Topological Regulation of Synaptic AMPA Receptor Expression by the RNA-Binding Protein CPEB3

Abstract

Synaptic receptors gate the neuronal response to incoming signals, but they are not homogeneously distributed on dendrites. A spatially defined receptor distribution can preferentially amplify certain synaptic inputs, resize receptive fields of neurons, and optimize information processing within a neuronal circuit. Thus, a longstanding question is how the spatial organization of synaptic receptors is achieved. Here, we find that action potentials provide local signals that influence the distribution of synaptic AMPA receptors along dendrites in mouse cerebellar stellate cells. Graded dendritic depolarizations elevate CPEB3 protein at proximal dendrites, where we suggest that CPEB3 binds to GluA2 mRNA, suppressing GluA2 protein synthesis leading to a distance-dependent increase in synaptic GluA2 AMPARs. The activity-induced expression of CPEB3 requires increased Ca(2+) and PKC activation. Our results suggest a cell-autonomous mechanism where sustained postsynaptic firing drives graded local protein synthesis, thus directing the spatial organization of synaptic AMPARs.

Keywords: AMPA receptor; CPEB3; GluA2; PKC; action potentials; calcium imaging; cerebellar stellate cell; dendritic gradient; subunit composition; topostatic plasticity.

Figure 1. Distance dependent distribution of synaptic AMPAR subtypes along dendrites

A. Left: Schematic showing that parallel fibers (vertical lines) extend orthogonally to the sagittal plane of stellate cell dendrites. The position of a stimulation electrode in a sagittal slice closely correlates with the location of a PF-stellate cell synapse. Middle: normalized I-V relationship for EPSCs evoked at each of the labeled sites. The dashed red line is the linear regression fit to the current amplitude at negative potentials (−60 to 0 mV). If EPSC amplitudes at positive potentials fall below the red line, these synaptic currents have an inwardly rectifying I-V relationship. Bottom: An example of the average current traces recorded in the soma (white circle) and evoked at several stimulation sites (colored circles) as labeled in the reconstructed image (Top right). Current amplitudes were normalized to the amplitude at −60 mV. At the distal sites the outward currents recorded at +40 mV relative to −60 mV increased. B. Left: The rectification index (RI) increases at distal synapses, indicating a greater presence of GluA2-containing AMPARs. Open circles represent the individual measurements (51 sites from 17 stellate cells and 16 mice) and the line shows the segmented average. Right: Average RI at the intermediate (40–60 μm) and distal synapses (>60 μm) is significantly larger than the RI at the proximal synapses (14–40 μm) (ANOVA, P < 0.00001). C. Left: The cumulative distribution of decay time constants for individual eEPSCs shows a significant increase in decay time at the intermediate (n=481 events) and distal synapses (n=373) compared to proximal sites (n=862) (#, P < 0.00005). Right: The decay time constant increases at distal synapses consistent with the presence of more GluA2-containing receptors (51 sites from 17 cells and 16 mice; ANOVA, P < 0.005). Bottom: The evoked EPSC amplitude at −60 mV (P < 0.05). Tukey post hoc test:*, P < 0.05; ***, P < 0.001. D. Bath application of IEM 1460 (100 μM), a GluA2-lacking AMPAR blocker, produced a greater inhibition at proximal synapses (5 sites, 1 site/cell, from 5 mice) than distal sites (6 sites, 1 site/cell from 6 mice, P = 0.005). Evoked synaptic currents were recorded before (black trace) and during the application of IEM 1460 (at 7.5–15 min, red trace) in the same stellate cells at each dendritic site. A time course of EPSC amplitude plot is shown in supplemental Figure 2A. E. Non-stationary fluctuation analysis (NSFA) of evoked EPSCs recorded at two dendritic locations. Left, example current traces of non-stationary fluctuation analysis of evoked EPSCs recorded at two dendritic locations (top, 35 μm; bottom, 45 μm). EPSCs (black lines) are superimposed to scaled average trace of 50 EPSCs (red lines). The difference between each EPSC and the scaled mean is shown as blue lines below. Middle and right, variance in the decay phase of the individual EPSCs around the mean is plotted against the averaged EPSC amplitude to produce the variance – mean current relationship. Red line is the parabolic fit from which mean single channel current was estimated (closed circles: fitted points of the data set). 50 (left) and 36 (middle) EPSCs were used for analysis. Right, the mean single channel conductance of EPSCs decreases at distal synapses (open circles present individual data; ANOVA, P < 0.0005; **, P < 0.002; ***, P < 0.0005). See also Figures S1 and S2.

