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Comparative Study
. 2005 Nov 16;25(46):10627-36.
doi: 10.1523/JNEUROSCI.1947-05.2005.

Rac1 induces the clustering of AMPA receptors during spinogenesis

Katie M Wiens  1 , Hang Lin, Dezhi Liao
Affiliations
Comparative Study

Rac1 induces the clustering of AMPA receptors during spinogenesis

Katie M Wiens et al. J Neurosci. .

Abstract

Glutamatergic synapses switch from nonspiny synapses to become dendritic spines during early neuronal development. Here, we report that the lack of sufficient Rac1, a small RhoGTPase, contributes to the absence of spinogenesis in immature neurons. The overexpression of green fluorescence protein-tagged wild-type Rac1 initiated the formation of dendritic spines in cultured dissociated hippocampal neurons younger than 11 d in vitro, indicating that Rac1 is likely one of the missing pieces responsible for the lack of spines in immature neurons. The overexpression of wild-type Rac1 also induced the clustering of AMPA receptors (AMPARs) and increased the amplitude of miniature EPSCs (mEPSCs). The expression of constitutively active Rac1 induced the formation of unusually large synapses with large amounts of AMPAR clusters. Also, our live imaging experiments revealed that the contact of an axon induced the clustering of Rac1, and subsequent morphological changes led to spinogenesis. Additionally, the overexpression of wild-type Rac1 and constitutively active Rac1 increased the size of preexisting spines and the amplitude of mEPSCs in mature neurons (>21 d in vitro) within 24 h after transfection. Together, these results indicate that activation of Rac1 enhances excitatory synaptic transmission by recruiting AMPARs to synapses during spinogenesis, thus providing a mechanistic link between presynaptic and postsynaptic developmental changes. Furthermore, we show that Rac1 has two distinct roles at different stages of neuronal development. The activation of Rac1 initiates spinogenesis at an early stage and regulates the function and morphology of preexisting spines at a later stage.

