of heatmap) highlighting axonal ZMAD calcium activity for each trial. Trial outcome is color coded (blue = Hit, yellow = Miss, orange = FA, brown = CR). Bottom Middle: Average axonal calcium activity of 150 trials for each session. Bottom: average pupil area (as a percentage) of 150 trials for each session. Data are mean ± SEM. Time scale = 1s.

Figure 3.. All Three Activity Parameters Exhibit Marked Increases Across Learning, But Only Axonal Calcium Activity Remains Unchanged, Irrespective of Trial Type or Outcome Segmentation

(A) Grand average axonal calcium activity at each behavioral time point. Data are mean ± SD. (B) Average of maximal axonal calcium amplitude markedly increases across learning before regressing to Shaping levels during the Reward session. ᴨ p < 0.01 Shaping vs. Early, ß p < 0.001 Shaping vs. Late, # p < 0.001 Shaping vs. Expert, ¶ p < 0.05 Early/Late vs. Reward, † p < 0.001 Expert vs. Reward. (C) Average area under the curve of the receiver-operator characteristic (auROC) also markedly increases across learning before regressing to Shaping levels during the Reward session. ^ p < 0.05, Shaping vs. Early; + p < 0.01, Shaping vs. Late; £ p < 0.001, Shaping vs. Expert; Ω p < 0.05, Early vs. Expert; @ p < 0.01, Late/Expert vs. Reward. (D) Grand average probability density function for licking-related activity at each time point. (E) Axonal calcium activity at Learning and Expert time points 2s pre and 2s post grand average RT. Data are mean ± SD. (F) Pre-RT axonal calcium activity is significantly larger than post-RT axonal calcium activity. (G) Grand average of normalized pupil area at each behavioral time point. (H) Representative cross-correlation of pupil area and axonal calcium activityI) Cross-correlation of pupil area and calcium activity plotted for each behavioral time point for each mouse. (J) Grand average of all licking (Top), calcium (Middle), and normalized pupil (Bottom) activity segmented by trial type: Go texture (Left) and NoGo texture (Right) presentation. (K) Average of maximal axonal calcium amplitude markedly increases across learning for both Go and NoGo texture presentation before regressing to Shaping levels during the Reward session. ¶ p = 0.0016 Go Shaping vs. Go Early, ᴨ p < 0.0001 Go Shaping vs. Go Late/Expert, @ p = 0.0265 Go Early vs. Go Late, ß p = 0.0154 Go Early vs. Go Expert, # p = 0.0005 Go Early vs. Go Reward, † p < 0.0001 Go Late/Expert vs. Go Reward. ^ p = 0.0128 NoGo Shape vs. NoGo Early, + p < 0.0001, NoGo Shape vs. NoGo Late/Expert, £ p = 0.0031 NoGo Early vs. NoGo Reward, Ω p = 0.0085 NoGo Early vs. NoGo Late, ? p = 0.0295 NoGo Early vs. NoGo Expert, ! p < 0.0001, NoGo Late/Expert vs. NoGo Reward. (L) Average auROC markedly increases across learning for both Go and NoGo texture presentation before regressing to Shaping levels during the Reward session. ^® p = 0.0041 Go Shaping vs. Go Early, © p < 0.0001 Go Shaping vs. Go Late/Expert, Δ p = 0.0041 Go Early vs. Go Late, ¿ p = 0.0003 Go Early vs. Go Expert, $ p = 0.0014 Go Early vs. Go Reward, & p < 0.0001 Go Late/Expert vs. Go Reward. ∞ p = 0.0462 NoGo Shaping vs. NoGo Early, Ø p < 0.0001 NoGo Shaping vs. NoGo Late/Expert, ∑ p = 0.0017 NoGo Early vs. NoGo Late, ¥ p = 0.0005 NoGo Early vs. NoGo Expert, ¢ p = 0.0142 NoGo Early vs. NoGo Reward, € p < 0.0001 NoGo Late/Expert vs. NoGo Reward. (M-P) Grand average of licking (Top), calcium (Middle), and normalized pupil (Bottom) activity segmented by trial outcomes: (M) Hit, (N) Miss, (O) FA, and (P) CR. (Q) Average of maximal axonal calcium amplitude of each mouse markedly increases across learning for all trials outcomes before regressing to Shaping levels during the Reward session. Data are mean ± SEM unless noted otherwise. * p < 0.05, ** p < 0.01, **** p < 0.0001. See also Figure S4.

Figure 4.. Photoinactivation Increases Number of Sessions Required For Expert Discrimination

(A) Schematic detailing pAAV-hSyn-JAWS-KGC-GFP-ER2 (JAWS) injection unilaterally into POm (Right) and a 200μm cannula implanted in the left posterior DLS (Left). For the No Stim cohort, only the cannula was implanted in the left posterior DLS. Activation of the inhibitory JAWS opsin was performed constantly on for 1s before and after texture arrival in the whisker field. JAWS activation probability per trial = 0.50. (B) Representative injection site in POm (Left), and the cannula placement in the posterior DLS along with ascending POm axons (Right). Scale = 1mm. Red inset shows ascending POm axons underneath the optic cannula. Inset scale = 200μm. (C) Top: Stimulation timing (constant illumination for 2s, centered around texture arriving at its endpoint) and texture movement representation during a trial. Note that no light is presented for the No Stim cohort as no stimulation occurred. Bottom: Outcomes for each stimulus-response pair. (D) Schematic representing texture movement and potential outcomes during a single trial. (E) Changes in Hit Rate, FA Rate, Sensitivity (d’), and Bias of all JAWS cohort mice (n = 4) as they transition from the Learning to the Expert phase in box-and-whisker plots. Note that mice are classified as Expert when they achieve a Hit Rate ≥ 0.80 and a FA Rate ≤ 0.30 for two consecutive sessions. Red line = 0. See Figure S5. (F) Probability density function for overall licking-related activity at each behavioral time point for the JAWS cohort. Vertical black line = sound cue representing trial start as the texture moves towards the whisker field. Vertical red lines = start (texture arrival at endpoint in whisker field) and end (texture departure towards starting point) of the PT window (time where mice can respond by licking). Colored boxes = 500ms grace period (licking does not trigger any outcomes). (G-H) Same as in E, F for the No Stim cohort. (I) JAWS cohort requires significantly more training sessions for expert discrimination compared to the FP and No Stim cohorts. (J) Longitudinal representation of sessions required for expert discrimination. (K) Comparison of Hit Rate, FA Rate, Sensitivity (d’), and Bias during the Learning and Expert phases. (L) Average RT is slower during photoinactivation than non-photoinactivated trials. Data are mean ± SEM. * p < 0.05.