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Originally published In Press as doi:10.1074/jbc.M404982200 on August 5, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43870-43878, October 15, 2004
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Local Control of AMPA Receptor Trafficking at the Postsynaptic Terminal by a Small GTPase of the Rab Family*

Nashaat Z. Gerges, Donald S. Backos, and José A. Esteban{ddagger}

From the Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632

Received for publication, May 5, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The delivery of neurotransmitter receptors into the synaptic membrane is essential for synaptic function and plasticity. However, the molecular mechanisms of these specialized trafficking events and their integration with the intracellular membrane transport machinery are virtually unknown. Here, we have investigated the role of the Rab family of membrane sorting proteins in the late stages of receptor trafficking into the postsynaptic membrane. We have identified Rab8, a vesicular transport protein associated with trans-Golgi network membranes, as a critical component of the cellular machinery that delivers AMPA-type glutamatergic receptors (AMPARs) into synapses. Using electron microscopic techniques, we have found that Rab8 is localized in close proximity to the synaptic membrane, including the postsynaptic density. Electrophysiological studies indicated that Rab8 is necessary for the synaptic delivery of AMPARs during plasticity (long-term potentiation) and during constitutive receptor cycling. In addition, Rab8 is required for AMPAR delivery into the spine surface, but not for receptor transport from the dendritic shaft into the spine compartment or for delivery into the dendritic surface. Therefore, Rab8 specifically drives the local delivery of AMPARs into synapses. These results demonstrate a new role for the cellular secretory machinery in the control of synaptic function and plasticity directly at the postsynaptic membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synaptic plasticity is critical for the establishment and maturation of functional neuronal circuits in the brain and is widely thought of as the cellular process responsible for learning and memory. An important mechanism controlling synaptic maturation and remodeling is the targeting and delivery of neurotransmitter receptors into synapses. However, very little is known of the exocytic processes that sort neurotransmitter receptors into the postsynaptic membrane. In addition, it remains to be determined how these specialized trafficking events are integrated with the intracellular membrane transport machinery. In fact, most of our current knowledge on membrane trafficking at the synapse derives from studies on neurotransmitter vesicle fusion at the presynaptic terminal (reviewed in Refs. 1 and 2).

The AMPA-type glutamatergic receptors (AMPARs)1 are highly dynamic components of excitatory synapses. They can traffic in and out of the synaptic membrane constitutively or in an activity-dependent manner, and this regulated trafficking is known to contribute to synaptic plasticity during brain development and in adulthood (3-6). AMPARs are heterooligomeric molecules composed of different combinations of glutamate receptor (GluR) 1 to GluR4 subunits (7). In hippocampus, AMPARs containing GluR1 or GluR4 subunits are delivered to synapses in an activity-dependent manner upon N-methyl-D-aspartate receptor (NMDA) receptor activation, leading to long-lasting synaptic potentiation (8-11). In contrast, AMPARs containing only GluR2 and GluR3 subunits cycle continuously in and out of synapses in a manner largely independent of synaptic activity (10, 12), but dependent on N-ethylmaleimide-sensitive factor (13-15) and Hsp90 (16) function. These two distinct trafficking routes have been coined as regulated and constitutive pathways, respectively (17). However, the cellular and molecular mechanisms mediating membrane transport in these pathways are largely unknown.

Members of the Rab family of small GTPases are important regulators of intracellular membrane sorting in eukaryotes. In particular, they are proposed to mediate membrane transport specificity (18-20). Rab proteins are likely to be important for neuronal function because alterations in Rab protein regulation can lead to mental retardation in humans (21). Rab3 is a presynaptic member of this family (22). It controls the fusion of neurotransmitter vesicles with the plasma membrane (23, 24) and mediates some presynaptic forms of synaptic plasticity (25, 26). In contrast, the potential role of Rab proteins in neurotransmitter receptor targeting and synaptic function at the postsynaptic terminal has never been tested.

Besides Rab3, three members of the Rab family are involved in exocytic delivery into the plasma membrane: Rab4, Rab8, and Rab11. In neurons, Rab8 has an exclusive somatodendritic distribution (27). In epithelial (28, 29) and photoreceptor (30, 31) cells, Rab8 mediates the transport of trans-Golgi network-derived membranes into specific compartments of the plasma membrane. Rab4 is involved in the recycling pathway from early endosomes back into the plasma membrane, whereas Rab11 mediates membrane transport from late endosomes and trans-Golgi network (32-39). These three Rab proteins act on late stages of the exocytic trafficking into the plasma membrane and therefore are attractive candidates to mediate receptor targeting into the postsynaptic membrane.

