Cortical Synaptic AMPA Receptor Plasticity during Motor Learning

Abstract

Modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) is a fundamental mechanism underlying synaptic plasticity, learning, and memory. However, the dynamics of AMPAR trafficking in vivo and its correlation with learning have not been resolved. Here, we used in vivo two-photon microscopy to visualize surface AMPARs in mouse cortex during the acquisition of a forelimb reaching task. Daily training leads to an increase in AMPAR levels at a subset of spatially clustered dendritic spines in the motor cortex. Surprisingly, we also observed increases in spine AMPAR levels in the visual cortex. There, synaptic potentiation depends on the availability of visual input during motor training, and optogenetic inhibition of visual cortex activity impairs task performance. These results indicate that motor learning induces widespread cortical synaptic potentiation by increasing the net trafficking of AMPARs into spines, including in non-motor brain regions.

Keywords: AMPA receptors; long-term potentiation; motor cortex; motor learning; synaptic clustering; synaptic plasticity; two-photon imaging; visual cortex.

Figure 1.. Daily Skilled Motor Learning Induces Increases in Spine GluA1 Levels

(A) Experimental timeline with schematic drawings of forelimb reaching task and in vivo two-photon imaging. (B) Representative imaging volume of sparsely labeled layer 5 neurons in the motor cortex expressing SEP-GluA1 (green), myc-GluA2, and dsRed (magenta). Left, 3D reconstruction, right, maximum intensity projections of apical dendrites. (C) Behavioral performance of mice trained on forelimb reaching task. Thin lines represent individual mice and bold line is the average. n = 20. Error bars, SEM. (D) Representative images of spines on layer 5 apical dendrites in trained mice across training sessions. Arrows indicate spines with gradually increasing SEP-GluA1 signal intensity; Arrow heads indicate a spine with a transient increase in SEP-GluA1. (E) Representative images of spines on layer 5 apical dendrites in control mice (exposed to the training chamber without reaching) across training sessions. (F) Average spine GluA1 intensity normalized to baseline levels in trained (blue) and control (gray) mice over the course of motor learning. (G) Average GluA1 intensity in the dendritic shaft of trained (blue) and control (gray) mice. (H) Average spine dsRed intensity of trained (blue) and control (gray) mice. (F-H) Line represents average across n = 74 dendrites with 1781 spines from 20 mice (trained) and n = 24 dendrites with 411 spines from 9 mice (control). Shading depicts bootstrapped 95% confidence intervals (see methods). *P < 0.05 indicates significant difference of mean fluorescence signal intensity from baseline. Right histograms represent distribution of bootstrapped mean values during training. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significant mean difference between groups.

Figure 2.. Spine GluA1 Levels Correlate with Increased Behavioral Performance

(A) Correlation of reaching performance and Spine GluA1 levels at individual training sessions. Small symbols represent individual mice and dashed lines are linear regressions for each mouse. Bold blue symbols represent average GluA1 levels and behavioral performance of n = 20 mice at each training session. Bold blue line is the linear regression for the average values. Error bars, SEM. (B) Correlation between slope of reaching performance learning curve and slope of spine GluA1 time course for individual mice. Line represents linear regression with 95% confidence interval (dotted lines). n = 20 mice. (C) Experimental timeline for imaging 24h after last training day. Imaging sessions that data in (D) is plotted from are indicated by blue lines. (D) Average spine GluA1 intensity at baseline, average of training session 7 and 8, and 24h after last training day. Only spines that increased in GluA1 levels during learning were included (average GluA1 at training session 7 and 8 was larger than baseline). n = 147 spines. Error bars, SEM. ns, not significant, **** P < 0.0001, Friedman test with Dunn’s multiple comparison test. (E) Experimental timeline for reaching performance testing and imaging 1 week after last training day. Imaging sessions that data in (G) is plotted from are indicated by blue lines. (F) Behavioral performance of mice at the beginning of training (average of sessions 1 and 2), end of daily training (average of sessions 7 and 8), and 1 week after the last training day (memory session). n = 13 mice. Error bars, SEM. ns, not significant, ** P < 0.01, **** P < 0.0001, Friedman test with Dunn’s multiple comparison test. (G) Average spine GluA1 intensity at baseline, average of training session 7 and 8, and after memory session. Only spines that increased in GluA1 levels during learning were included (average GluA1 at training session 7 and 8 was larger than baseline). n = 351 spines. Error bars, SEM. **** P < 0.0001, Friedman test with Dunn’s multiple comparison test.

