Functional compartmentalization of endosomal trafficking for the synaptic delivery of AMPA receptors during long-term potentiation

Abstract

Endosomal membrane trafficking in dendritic spines is important for proper synaptic function and plasticity. However, little is known about the molecular identity and functional compartmentalization of the membrane trafficking machinery operating at the postsynaptic terminal. Here we report that the transport of AMPA-type glutamate receptors into synapses occurs in two discrete steps, and we identify the specific endosomal functions that control this process during long-term potentiation. We found that Rab11-dependent endosomes translocate AMPA receptors from the dendritic shaft into spines. Subsequently, an additional endosomal trafficking step, controlled by Rab8, drives receptor insertion into the synaptic membrane. Separate from this receptor delivery route, we show that Rab4 mediates a constitutive endosomal recycling within the spine. This Rab4-dependent cycling is critical for maintaining spine size but does not influence receptor transport. Therefore, our data reveal a highly compartmentalized endosomal network within the spine and identify the molecular components and functional organization of the membrane organelles that mediate AMPA receptor synaptic delivery during plasticity.

Figure 1.

Rab8 and Rab11 are strictly required for LTP. a, Organotypic slice cultures were infected with viruses expressing Rab4dn (green), Rab11dn (red), Rab8dn (blue), or Rab5dn (gray). Whole-cell recordings were established from neurons expressing the Rab protein dominant-negative or uninfected cells. LTP was induced by pairing 3 Hz presynaptic stimulation with 0 mV postsynaptic depolarization (black arrow), as described in Materials and Methods. Inset, Sample trace of evoked AMPA receptor-mediated synaptic responses recorded at −60 mV before pairing (thin line) and 30 min after pairing (thick line) from control (uninfected) neurons or Rabdn-infected cells, as indicated. b, Normalized average potentiation of AMPAR-mediated responses collected between 30 and 35 min after LTP induction in paired (LTP) and unpaired (Control) pathways from neurons expressing different Rabdn constructs or uninfected, as indicated. Error bars represent SEM in all figures. n represents number of cells. c, d, Average values of input resistance (c) and holding current (d) from the LTP recordings shown in a and b.

Figure 2.

Rab protein requirement for the regulated trafficking of GluR1 into spines. a, Representative confocal images of a spine and the adjacent dendritic shaft from neurons transfected with GFP–GluR1 and tCaMKII plus RFP (top) or Rabdn–RFP constructs (bottom 3 panels), as indicated. GFP fluorescence signal (green) represents GluR1 partition between spine and dendrite. Scale bars, 1 μm. b, Average spine/dendrite ratio of GFP fluorescence intensities across spine–dendrite pairs from neurons transfected with GFP–GluR1 and tCaMKII plus RFP or Rabdn–RFP constructs, as indicated. GFP–GluR1 accumulation was calculated as described in Materials and Methods. n represents the number of spine–dendrite pairs from six different neurons per condition. c, Cumulative probability distributions of spine/dendrite ratios from the same data plotted in b.

Figure 3.

Rab dominant-negative effect on spine size and length. a, Representative confocal images of spines and adjacent dendritic shafts from neurons transfected with GFP and RFP (top) or with GFP and RFP–Rabdn constructs (bottom 4 panels), as indicated. GFP fluorescence signal (green) represents total GFP distribution to indicate volume of the spine and adjacent dendritic shaft. Scale bars, 1 μm. b, Average spine/dendrite ratio of GFP fluorescence was calculated and analyzed as described for Figure 2. c, Average spine length was calculated as the distance between the peak of GFP fluorescence intensity in the spine and in the adjacent dendritic shaft from neurons expressing GFP and different RFP-tagged constructs, as indicated. n represents number of spine–dendrite pairs from five to seven neurons per condition.

Figure 4.

Dominant-negative Rab proteins do not alter basal levels of GluR1 phosphorylation. a, Western blot representative example of the effect of Rabdn expression on GluR1 phosphorylation at Ser845 and Ser831. Protein extracts were prepared from microdissected CA1 regions expressing the indicated Rabdn protein fused to GFP. b, c, Quantification of GluR1 phospho-serine 845 (P-S845; b) or phospho-serine 831 (P-S831; c) relative to total GluR1. Values are normalized to the ratio of phospho/total GluR1 from uninfected slices processed in parallel. n represents number of independent experiments.