Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors

Abstract

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (less than 1 h) had initially been explained either by presynaptic increases in glutamate release or by direct modification of postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional postsynaptic AMPA receptors (AMPARs), sourced either from an intracellular reserve pool by exocytosis or from nearby extra-synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during early LTP remains unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Here, using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion markedly impairs synaptic potentiation of Schaffer collaterals and commissural inputs to the CA1 area of the mouse hippocampus in cultured slices, acute slices and in vivo. Our data also identify distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus inhibited fear conditioning, indicating that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein-protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning.

Extended Data Figure 1. X-link of AMPA receptors in cultured hippocampal neurons measured by quantum dot tracking.

Neutravidin (NA) and anti-GluA2 IgG (clone 14B11) X-linking, to a similar extent, reduce the surface diffusion of bAP::SEP::GluA2 expressed in rat hippocampal neurons as measured by quantum dot tracking. Relative frequency histogram (left) of log-transformed diffusion coefficients (D), bar graph of mobile fraction (middle) and a plot of group data for mean-squared displacement (MSD) curves of all trajectories (right). Control was no antibody. Bar graph shows mean with s.e.m. error bars and data points. Statistical significance was assessed by 1-way ANOVA with Holm-Bonferroni post-tests (ns = not significant, ** P < 0.01; *** P < 0.001).

Extended Data Figure 2. Controls relating to X-link by pre-treating bAP::SEP::GluA2-transfected slices cultures with NA.

a, Biotin-binding proteins diffuse through living organotypic slices and bind specifically to bAP::SEP::GluA2-expressing cells. Images show a maximum projection of an example 6.6 micron Z-stack in a transfected Gria2^-/- organotypic slice. CA1 neurons were cotransfected with tdTomato and bAP::SEP::GluA2. Streptavidin AF-633 staining was observed in all 12 bAP::SEP::GluA2-transfected CA1/3 cells observed and imaged. In contrast, no surface staining was observed in 5 CA1/3 cells transfected with myc::SEP::GluA2. b-c Whole-cell recordings of bAP::SEP::GluA2 replacement CA1 neurons reveal stable baseline synaptic transmission (without high-frequency stimulation, i.e. pseudo (p)HFS) in slices either with (c) or without NA-pretreatment (b). Summary plots of mean normalised EPSP slope ± s.e.m. (left) and cumulative histograms of STP and LTP (right). d, Top, Superimposed post-synaptic response of each cell during the first HFS train (grey) for Ctl and NA pre-treatment groups; the average responses (after spikes were removed using a median filter with window width ranging from 2-6 ms) are shown in black and blue respectively. Bottom, Bar graph showing no significant effect detected of NA pre-treatment on the area-under-curve (AUC) of the post-synaptic depolarization recorded across the three HFS trains used to induce synaptic potentiation. e, Bar graph showing no significant effect of NA pre-treatment on the EPSP slope during the baseline recording. f, No significant effect of NA pre-treatment on the amplitude and time course of NMDAR currents is detected in bAP::SEP::GluA2 replacement cells. Top, Evoked NMDAR excitatory postsynaptic currents (EPSCs) were simultaneously recorded in bAP::SEP::GluA2-transfected and neighbouring untransfected cells of Gria2^-/- slices. Example NMDAR-mediated EPSC recordings from transfected cells in Ctl (left) and NA pre-treatment (right). Two-exponent fits to the decay (dashed red lines) and the weighted average (bold red line) are superimposed over the traces. Bottom, Scatter plots of NMDAR EPSC amplitude (above) and decay time constant (below) for transfected and untransfected cells from the Ctrl (left) and NA pre-treatment (right). Bold line represents the line of unity. Dashed lines represent 95 % confidence bands from linear fits through the origin. The line of unity is between the confidence bounds in all cases. All bar graphs show means with s.e.m. error bars and data points (d-e). Statistical significance was assessed by mixed model nested ANOVA (d) or 2-way ANOVA without interaction (e; ns = not significant, * P < 0.05).

