Differential trafficking of AMPA and NMDA receptors during long-term potentiation in awake adult animals

Abstract

Despite a wealth of evidence in vitro that AMPA receptors are inserted into the postsynaptic membrane during long-term potentiation (LTP), it remains unclear whether this occurs in vivo at physiological concentrations of receptors. To address the issue of whether native AMPA or NMDA receptors undergo such trafficking during LTP in the adult brain, we examined the synaptic and surface expression of glutamate receptor subunits during the early induction phase of LTP in the dentate gyrus of awake adult rats. Induction of LTP was accompanied by a rapid NMDA receptor-dependent increase in surface expression of glutamate receptor 1-3 (GluR1-3) subunits. However, in the postsynaptic density fraction only GluR1 accumulated. GluR2/3-containing AMPA receptors, in contrast, were targeted exclusively to extrasynaptic sites in a protein synthesis-dependent manner. NMDA receptor subunits exhibited a delayed accumulation, both at the membrane surface and in postsynaptic densities, that was dependent on protein synthesis. These data suggest that trafficking of native GluR1-containing AMPA receptors to synapses is important for early-phase LTP in awake adult animals, and that this increase is followed homeostatically by a protein synthesis-dependent trafficking of NMDA receptors.

Figure 1.

Characterization of membrane subfractions prepared from dentate gyrus synaptoneurosomes. a, b, Western blot and densitometric quantitation showing that biotin-neutravidin precipitation could recover a proportional amount of GluR1 (106 kDa) subunits from increasing amounts (50–300 μg) of synaptoneurosomal protein (a) and equivalent amounts of GluR1 subunit (b, top) were recovered from each of paired matched hemispheres (1:Left and 2:Right) when equivalent amounts (200 μg) of synaptoneurosomal protein were used in the biotin-neutravidin precipitation. “Neurosome” is 20 μg of synaptoneurosomal protein (without precipitation), and “biotin” is 20% of the total yield of protein eluted from the biotin-neutravidin beads. b, Bottom, No calnexin (90 kDa) or tubulin (50 kDa) were recovered in the biotin-neutravidin precipitated fractions, but were readily detected in the synaptoneurosome fractions. c, Western blot analysis demonstrating that the Triton-X insoluble fraction (Insol.) recovered from synaptoneurosomes contained GluR1 subunits and the postsynaptic density marker protein, PSD95, but not the extrasynaptic receptor GABA_BR (105 kDa) or tubulin. The Triton-X soluble fraction (Sol.) in contrast contained just detectable levels of GluR1 as well as readily detectable amounts of GABA_BR and tubulin.

Figure 2.

AMPAR subunit expression after the induction of LTP in the dentate gyrus of awake rats. a, Induction of LTP by HFS in a group of 16 rats used for Western blot analyses at the 20 min time-point. Data are mean ± SEM fEPSP slopes are expressed as a percentage of the baseline recordings made 5 min before HFS (bar). Waveforms are averages of 20 sweeps taken at the times indicated for a single animal. Calibration: 5 mV, 5 ms. Western blot analysis was used to assess the relative levels of AMPAR subunits in cell-surface and synaptic extracts. b, Sample Western blots showing GluR1–3 levels in cell-surface extracts isolated 20 min post-HFS from matched control (C) and tetanized (T) hemispheres from a single animal. c, Histogram of average optical densities (ODs) showing a rapid increase in cell-surface expression of GluR1–3 subunits at various times post-HFS (n = 6–14). d, Histogram of average ODs showing GluR1–3 subunit levels in synaptoneurosomes at various times post-HFS. GluR subunits did not increase in synaptoneurosomes 0 or 20 min post-HFS (n values = 4–8), apart from GluR1 which showed a small increase at 20 min post-HFS (n = 11) and GluR3 which showed a small decrease 4 h post-HFS. e, Histogram of average ODs showing no increase in GluR1–3 subunit levels in whole cell extracts isolated 20 min and 4 h post-HFS (n values = 4–6). A modest reduction in GluR3 levels was found 4 h post-HFS. All data in this and all following figures reflect the relative percentage difference in optical density between tetanized and control hemispheres on the same Western blot. Asterisks in all figures signify a significant difference by paired t test, p ≤ 0.05, from the control hemisphere.

Figure 3.

NMDAR subunit expression at the cell-surface of synaptoneurosomes after LTP in awake rats. There was a significant increase in NR1 and NR2B at 20 min and a trend toward an increase in NR2A levels, but no significant changes at 0 min post-HFS, relative to the control hemisphere (n = 4–9). By 4 h post-HFS, only NR2B remained elevated. Sample Western blots comparing control and tetanized hemispheres, as per Figure 2, are shown for the NMDAR subunits, 20 min post-HFS.

Figure 4.

NMDAR- and protein synthesis-dependency of glutamate receptor subunit expression at the cell-surface. a, The NMDAR antagonist CPP completely blocked LTP induction (n = 8). The protein synthesis inhibitor cycloheximide (CXM) did not affect LTP over the first 20 min of expression (n = 11). Waveforms are as in Figure 2. b, CPP completely blocked GluR1–3 increases at the cell-surface, 20 min post-HFS (n = 4–6). Cycloheximide blocked the increases in GluR2 and GluR3 but not GluR1 (n = 4–8). c, Both CPP and cycloheximide blocked NR1 and NR2B increases at the cell-surface of synaptoneurosomes 20 min post-HFS (n = 3–7). NR2A levels were not determined. Sample Western blots show glutamate receptor levels in cell-surface extracts isolated 20 min post-HFS from matched control (C) and tetanized (T) hemispheres from a single animal.

Figure 5.

LTP induction at perforant path synapses in awake animals results in an increase in zif/268 but not nr2b mRNA levels, as determined by quantitative PCR. A robust 11-fold increase in zif/268 mRNA levels (p < 0.0001) was detected 20 min post-HFS in experimental animals (LTP), whereas no significant change was detected for nr2b. No difference in zif/268 or nr2b mRNA levels was detected between hemispheres of unstimulated, control rats.

Figure 6.

Glutamate receptor subunit expression in a PSD-enriched fraction from synaptoneurosomes isolated after LTP in awake rats. a, b, Average results for AMPAR subunits (a) and NMDAR subunits (b). GluR1 showed a significant increase at 0 min post-HFS (n = 6). GluR1 and NR1 showed significant increases 20 min post-HFS (n = 8). No increase was observed for GluR2, GluR3, NR2A or NR2B (n = 4–8) at 0 or 20 min. Sample Western blot, as per Figure 2, shows glutamate subunit expression 20 min post-HFS for control versus tetanized hemispheres.