Differential redistribution of native AMPA receptor complexes following LTD induction in acute hippocampal slices

Abstract

AMPAR trafficking is crucial for the expression of certain forms of synaptic plasticity. Here, using surface biotinylation of hippocampal slices and subsequent synaptosome isolation we assessed AMPAR surface expression in synaptosomes following NMDA-evoked long-term depression (NMDA-LTD). Surface levels of GluR1, GluR2 and GluR3 in synaptosomes were markedly reduced 90 min after NMDA-LTD induction. Consistent with endocytosis and degradation, whole-cell surface and total expression levels of GluR2 and GluR3 were also reduced. In contrast, whole-cell surface levels of GluR1 were unaltered at 90 min suggesting that AMPARs with different subunit composition are redistributed to different non-synaptic compartments following LTD induction in acute hippocampal slices.

Fig. 1

AMPA receptor surface expression in hippocampal slices. Acute hippocampal slices from P21 to 23 male Wistar rats were biotinylated and the surface expression of each subunit was determined by quantitative immunoblotting (see Section 2). (A) Representative blots for GluR1, GluR2, GluR3 and β-tubulin. (B) Proportion of AMPAR subunits that are surface expressed (mean ± SEM of three independent experiments).

Fig. 2

Characterisation of synaptosome preparation. Acute hippocampal slices from P21 to 23 male Wistar rats were biotinylated, synaptosomes were isolated and the surface expression of each subunit was determined by quantitative immunoblotting (see Section 2). (A) Synaptotagmin, PSD-95 and GluR1 were enriched in the synaptosome fraction compared to the whole-cell membrane fraction. (B) GluR1 and PSD-95 but not synaptotagmin were enriched in the PSD fraction compared to the synaptosome fraction. (C) Representative blots for GluR1, GluR2, GluR3 and β-actin. (D) Proportion of AMPAR subunits that are surface expressed in synaptosomes (mean ± SEM of three independent experiments).

Fig. 3

Down-regulation of synaptosomal AMPARs 90 min following NMDA-LTD induction. (A) Example traces at t₁ = −5, t₂ = 20, t₃ = 30 and t₄ = 90 min (labelled 1–4). (B, C) NMDA application (5 min, 20 μM) causes a prolonged reduction in the fEPSP slope. No long-term changes in fEPSP fibre volley amplitude were detected. Data represent the mean ± SEM of five independent experiments. (D–F) In the absence of NMDA application no change was observed in the fEPSP slope or fibre volley amplitude. Data represent the mean ± SEM of three independent experiments. Example traces (D) represent t₁ = −5, t₂ = 20, t₃ = 30 and t₄ = 90 min. Calibration bars 10 ms, 0.5 mV. (G) Representative blots for surface-synaptic and total-synaptic GluR1, GluR2 and GluR3. There were significant decreases in the levels of surface-synaptosomal (H) and total-synaptosomal (I) GluR1, GluR2 and GluR3 in the NMDA-treated slices compared to control. No change was observed in the levels of surface-synaptosomal N-cadherin or total-synaptosomal β-actin. Data represent the mean ± SEM of at least four independent experiments (*P < 0.05).

Fig. 4

Subunit specific reduction in whole-cell surface and total expression 90 min following NMDA-LTD induction. (A, D) Representative blots for whole-cell surface and total AMPAR subunits at 15 and 90 min. (B, C) At 90 min there were significant decreases in the surface and total levels of GluR2 and GluR3 in the NMDA-treated slices compared to control. No significant differences were detected for GluR1 at 90 min. At 15 min there were significant decreases in the levels of surface, but not total, GluR1, GluR2 and GluR3 in the NMDA-treated slices compared to control (D–F). No differences were detected for β-tubulin at 15 or 90 min. Data represent the mean ± SEM of at least three independent experiments (*P < 0.05).