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Figures

Figure 1.
Figure 1.
Expression of wild-type Rac1 induced the formation of dendritic spines early in development. A, Left, An 11-d-old neuron expressing Racw was photographed when both illumination light and green fluorescent light were on. Middle, Only fluorescent light was on. The arrowhead denotes the enlarged area of the right panel. Right, A zoom-in image of the middle panel. The arrows point to dendritic spines, and the arrowhead indicates Racw clustering at the periphery of the dendrite. DIC, Differential interference contrast; Fluo, fluorescence. B-E, Neurons expressing Racw (B, C; B is the fixed neuron from A), GFP (D), and Rac- (E) were stained with antibodies against AMPARs (anti-GluR1 and anti-GluR2/3 were added together; B) or synaptophysin (C-E). The arrows indicate AMPAR (B) or synaptophysin (C-E) clusters. F, Ratio of the fluorescence (Flu) intensity in the center of a spine versus the center of the dendrite from which the spine grew (Sp/D). A value >100% means that the spine is brighter than the dendrite. Inset, The middle of the dendrite is brighter than spines in a 3-week-old GFP-labeled neuron. G, The density of dendritic protrusions and spines per 100 μm dendrite (Drt) was measured in neurons expressing GFP, Racw, and Rac-. Error bars represent SE. ***p < 0.001.
Figure 2.
Figure 2.
The clustering of Rac1 recruited functional AMPARs to synapses in immature neurons. A, Averaged traces recorded from four individual neurons for 20 min. B, A comparison between an individual trace from a GFP-expressing neuron and an unusually large mEPSC response from a Rac+-expressing neuron. C-F, Various parameters of mEPSCs were compared among four groups of neurons expressing GFP, Racw, Rac-, and Rac+. Rise T, Rise time; Decay T, decay time. Error bars represent SE. *p < 0.05; **p < 0.01; ***p < 0.001. G, Neuron expressing Rac+ and stained for synaptophysin. The arrow indicates that the soma and proximal dendrites had intense clusters of Rac+ and were innervated by many presynaptic termini. The arrowhead denotes that lamellipodia had almost no presynaptic termini. H, Neuron expressing Rac+ and stained for AMPARs (anti-GluR1 and anti-GluR2/3 antibodies combined). The AMPAR clusters in nearby untransfected neurons (arrows) were much smaller than the clusters in neurons expressing Rac+ (arrowheads). This result together with the above electrophysiological data indicates that activation of Rac1 can recruit AMPARs to synapses. I, Enlarged image of a neuron expressing Rac+ and stained for synaptophysin. The arrows denote that one Rac+ cluster was innervated by many presynaptic termini.
Figure 3.
Figure 3.
Rac1 induces the clustering of surface AMPARs. A, Neurons expressing Racw were immunostained for surface AMPARs using an antibody against the extracellular N terminus of the GluR1 subunit. The arrowhead indicates the enlarged area for B. B, Enlarged image from A showing that Racw clusters colocalized with AMPAR clusters (arrows). C-E, Neurons expressing GFP, Rac-, and Rac+ were immunostained with the same antibody used in A. The arrows in E indicate that the soma and proximal dendrites in neurons expressing Rac+ contained numerous large AMPAR clusters. N-GluR1, N terminus of the GluR1 subunit. F, Quantification of the number of surface AMPAR clusters per 100 μm dendrite (Drt). AMPARs were counted in the proximal dendrites of neurons expressing Rac+. G, Quantification of the percentage of protrusions that colocalized with surface AMPAR staining indicates that Racw is also able to induce the clustering of AMPARs at dendritic spines. Error bars represent SE. ***p < 0.001.
Figure 4.
Figure 4.
Temporal steps in Rac1-dependent formation of dendritic spines. A, Images of neurons were taken 8 h after transfection. Racw and Rac+ were concentrated in the periphery of dendrites, whereas Rac- was concentrated in the center (arrowheads). Racw induced spinogenesis and clustered in spine heads, whereas Rac+ induced random lamellipodia and clustered in their edges (arrows). B, A neuron 3 d after the transfection of Rac-. Rac- clustered in discrete granules at the proximal (arrowhead) and distal (arrow) dendrites. C, The ratio of fluorescence (F) intensity in the periphery of dendrites versus the middle of dendrites (Per/Ctr) in four groups of neurons. D, The proportion of neurons that contained granules versus the total number of neurons expressing Rac- in various days after transfection. E, The density of spines in neurons expressing Racw (at 8 and 32 h after transfection), GFP (15-21 DIV), and Rac- (15-28 DIV). Trsf, Time after transfection. Error bars represent SE. **p < 0.01; ***p < 0.001. F, Time-lapse images of a neuron expressing Racw showing how the contact of an axon led to spinogenesis. G, Spines were still formed in the presence of TTX in neurons 32 h after transfection of Racw. Right, Spine density in the presence (+) and absence (-) of TTX. H, A model based on results in this figure shows how Rac1 initiates the formation of dendritic spines.
Figure 5.
Figure 5.
Developmental changes in endogenous Rac1 expression. A, Left, Western blots of Rac1 (bottom bands; ∼22 kDa) from neurons at 4-21 DIV. Actin was used as control (top bands). Right, Graphical representation calculated by dividing the expression of Rac1 by the expression of actin at each age. Ctl, Control. B, C, Endogenous Rac1 was detected using an anti-Rac1 antibody in immunocytochemistry at 7 DIV (B) and 21 DIV (C). Rac1 colocalizes with AMPARs and synaptophysin (arrows) at 21 DIV.
Figure 6.
Figure 6.
Rac1 regulates both the morphology and function of preexisting spines. A, Images of neurons at 21-22 DIV were taken 1 d after transfection with a gene gun. B, Ratio of fluorescence (F) intensity in spines versus dendrites (Sp/D) in four groups of neurons. Open bar, GFP; hatched bar, Rac-; dotted bar, Racw; black bar, Rac+. C, The size of dendritic spines in four groups of neurons. D, Representative traces recorded from neurons transfected using the gene gun. E-H, The amplitude, frequency, rise time, and decay time of mEPSCs in four groups of neurons. I, Averaged traces of neurons transfected at 5-7 DIV with GFP, Rac-, and Rho- and recorded at 17-21 DIV. J-M, The amplitude, frequency, rise time, and decay time of neurons represented in I. Rac- was able to decrease the mEPSC response, whereas Rho- was not. Error bars represent SE. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7.
Figure 7.
Rac1 acts via CRIB motif-containing proteins to induce the formation of dendritic spines and the clustering of AMPARs. A, Left, An 8 DIV neuron expressing a Rac1 mutation (Rac1Y40K) that disrupts the activation of CRIB motif-containing proteins such as PAK. The arrowhead indicates the enlarged area in the middle panel. Middle, Enlarged image of the area at the arrowhead in the left panel. Right, Enlarged image of a 28 DIV neuron expressing Rac40. Note the unusually long filopodia. B, Rac40 increased the length of protrusions, whereas Racw decreased the length in neurons 7-11 DIV. C, AMPAR (GluR1 and GluR2/3) staining of a neuron expressing Rac40 at 8 DIV. The arrows denote the lack of AMPARs in the protrusions from the dendrite. D, Number of protrusions and spines [normalized to 100 μm length of dendrite (Drt)] of neurons expressing GFP, Racw, and Rac40. E, Percentage of protrusions containing AMPAR clusters. Open bars, Total AMPARs were detected with two antibodies against GluR1 and GluR2/3. Solid bars, Surface AMPARs were detected with an antibody against N-GluR1. F, Average traces of mEPSC recordings of GFP, Racw, and Rac40. G-I, mEPSC amplitude, frequency, rise time, and decay time for neurons expressing GFP, Racw, and Rac40. Error bars represent SE. *p < 0.05; **p < 0.01; ***p < 0.001. J, A model that incorporates previously reported studies indicates that the cycling of Rac1 between active and inactive states is important for normal spinogenesis (Symons and Settleman, 2000). Active Rac1 molecules are mobilized to submembrane spaces in the dendrites (step 1), and these molecules might be in activated after they bind to membrane proteins (step 2). Ephrin B from an axon activates Eph B receptors and Kalirin, which then cluster and activate Rac1 (step 3). Finally, the clustering of activated Rac1 activates downstream effector proteins that have CRIB motifs (e.g., PAK) and leads to the formation of spines and an increase in the anchoring sites for AMPARs (step 4).
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