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1 Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland2 Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland3 Department of Physiology, University of Wisconsin, Madison, WI 53706, USA
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(Received 29 January 2004;
accepted after revision 25 May 2004;
first published online 28 May 2004)
Corresponding author D. A. Richards: Department of Physiology, SMI 129, University of Wisconsin, 1300 University Ave, Madison, WI 53706, USA. Email: richards{at}physiology.wisc.edu
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Introduction |
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Over the last decade it has become clear from many reports that actin can cause movement. The chief experimental models for this are the membrane ruffles and lamellipodia seen in fibroblasts. These studies have demonstrated that actin polymerization and branching can ‘push’ the plasma membrane forwards (reviewed in Pollard & Borisy, 2003). In some of these studies, it has been proposed that an important element is actin nucleation at lipid rafts in the inner leaflet of the plasma membrane (reviewed in Caroni, 2001). This occurs via the neural Wiskott-Aldrich syndrome protein–Arp 2/3 complex and it is thought that the rapid formation of a dense meshwork of short actin filaments at the membrane gives rise to the protrusive force which moves the membrane forwards, while deeper within the cytoplasm, the actin polymers slowly disassociate to preserve the concentration of free actin monomers (Pollard & Borisy, 2003). Since the membrane is a fluid matrix of diffusible lipids and immobilized rafts, the formation of a physical connection between the plasma membrane and the cytoskeleton will result in changes in the laminar diffusion constant of membrane-bound macromolecules. At its simplest, this can be viewed as a change in the viscosity of the milieu immediately adjacent to the membrane. It is possible to test for such an effect by monitoring the rate of diffusion of a membrane-bound probe.
We have investigated the diffusion of a membrane marker in dendritic spines using fluorescence recovery after photobleaching (FRAP). This technique has proven useful for the study of membrane-limited diffusion (Saxton & Jacobson, 1997), and has been used previously to monitor cytoplasmic coupling between dendritic spines and the dendritic shaft (Svoboda et al. 1996; Majewska et al. 2000). Our chosen probe was an enhanced green fluorescent protein (GFP) linked to the membrane via a short palmitoylated peptide sequence. The use of a surface-bound form of GFP enables us to accurately track the boundaries of individual spines, rather than focusing simply on the deepest areas of cytoplasm (which would be the case if we used cytoplasmically localized GFP). Our membrane-bound GFP is tagged to the membrane via the N-terminal fragment of a myristoylated alanine rich C kinase substrate (MARCKS) mutant where the myristoylation site has been exchanged for a palmitoylation site (De Paola et al. 2003). Use of transgenic mice also enables us to avoid perturbing the system to incorporate our marker. This would ordinarily lead to very high background fluorescence from all the GFP labelled cells, but we have avoided this problem by expressing our membrane GFP in the Thy1.2 expression cassette, which results in only a very small subset of cells being labelled (Caroni, 1997; De Paola et al. 2003). This enables us to visualize a particular cell without interference from high surrounding levels of mGFP fluorescence.
Our analysis of the diffusion of a fluorophore bound to the inner leaflet of the plasma membrane indicated that fluorescence recovery rates differed greatly between the main dendritic shaft, and the heads of dendritic spines, consistent with the bulk of the cytoskeletal contacts being in the spine heads. More interestingly, we found that activation of postsynaptic glutamate receptors induced a change in the rate of fluorescence recovery. This effect directly correlated with the ability of glutamate receptor antagonists and agonists to activate and inhibit spine motility, such that more motile spines exhibited a slower recovery of fluorescence. These data provide the first evidence for a link between cell motility and diffusion within the membrane, and suggest a model in which connections between actin and the plasma membrane are responsible for spine motility; glutamate receptor activation results in a severing of these connections between cytoskeleton and membrane. This glutamatergic regulation of membrane dynamics is likely to have important implications for the clustering of postsynaptic receptors, promoting or inhibiting the exchange of extrasynaptic receptors bound within the post-synaptic density (PSD).
