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Visualization of NMDA receptor–dependent AMPA receptor synaptic plasticity in vivo

Nature Neuroscience volume 18, pages 402–407 (2015)Cite this article

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Abstract

Regulation of AMPA receptor (AMPAR) membrane trafficking is critical for synaptic plasticity, as well as for learning and memory. However, the mechanisms of AMPAR trafficking in vivo remain elusive. Using in vivo two-photon microscopy in the mouse somatosensory barrel cortex, we found that acute whisker stimulation led to a significant increase in the intensity of surface AMPAR GluA1 subunit (sGluA1) in both spines and dendritic shafts and a small increase in spine size relative to prestimulation values. Interestingly, the initial spine properties biased spine changes following whisker stimulation. Changes in spine sGluA1 intensity were positively correlated with changes in spine size and dendritic shaft sGluA1 intensity following whisker stimulation. The increase in spine sGluA1 intensity evoked by whisker stimulation was NMDA receptor dependent and long lasting, similar to major forms of synaptic plasticity in the brain. In this study we were able to observe experience-dependent AMPAR trafficking in real time and characterize, in vivo, a major form of synaptic plasticity in the brain.

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Figure 1: Expression of SEP-GluA1 in layer 2/3 barrel cortex neurons in vivo.
Figure 2: Acute whisker stimulation led to an increase in spine sGluA1 intensity in vivo in apical dendrites of layer 2/3 neurons in the barrel cortex.
Figure 3: Acute whisker stimulation led to an increase in intensity in spine sGluA1 and shaft sGluA1 but had little effect on spine size.
Figure 4: Changes in spine sGluA1 intensity were positively correlated with changes in shaft sGluA1 intensity and spine size following whisker stimulation, and neighboring spines cooperatively increased their spine sGluA1 intensity.
Figure 5: Increases in spine sGluA1 intensity following whisker stimulation were NMDA receptor dependent.
Figure 6: The increase in spine sGluA1 intensity following whisker stimulation was stable for 48 h, whereas the increase in shaft sGluA1 intensity receded over time.

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Acknowledgements

We thank L.J. Volk and N.K. Hussain for manuscript comments, T. Shelley for technical support and L. Hamm for management support. This work was supported by NIH grants P50MH100024, R01NS036715 (both to R.L.H.) and R01MH051106 (to D.J.L.) and by the Howard Hughes Medical Institute (to R.L.H.).

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Author notes

  1. Da-Ting Lin

    Present address: Present address: National Institute on Drug Abuse, Baltimore, Maryland, USA.,

  2. Yong Zhang and Robert H Cudmore: These authors contributed equally to this work.

Authors and Affiliations

  1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Yong Zhang, Robert H Cudmore, Da-Ting Lin, David J Linden & Richard L Huganir

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Contributions

Y.Z., D.-T.L. and R.L.H. designed experiments. Y.Z. performed experiments and analyzed data. R.H.C. developed analytic tools and designed the software. Y.Z., R.H.C., D.J.L. and R.L.H. wrote the manuscript.

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Correspondence to Richard L Huganir.

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Competing interests

Under a licensing agreement between Millipore Corporation and The Johns Hopkins University, R.L.H. is entitled to a share of royalties received by the University on sales of products described in this article. R.L.H. is a paid consultant to Millipore Corporation. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict-of-interest policies.

Integrated supplementary information

Supplementary Figure 1 SEP-GluA1 is moderately overexpressed in electroporated animals.

a, Immunostaining of GluA1 in brain slices of GluA1 knockout (left) and wild type animals (middle). High-magnification view of punctate GluA1 staining in wild type animals (right). b, Immunostaining of GluA1 (left) and GFP (right) to reveal SEP-GluA1 in brain slices of electroporated pups. Note that the level of total GluA1 is similar between transfected and untransfected neurons. Arrows mark neurons transfected with SEP-GluA1. c, Quantification of relative GluA1 expression level in transfected (red) and untransfected (black) neurons from b, GluA1 expression level in untransfected neurons are normalized to 100%. p > 0.08, student’s t test.

