Locally coordinated synaptic plasticity of visual cortex neurons in vivo

Abstract

Plasticity of cortical responses in vivo involves activity-dependent changes at synapses, but the manner in which different forms of synaptic plasticity act together to create functional changes in neurons remains unknown. We found that spike timing-induced receptive field plasticity of visual cortex neurons in mice is anchored by increases in the synaptic strength of identified spines. This is accompanied by a decrease in the strength of adjacent spines on a slower time scale. The locally coordinated potentiation and depression of spines involves prominent AMPA receptor redistribution via targeted expression of the immediate early gene product Arc. Hebbian strengthening of activated synapses and heterosynaptic weakening of adjacent synapses thus cooperatively orchestrate cell-wide plasticity of functional neuronal responses.

Fig. 1.. Induction of receptive field plasticity in V1 neurons.

(A) Pairing protocol. (B) Effect of pairing on a neuron’s receptive field and its dendritic spines (S₁-S₄). (C) Whole-cell recording during sparse noise stimuli. (D) Top: Excitatory current trace of a recorded neuron. Bottom: Average EPSC for each stimulus location. (E) Receptive field obtained by averaging EPSCs between 50–150ms. (F) EPSC in z-score averaged over all neurons and stimulus locations (n=8 neurons). Dashed blue line: 150ms. (G) Single-cell electroporation in vivo. (H) Neuron expressing mRuby2-P2A-GCaMP6s and ChR2-mCherry. (I) Loose-patch recording of a ChR2+ neuron spiking to single blue light pulses. Spike waveforms are shown (grey: standard deviation). (J) Calcium df/f traces obtained from soma. Arrows: onset of visual stimuli, presented pre-pairing at the preferred location (black) and post-pairing at the target location (red). (K) Top: Receptive fields from J traces. Preferred stimulus locations shown with black (pre) and red (post) squares. Black dots: center-of-mass. Red crosses: target stimulus. Bottom: Response time course in squared locations. Shaded areas: s.e.m. (L) Distance between target and receptive field center-of-mass pre- and post-pairing (black, n=22 neurons, N=23 mice, paired Wilcoxon test, ** p<0.01). Red dot: example in panel K. Cross: average shift. Control neurons with unpaired ChR2 visual stimulation shown in gray (n=21 neurons, N=11 mice, p=0.06, N.S. Not significant). Receptive field shifts were significantly different between paired and unpaired populations (unpaired Kruskal-Wallis test, ***p<0.001).

Fig. 2.. Hebbian potentiation and heterosynaptic depression in stretches of dendrite.

(A) Time-lapse dendritic imaging. (B) Spine volumes pre- and post- (>2h) pairing for ChR2+ (n=1987 spines) and ChR2− (n=845 spines) neurons. (C) Proportion of enlarged (δV+) and shrunken (δV−) spines above different δV values (variance F-test between ChR2+ and ChR2−, p<0.001). Black dashed line: sLTP and sLTD threshold δV= ±0.25. (D) Left: average normalized volume change over time for sLTP (n=110) and sLTD (n=98) spines. Dashed lines: threshold. Right: sLTP-to-sLTD volume change ratio. (E) sLTP and sLTD spine density per dendrite (n=20 dendrites; Pearson coefficient: 0.55, p<0.05; paired Wilcoxon test, *p<0.05). (F) sLTD spine density variation relative to mean as a function of distance from sLTP spines (n=103 sLTP spines, one-way ANOVA test, p<0.01, unpaired Kruskal-Wallis test, ***p<0.001 and *p<0.05 with Bonferroni correction). (G) ChR+ dendrite imaged pre- and post-pairing and reconstructed by EM after the experiment, post-pairing. (H) Spine volume measured with EM compared to two-photon fluorescence signal (n = 36 spines, EM versus post: r² = 0.85, EM versus pre: r² = 0.57). Lines depict best-fit power functions. Inset: distribution of fit residuals (unpaired variance F-test, **p<0.01). (I) Spine volume versus synaptic surface area (n=36, r² = 0.91). Note that some small spines lacked synapses, as previously reported (33). (J-L) Same as G-I for a ChR- neuron (n = 33 spines, EM versus post: r² = 0.88, EM versus pre: r² = 0.85, unpaired variance F-test, N.S. Not significant). Spine volume versus synapse area (n=39, r² = 0.92). Error bars: s.e.m.