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Methods |
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Variegated mice were generated using standard techniques. A construct was generated where the cDNA for enhanced green fluorescent protein was fused to the membrane-anchoring domain (first 41 amino acids) of a double palmitoylated mutant of MARCKS (Laux et al. 2000), to form a membrane-targeted marker (mGFP) under the Thy1 promoter. Twenty-five distinct lines were generated, each with subtly different patterns of expression. Of these, L15 mice were chosen as they had low but consistent levels of mGFP positive cells within area CA1 of the hippocampus.
Slice cultures
Organotypic slice cultures were used for these experiments as they provide the major advantage of exhibiting preserved tissue-specific organization of synaptic connections, in an in vitro preparation suitable for imaging studies. Slices were prepared from the hippocampi of 6-day-old L15 mice killed by decapitation following a protocol approved by the Veterinary Department of the Canton of Zurich. The slices (400 µm) were maintained in roller tubes for 3–6 weeks before use, as previously described for rat (Gahwiler et al. 1997).
Confocal imaging
Slice cultures were transferred to a recording chamber mounted on an upright microscope (Leica DM LFSA, Leica microsystems, Heidelberg, Germany) equipped with a heated (30°C) submersion chamber where slices were continually perfused with a solution comprising (mM): NaCl, 137; KCl, 2.7; CaCl2, 2.5; MgCl, 2; NaHCO3, 11.6; NaH2PO4, 0.4; and glucose, 5.6. The confocal scanhead was a Leica SP2. mGFP was imaged using the 488 nm laser line, with an XY dimension of 26 nm pixel–1, and a Z dimension of 200–250 nm (12–18 images per stack). Stack acquisition took 15–20 s. The objective was a Leica water immersion HCX APO 63x 0.9 NA. objective. Bleaching was minimal for acquisition series of < 30 stacks.
Spine motility
Image stacks (4D) were deconvolved using Huygens Pro (Scientific Volume Imaging B.V., Hilversum, the Netherlands) running on a Silicon Graphics Octane workstation (Mountain View, CA, USA), using a full maximum likelihood extrapolation algorithm. Volume rendering and quantification were carried out using Imaris Surpass software (Bitplane AG, Zurich, Switzerland) running on a Windows 2000 Professional workstation. The same parameters were used for all time points of an experimental series. All spines were tertiary apical dendrites of CA1 pyramidal cells.
Spine motility was assessed by measuring the volume of 3D reconstructed spines over time. Averaging all spines, spine volume did not change over the duration of an experiment. This allowed us to define motility as the mean deviation of spine volume from its average value, over time. Quiescent spines were defined as those whose volume changes did not exceed those seen in latrunculin A-treated preparations (where actin-dependent motility is de facto blocked). In Supplementary material Fig. 1, we show repesentative time series of spines with motility measured according to our volume-based method, and the shape factor method (length/width) of Fischer et al. (1998, 2000). Both analyses show a clear time-dependent effect of AMPA (open bar) on motility.
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Fluorescence recovery after photobleaching (FRAP) experiments were carried out by defining a spot (centred on a single pixel) within a spine of interest, and bleaching that spot with 100% laser intensity for 1 s. Recovery was measured by rapid imaging of a single image plane in letter box mode (64 x 256), with a line scanning frequency of 800 Hz. Experiments were analysed in ImageJ (NIH; http://rsb.info.nih.gov/ij). Two trials were carried out per spine, and averaged. Each curve was then individually fitted either with a single exponential, to derive a value for 
, and to derive D the simple diffusion coefficient assuming 1D diffusion along the dendritic spine shaft, or with the equation:
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is the time exponent for anomalous subdiffusion. For comparison of diffusion values between treatments, statistical significance was assessed using Student's t test. Reagents
AMPA and NBQX were supplied by Tocris Cookson (Bristol, UK), latrunculin A by Molecular Probes (Eugene, OR, USA) and other reagents by Sigma (Bucks, Switzerland).