In this study, we have explored the role of these exocytic Rab proteins in synaptic function and plasticity and, in particular, in the late trafficking events that deliver AMPARs into synapses. Using a combination of molecular biology, electrophysiology, and imaging techniques on organotypic hippocampal slice cultures, we have found that Rab8, but not Rab4 or Rab11, is required for the late stages of AMPAR synaptic delivery into the postsynaptic membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs of Recombinant Proteins and Expression—Rab8a, Rab4a, and Rab11a coding sequences were cloned by PCR from a commercial rat brain cDNA preparation (catalog no. 7150; Clontech). The rat Rab8a sequence that we cloned has 96% and 97% identity to the mouse and human sequences, respectively. The reported Rab8a rat sequence in the NCBI data base (accession numbers M83675 [GenBank] and NM_053998 [GenBank] ) has a significant N-terminal truncation (83 amino acids) with respect to the mouse and human sequences and with respect to the rat sequence we cloned. This is probably due to a sequencing error in the rat sequence submitted originally. Our cloned Rab4a sequence was identical to the reported rat Rab4a sequence in the NCBI data base (accession number P05714 [GenBank] ) with the exception of two differences: Q47S and T75R. These two amino acids in our sequence (Ser47) and (Arg75) are conserved in the mouse and human sequences reported in the NCBI data base (accession numbers NP_033029 [GenBank] and NP_004569 [GenBank] , respectively). Therefore, these two differences probably correspond to sequencing errors in the previously reported rat sequence. Our cloned Rab11a sequence was identical to the reported one in the NCBI data base (accession number NM_031152 [GenBank] ). Rab8a, Rab4a, and Rab11a coding sequences were cloned as fusion proteins downstream from enhanced green fluorescence protein using the pEGFP-C1 plasmid (catalog no. 6084; Clontech). The dominant negative mutants (GDP-bound form) were generated by PCR introducing a single-amino acid substitution described previously (T22N for Rab8dn, S22N for Rab4dn, and S25N for Rab11dn). The RFP fusion construct of Rab8dn was generated with a red fluorescence protein variant (tdimer2 (12, 40)) generously provided by Dr. Roger Tsien (University of California San Diego). All constructs were recloned into pSinRep5 for Sindbis virus preparation (41). Hippocampal slices are prepared from young rats (postnatal day 5 to 7) and placed in culture on semiporous membranes (42). After 4-7 days in culture, the recombinant gene is delivered into the slices. For the experiments shown in Figs. 3 (A and B), 4, 5 (B and C), and 7, we used the biolistic delivery method (43), which allowed us to co-express several proteins with plasmids bearing the cytomegalovirus promoter. For the rest of the experiments, we use the Sindbis virus expression system (44). This is a replication-deficient, low-toxicity, neurotropic virus, allowing the expression of recombinant proteins exclusively in neurons by injecting the viral solution extracellularly in the desired area of a hippocampal slice (41). The recombinant proteins were expressed for 36 h when AMPA receptor subunits were expressed (Figs. 3, A and B, 4, and 7) or for 15 h in the rest of the cases. Neurons remain morphologically and electrophysiologically intact during these expression times. All biosafety procedures and animal care protocols were approved by the University of Michigan.



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  		FIG. 3.
Immunohistochemical localization of surface AMPA receptors in slices expressing Rab8dn. A, confocal imaging of GluR2-GFP-transfected neuron. GFP fluorescence signal (top panel) and surface anti-GFP immunoreactivity of the same neuron in nonpermeabilized conditions (bottom panel). B, confocal imaging for a neuron co-transfected with GluR2-GFP and Rab8dn-GFP. GFP signal and surface anti-GFP immunoreactivity are as described in A. C, control neurons expressing Rab8dn alone. Negative surface immunostaining (bottom panel) is expected, given the intracellular distribution of Rab8-GFP.

 



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  		FIG. 4.
Rab8dn decreases AMPAR surface delivery locally at spines. A, representative confocal image of a neuron co-transfected with GluR2-GFP and Rab8dn-RFP. Left panel, GFP fluorescence signal showing total GluR2 receptor distribution. Middle panel, surface GluR2-GFP receptors assayed by anti-GFP immunoreactivity of the same neuron in nonpermeabilized conditions. Right panel, RFP fluorescence signal from the same neuron indicating expression of the co-transfected Rab8dn-RFP (neurons transfected only with GluR2-GFP do not show any RFP signal above background; data not shown). Scale bar, 30 µm. B, representative line plot analysis of total (first and third panels, GFP signal) and surface (second and fourth panels, Cy5 signal) receptor across a spine and the adjacent dendritic shaft of GluR2-transfected or GluR2 + Rab8dn-transfected neurons, as indicated. Values for surface and total receptors were taken from the fluorescence intensity peaks after background subtraction (dashed line). Scale bar, 2 µm. C, surface ratio from pairs of spines and dendrites in GluR2-transfected neurons (left panel) or in GluR2 + Rab8dn-transfected neurons (middle panel). Surface ratios for spines and dendrites were calculated by dividing the corresponding background-subtracted Cy5 and GFP values. Surface ratios are normalized by the mean dendrite value (0.31 ± 0.05 for GluR2 alone and 0.31 ± 0.04 for GluR2 + Rab8dn). Cumulative probabilities (right panel) of spine/dendrite ratios show a significant difference between the surface ratio distribution of GluR2-transfected and GluR2 + Rab8dn-transfected neurons. For comparison, dashed lines indicate that 50% of the spine-dendrite pairs have less surface ratio in the spine than in the dendrite for cells transfected with GluR2 alone (black line, cumulative probability = 0.5 for spine/dendrite = 1). In contrast, 75% of the spine-dendrite pairs have less surface ratio in the spine than in the dendrite for cells co-transfected with Rab8dn (red line, cumulative probability = 0.75 for spine/dendrite = 1). D, total receptor amount (GFP signal) in spines and dendrites are calculated in GluR2-transfected neurons (left panel) and in GluR2 + Rab8dn-transfected neurons (middle panel). Values are normalized by the mean obtained at dendrites (50 ± 4 for GluR2 alone and 54 ± 3 for GluR2 + Rab8dn). The cumulative probability (right panel) indicates that Rab8dn does not significantly change the total receptor distribution in spines versus dendrites.