Extended Data Figure 3. Controls relating to manipulations preventing AMPAR diffusion and exocytosis.

Left, Summary plots of mean normalized EPSP slope ± s.e.m. (HFS = high-frequency stimulation). Right, Cumulative histograms for average normalized EPSP slope during STP and LTP. a, Robust STP and LTP following HFS in myc::SEP::GluA2 replacement cells following NA pre-treatment. b Rundown of basal transmission by 0.5 μM intracellular TeTx during pseudo (p)HFS recordings of bAP::SEP::GluA2 replacement cells. c, Absent potentiation following HFS in bAP::SEP::GluA2 replacement cells when NA pre-treatment is combined with intracellular 500 μM NEM. Statistical significance was assessed by RM- ANOVA (a-c; ns = not significant, * P < 0.05; ** P < 0.01).

Extended Data Figure 4. Presynaptic plasticity controls for NA X-linking of bAP::SEP::GluA2.

a NA has effect on paired-pulse ratio (PPR) of: a, the slope of AMPA-mediated EPSPs evoked at 50 ms intervals in untransfected CA1 pyramidal neurons. b, the amplitude of NMDA-mediated EPSCs evoked at 50 ms intervals in bAP::SEP::GluA2 replacement cells. All bar graphs show means with s.e.m. error bars and data points. Statistical significance was assessed by unpaired t-tests (a-b; ns = not significant).

Extended Data Figure 5. Statistical comparison between all the different treatments for STP and LTP.

Bar graph summarizing statistical comparison of the data for the manipulations in Fig. 2a-e, Extended Data Fig. 2b-c and Extended Data Fig. 3a-c. Different AMPAR trafficking manipulations have distinct effects on synaptic potentiation. The results demonstrate that HFS-dependent STP is only significantly different from control when surface diffusion of existing surface AMPARs is prevented. In contrast, HFS-dependent LTP is significantly different only for manipulations that prevent the delivery of newly exocytosed receptors. Bar graph shows marginal means and Least Significant Difference (LSD) error bars. Data points for all conditions are plot in the cumulative histograms of Fig. 2a-e, Extended Data Fig, 2b-c and Extended data Figure 3a-c. Statistical significance was assessed by 2-way RM-ANOVA with Benjamini and Hochberg post-tests (ns = not significant, * P < 0.05; ** P < 0.01; *** P < 0.001).

Extended Data Figure 6. Control experiments for antibody-mediated X-link of AMPA receptors in cultured hippocampal neurons.

a Anti-GluA2 IgG (bivalent) but not Fab (monovalent) prevents normal FRAP of spine SEP-GluA2. Graphs show ensemble grand mean FRAP curves, fits and standard error bands for experiments using transfected cultures pre-treated for half an hour with 80 mg/L anti-GluA2 Fab clone 15F1 (Top), 80 mg/L anti-GluA2 IgG clone 15F1 (Middle) or vehicle (Bottom). b-c Relative frequency histogram of log-transformed diffusion coefficients (D, left) and bar graph of percentage mobile fractions obtained from single-particle tracking (SPT) experiments. b U-PAINT single particle tracking of endogenous GluA2 in the presence or absence of anti-GluA2 IgG clone 14B11. c PALM single particle tracking of expressed mGluR5::mEOS in the presence or absence of anti-GluA2 IgG clone 14B11. All bar graphs show means with s.e.m. error bars and data points. Statistical significance was assessed by unpaired t-tests (b-c, ns = not significant, *** P < 0.001).