Fig. 3.. Functional identification of sLTP and sLTD dendritic spines.

(A) Neuron expressing mRuby2-P2A-GCaMP6s. (B) Top: Dendritic stretch from A. Bottom: Calcium traces for two spines and branch. Arrows: onset of preferred stimuli. (C) Receptive fields for spines in B. Shaded gray: s.e.m. Colormap: time-averaged response. (D) Dendritic segment with nearby sLTP (1) and sLTD (2) spines. (E) Left: Profiles from dashed red line in D. Middle: receptive field of the spine. Red cross: target position. Right: Response time course for the preferred stimulus. Shaded areas: s.e.m (F) Same as E for sLTD spine in D. Right: Response time course for the preferred stimulus (gray) and for the target stimulus location (red). (G) Average normalized receptive field centered on target for sLTP (n=94) and sLTD (n=87) spines. Bottom: difference between distributions. (H) Top: Distribution of receptive field distances from target. Black distribution: randomized spine identity. Shaded areas: standard deviation. Bottom: Average normalized volume change as a function of receptive field distance from target for all sLTP and sLTD spines (n=181 spines, one-way ANOVA test, p<0.01, * p<0.05 and ** p<0.01 with Bonferroni correction). Error bars: s.e.m.

Fig. 4.. Role of Arc in regulating plasticity of V1 neurons.

(A) Neuron expressing ChR2-mCherry, DsRed2 and SEP-GluA1 in close proximity to a neuron expressing GCaMP6s. (B) Dendrites expressing SEP-GluA1 (left) or Arc-EGFP (right). (C) Spines corresponding to white rectangles in B. (D) Comparison between normalized change in volume and SEP-GluA1 (left) or Arc-EGFP (right) enrichment (SEP-GluA1: n=4354 spines from 17 neurons, N=12 mice, Pearson coefficient=0.22, *** p<0.001; Arc-EGFP: n=1719 spines from 16 neurons, N=8 mice, Pearson coefficient=−0.37, *** p<0.001). (E) Relative volume changes compared to relative SEP-GluA1 (top, n=122 dendrites) or Arc-EGFP (bottom, n=81 dendrites) enrichment changes averaged over all spines or measured in the dendritic shaft (Pearson coefficient: 0.27, ** p<0.01 for SEP-GluA1 and −0.29, ** p<0.01 for Arc-EGFP, N.S. Not Significant). Purple and orange lines: best linear fits. Circles: average values. (F) Dendrite expressing SEP-GluA1 and Arc shRNA-DsRed2. (G) Distributions of spine SEPGluA1 average intensity when Arc is endogenously expressed or knocked down (n=960 spines from 3 neurons for KD, n=2669 spines from 11 neurons for Ctrl, Kruskal-Wallis test, *** p <0.001). (H) SEP-GluA1 intensity correlation with neighboring spines. (I-K) Same as F-H in control or CaMKIIβ KD dendrites expressing Arc-EGFP (n=2053 spines from 4 neurons for KD, n=2550 spines from 11 neurons for Ctrl, Kruskal-Wallis test, *** p <0.001). (L) Distance between target and receptive field pre and post pairing for the KD condition (n=9 neurons, N=7 mice, paired Wilcoxon test, N.S. not significant). (M) sLTP and sLTD spine density per dendrite for control (n=66 dendrites; Pearson coefficient: 0.31, p<0.05; paired Wilcoxon test, *** p<0.001) and Arc KD neurons (n=39 dendrites; Pearson coefficient: −0.37, p<0.05; paired Wilcoxon test, N.S. Not Significant) conditions. (N) sLTD spine density variation relative to the mean as a function of distance from sLTP spines for the control (average over n=253 sLTP spines, one-way ANOVA test, p<0.001, unpaired Kruskal-Wallis test, ***p<0.001 and *p<0.05 with Bonferroni correction) and Arc KD conditions (average over n=142 sLTP spines, one-way ANOVA test, N.S. Not significant). Dendrites in the control condition either express a scrambled Arc shRNA plasmid fused with DsRed (n=46 dendrites) or mRuby2-P2A-GCaMP6s (n=20 dendrites).