 



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  		FIG. 5.
Rab8 is necessary for both the constitutive and regulated delivery of AMPA receptors into synapses. A-C, top, sample trace of evoked AMPA receptor-mediated synaptic responses recorded at -60 mV from uninfected/nontransfected and infected/transfected cells. Scale bars, 20 pA and 10 ms. A, bottom, Rab8dn significantly reduced AMPA transmission in the presence of high Mg2+ (12 mM MgCl2). Average current amplitude at -60 mV from uninfected and infected cells was 46.9 ± 9.9 and 26.7 ± 4.5 pA, respectively. n represents the number of pathways from cell pairs. p = 0.03, according to the Wilcoxon test. B, bottom, expression of t-CaMKII significantly increases AMPAR-mediated transmission. Nontransfected, 32.5 ± 6.1 pA; transfected with t-CaMKII, 81.5 ± 14.4 pA. p = 0.001, according to the Wilcoxon test; n represents the number of the pathways from cell pairs. C, bottom, Rab8dn blocked t-CaMKII-induced potentiation of AMPA responses. Nontransfected, 25.0 ± 4.2 pA; transfected with t-CaMKII + Rab8dn, 19.7 ± 3.8 pA. D, Rab8dn does not change the phosphorylation levels of CaMKII. Left panels, Western blot analysis showing the P-Thr286 {alpha}CaMKII/total {alpha}CaMKII ratios in Rab8dn-infected slices and uninfected slices. Each lane in the Western blot is the result of pooling together extracts from three dissected CA1 regions. Right panel, quantification by densitometric scanning of four independent experiments as the one shown in the left.

 



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  		FIG. 7.
Rab8 is necessary for the constitutive delivery of GluR2 and for the delivery of GluR1 triggered by t-CaMKII or by PSD95 into synapses. A and B, bottom, rectification values were calculated as the ratio between the amplitude of the synaptic response at -60 mV over the amplitude at +40 mV. Endogenous receptors conduct current at -60 and +40 mV, whereas recombinant receptors conduct only at negative membrane potentials. Therefore, delivery of the recombinant homomeric receptors is accompanied by an increase in the rectification value (a decrease in the outward current, depicted by the arrowhead in A, with respect to the inward current). A, average rectification values were as follows: control (uninfected), 1.9 ± 0.2; GluR2-(R586Q), 3.3 ± 0.2; GluR2-(R586Q) + Rab8dn, 2.1 ± 0.3; GluR2-(R586Q) + Rab8wt, 3.3 ± 0.2; and Rab8dn, 2.3 ± 0.3. B, average rectification values were as follows: GluR1 + t-CaMKII, 3.9 ± 0.7; GluR1 + t-CaMKII and Rab8dn, 1.8 ± 0.2; GluR1 + PSD95, 3.5 ± 0.5; GluR1 + PSD95 and Rab8dn, 2.0 ± 0.2; and GluR1 + PSD95 and Rab8wt, 3.5 ± 0.4. n represents the number of pathways; p is the probability value according to Student's t test. A and B, top, sample traces of evoked AMPAR-mediated synaptic responses recorded at -60 and +40 mV from control or transfected cells as indicated. Scale bars, 20 pA and 20 ms.

 
Electrophysiology—Simultaneous double whole-cell recordings were obtained from nearby pairs of infected and uninfected CA1 pyramidal neurons under visual guidance using fluorescence and transmitted light illumination. The recording chamber was perfused with 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl2, 4 mM MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose, 0.1 mM picrotoxin, 10 µM bicuculline, and 2 µM 2-chloroadenosine, at pH 7.4, and gassed with 5% CO2/95% O2. Patch recording pipettes (3-6 megaohms) were filled with 115 mM cesium methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, and 0.6 mM EGTA, at pH 7.25. In the rectification experiments (i.e. Fig. 7), 0.1 mM spermine was added. Voltage-clamp whole-cell recordings were carried out with multiclamp 700A amplifiers (Axon Instruments, Union City, CA). Synaptic responses were evoked with two bipolar electrodes with single voltage pulses (200 µs, up to 20 V). The stimulating electrodes were placed over Schaffer collateral fibers between 300 and 500 µm from the recorded cells. Because only CA1 cells and not CA3 cells are infected, this configuration ensures that recombinant proteins are always expressed exclusively in postsynaptic cells. Synaptic AMPA receptor-mediated responses were measured at -60 mV, and NMDA receptor-mediated responses were measured at +40 mV, and they were averaged over 50-100 trials. In the rectification experiment (Fig. 7), NMDA receptor-mediated responses were blocked pharmacologically using 0.1 mM DL-2-amino-5-phosphonovaleric acid. Synaptic AMPA receptor-mediated responses were measured at -60 and +40 mV, and the ratio of these responses was used as an index of rectification. Long-term potentiation (LTP) experiments were carried out as described previously (16), by pairing 0 mV postsynaptic depolarization with 3 Hz presynaptic stimulation (300 pulses).