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Results |
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270 nm, in good agreement with the theoretical maximum (
emission/2 = 270 nm). These values reflect the light-limited resolution of a point source. The motility of dendritic spines is known to be both regulated by glutamate receptors, and dependent on actin polymerization. How does activation of glutamate receptors cause a down-regulation of actin-mediated motility? Cell motility in many cell types involves actin nucleation at the membrane, followed by polymerization, which has the effect of pushing the membrane forwards (Pollard & Borisy, 2003). We speculated that a similar mechanism might be involved in spine motility. Fluorescence recovery after photobleaching (FRAP) is an optical technique that can be used to measure the mobility of fluorescent molecules. If the fluorescent molecules are tethered to the membrane, then FRAP measures the laminar diffusion within the membrane leaflet. We reasoned that if spine motility involves interactions between the spine membrane and the actin cytoskeleton, then changes in motility should produce corresponding changes in the ability of mGFP to diffuse within the membrane. Figure 2 shows an example of FRAP of mGFP in a dendritic spine. The images in Fig. 2A show a spine before, and at intervals after, photobleaching. An example of a FRAP curve is shown in Fig. 2B, where fluorescence from the spine head, the spine shaft, and the dendritic shaft are plotted. Fluorescence in the spine head is greatly reduced immediately after photobleaching, but recovers within 10 s. As might be expected, this time course contrasts with that seen using cytosolic indicators such as fluorescent dyes and GFP, where subsecond recovery curves have been observed (Svoboda et al. 1996; Majewska et al. 2000). We also note that the fluorescence within the dendrite itself did not change as a result of the bleach, indicating that membrane diffusion occurs rapidly within this compartment.
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= 0.89 ± 0.09 s) compared to controls (= 3.4 ± 0.1 s), while addition of 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX; 10 µM), an AMPA/kainate-receptor antagonist, caused a slowing of fluorescence recovery (= 4.38 ± 0.23 s). Since AMPA also blocks the motility of spines, and NBQX enhances it, we wondered if the effects of AMPA and NBQX would correlate with mGFP diffusion in static and motile spines which were otherwise untreated. To investigate this possible link between motility and laminar diffusion of mGFP, we measured the motility of individual spines, and then subjected them to FRAP analysis of the diffusion of mGFP. The motility of dendritic spines was examined by imaging 3D stretches of dendrite at high resolution. We then analysed the motility of spines as the mean deviation of volume for the spines at each time point from the average volume over the entire time course of the experiment. We then picked motile or quiescent spines (see Methods) and carried out FRAP experiments upon them. Figure 3B shows the result when spines are grouped according to their motility; motile spines show a slower time course of FRAP (= 4.17 ± 0.33 s) than quiescent spines (= 2.18 ± 0.21 s).
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of 0.85 ± 0.11 s, and coapplication with NBQX did not significantly change this (= 0.89 ± 0.12 s). In Fig. 4A we plot the t
(which, unlike exponential fits, makes no assumptions as to the shape of the data) of fluorescence recovery of individual spines, plotted against the motility measured for those same spines. A strong linear correlation was observed (r= 0.81).
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, D, the diffusion coefficient, and L, the length of the spine neck, where =L2/2D. Clearly this only provides an approximation for the diffusion constant, but the value is useful for comparison between groups. Most importantly, this model allows the length of the spine to be directly taken into account.
In Fig. 4B we plot data from all groups where we measured both the motility (see Methods) and FRAP of individual spines. There is a strong inverse correlation between the diffusion coefficient D and motility (r=–0.91), such that spines which are more motile have a smaller value of D than more quiescent ones. This means that motile spines have more restricted laminar diffusion in the inner leaflet of the plasma membrane. The model is justified, since the data are more tightly grouped than in Fig. 4A, where only the empirical t is used. A major factor in this improvement is the spine length – time for diffusion is dependent on distance, whereas D is independent of distance.
