PICK1 regulates incorporation of calcium-permeable AMPA receptors during cortical synaptic strengthening

Abstract

While AMPA-type glutamate receptors (AMPARs) found at principal neuron excitatory synapses typically contain the GluR2 subunit, several forms of behavioral experience have been linked to the de novo synaptic insertion of calcium-permeable (CP) AMPARs, defined by their lack of GluR2. In particular, whisker experience drives synaptic potentiation as well as the incorporation of CP-AMPARs in the neocortex. Previous studies implicate PICK1 (protein interacting with C kinase-1) in activity-dependent internalization of GluR2, suggesting one potential mechanism leading to the subsequent accumulation of synaptic CP-AMPARs and increased synaptic strength. Here we test this hypothesis by using a whisker stimulation paradigm in PICK1 knock-out mice. We demonstrate that PICK1 facilitates the surface expression of CP-AMPARs and is indispensable for their experience-dependent synaptic insertion. However, the failure to incorporate CP-AMPARs in PICK1 knock-outs does not preclude sensory-induced enhancement of synaptic currents. Our results indicate that synaptic strengthening in the early postnatal cortex does not require PICK1 or the addition of GluR2-lacking AMPARs. Instead, PICK1 permits changes in AMPAR subunit composition to occur in conjunction with synaptic potentiation.

Figure 1.

Single-row experience drives PICK1-dependent increase in AMPAR-EPSC rectification at layer 4–2/3 synapses. A, Plasticity was induced in PICK1 knock-out mice by single-row experience, which entailed the removal of all but a unilateral D row of whiskers for 48 h. Middle, Right, Stimulus and recording electrode configuration for examination of cortical layer 4–2/3 EPSCs in spared (middle, D column) and deprived (right, C and E columns, pooled) barrel columns after single-row experience. B, Overlay of layer 4–2/3 AMPAR-EPSCs evoked at the following holding potentials in the presence of internal spermine (100 μm): −70, −60, −40, −20, 0, +20, +40, +50 mV. Responses have been normalized to amplitude at −70 mV. C, D, Normalized amplitude of AMPAR-EPSCs as a function of holding potential (I–V plot) for PICK1+/+ (C) and PICK1−/− (D) mice. E, Mean rectification index for control and single-row experience mice. *p < 0.001 by ANOVA followed by Bonferroni post hoc comparison.

Figure 2.

Sensory-driven enhancement of AMPA:NMDA ratio does not require PICK1. A, Overlay of EPSCs evoked at −70, 0, and +40 mV. B, Mean AMPA:NMDA ratio for EPSCs. *p < 0.001 by ANOVA followed by Bonferroni post hoc comparison.

Figure 3.

Quantal AMPAR-EPSCs are potentiated by single-row experience independent of PICK1 or CP-AMPARs. A, Mean AMPAR miniature EPSCs evoked by stimulation in the presence of Sr²⁺ (Sr²⁺-mEPSCs) at layer 4–2/3 inputs. B, Mean amplitude of Sr²⁺-mEPSCs. C, Cumulative distribution of Sr²⁺-mEPSC amplitudes. D, Overlay of Sr²⁺-mEPSCs, illustrating faster decay of AMPAR currents after single-row experience in spared barrel columns of PICK1+/+ but not PICK1−/− mice. E, Mean rise time (20–80%) of Sr²⁺-mEPSC s. F, Mean τ_decay of Sr²⁺-mEPSC s. B, F, *p < 0.01 by ANOVA followed by Bonferroni post hoc comparison. C, *p < 0.0001, Kolmogorov–Smirnov test comparing spared to control.

Figure 4.

PICK1 facilitates the surface expression of GluR2-lacking AMPARs. A, Example experiment in which acute barrel cortex slices from PICK1−/− mice and their PICK1+/+ littermates were subjected to surface biotinylation assay. The relative level of surface and total AMPARs was assessed by Western blot using specific antibodies against GluR1 and GluR2 subunits after first normalizing to α-tubulin (see Materials and Methods). B, Total AMPAR subunit levels (input) and surface: total (input) ratios of GluR1 and GluR2 in PICK1−/− mice, expressed as a percentage of wild-type littermates. C, Example immunodepletion of GluR2-containing AMPARs from cortical lysates to examine the fraction of GluR1 that is unassociated with GluR2. Slices were biotinylated to enable subsequent affinity purification of surface AMPARs. I, Input; T, total unbound fraction; S, surface unbound fraction following two rounds of GluR2 immunodepletion. Surface unbound fraction was loaded at 30× input concentration. D, E, AMPAR levels in the total unbound (D) and surface unbound (E) fractions following GluR2 immunodepletion (refer to Materials and Methods). *p < 0.05, Student's two-tailed t test comparing PICK1+/+ and PICK1−/− mice.