Figure 2. The amplitude of Ca²⁺ transients generated in response to somatic action potentials decreases with the distance along the dendrites of stellate cells

A. two-dimensional projection of a two-photon 3D Z-stack for a stellate cell filled with Oregon Green BAPTA-1 (100 μM). The grey lines represent the dendritic regions where the Ca²⁺ transients were measured using line scans. B. Ca²⁺ transients (expressed as ΔF/F) generated in response to a 100 Hz-train of five somatic action potentials (lower voltage trace). The numbers on the left of the traces refer to the locations of the line scans in (A). C. Plot of the mean amplitude of the peak of the Ca²⁺ transients associated with the train of five back propagating APs as a function of the distance from the soma. The values of ΔF/F were binned into 10 μm intervals (20 μm for the most distal locations, ≥ 70 μm) and averaged. Open circles represent the individual measurements (63 sites from 11 stellate cells and 8 mice). D. The decrease in dendritic Ca²⁺ transients is spatially correlated with an increase in the rectification index of EPSCs.

Figure 3. Ca²⁺ entry during action potentials determines synaptic AMPAR phenotype along dendrites

A. Cerebellar slices were incubated with 1 mM tetraethylammonium (TEA) in the presence of 25 μM actinomycin D (actD), 100 μM picrotoxin (PTX) and 1 mM kynurenic acid (KYNA) for 3 hrs to increase the duration of APs and enhance Ca²⁺ entry (Liu et al., 2010). Left: Example of recordings at 2 locations and corresponding I-V relationship shows a decrease in outward current (at +40 mV) relative to current at −60 mV at the intermediate stimulation distance. Middle: The average RI and decay time constant of EPSCs did not increase at 40–60 μm (23 sites from 8 cells and 8 mice). Right: Increasing AP duration by TEA treatment shifted the dendritic GluA2 gradient toward more distal regions. Data from actD (+PTX+KYNA) treated and control in Fig. 1B were not different from each other (suppl Fig. 4A and 4B) and were shown as a combined control. Adjacent bins’ data points are labeled red/purple. B. The decay time constants for individual EPSCs were significantly reduced at the intermediate dendrites (40–60 μm) following TEA + ActD treatment relative to ActD control (#, P < 0.01, 203 and 248 events respectively), consistent with a decrease in synaptic GluA2. C. The rectification index and decay time constant of EPSCs increased at intermediate synapses in ActD treated control (20 sites from 8 cells and 8 animals). D. Following TEA treatment the mean single channel conductance of EPSCs at 40–60 μm was no longer reduced compared to proximal synapses. E and F. Cerebellar slices were treated with an N-type Ca²⁺ channel blocker, ω-conotoxin GVI-A (ω-CTX, 500 nM) for 3 hrs. I-V relationship for EPSCs evoked at the proximal sites became more linear following ω-CTX treatment (17 proximal sites and 12 distal sites from 12 cells and 9 mice). Open circles represent the individual measurements, adjacent bins’ data points are labelled blue/green. P < 0.05. See also Figure S4.

Figure 4. Disruption of the CPEB3-GluA2 mRNA interaction enhances synaptic GluA2 expression at the proximal dendrites in a protein synthesis-dependent manner