Studies of membrane diffusion tend to use either single particle tracking or FRAP measurements, and these tend to give rise to different values for D. This has been explained by the existence of lipid microdomains, which constrain diffusion to limited regions. As a result, diffusion over very short time scales (single particle tracking) tends to be fast relative to the bulk measurements on longer time scales which are measured by FRAP data. This has been formally shown by Feder et al. (1996) in work where they derived an approximation of anomalous diffusion which fitted both FRAP data and single particle tracking data. The retardation in mGFP diffusion observed following AMPA/kainate- receptor blockade could be due to changes in the diffusion coefficient, in the immobile fraction, or in the extent of anomalous diffusion. To answer this, we fitted each individual FRAP curve (see Methods) to determine which parameters changed under each condition. The results are summarized in Table 1. Anomalous diffusion is seen as a slowing of the slow component of diffusion, with little change in the fast component (which lies within the lipid microdomain). Figure 5 shows a graphical representation of some of the data in Table 1. As illustrated in Fig. 5A, there is a modest correlation between the level of fluorescence recovery, and motility, but this disappears when the anomalous diffusion model is used. Anomalous diffusion which is reflected in both the ratio of fast and slow components of diffusion, and the value of did not appear to change in the conditions examined. Values for remained largely consistent (see Fig. 5B; = 0.82 ± 0.11 for AMPA-treated spines compared to 0.72 ± 0.12 for NBQX-treated spines.) As shown in Table 1 and Fig. 5C and D both fast and slow components of diffusion changed in parallel; this implies that anomalous diffusion itself was unchanged by the various treatments. As a result, our data are equally well fitted by the simple model where D is the only value to change. The overall fit to the data is improved, however, as evidenced by the change in R, the mobile fraction (80% in simple diffusion fits, compared to 96% in the anomalous subdiffusion model).
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Discussion |
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The diffusion rates of lipids, lipid-linked proteins and integral membrane proteins have been extensively studied over the last 30 years. (Axelrod et al. 1976; reviewed in Saxton & Jacobson, 1997). These studies, initially dependent on FRAP alone, have been supplemented by single particle fluorescence measurements. Diffusion coefficients measured in these ways have indicated that the rate of diffusion scales according to time and distance from origin (known as anomalous diffusion). Despite this, characteristic values have emerged for rates of diffusion in membranes; lipids and lipid-linked proteins typically have rates of diffusion in the range of 1 µm2 s–1, whereas integral membrane proteins such as receptors typically have slower rates of diffusion, in the range 0.01–0.4 µm2 s–1 (Saxton & Jacobsen, 1997). In this study we have characterized the diffusion of a lipid-linked (palmitoylated) GFP in the membrane of dendritic spines, the postsynaptic specializations which are the site of the majority of excitatory connections in the hippocampus.
FRAP analysis of mGFP reveals that fluorescence recovers smoothly following bleach, indicating that mGFP can diffuse in the inner leaflet of the plasma membrane in which it is anchored by the N-terminal double palmitation. Activation or blockade of glutamate receptors leads to a speeding or slowing, respectively, of the rate of fluorescence recovery. This most likely reflects diffusion of unbleached mGFP into the spine from the dendrite.
