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Filopodia are a structural substrate for silent synapses in adult neocortex

Nature volume 612, pages 323–327 (2022)Cite this article

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Abstract

Newly generated excitatory synapses in the mammalian cortex lack sufficient AMPA-type glutamate receptors to mediate neurotransmission, resulting in functionally silent synapses that require activity-dependent plasticity to mature. Silent synapses are abundant in early development, during which they mediate circuit formation and refinement, but they are thought to be scarce in adulthood1. However, adults retain a capacity for neural plasticity and flexible learning that suggests that the formation of new connections is still prevalent. Here we used super-resolution protein imaging to visualize synaptic proteins at 2,234 synapses from layer 5 pyramidal neurons in the primary visual cortex of adult mice. Unexpectedly, about 25% of these synapses lack AMPA receptors. These putative silent synapses were located at the tips of thin dendritic protrusions, known as filopodia, which were more abundant by an order of magnitude than previously believed (comprising about 30% of all dendritic protrusions). Physiological experiments revealed that filopodia do indeed lack AMPA-receptor-mediated transmission, but they exhibit NMDA-receptor-mediated synaptic transmission. We further showed that functionally silent synapses on filopodia can be unsilenced through Hebbian plasticity, recruiting new active connections into a neuron’s input matrix. These results challenge the model that functional connectivity is largely fixed in the adult cortex and demonstrate a new mechanism for flexible control of synaptic wiring that expands the learning capabilities of the mature brain.

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Fig. 1: Filopodia account for a large fraction of dendritic protrusions in L5 and L2/3 pyramidal neurons of adult mouse primary visual cortex.
Fig. 2: Filopodia exhibit AMPAR-immunonegative and NMDAR-immunopositive synapses.
Fig. 3: Filopodia lack AMPAR- but exhibit NMDAR-mediated transmission and release-competent presynaptic partners.
Fig. 4: Silent synapses at filopodia can be unsilenced by Hebbian pairing.

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Data availability

The data generated and analysed in the current study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Code for image analysis can be accessed at https://github.com/harnett/FilopodiaStructuralSubstrateSilentSynapses.

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Acknowledgements

We thank D. H. Yun for technical assistance with eMAP, K. Tsimring and A. Krol for technical assistance with perfusions, and C. Yaeger, M. Tadross, E. Nedivi and M. Bear for constructive criticism of the manuscript. We thank H. Umemori for the donation of Thy1-GFP-M+ mouse pups. Financial support was provided by the Boehringer Ingelheim Fonds (D.V.), National Institutes of Health RO1NS106031 (M.T.H.), the James W. and Patricia T. Poitras Fund at MIT (M.T.H.), a Klingenstein-Simons Fellowship (M.T.H.), a Vallee Foundation Scholarship (M.T.H.) and a McKnight Scholarship (M.T.H.).

Author information

Authors and Affiliations

  1. McGovern Institute for Brain Research, MIT, Cambridge, MA, USA

    Dimitra Vardalaki & Mark T. Harnett

  2. Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA

    Dimitra Vardalaki, Kwanghun Chung & Mark T. Harnett

  3. Picower Institute for Learning and Memory, MIT, Cambridge, MA, USA

    Kwanghun Chung

  4. Institute for Medical Engineering and Science, MIT, Cambridge, MA, USA

    Kwanghun Chung

  5. Department of Chemical Engineering, MIT, Cambridge, MA, USA

    Kwanghun Chung

  6. Broad Institute of Harvard University and MIT, Cambridge, MA, USA

    Kwanghun Chung

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Contributions

D.V. performed all experiments, analysed all data and prepared the figures. K.C. provided eMAP resources. M.T.H supervised all aspects of the project and wrote the manuscript with D.V.

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Correspondence to Mark T. Harnett.