Biochemistry—Hippocampal extracts were prepared in homogenization buffer containing protease inhibitors (10 mM HEPES, 500 mM NaCl, 10 mM EDTA, 4 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml chymostatin, 2 µg/ml leupeptin, 2 µg/ml antipain, 2 µg/ml pepstatin and 1% Triton X-100) as described previously (8). Expression of Rab proteins was analyzed by Western blot with anti-Rab4 (BD Biosciences), anti-Rab8 (Pharmingen), and anti-Rab11 (Zymed Laboratories) antibodies. Phosphorylation of {alpha} calmodulin-dependent protein kinase (CaMK) II at Thr286 was analyzed with phospho-specific and regular anti-{alpha}CaMKII antibodies (Upstate Biotechnology) using the homogenization buffer described above supplemented with phosphatase inhibitors (10 mM NaF, 1 µM microcystin LR, and 0.5 µM calyculin A).

Fluorescence Immunohistochemistry—Immunohistochemical detection of recombinant AMPA receptors was carried out with anti-GFP mouse antibody (Roche Applied Science). Fluorescence labeling was achieved with anti-mouse biotinylated secondary antibody (Sigma) and streptavidin tagged with either AlexaFluor 594 (Molecular Probes; Fig. 3) or Cy5 (Amersham Biosciences; Fig. 4). Detergents were omitted in all incubations to evaluate surface expression. Images were taken using Olympus FV 500 confocal microscopy. A x60 lens with water immersion interface was used. FluoView software was used for acquiring the images. ImageJ was used for three-dimensional reconstruction and quantification of fluorescence intensities. Analysis of surface immunostaining at spines and dendrites was carried out as follows. Line plots of fluorescence intensity were generated across spine heads and the adjacent dendritic shafts. Fluorescence intensity at each compartment was quantified from the peaks corresponding to the spine and the dendrite after background subtraction (Fig. 4B). Then, surface ratios were calculated as the ratio between the GFP signal (total receptor) and the Cy5 signal (surface receptor).

Immunogold Electron Microscopy—Hippocampal slices were fixed and processed for osmium-free post-embedding immunogold labeling, essentially as described previously (45). Rab8 was labeled with anti-Rab8 antibody (Pharmingen) and an anti-mouse antibody coupled to 6-nm gold particles (Electron Microscopy Sciences). Electron micrographs were obtained with a Philips CM-100 transmission electron microscope and a Kodak 1.6 Megaplus digital camera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Role of Rab4, Rab8, and Rab11 in AMPA Receptor-mediated Synaptic Transmission—As a first step to evaluate the role of these three exocytic Rab proteins in synaptic transmission, we blocked the function of these proteins individually by expressing the corresponding dominant negative (Rab-dn) forms as GFP fusion proteins in organotypic hippocampal slice cultures (see "Experimental Procedures"). Point mutations known to confer dominant negative (GDP-bound) phenotypes to these proteins have been employed in multiple occasions: Rab4-(S22N) (46-48), Rab8-(T22N) (31, 49-51), and Rab11-(S25N) (36-39, 52-55). Also, GFP-Rab fusions have been used successfully in numerous studies to study membrane trafficking in living cells (31, 33, 56-58). Rab4dn-, Rab8dn-, and Rab11dn-GFP were expressed efficiently in hippocampal slice cultures, as assayed by Western blot analysis (see the left panels in Fig. 1).



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  		FIG. 1.
Rab8 is necessary for AMPA receptor delivery into synapses. A-D, left panels, Western blot analysis of the expression of recombinant Rab-GFP in hippocampal slices using the Sindbis virus method. Large arrowheads indicate recombinant Rab-GFP proteins (about 52 KDa); small arrows indicate endogenous Rab proteins (about 25 KDa). Sample trace of evoked AMPAR- and NMDAR-mediated synaptic responses recorded at -60 and +40 mV, respectively, from uninfected and infected cells. Scale bars, 20 pA and 40 ms. Left graphs, average AMPAR-mediated current amplitude (i.e. the peak of the response recorded at -60 mV) from infected (Inf.) neurons expressing Rab8dn (A), Rab4dn (B), Rab11dn (C), or Rab8wt (D) and control neighboring cells not expressing the recombinant protein (Uninf.). n represents the number of pathways from cell pairs; p is the probability value according to the Wilcoxon test. Middle graphs, average NMDAR-mediated current amplitude (recorded at +40 mV at a latency when AMPA responses are fully decayed, 60 ms) from uninfected and infected cells (n also represents the number of pathways from cell pairs). Right graphs, average AMPA/NMDA ratios for uninfected and infected cells (n represents the number of pathways).

 
The effect of these dominant negative proteins on AMPAR and NMDA receptor (NMDAR) function was evaluated by recording simultaneously from nearby infected and uninfected CA1 neurons, allowing direct comparison of synaptic responses evoked by stimulating the Schaffer collateral pathway from the CA3 region. Importantly, only CA1 cells (but not CA3 cells) express the recombinant dominant negative protein; therefore, the site of action of these proteins when monitoring CA3 to CA1 synaptic transmission is necessarily postsynaptic. As shown in Fig. 1A, Rab8dn significantly depressed AMPAR-mediated currents, without affecting NMDAR-mediated currents. Other electrophysiological parameters were not significantly different between uninfected and Rab8dn-infected neurons (input resistance, 168 ± 23 (uninfected) and 192 ± 25 megaohms (infected); holding current, -68 ± 13 (uninfected) and -57 ± 9 pA (infected)). This result suggests that Rab8 function is required for AMPAR delivery into synapses. In contrast, neither Rab4dn (Fig. 1B) nor Rab11dn (Fig. 1C) significantly changed AMPAR- or NMDAR-mediated synaptic responses. Furthermore, a dominant negative form of the endocytic Rab5 protein (S34N) did not alter AMPA or NMDA transmission either.2 These results serve to verify that the depression of AMPAR responses by Rab8dn was not due to virus infection or nonspecific sequestration of regulatory proteins, such as GDP/GTP exchange factors or GDP dissociation inhibitors.