The influence of microdomains
Microdomains within the plasma membrane have been shown to greatly influence the motility of membrane markers, especially in studies using single particle tracking. These microdomains could be due to recruitment and retention of components to lipid rafts, and also to corralling; retention of markers within regions as a result of cytoskeletal binding to membranes (Sheetz, 2001). To take microdomains into account in our analysis, we used the method proposed by Feder et al. (1996), and looked for evidence of a change in , the coefficient of anomalous diffusion. Instead, we found changes in other parameters, indicating that the retardation of diffusion we have observed is not due to an increase in the number of microdomains crossed by the diffusing marker. Instead we interpret the slowing of diffusion seen in parallel with spine motility as being due to the increased viscosity of the cytoplasm immediately beneath the inner leaflet of the plasma membrane. The increased viscosity we observe might be due to increased filamentous actin networks, and perhaps actin nucleation at the membrane itself. This is an important point which demonstrates that variations in the effective viscosity of the cytoplasm provide a source of diffusional anomalies in addition to membrane subdomains. In the light of these findings, it is particularly interesting that other results suggest that the spine head itself may constitute a lipid microdomain. Since the entire spine head is bleached within 1 s, the apparent value for D within this area is much faster than the value observed during recovery (trecovery is between 2 and 10 s). Thus potential energy barriers exist which resist the exchange of lipids from the spine head to the dendrite. It would not be startling if spine heads did prove to contain lipid microdomains, since in other preparations they typically have areas of 0.5–1 µm2, which compares well to the surface area of a spine head, 1 µm2.
Relating motility to diffusion within the membrane
In this study we relate membrane diffusion, actin dynamics, and dendritic spine motility. Although mGFP diffusion and spine motility are correlated, it is necessary to ask whether this relationship is informative or merely coincidental. How does the actin cytoskeleton cause spines to move? From our data, it seems likely that a connection is established between the actin cytoskeleton and the membrane when motility is up-regulated, as it is following AMPA receptor blockade. Dendritic spines contain F-actin which can be considered to be in two forms (Halpain, 2000): a central core which is extremely resistant to turnover (hence the fact that latrunculin A does not cause spines to disappear), and dynamic actin which appears to be responsible for spine motility (Fischer et al. 1998, 2000; Matus, 2000). Different models have been proposed for actin-mediated motility. In Listeria and Shigella, actin-based motility can be reconstituted using actin together with activated ARP2/3 complex, cofilin and capping protein (Loisel et al. 1999); this is as yet not possible for eukaryote systems, suggesting that a more complex regulation is involved. What appears clear is that a combination of factors, including Wiskott-Aldrych syndrome Protein, Arp2/3, cofilin, profilin and other proteins, all interact to shape the directionality and branching of actin polymerization.
At the plasmalemma, this situation is complicated by the need to manipulate the bilayer itself. Likely intermediaries in this role are phosphatidyl inositol (PI) and lipid rafts (Caroni, 2001). Phosphatidyl inositol 4,5-bisphosphate (PIP2) molecules have been shown to modulate actin dynamics (Martin, 1998), actin nucleation (Hill et al. 2000) and its interaction with lipid bilayers (Takenawa & Itoh, 2001); it is therefore plausible to suggest that it is actin polymerization adjacent to lipid microdomains enriched in PIP2 which drives the membrane movements seen in the motility of dendritic spines. A central role for PIP2 would leave spine motility in line with other forms of motility (Sheetz, 2001), and also endocytosis (Schafer, 2003). An important factor in such interactions is the relative abundance of PIP2 in the membrane; although cytoskeletal elements bind PIP2 with relatively low affinity, the concerted action of thousands of such interactions (there are thought to be 103 or 104 PIP2 molecules per µm2 of membrane) has a marked effect. Consequently, reorganization of the cytoskeleton adjacent to the membrane will exert complementary changes on the behaviour of the membrane.
One model for motility suggests that movement can be regulated through the tension exerted on the membrane by the cytoskeleton (Sheetz, 2001). In this model, based on biophysical observations made on membrane ‘blebs’, loss of cohesion between the cytoskeleton and the membrane results in protrusion of the membrane into the extracellular space (Dai & Sheetz, 1999). A similar phenomenon on a much smaller scale could account for the swelling and shrinkage of spine heads observed during dendritic spine motility.