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Extended data figures and tables

Extended Data Fig. 1 Morphological measurements of dendritic protrusions.

a, Illustration of a dendritic protrusion and corresponding measurements: head diameter. (dhead), neck diameter (dneck), length (l). b–d, Population histograms of morphological characteristics across all dendritic protrusions (n = 2234). e, Population histogram of the relationship between dhead and dneck. Shaded area indicates a ratio below 1.3, the first criterion used to classify filopodia versus spines. f, Population histogram of dhead/l for protrusions with dhead/dneck below 1.3 (shaded area in e). Protrusions with dhead/l above 3 were classified as filopodia, those below 3 were likely short stubby spines and were not analyzed further (shaded area= (dhead /dneck < 1.3) ∩ (l/ dhead>3)). g, Same as e but for each of the 4 mice. h, Fraction of dendritic protrusions classified as filopodia per mouse (n = 527, 944, 435, 328 dendritic protrusions and 30, 47, 25, 21 dendritic branches for mouse 1, 2, 3, 4 respectively). Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers. ns P = 0.093, Kruskal-Wallis test.

Source data

Extended Data Fig. 2 Filopodia in L2/3 pyramidal neurons exhibit AMPAR immune-negative and NMDAR immune-positive synapses.

a, Example confocal image of a V1 L2/3 neuron (green arrowhead) expressing GFP after viral transfection in V1 in an originally 45 μm thick slice. Scale bar: 100 μm expanded/ 59 μm original. Image was taken after reshrinking the tissue from 4x expansion to 1.7x expansion. b, Box plot (left) and kernel density estimate (right) of signal intensity in Bassoon (blue), NMDAR (yellow), and AMPAR (red) channels for L2/3 pyramidal neuron spines (n = 275). Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers. Signal in each channel is shown for all dendritic protrusions, each represented by one dot. c, As in b, but for filopodia (n = 134).

Source data

Extended Data Fig. 3 Anti-Bassoon signal intensity threshold for presence of presynaptic partner.

a, Cumulative density function of Bassoon signal intensity in spines (red) and filopodia (yellow). Vertical line at the choosen threshold (anti-Bassoon signal = 0). b, Magnified plot of a around 0. c-d, Example filopodia with anti-Bassoon signal intensities below above the threshold. Scale bar: 5 μm expanded/1.25 μm original.

Source data

Extended Data Fig. 4 Representatitive examples of eMAP at dendritic protrusions.

Example four channel images of dendritic protrusions with different dhead/dneck values (increasing from left to right). From top to bottom: cell-filling GFP stained with Alexa Fluor 488 (green) at lower magnification to show full protrusion shape, presynaptic protein Bassoon stained Alexa Fluor 405 (blue), NMDAR subunit NR1(GluN1) stained with Alexa Fluor 555 (yellow), and AMPAR subunit GluR1(GluA1) stained with Alexa Fluor 647 (red), all at higher magnifiaction to show synaptic localization. Scale bar: 2 μm expanded/0.5 μm original.

Extended Data Fig. 5 Anti-GluA1 signal increases with spine size.

a, Anti-GluA1 signal intensity as a function of head diameter for spines (red) and filopodia (yellow). b, Correlation between head diameter and anti-GluA1 signal intensity for spines. The data are fitted with a line of slope 421+/−22 using linear regression. c, Correlation between diameter of filopodium head and anti-GluaA1 signal intensity. The data are fitted with a line of slope 95+/−39 using linear regression. Correlation coefficients (r) and p-values were obtained from a two-tailed, non-parametric Spearman correlation.

Source data

Extended Data Fig. 6 Spatial resolution of two-photon MNI-glutamate uncaging at adult mouse cortical protrusions.

a, Left: Two-photon z-stack of a V1 L5 pyramidal neuron filled with Alexa-488 via somatic patch pipette. Basal branch segment of interest indicated by yellow box. Right: Magnified view of basal branch of interest. b, (top) Voltage response for the spine at lateral uncaging locations shown in a. (bottom) plot of lateral uncaging resolution. Continuous line is the Gaussian fit of the amplitudes of two-photon glutamate uncaging along lateral steps (circles). c, (top) Voltage response for the spine at axial locations shown in a. Each voltage trace is an average of the voltage traces evoked at a specific axial step above and below of the spine. (bottom) plot of axial uncaging resolution (see b). d, Magnified view of a filopodium of a basal branch of a L5 pyramidal neuron. All uncaging experiments shown in e and f were performed in Mg2+ free ASCF with AMPA blocked (DNQX, 20 μM). e, As in b, for the filopodium shown in d. f, As in c, for the filopodium shown in d.