Extended Data Figure 7. No detectable effect of incubation with X-linking anti-GluA2 IgG on basal endocytosis or phosphorylation of GluA1-containing AMPA receptors.

a Schematic of the experimental protocol performed on DIV 17 cultured hippocampal neurons and data summary of fluorescence (normalized to the mean fluorescence at time zero) for anti GluA1 antibody feeding 30 minutes following 15 minute X-link by 10 μg/ml anti-GluA2 IgG (clone 15F1). Note that most GluA1 AMPA receptors in pyramidal neurons exist as GluA1/2 heteromers. Similar results were obtained from two experiments and combined, where the control was either: 1) no antibody; or 2) anti-GFP. The images are all scaled the same. b Schematic of experimental protocol (top), images of example Western blots (middle) and data for phosphorylation at GluA1 serine 845 and 831 after 15 minute X-link by 10 μg/ml anti-GluA2 IgG (clone 15F1) or control IgG (anti-GFP). Phosphorylation was unaffected by the X-link manipulation. c. Schematic of experimental cLTP protocol (top), images of example Western blots (middle) and data for phosphorylation at GluA1 serine 845 after 15 minute X-link by 10 μg/ml anti-GluA2 IgG (clone 15F1) or control IgG (anti-GFP) followed by chemical LTP (cLTP) or control treatment. The X-link manipulation had little impact on S845 phosphorylation induced by cLTP. We achieved similar phosphorylation results for AMPARs isolated by surface biotinylation and eluting from streptavidin beads. All bar graphs show means with s.e.m. error bars and data points. Note that in b and c the data points for each experiment were recentered on the grand mean; thus the error bars approximate within-experiment s.e.m. Black arrow heads adjacent to the molecular weight (MW) lane in b and c denote the 95 kDa size marker. Statistical significance was assessed by mixed model nested ANOVA (a), 2-way ANOVA without interaction (b) or 2-way RM-ANOVA (ns = not significant, *** P < 0.001). For gel source data, see Supplementary Figure 1.

Extended Data Figure 8. Effect of X-linking AMPA receptors on synaptic potentiation and basal transmission in acute hippocampal slices.

a Schematic diagram illustrating the protocol for pre-injection antibody X-link experiments in acute slices. b Summary plots of mean normalised fEPSP slope (top) and paired-pulse ratio (PPR, 200 ms interval) of the slope (bottom) ± s.e.m. c Input-output curves of the field EPSP slope are unaffected by antibody infusion. The fiber volley varied linearly over the range of stimulation intensities (data not shown). d Input-output curves of evoked NMDAR-mediated EPSCs are unaffected by antibody infusion. e Spontaneous EPSC frequency (left) and amplitude (right) are unaffected by antibody infusion. f NMDA/AMPA ratios are unaffected by antibody infusion. ANOVA on log₁₀(ratio _NMDA/AMPA), F(2,54) = 0.53, P = 0.5942. Numbers in brackets indicate the number of cells (f) or the number of slices (c-e), where measurements from whole cell recordings within the same slice were averaged (d-e). All bar graphs show means ± s.e.m. error bars and data points. Statistical significance was assessed by 1-way ANCOVA (c-d) or 1-way ANOVA (e-f).

Extended Data Figure 9

Cumulative histograms for the average normalized EPSP slope during STP and LTP from the HFS- and TBS-induced synaptic potentiation experiments summarised in Fig 3b and c respectively.

Figure 1. Biotin-tethered AMPARs are effectively X-linked by neutravidin to prevent their surface diffusion.

a, Construct for dual expression of AP::SEP::GluA and BirA-ER. b, Strategy to X-link bAP::SEP::GluA (AP = acceptor peptide; SEP = Super-Ecliptic pHluorin; IRES = internal ribosome entry site). c, Example images (top) and graph showing mean FRAP curves, fits and standard error bands (bottom) for control and pre-treatment with NA (50 nM for 2 min). Inverted image lookup table. d, Receptor mobile fraction in spines of cells expressing AP::SEP::GluA1 and AP::SEP::GluA2 is reduced by NA pre-treatment and depends on the AP tag and BirA-ER. e, Molecular replacement with bAP::SEP::GluA2 in CA1 neurons. Example AMPAR current traces from Schaffer collateral (SC) synapse stimulation (top) or by 1-photon glutamate uncaging on the soma (bottom). f, Pre-treating slices with NA (100 nM for 45 min) caused no detectable effect on: Top. AMPA/NMDA ratios, Middle, evoked excitatory postsynaptic conductances, or Bottom, glutamate uncaging responses. Bar graphs show marginal means with 83% confidence intervals (d) or mean with s.e.m. error bars and data points (e-f). Statistical significance was assessed by mixed model nested ANOVA (d), 1-way ANOVA (e) or 2-way ANOVA with Holm-Bonferroni post-tests (f; ns = not significant, ** P < 0.01; *** P < 0.001).