Supplementary Figure 2 Chronic cranial window surgery does not cause astrocyte proliferation.

Immunostaining of astrocyte marker GFAP under a chronic cranial window after animals are finished with two-photon imaging. Surgery hemisphere is displayed in the upper panel, control hemisphere in the middle panel, and hemisphere from intentionally injured animals in the lower panel.

Supplementary Figure 3 Acute whisker stimulation evokes a significant increase in average spine sGluA1 intensity and average shaft sGluA1 intensity, with little effect on average spine size.

a, Absolute changes in spine structure intensity for control and stimulated animals (0.01 ± 0.005 vs. 0.004 ± 0.005 at hour 1, 0.02 ± 0.006 vs. 0.02 ± 0.006 at hour 2, 0.025 ± 0.007 vs. 0.016 ± 0.007 at hour 3). b, Percent change in spine structure intensity for control and stimulated animals with raw data as in Fig. 1c (107.05% ± 1.36% vs. 105.92% ± 1.3% at hour 1, 108.96 ± 1.54% vs. 111.59% ± 1.5% at hour 2, 112.11% ± 1.68% vs. 110.61% ± 1.81% at hour 3). c, Absolute changes in spine sGluA1 for control and stimulated animals ((–0.10 ± 0.04 vs. 0.35 ± 0.05 at hour 1, 0.03 ± 0.05 vs. 0.41 ± 0.05 at hour 2, 0.17 ± 0.06 vs. 0.41 ± 0.05 at hour 3). d, Percent change in spine sGluA1 intensity for control and stimulated animals with raw data as in Fig. 1e (96.19% ± 1.34% vs. 128.55% ± 2.91% at hour 1, 100.74% ± 1.55% vs. 133.75% ± 3.15% at hour 2, 106.47% ± 1.75% vs. 135.75% ± 3.29% at hour 3). e, Absolute changes in shaft sGluA1 for control and stimulated animals ((–0.19 ± 0.03 vs. 0.58 ± 0.05 at hour 1, 0.005 ± 0.03 vs. 0.65 ± 0.05 at hour 2, 0.18 ± 0.04 vs. 0.65 ± 0.05 at hour 3). f, Percent change in shaft sGluA1 intensity for control and stimulated animals with raw data as in Fig. 1g (94.19% ± 0.68% vs. 127.52% ± 2.42% at hour 1, 97.09% ± 0.73% vs. 130.07% ± 2.59% at hour 2, 99.41% ± 0.95% vs. 129.35% ± 2.34% at hour 3). ***p < 0.001, **p < 0.01, two-way ANOVA with Bonferroni posttests. a.u., artificial unit. Error bars = s.e.m.

Supplementary Figure 4 Determining regions of interest for intensity analysis.

a, Each spine was assigned three different non-overlapping regions of interest (ROIs): spineROI, shaftROI and backgroundROI. The spineROI enclosed the spine head and did not include pixels within the radius of the dendritic backbone. The shaftROI was constructed from the backbone line and radius of the dendrite, centered on each spine and symmetrically expanded along the dendrite to match the number of pixels in the spineROI. The backgroundROI (same shape and number of pixels as the spineROI) was translated in x/y to a nearby region of the image that was representative of the background fluorescence. b, To obtain intensity measurements for the GluA1 channel, the three ROIs created using the backbone and spine markings in the structural channel (panel a), were placed in the same position on the GluA1 channel and the backgroundROI was translated again to minimize intensity. Note, all spine markings and intensity analysis were performed on the raw 3D image stacks.