Figure 3.. Diverse Synaptic GluA1 Plasticity Between Individual Spines

(A) Heatmap showing amount of spine GluA1 in individual spines of trained mice. Each row represents a single spine. The GluA1 level of each spine is normalized to its average baseline levels and rows are sorted by average GluA1 change during motor learning. n = 1781 spines. (B) Same as (A) showing heatmap of individual spines for control mice. n= 411 spines. (C) Distribution histogram and cumulative distribution (inset) of average GluA1 change during motor learning. P < 0.0001 (Two-sample Kolmogorov-Smirnov test). (D) Fraction of spines showing an average increase (>26% increase over baseline), decrease (>26% decrease), or being stable (<26% change) during learning. Error bars, 95% confidence intervals. **** P < 0.0001, Chi-Square test with Bonferroni correction. (E) Comparison of GluA1 change in early training sessions (average of training sessions 1 and 2) and late training sessions (average of training sessions 7 and 8) with linear fits. n = 1154 spines.

Figure 4.. Spine GluA1 Plasticity is Locally Clustered

(A) Dendrospinogram of individual dendritic segments from trained mice indicating the location of all spines (black vertical line) along the segment. Location of spines showing an average increase (>26%) in spine GluA1 during learning are indicated in magenta and spines showing an average decrease (>26%) are indicated in blue. (B) Schematic drawing of nearest neighbor spines. (C) Average changes in spine GluA1 intensity during learning in neighboring spines in trained mice (top) and control mice (bottom) with linear fits. Changes in spine sGluA1 intensity in the same direction in neighboring spines are highlighted in turquois (both spines increase >26%), and changes in spine sGluA1 intensity in opposite directions in neighboring spines are highlighted in dark blue (one spine increases >26%, one spine decreases >26%). Dashed vertical lines: left, 0.74, right, 1.26. Dashed horizontal lines: upper, 1.26, lower, 0.74. n = 631 spine pairs (trained), n=139 spine pairs (control). (D) Same as (C) with randomized spine pairing. (E) Distribution of fraction of spine pairs where both spines increase from 10,000 random spine pairings. Magenta vertical line marks the observed fraction of spines (13.6% for trained mice (top) and 3.6% for control mice (bottom), see (C)). Monte Carlo P value was calculated by summing the tail of the shuffled histogram. (F) Distribution of fraction of spine pairs where spines show changes in opposite directions from 10,000 random spine pairings. Magenta vertical line marks the observed fraction of spines (3.5% for trained mice (top) and 4.3% for control mice (bottom), see (C)). Monte Carlo P value was calculated by summing the tail of the shuffled histogram. (G) Average spine GluA1 levels of all dendritic segments within individual neurons (light blue) and average of all neurons (dark blue) over the course of motor learning. (H) Distribution of average neuronal spine GluA1 levels during motor learning. (I) Left: Schematic of neuronal tracing to identify dendrite order. Dendrite in magenta exemplifies 4’ dendrite. Right: Average spine GluA1 levels during motor learning in dendritic segments of different orders. Error bars, SEM. ns, not significant, one-way ANOVA. (J) Top: Separation of two sibling dendritic branches into a low plasticity branch (LPB, light blue) and high plasticity branch (HPB, dark blue) based on average spine GluA1 change during motor learning. Bottom: Within sibling branch pairs average spine GluA1 is significantly higher in HPB compared to LPB. Gray lines, individual branch pairs. n = 10 branch pairs. Error bars, SEM. ** P < 0.01, paired t-test. (K) Distribution of mean ratio between spine GluA1 levels in HPB and LPB in 10,000 times randomly paired dendritic segments. Observed mean ratio difference (J) was 1.1. P = 0.003