Figure 2. X-link reveals surface diffusion as a critical step in the synaptic delivery of AMPARs during synaptic potentiation.

Top, Scheme illustrates experimental protocols on organotypic Hippocampal slices. a-e, Left, Example whole-cell voltage traces and summary plots of mean normalized EPSP slope ± s.e.m. (HFS = high-frequency stimulation). Middle, Cumulative histograms for average normalized EPSP slope during STP and LTP. Right, Models of experimental manipulations. a, Robust STP and LTP following HFS under control conditions (4 experiments) b, Detectable HFS-induced LTP but not STP in slices pre-treated with 100 nM NA (5 experiments). c, Severe attenuation of LTP but not STP with 0.5 μM intracellular TeTx. d-e, No detectable change in EPSP slope after HFS when 100 nM NA pre-treatment is combined with either: d, TeTx in intracellular recording solution, or e, continuous infusion of 10 pM NA in the external recording solution (3 experiments). Statistical significance was assessed by repeated measures (RM)-ANOVA with Holm-Bonferroni post-tests (a-e; ns = not significant, * P < 0.05; ** P < 0.01).

Figure 3. Antibody X-link of endogenous GluA2 attenuates LTP of CA1 fEPSPs in vitro and in vivo.

a Acute slice experimental setup and antibody labelling controls. b-c Protocol (top), example traces (middle) and summary plots of mean normalized fEPSP slope ± s.e.m. (Ab = antibody). No stable synaptic potentiation following high frequency stimulation (HFS, b) or theta-burst stimulation (TBS, c) when α-GluA2 IgG pre-injection is combined with continuous infusion of the antibody. Cumulative histograms for STP and LTP are plot in Extended Data Fig. 8. d In vivo experimental protocol and histological controls. (VHC = ventral hippocampal commissure; Ab = antibody). e-h LTP recordings following injection of: e, anti-GluA2 Fab; f, anti-GluA2 IgG or g, control IgG. Left. Mean normalized fEPSP slope ± s.e.m. Right. Example voltage traces before and after HFS. h, Bar graph of the means with s.e.m. error bars and data points for the normalized fEPSP slope potentiation calculated from the data in Fig. 3e-g. Statistical significance was assessed by 1-way ANOVA with Holm-Bonferroni post-tests (h, ns = not significant, * P < 0.05; ** P < 0.01)

Figure 4. Impairment of a hippocampal-dependent learning task by infusion of X-linking anti-GluA2 IgG.

Shaded data are controls for baseline freezing levels. Unshaded data were used for hypothesis testing. a-b, Selective effects on contextual (a) versus cued (b) fear learning. a, Left, Antibody infusion sites. Right, Pre-conditioning infusion of anti-GluA2 IgG reduces freezing to conditioned context (A). b, Cued fear learning was robust for all antibodies. c, No detectable difference in freezing to conditioned context between antibodies for pre-test infusions. All bar graphs show means ± s.e.m. error bars. Data points are shown where data is compared using statistics. Statistical significance was assessed by 2-way repeated measures (RM)-ANOVA with Holm-Bonferroni post-tests (ns = not significant, ** P < 0.01; *** P < 0.001). The mouse brain drawing is reproduced with permission from ‘The mouse brain atlas’, Franklin K.B.J. and Paxinos G., p74, Copyright Elsevier (2007).