To determine whether Rab8 is a rate-limiting factor for AMPAR delivery, we overexpressed Rab8wt (Fig. 1D, left panel) and compared AMPAR- and NMDAR-mediated responses from control and infected nearby neurons. As shown in the graphs in Fig. 1D, overexpression of Rab8wt did not produce any significant effect on AMPAR- or NMDAR-mediated synaptic transmission. Because Rab8wt overexpression did not increase AMPA currents, this result suggests that Rab8 is not rate-limiting for AMPAR delivery into synapses.

Expression and Ultrastructural Localization of Rab8 in Hippocampal Neurons—The electrophysiological experiments shown above suggest a role for Rab8 in AMPA receptor synaptic trafficking. To further investigate this possibility, we determined the ultrastructural localization of Rab8 in hippocampal neurons from brain tissue. Although previous studies have localized Rab8 at the somatodendritic compartment in dissociated neuronal cultures (27), the localization of Rab8 relative to synaptic contacts has not been addressed before. To this end, we used post-embedding anti-Rab8 immunogold labeling on the synaptic region of the CA1 hippocampus (stratum radiatum; see "Experimental Procedures"). Rab8 was abundant in postsynaptic terminals (Fig. 2A, arrows; Fig. 2B, Postsynaptic), whereas the presynaptic labeling was marginal (Fig. 2B, Pre-). Quantification of the immunogold particles in the postsynaptic terminal indicated that Rab8 accumulates in intracellular compartments in close proximity to the synapse (Fig. 2B, Intra) and, in some cases, directly at the postsynaptic density (Fig. 2B, PSD). This result supports a potential role for Rab8 in the local transport of membrane proteins into synapses.



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  		FIG. 2.
Localization of Rab8 at postsynaptic terminals. A, ultrastructural localization of Rab8 at CA1 hippocampal synapses by electron microscopy. Rab8 immunogold particles (arrows) were found at the postsynaptic terminal, including the postsynaptic density. Labeling was also detected in dendritic shafts (data not shown). Presynaptic terminals are labeled with asterisks. Scale bar, 100 nm. B, quantification of immunogold labeling. Most of the gold particles were found postsynaptically, with marginal presence presynaptically (Pre) indicating specificity for the immunogold labeling. The majority of the gold particles were found intracelullarly (Intra). Gold particles were also found at the postsynaptic density (PSD) as well as extrasynaptic membranes (Extra). For this quantification, only gold particles within 600 nm of the synapse were included. C, Rab8-GFP is present at dendritic spines. Representative confocal images showing the distribution of the recombinant Rab8-GFP in hippocampal neurons from slices. Rab8 is present in cell body and dendrites (left panel), as well as in dendritic spines (right panels).

 
We also examined Rab8wt-GFP distribution in CA1 neurons from hippocampal slices by imaging GFP fluorescence with confocal microscopy. Rab8wt-GFP was broadly expressed in the cell (Fig. 2C, left panel), including distal dendrites and spines (Fig. 2C, right panels). Although these results do not show specific targeting of Rab8-GFP to spines, they do suggest that the recombinant protein has access to membrane compartments in close proximity to synapses.

Rab8 Is Not Required for the Large-scale Transport of AMPA Receptors along Dendrites—Early studies using antisense oligo-nucleotides in dissociated neuronal cultures suggested that Rab8 may be involved in the export of membrane proteins from the neuronal cell body into dendrites (27). Therefore, it is conceivable that Rab8dn may depress AMPAR-mediated transmission by interfering with AMPAR transport into dendrites. We tested this possibility by co-expressing GFP-tagged GluR2 and Rab8dn using biolistic gene delivery (the same constructs and transfection method used for the electrophysiological experiments shown in Fig. 7A; see below). The surface distribution of the recombinant receptor was monitored by immunostaining using an anti-GFP antibody in nonpermeabilized conditions (the GFP tag is placed at the N terminus of GluR2, and it is therefore exposed to the extracellular side of the plasma membrane). As shown in Fig. 3B, Rab8dn did not qualitatively alter the export of GluR2-GFP into the dendritic surface, including secondary and tertiary distal dendrites (similar to the immunostaining distribution obtained for GluR2-GFP expressed alone, Fig. 3A; see Fig. 4 for a quantitative analysis of surface receptor delivery at spines and dendrites). As control, neurons expressing Rab8dn-GFP alone did not show any surface immunostaining (Fig. 3C), consistent with the intracellular distribution of Rab8. These results suggest that Rab8dn depresses AMPA transmission not by globally altering AMPAR transport into dendrites, but by interfering with a local trafficking step, possibly the synaptic delivery of the receptor.