Figure 5.. Increased Spine GluA1 levels in the Visual Cortex during Motor Learning

(A) Representative images of spines on layer 5 apical dendrites in the visual cortex of trained mice over the course of motor training. (B) Average spine GluA1 intensity normalized to baseline levels in the visual cortex of trained mice over the course of motor training and in the visual cortex of control mice. n= 14 dendrites with 251 spines from 4 mice (trained) and n = 10 dendrites with 154 spines from 3 mice (control). Line represents averages, shading depicts bootstrapped 95% confidence intervals (see methods). *P < 0.05 indicates significant difference of mean fluorescence signal intensity from baseline. Right histograms represent distribution of bootstrapped mean values during training. *P < 0.05 indicate significant mean difference between groups. (C) Immunoblots of PSDs from motor cortex and visual cortex of control and trained mice. (D) Quantification of synaptic GluA1 normalized to GluN1 in the motor cortex (bilateral average) of trained and control mice. n = 6 each. Error bars, SEM. ** P < 0.01, unpaired t-test. (E) Quantification of synaptic GluA1 normalized to GluN1 in the visual cortex (bilateral average) of trained and control mice. n = 6 each. Error bars, SEM. *** P < 0.001, unpaired t-test.

Figure 6.. Training Mice in Darkness Reduces Spine GluA1 Plasticity

(A) Schematic of mouse trained on forelimb reaching task in darkness with IR light illumination. (B) Behavioral performance of mice trained on forelimb reaching task in light or dark conditions. n = 10–12 mice. Error bars, SEM. No significance difference between groups, repeated measure two-way ANOVA. (C) Representative images of spines on layer 5 apical dendrites in the visual cortex of mice trained in darkness over the course of motor training. (D) Average spine GluA1 intensity normalized to baseline levels in mice trained under light (magenta, same as Figure 5B) and mice trained in darkness (gray) over the course of motor learning. Line represents average across n = 14 dendrites with 251 spines from 4 mice (trained in light) and n = 13 dendrites with 229 spines from 4 mice (trained in dark). Shading depicts bootstrapped 95% confidence intervals. Shading depicts bootstrapped 95% confidence intervals (see methods). *P < 0.05 indicates significant difference of mean fluorescence signal intensity from baseline. Right histograms represent distribution of bootstrapped mean values during training. *P < 0.05 indicate significant mean difference between groups. (E) Fraction of spines showing an average increase (>26% increase over baseline), decrease (>26% decrease), or are stable (<26% change) during learning. Error bars, 95% confidence intervals. **** P < 0.0001, Chi-Square test with Bonferroni correction.

Figure 7.. Training Mice in Darkness Dissociates Visual Cortex from Reaching Performance

(A) Schematic of mouse trained on forelimb reaching task with bilateral optogenetic fiber implanted on top of cortex. (B) Experimental timeline for mouse training with two additional behavior sessions on days 9 and 10 during which in a subset of trials either the motor or visual cortex was optogenetically inhibited. (C) Wide-field brain imaging of cortex in VGAT-ChR2-YFP mice with photobleaching of regions targeted for optogenetic inhibition. (D) Reaching performance of mice trained in light comparing trials with (blue) or without (gray) optogenetic inhibition of motor or visual cortex or with the optic fiber placed in the training chamber (control). n = 3 mice each. Error bars, SEM. *P < 0.05, repeated measure two-way ANOVA, ns, not significant. (E) Reaching performance of mice trained in darkness comparing trials with (blue) or without (gray) optogenetic inhibition of motor or visual cortex. n = 3 mice each. Error bars, SEM. *P < 0.05, ns, not significant, repeated measure two-way ANOVA.