A. Cerebellar slices were incubated with 100 μM cycloheximide (CHX) for 3 hrs and then synaptic currents were recorded and evoked at the stimulation sites labeled in the left panel. Spermine (100 μM) was included in the pipette solution to block synaptic GluA2-lacking receptors at positive potentials. Right: Average EPSC traces recorded at +40 and −60mV and corresponding I-V relationship. B. The increase in the RI of EPSCs at the distal synapses was abolished following CHX treatment (7 sites for proximal; 6 for intermediate; 4 sites for distal from 6 cells; 6 mice) and EPSC RI at > 40 μm was reduced compared to control (P < 0.05). C. SELEX1904 is an RNA oligomer that competes with GluA2 mRNA for binding to CPEB3 (Huang et al., 2006). If CPEB3 binding with GluA2 mRNA prevents GluA2 protein synthesis at proximal synapses, SELEX1904 should increase synaptic GluA2 expression in stellate cells. D. Left: Example of evoked EPSCs at a proximal synapse. Including SELEX1904 (10 μM) in the pipette solution increased EPSC amplitude at +40 mV and the I-V relationship of EPSCs became more linear with time, indicating an increase in synaptic GluA2 receptors. Control oligo sequence was identical to SELEX1904, but was missing several terminal nucleotides needed to stabilize the hairpin 3D conformation that disrupts CPEB3/GluA2 mRNA interaction, see Methods. Right: Anisomycin (ANS), when added to ACSF prior to and during recording, prevented the SELEX-induced increase in EPSC RI. E. RI of EPSCs remained unaltered during the first 30 minutes and then gradually increased at proximal dendrites (< 40 μm, n = 6). A control oligomer (10 μM) did not alter the amplitude (B) and RI of EPSCs (n = 5). (Two-way ANOVA, P < 0.0005 for treatment groups and for time bins; Tukey means comparison vs either control, *, P < 0.01; **: P < 0.005; or vs time bin 1, ***: P < 0.0005). F. The initial RIs are those recorded within 15 min (4 cells) and between 15–30 min (2 cells in which we were unable to find the inputs within 15 min) after obtaining the whole cell configuration. Change in RI (Δ RI) was calculated as a difference between the average RI after it reached a plateau and the initial RI (linear regression for SELEX, R² = 0.62 and for CTL oligo, R² = 0.11). See also Figure S4.

Figure 5. TTX treatment enhances the protein synthesis-dependent expression of GluA2-containing AMPARs at synapses and reduces the level of CPEB3 expression in stellate cells

A. Slices were pretreated with 1 mM kynurenic acid (KYNA) and 100 μM picrotoxin (PTX) without (control) or with 0.5 μM tetrodotoxin (TTX) for 3 hrs. Right: Slices were treated with TTX in the presence of a protein synthesis inhibitor (100 μM cycloheximide, CHX) for 3 hrs. Average current traces recorded at +40 and −60 mV and the I-V relationship of AMPAR-mediated spontaneous EPSCs in stellate cells. TTX treatment increased rectification of EPSCs, which was prevented by cycloheximide. B. Summary data. Left: RI of individual cells (open circles) and average value (filled circle). Middle and right: EPSC amplitudes at +40 and −60 mV (cont, n = 8; TTX, n = 10; TTX + CHX, n = 6; CHX, n = 4;). ANOVA, P < 0.0001; Tukey post-hoc, *, P < 0.05; **, P < 0.005; ***, P < 0.0005. C. Cerebellar cultures were prepared from GAD-65 mice, in which stellate cells expressed GFP and GFP negative neurons were mainly granule cells. TTX treatment for 3 hrs reduced CPEB3-ir in GFP positive cells. Left, CPEB3-ir. Inserts show GFP. Right, comparison of somatic CPEB3-ir in TTX treated vs control stellate cells (GFP+) and granule cells (GFP−, bottom) (GFP+, >120 cells, P < 0.00001; GFP−, 67 control and 100 TTX-treated cells from 3 cultures). D. Effects of TTX treatment on CPEB3-ir levels in GFP+ cells and GFP− cells. Bottom: Prolonged incubation with TTX for 24 hrs did not further reduce the CPEB3 levels in GFP+ cells (3 cultures). ANOVA, P < 0.002; post-hoc test, **, P < 0.005; Scale bar: 12 μm. See also Figures S5, S6 and S7.