Rab8 Is Required for Local AMPA Receptor Delivery into the Spine Surface—To test whether Rab8 is involved in a local step of AMPAR trafficking, we quantitatively evaluated the surface delivery of AMPARs at dendritic spines and the adjacent dendritic shafts. For this experiment, we substituted the GFP tag on Rab8dn with a red fluorescence protein (Rab8dn-RFP; see "Experimental Procedures"), so the fluorescence of Rab8dn can be separated from the co-expressed GluR2-GFP (the functionality of Rab8dn-RFP was confirmed electrophysiologically by co-expressing it with GluR2-(R607Q)-GFP and monitoring receptor delivery into synapses using a rectification assay; see description of Fig. 7). Surface immunostaining was then carried out with an anti-GFP antibody under nonpermeabilized conditions, as described above. In this case, the labeling was visualized with an infrared fluorophore (Cy5). Therefore, this experimental design allows us to monitor the total amount of receptor (GFP channel), the fraction exposed to the cell surface (Cy5 channel), and the presence of the co-expressed Rab8dn (RFP channel) (see Fig. 4A as an example).

Line plots of fluorescence intensity were generated across the spine head and the adjacent dendritic shaft. Surface ratios for spine and dendrites were then calculated as the ratio between the corresponding peaks in the Cy5 channel (surface receptor) and the GFP channel (total receptor) after background subtraction (see Fig. 4B). Spines are solely selected from their GFP image, precluding any bias with respect to their surface immunostaining. The results of this analysis are shown in Fig. 4C. When expressing GluR2-GFP alone, surface ratios were similar in spine and dendrites. In contrast, surface ratio was significantly lower in the spines as compared with the adjacent dendrites when Rab8dn was co-expressed. Importantly, absolute surface ratio at dendrites was not altered by Rab8dn (Cy5/GFP ratios were 0.31 ± 0.05 for GluR2 alone versus 0.31 ± 0.04 for GluR2 + Rab8dn). These data indicate that Rab8dn specifically impairs the delivery of AMPARs to the surface of the spine. Interestingly, Rab8dn did not decrease the total amount of receptor (GFP channel) in the spine compared with the dendrite (Fig 4D) or total receptor abundance at dendrites (absolute GFP values were 50 ± 4 for GluR2 alone versus 54 ± 3 for GluR2 + Rab8dn). These results support the interpretation that Rab8 is involved in a local trafficking step from an intracellular membrane compartment inside the spine to the postsynaptic plasma membrane.

Rab8 Is Necessary for Both the Constitutive and Regulated Delivery of AMPA Receptors into Synapses—As described above, AMPARs may reach synapses through a constitutive pathway in an activity-independent manner and through a regulated pathway triggered by NMDA receptor opening and CaMKII activation (17). To test the role of Rab8 in the constitutive pathway, we expressed Rab8dn in slices kept in conditions of reduced neuronal activity (12 mM Mg2+ was added to the culture medium immediately after infection with the virus expressing Rab8dn; recordings were carried out in the presence of the standard 4 mM Mg2+). As shown in Fig. 5A, Rab8dn depresses AMPAR-mediated responses in conditions of reduced neuronal activity, suggesting that Rab8 is necessary for the constitutive (activity-independent) delivery of AMPARs into synapses.

The role of Rab8 in the activity-dependent delivery of AMPARs was tested with a truncated form of {alpha}CaMKII (t-CaMKII) that is constitutively active (8, 59). Expression of t-CaMKII potentiated AMPAR-mediated responses (Fig. 5B), as described previously (8, 59). However, this potentiation was fully blocked when Rab8dn was co-expressed with t-CaMKII (Fig. 5C). This result is consistent with Rab8 also being necessary for the regulated delivery of AMPARs triggered by CaMKII. To test whether the absence of potentiation is due to the interference of Rab8dn with CaMKII activity, we quantified the autophosphorylation levels of {alpha}CaMKII at Thr286 in control and Rab8dn-expressing neurons. As shown in Fig 5D, phospho-CaMKII levels from CA1 dissected regions were similar between infected and uninfected slices.

Rab8 Is Necessary for Synaptic Plasticity—LTP in the CA1 hippocampus is one of the most thoroughly studied forms of synaptic plasticity, and it is accompanied by the synaptic delivery of GluR1-containing AMPARs (8). To test whether Rab8 is necessary for the synaptic delivery of AMPARs during synaptic plasticity, we examined pairing-induced LTP in CA1 neurons infected with Rab8dn or Rab8wt. Consistent with the effect of Rab8dn on CaMKII-induced AMPAR delivery, Rab8dn blocked LTP expression (Fig. 6). Unexpectedly, Rab8dn led to synaptic depression. This late depression upon LTP induction has been reported previously in situations in which AMPAR delivery was impaired (8). Importantly, LTP was normal in cells expressing Rab8wt, as compared with uninfected cells (Fig. 6). These results indicate that Rab8 function is necessary for LTP.



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  		FIG. 6.
Rab8 is necessary for LTP. Organotypic slice cultures were infected with virus expressing either Rab8wt or Rab8dn. Whole-cell recordings were established from neurons expressing the recombinant proteins or uninfected cells, and LTP was induced by pairing 3 Hz presynaptic stimulation with 0 mV postsynaptic depolarization, as described previously (8). A, bottom, pairing significantly increased AMPAR-mediated responses in uninfected (p = 0.002, paired t test) and Rab8wt-infected (p = 0.001, paired t test) neurons. However, pairing significantly decreased synaptic responses in Rab8dn-expressing neurons (p = 0.001, paired t test). A, top, sample trace of evoked AMPAR-mediated synaptic responses recorded at -60 mV before pairing (thin line) and 30 min after pairing (thick line) from control (uninfected) neurons or infected cells, as indicated. Scale bars, 20 pA and 10 ms. B, normalized steady-state AMPAR response amplitudes in paired (LTP Induction) and control (Unpaired pathway) pathways in cells expressing Rab8wt-GFP, cells expressing Rab8dn-GFP, and uninfected control cells. AMPAR responses from control pathways were not statistically different from baseline responses.