It is therefore particularly interesting that treatments which impair actin dynamics also affect both normal synaptic transmission (Kim & Lisman, 1999) and synaptic plasticity (Kim & Lisman, 1999; Krucker et al. 2000), and inhibition of PI metabolism produces an impairment in both synaptic plasticity (Lin et al. 2001; Sanna et al. 2002) and associative fear conditioning (Lin et al. 2001). Additional evidence links spine motility to synaptic plasticity: growth of dendritic spines has been associated with long-term potentiation in area CA1 of the hippocampus (Maletic-Savatic et al. 1999; Engert & Bonhoeffer, 1999), and in vivo studies support experience-driven changes in spine morphology (Lendvai et al. 2000; Shepherd et al. 2003).
Implications of modulating membrane diffusion
The finding that the diffusion of macromolecules in the inner leaflet of the plasma membrane can be modulated has important implications for receptor clustering. It has become clear that some receptors, such as a subpopulation of NMDA receptors, are able to freely diffuse in the postsynaptic membrane (Tovar & Westbrook, 2002). The importance of this has been directly shown at the neuromuscular junction; loss of presynaptic activity rapidly leads to dispersal of postsynaptic acetylcholine receptors (Akaaboune et al. 2002). A variation of this phenomenon may play a role in synaptic plasticity at central synapses. The glutamate-dependent lowering of diffusion barriers described in our study, combined with a stronger potential energy trap for receptors, could lead to an increased receptor density. Equally, such a mechanism allows for exchange of receptor subunits. Such a diffusion-mediated shuffling of receptors has advantages over receptor endocytosis since it can very rapidly alter postsynaptic responses (as receptors diffuse from the post synaptic density they will be activated increasingly slowly and weakly as the apparent glutamate concentration drops). A detailed discussion of diffusion-mediated receptor shuffling can be found in Choquet & Triller (2003). This selective trapping of receptors may underlie the recently described ability of stargazin to control the clustering of AMPA receptors (Schnell et al. 2002).
The results presented here demonstrate that the diffusion of a lipid-tethered molecule can be regulated by physiologically relevant stimuli, an important advance in the understanding of how membrane biophysics can affect cellular and physiological function. Our results extend our knowledge of the mechanisms of spine motility, and the role played by the actin cytoskeleton in this process. This may represent a phenomenon specific to these neuronal specializations, or be found in other motile structures. In Fig. 6 we present a summary scheme for the relationship between glutamate receptor activation, actin dynamics, and spine motility. We propose that periods of synaptic inactivity lead to actin nucleation and branching adjacent to, or at, the plasma membrane, leading to an increase in spine motility. Synaptic activity, probably acting through intracellular calcium, reverses this process, leading to the breakdown of branched actin filaments at the membrane, and giving rise to a rounded, static spine.
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Supplementary material |
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DOI: http://jp.physoc.org/cgi/content/full/jphysiol.2004.062091v1/DC1 and contains supplementary material consisting of a figure entitled: The effect of AMPA measured by volumetric and shape factor analyses.
This material is also available at:
http://blackwellpublishing.com/products/journals/suppmat/tjp/tjp351/tjp351sm.htm
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References |
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 D. A. Richards, J. M. Mateos, S. Hugel, V. de Paola, P. Caroni, B. H. Gahwiler, and R. A. McKinney Glutamate induces the rapid formation of spine head protrusions in hippocampal slice cultures PNAS, April 26, 2005; 102(17): 6166 - 6171. [Abstract] [Full Text] [PDF]
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 K. Okamura, H. Tanaka, Y. Yagita, Y. Saeki, A. Taguchi, Y. Hiraoka, L.-H. Zeng, D. R Colman, and N. Miki Cadherin activity is required for activity-induced spine remodeling J. Cell Biol., December 6, 2004; 167(5): 961 - 972. [Abstract] [Full Text] [PDF]
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, static spines, 
, motile spines; •, AMPA treatment; 
, NBQX treatment; 
, latrunculin treatment; 
, spines treated with both latrunculin and NBQX. A strong correlation between spine motility and FRAP t