Source data

Extended Data Fig. 7 Responses to focal extracellular synaptic stimulation for the filopodium shown in Fig. 3e.

a, Superimposed traces of somatic voltage recordings (left) and corresponding changes in local Ca2+ (measured via Fluo-4 fluorescence; ΔF/F) at the parent dendritic branch (middle) and at the tip of the filopodium (right) in response to focal extracellular synaptic stimulation in Mg2+-free aCSF with AMPA blocked (via DNQX, 20 μΜ). All synaptic stimulation successes and failures for the filopodium in Fig. 3e are shown. Synaptic stimulation driven backpropagating action potential (bAP) shown in red. b, Same as in a with traces spaced apart. Grey dashed line indicates the onset of synaptic stimulation.

Source data

Extended Data Fig. 8 Length of protrusions before and after induction protocols.

Length of protrusions before (grey) and after (red) induction in filopodia and spines. Three different induction protocols were tested in filopodia: i- Pairing protocol (n = 15 filopodia from 13 slices and 10 mice); ii- Somatic action potentials without any caged glutamate present (Post alone; n = 7 filopodia from 7 slices and 6 mice); iii- Glutamate uncaging without somatic action potential (Pre alone; n = 7 filopodia from 7 slices and 6 mice); ns P > 0.15. Two-sided Wilcoxon signed-rank test. Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers.

Source data

Extended Data Fig. 9 Spiny synapses do not exhibit changes in synaptic strength or length in response to the STDP protocol.

a, Schematic of the experiment. A control spine on a different branch than the branch of the test spine was always present. 40 and 90 repetitions of the pairing protocol were used for spines. b, Relative change of peak somatic uEPSP amplitude after pairing. P = 0.5781 (40 repetitions, n = 7 test and 7 control spines from 7 slices and 4 mice), P = 0.9375 (90 repetitions, n = 7 test and 7 control spines from 7 slices and 3 mice), two-sided Wilcoxon signed-rank test. Box plot represents median and IQR with whiskers extending to the 95% CI. c, Relative change of spine length after pairing. P = 0.4688 (40 repetitions, n = 7 test and 7 control spines from 7 slices and 4 mice), P = 0.8125 (90 repetitions, n = 7 test and 7 control spines from 7 slices and 3 mice), two-sided Wilcoxon signed-rank test. Box plot represents median and IQR with whiskers extending to the 95% CI.

Source data

Extended Data Fig.10 Super-resolution characterization of synapses in developing mouse visual cortex.

a, Example confocal image of a postnatal day (P) 13 Thy1- GFP-M+ L5 pyramidal neuron dendritic segment after 4x expansion. Scale bar: 10 μm expanded/2.5 μm original. b, Fraction of dendritic protrusions classified as filopodia in P13 L5 PNs (n = 371 dendritic protrusion, 18 dendritic branches, 3 mice). Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers. c, Fraction of total synapses in the three dendritic locations in P13 L5 PNs (n = 397 synapses, 18 dendritic branches, 3 mice). Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers. d, (left) Box plot and individual data for signal intensity in Bassoon (blue), NMDAR (yellow), and AMPAR (red) channels for spines (n = 236). (right) example four channel images of a representative spine. Box plot represents median and IQR with whiskers extending to the most extreme points not considered outliers. Scale bar: 5 μm expanded/1.25 μm original (GFP), 1 μm expanded/0.25 μm original (Bassoon). e, As in b, but for filopodia (n = 79). f, As in b, but for shaft synapses (n = 82). Example images show a shaft synapse that lacks AMPARs (top) and a shaft synapse that exhibits AMPARs (bottom). g, Comparison of dendritic protrusion types in P13 (n = 371) and adult mice (n = 2234). h, Comparison of synapse distribution in P13 (n = 397) and adult mice (n = 2188).

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Vardalaki, D., Chung, K. & Harnett, M.T. Filopodia are a structural substrate for silent synapses in adult neocortex. Nature 612, 323–327 (2022). https://doi.org/10.1038/s41586-022-05483-6

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