 
Rab8 Is Necessary for the Constitutive Delivery of GluR2 and for the CaMKII-triggered Delivery of GluR1 into Synapses—To directly test the role of Rab8 in the pathway-specific delivery of different AMPAR populations into synapses, we co-expressed Rab8dn with individual recombinant AMPAR subunits and monitored their delivery taking advantage of their rectification properties (electrophysiological tagging) (8-11). The constitutive delivery of GluR2 can be assessed using the inwardly rectifying GluR2 mutant GluR2-(R607Q), which does not conduct outward current at positive membrane potentials. Expression of this recombinant receptor produced inward rectification (Fig 7A; compare uninfected with GluR2(Q)), as described previously (10), indicating the delivery of the homomeric receptor. Co-expression of Rab8dn with GluR2-(R607Q) blocked this rectification (Fig. 7A), indicating that Rab8 is necessary for the constitutive delivery of GluR2 (similar rectification values were obtained with Rab8dn-GFP (2.1 ± 0.3; n = 12) and Rab8dn-RFP (2.3 ± 0.2; n = 10)). As a control, co-expression of Rab8wt with GluR2-(R607Q) did produce rectification. This control indicates that the effect of Rab8dn was not due to insufficient co-expression of two recombinant proteins (see also Rab8wt control in Fig. 7B). In addition, Rab8dn did not produce any change in rectification if no recombinant AMPAR subunit was co-expressed (Fig. 7A).

The activity-dependent delivery of GluR1-containing AMPARs can be evaluated by using recombinant GluR1 and t-CaMKII to trigger GluR1 delivery. Co-expression of GluR1 and t-CaMKII produced inward rectification (Fig. 7B), as described previously (8), revealing the delivery of homomeric GluR1. Co-expression of Rab8dn with GluR1 and t-CaMKII blocked rectification (Fig. 7B), indicating that Rab8 is necessary for the regulated delivery of GluR1-containing AMPARs.

Rab8 Is Necessary for the Delivery of AMPARs Induced by PSD95—It has been described recently that overexpression of PSD95 leads to potentiation of AMPAR-mediated responses (60, 61) in a process that is accompanied by the synaptic delivery of GluR1-containing receptors (62). Although the mechanisms are not completely understood, it has been proposed that this synaptic delivery is mediated by stargazin, which binds both PSD95 and AMPARs (63, 64). We wished to determine whether PSD95-induced delivery of AMPARs also requires Rab8. To this end, we co-expressed recombinant GluR1, PSD95, and Rab8dn. As reported previously (62), co-expression of PSD95 and GluR1 leads to the delivery of recombinant, homomeric GluR1 receptors, producing inward rectification (Fig. 7B). However, when Rab8dn was co-expressed with GluR1 and PSD95, the increase in rectification was blocked, indicating that Rab8 is also necessary for this AMPAR delivery pathway. As control, co-expression of GluR1 with PSD95 and Rab8wt did show rectification (Fig. 7B), indicating that the absence of rectification observed with the Rab8dn is not due to inefficient co-expression of three proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Rab family of small GTPases is known to confer directionality to intracellular membrane trafficking in several cell types (reviewed in Ref. 20). However, it is surprising that very few studies have addressed the role of Rab proteins in neuronal function, where polarized membrane traffic is essential to maintain presynaptic and postsynaptic function. The notable exception is Rab3, which is known to regulate neurotransmitter vesicle fusion and short-term plasticity at the presynaptic terminal (26), although the precise mechanisms underlying this regulation are still being elucidated (65, 66).

We have now tested the role of the other three exocytic Rab proteins, namely, Rab4, Rab8, and Rab11, at the postsynaptic terminal. These studies have identified Rab8 as a critical player in postsynaptic membrane trafficking and plasticity. Rab8 was proposed to mediate dendritic membrane trafficking and neurite outgrowth nearly a decade ago (27, 67). These early studies, using neuronal dissociated cultures, suggested the involvement of Rab8 in early stages of the secretory pathway. Since then, no reports have tested whether Rab8 plays a more direct role in synaptic function. Here we have shown that Rab8 is a necessary mediator of the local delivery of AMPARs into excitatory synapses. Using quantitative surface immunostaining, we show that Rab8 is required for the delivery of AMPARs into the spine surface, but not for transport of AMPARs from the dendritic shaft into the spine compartment or for AMPAR delivery into the dendritic plasma membrane. In addition, by monitoring electrophysiologically the synaptic targeting of endogenous and recombinant AMPARs, we demonstrate that Rab8 is necessary for both the constitutive cycling of AMPARs and their regulated synaptic delivery, as triggered by CaMKII activation, PSD95 overexpression, or LTP induction. Therefore, we propose that Rab8 drives the local transport of AMPARs from an intracellular membrane compartment, possibly in the dendritic spine, to the synaptic membrane.