Supplementary Figure 5 Whisker stimulation does not alter spine turnover.

a, Example time series with 4 persistent spines. Dendritic backbone (cyan), manually selected spines (yellow numbers and arrows) and the line connecting the spine head to the dendritic backbone (blue). b, Pooled data of spine turnover for dendrites imaged in stimulated and control animals (stimulated: n = 38 dendrites, 5 animals; control n = 38 dendrites, 4 animals). In stimulated animals, whisker stimulation is given between hour 0 and 1. All analysis is performed on individual dendrites and then pooled across each condition. Spine density (spines/μm) is the number of spines on a given dendrite divided by the total dendritic length (μm) that was scored for that dendrite. Percent added is the number of spines categorized as added (on a given dendrite) divided by the total number of spines for the same dendrite in the previous time-point. Likewise, percent subtracted is the number of spines categorized as subtracted on a given dendrite divided by the total number of spines for the same dendrite in the previous time-point. Turn over ratio (TOR) is the sum of added and subtracted spines within a dendrite divided by two times the total number of spines for the same dendrite in the previous time-point. There is no significant difference between dendrites from stimulated and control animals in any of the spine turnover measurements at any of the time-points (Two-way ANOVA).

Supplementary Figure 6 Initial spine and shaft properties bias spine changes following whisker stimulation.

a, Scatter plots of absolute change in spine structure intensity at 1 hour versus initial spine structure intensity at 0 hour with linear fits. (r = –0.2546, p < 0.001). b, Scatter plots of absolute change in spine sGluA1 intensity at 1 hour versus initial spine structure intensity at 0 hour with linear fits. (r = –0.1057, p = 0.0124). c, Scatter plots of absolute change in shaft sGluA1 intensity at 1 hour versus initial spine structure intensity at 0 hour with linear fits. (r = 0.0686, p = 0.1052). d, Scatter plots of absolute change in spine sGluA1 intensity at 1 hour versus initial spine sGluA1 intensity at 0 hour with linear fits. (r = 0.0446, p = 0.2927). e, Scatter plots of absolute change in spine sGluA1 intensity at 1 hour versus initial shaft sGluA1 intensity at 0 hour with linear fits. (r = 0.0647, p = 0.1262). f, Scatter plots of absolute change in shaft sGluA1 intensity at 1 hour versus initial shaft sGluA1 intensity at 0 hour with linear fits. (r = 0.1608, p < 0.001). In each panel, center solid black line is linear fit, enclosing solid black lines are 95% confidence intervals, horizontal dashed line is 0. a.u., artificial unit. r, Pearson’s linear correlation coefficient. p, Pearson’s correlation t test.

Supplementary Figure 7 Correlation between changes in intensity in spine sGluA1 and shaft sGluA1.

No significant correlation was detected between spine sGluA1 and spine size at 1 hour in the subset of spines that showed a > 30% increase in spine sGluA1 after whisker stimulation. 206 spines, r, Pearson’s linear correlation coefficient.

Supplementary Figure 8 Correlation between changes in intensity in spine sGluA1 and shaft sGluA1 over time in stimulated animals.

One hour after stimulation, the changes in spine sGluA1 are correlated with shaft sGluA1 (upper left). This correlation diminishes by hour 48 (lower right). 105 spines, 13 dendrites in 3 stimulated animals. r, Pearson’s linear correlation coefficient, p value is from Monte-Carlo shuffling.

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Supplementary Figures 1–8 (PDF 4812 kb)

Supplementary Methods Checklist (PDF 421 kb)

41593_2015_BFnn3936_MOESM36_ESM.mov

Z-stack two-photon images of layer 2/3 pyramidal neurons in the barrel cortex of electroporated animals (SEP-GluA1 in green, dsRed2 in magenta). (MOV 5689 kb)

3D reconstruction of the Z-stack image in Supplementary Video 1. (MOV 17897 kb)

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Zhang, Y., Cudmore, R., Lin, DT. et al. Visualization of NMDA receptor–dependent AMPA receptor synaptic plasticity in vivo. Nat Neurosci 18, 402–407 (2015). https://doi.org/10.1038/nn.3936

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