Figure 6. Immunohistochemical gradients of CPEB3 and GluA2 proteins in stellate cell dendrites

A. Examples of dendritic CPEB3-ir in GFP+ neurons in cerebellar cultures. CPEB3/GFP ratio was calculated by dividing the corresponding signals in each ROI (see Methods for details). Individual channels (GFP-ir, CPEB3-ir, color-coded ratio) are diagonally offset for illustration purposes. Left: Control CPEB3 staining showed a distance-dependent decrease towards the distal dendritic regions (3 hrs incubation in PTX+KYNA). Right: the CPEB3-ir decrease was attenuated by incubating cells in 500 nM ω-conotoxin GVIA (ω-CTX) prior to immunostaining for CPEB3 (3 hrs incubation in ω-CTX+PTX+KYNA). B. The ratio of CPEB3-ir/GFP fluorescence intensity was used to quantify the CPEB3 expression at various segments along the dendrites and was normalized to the value at 5–14 μm from the soma for each dendritic process. Right: group data, represented as a color gradient of an “average” schematic dendrite. C. CPEB3-ir decreased at more distal dendrites (>60 μm) in control (n = 19 from 7 cultures, one-way ANOVA, P < 0.05, Tukey post-hoc, 14–40 vs >60 μm, P<0.05), which was abolished following ω-CTX treatment (n = 14 from 6 cultures). *, P < 0.05; ***, P < 0.005. Right: dendritic GFP fluorescence did not change after ω-CTX treatment. D. Effects of TEA treatment on the CPEB3-ir along dendrites. Cultures were treated with ActD, (+KYNA and PTX) as control or with the addition of 1 mM TEA for 3 hrs. Left: CPEB3-ir decreases at intermediate (40–60 μm) and distal (>60 μm) dendrites relative to proximal dendrites (14–40 μm) in control (n = 13; repeated measures ANOVA, P < 0.002; Tukey post hoc test: *, P < 0.05; **, P < 0.005). However following TEA treatment CPEB3-ir did not decrease at intermediate sites (n = 11; RM-ANOVA, P = 0.055). Right: changes in CPEB3-ir at 40–60 μm relative to 14–40 μm of individual dendritic processes. One sample Wilcoxon signed rank test; †, P < 0.05. E. Left: Distal dendrites of stellate cells show an increase in GluA2-ir staining. Top: a representative example of surface GluA2 staining in GAD65::eGFP stellate cell. Bottom: the dendritic GluA2 puncta were color-coded for total GluA2-ir fluorescence (area × average intensity), corresponding to the total number of GluA2 molecules present at the synapse. Right: Artificially constructed image showing predicted distribution of GluA2 puncta along an “average” dendrite, modelled on an increase in 30% puncta size and 20% intensity (suppl. Fig. 8C). This example illustrates that it is difficult to visually observe the resulting 60% GluA2 increase at distal synapses “by eye,” yet it is amenable to computer-assisted detection (see Methods for explanations). F. Left, total GluA2-ir fluorescence along a dendritic process increased with the distance from the soma (11 processes from 5 cultures). Middle, Total GluA2-ir of individual processes for proximal vs distal puncta (paired Wilcoxon signed rank test, P < 0.005). Right, both RI in slices (see Fig. 1) and GluA2-ir showed similar increase with the dendritic distance. See also Figure S8.

Figure 7. Spontaneous action potentials regulate the expression of CPEB3 via activation of PKC

A. Quantification of PKC-ir. Left, confocal images of a GABAergic interneuron (GFP). Dashed lines outline each compartment. Right, line scans showing the levels of PKC-ir, GFP and RedDot2 (RD2 for nuclear staining) across the soma. B. Translocation of PKC to the cytoplasm after TTX treatment. Top: PKC-ir in GFP neurons treated with TTX or PKC modulators. Bottom: distribution of PKC-ir in each compartment. C. Left, mean membrane and cytoplasmic PKC-ir levels. Right, the PKC _memb/PKC _cyto ratio. TTX treatment reduced membrane staining and increased cytoplasmic CPEB3-ir, thereby lowering the PKC _memb/PKC _cyto ratio. These changes were reversed by co-application of PMA and TTX. Chelerythrine (CHE) or bisindolylmaleimide (BIS), PKC inhibitors, also reduced cytoplasmic translocation of PKC and the PKC _memb/PKC _cyto ratio. D. Effect of the same treatments on CPEB3-ir level (> 3 cultures). Scale bars: 5 (A, B). C (ANOVA, P < 0.002), D (repeated measures ANOVA, P < 0.02), post-hoc test, *, P < 0.05; **, P < 0.01 ***, P < 0.005. See also Figure S7.