Rab8 is a known mediator of exocytic transport from the trans-Golgi network into the plasma membrane. As such, it is possibly involved in the surface delivery of a variety of membrane proteins in neurons. However, an unexpected result of the present study is that Rab8 drives the local targeting of AMPARs into the highly specialized compartment that constitutes the postsynaptic membrane. Several lines of evidence support this role of Rab8 in the short-range synaptic delivery of AMPARs. Firstly, Rab8, besides being present in the dendritic shaft, is localized in very close proximity to the synapse, including the postsynaptic density. Secondly, Rab8 is necessary for the continuous synaptic cycling of GluR2/3-containing AMPARs. Although the molecular details of this activity-independent pathway are not well understood, the fast kinetics of this pathway (13, 15) suggests that it occurs in close proximity to the synapse. And thirdly, quantitative immunohistochemical analysis showed that blockade of endogenous Rab8 function by expression of Rab8dn does not affect the transport of AMPARs in dendrites, but it does impair receptor delivery into the spine surface. This result is in contrast with a previous publication showing that Rab8 was necessary for the dendritic transport of a recombinant viral glycoprotein (27). Although we do not know the reason for this discrepancy, it is possible that Rab8 plays a more general role in membrane trafficking in developing neurons, such as those in dissociated primary cultures, as compared with the more differentiated neurons present in our organotypic slice cultures. This interpretation is consistent with the proposed role of Rab8 in neurite outgrowth before axons and dendrites are differentiated (67).

As mentioned above, Rab8 mediates exocytic transport from the trans-Golgi network. Interestingly, it has been described recently that distal dendrites contain intracellular compartments belonging to the trans-Golgi network (68, 69). Our functional results fit very well with these morphological observations and suggest that AMPARs are driven locally from distal trans-Golgi network outposts into synapses via a Rab8-mediated process. Exocytic delivery of membrane proteins from the trans-Golgi network can proceed directly into the plasma membrane or through intermediate recycling endosomes (29, 70). Rab8 is present in both trans-Golgi network membranes and recycling endosomes (29), and to date, a possible role of Rab8 in controlling membrane sorting from both compartments has not been ruled out. Interestingly, we have found that Rab8 mediates both the continuous cycling of GluR2/3 receptors and the transient, activity-dependent delivery of GluR1/2 receptors. At this point, we cannot resolve whether these two populations of receptors are delivered from different intracellular compartments or whether a single but subcompartmentalized storage place is the source for both types of exocytic events. However, biochemical membrane fractionations (71) and imaging experiments (10, 72) suggest that GluR1/2 and GluR2/3 receptors have different subcellular distributions. Therefore, we propose that the synaptic delivery of GluR1/2 receptors during synaptic plasticity is regulated from the trans-Golgi network, whereas the activity-independent cycling of GluR2/3 receptors occurs from a specialized recycling compartment in close proximity to the synapse (see Fig. 8).



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  		FIG. 8.
Membrane sorting model for the control of AMPAR synaptic delivery by Rab8. The regulated (activity-dependent) delivery of GluR1/2 receptors would be mediated by Rab8 from a membrane compartment belonging to the trans-Golgi network (TGN). In contrast, the continuous (activity-independent) delivery of GluR2/3 receptors would occur from specialized recycling endosomes (RE), whose sorting is also controlled by Rab8.

 
In this sense, it is important to note that neither Rab4 nor Rab11 seems to be involved in the continuous cycling of AMPARs (the corresponding dominant negative mutants did not affect basal AMPAR-mediated synaptic transmission). This observation is somehow surprising because these Rab proteins are known to mediate the trafficking of various populations of recycling endosomes (32-36, 52). Our results, then, support the interpretation that GluR2/3 synaptic delivery occurs from a specialized membrane compartment, whose cycling is controlled by Rab8 but not by Rab4 or Rab11. This interpretation does not rule out a role for Rab4 and/or Rab11 in other steps of AMPAR trafficking. For instance, it has been reported that AMPARs that return to the plasma membrane after activity-dependent internalization co-localize with Rab4-positive compartments (73, 74). However, our results indicate that Rab8 is the Rab family member that directly mediates the late stages of membrane transport that result in the synaptic delivery of AMPARs during both constitutive receptor cycling and activity-dependent delivery (LTP).

In conclusion, these results expand our view of the cellular functions accomplished by the Rab protein-driven exocytic machinery in neurons and shed light on the membrane trafficking events that control the dynamic organization of the postsynaptic terminal.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health (Grant MH070417), National Alliance for Research on Schizophrenia and Depression, and Alzheimer's Association (to J. A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Tel.: 734-615-2686; Fax: 734-763-4450; E-mail: estebanj{at}umich.edu.

1 The abbreviations used are: AMPAR, AMPA-type glutamatergic receptor; NMDA, N-methyl-D-aspartate receptor; NMDAR, NMDA receptor; GluR, glutamate receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; LTP, long-term potentiation; RFP, red fluorescence protein; GFP, green fluorescence protein. Back

2 T. C. Brown, I. C. Tran, D. S. Backos, and J. A. Esteban, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Roberto Malinow, Ronald Holz, Lori Isom, Edward Stuenkel, and María S. Soengas for critical reading of the manuscript. We also thank Kristen Verhey for helpful discussions.



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I. J. Rampil, D. H. Moller, and A. H. Bell
Isoflurane modulates genomic expression in rat amygdala.
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