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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Curr Opin Neurobiol. 2010 Sep 9;21(1):175–181. doi: 10.1016/j.conb.2010.08.013

The Dynamic Cytoskeleton: Backbone of Dendritic Spine Plasticity

Erik W Dent 1,2, Elliott B Merriam 1, Xindao Hu 1
PMCID: PMC3010448  NIHMSID: NIHMS235541  PMID: 20832290

Summary

Dendritic spines are small actin-rich protrusions on the surface of dendrites whose morphological and molecular plasticity play key roles in learning and memory. Both the form and function of spines are critically dependent on the actin cytoskeleton. However, new research, using electron microscopy and live-cell super-resolution microscopy indicates that the actin cytoskeleton is more complex and dynamic than originally thought. Also, exciting recent studies from several labs indicate that microtubules, once thought to be restricted to the dendrite shaft, can make excursions into the most distal regions of dendritic spines. Moreover, microtubule invasions of spines appear to be associated with changes in synaptic activity. Thus, it is likely that dynamic interactions between microtubules and actin filaments within dendritic spines play important roles in dendritic spine plasticity.

Introduction

Organisms must be able to modify their synaptic connections during development and in adulthood. This synaptic plasticity is essential for the refinement of connections during the construction of a functional nervous system as well as for learning and memory throughout life. When processes involved in synaptic plasticity go awry, either in disease or during aging, dysfunction of the organism results. Myriad studies have provided evidence for both short- and long-term changes in synaptic efficacy. Both types of synaptic changes can be regulated at the level of either the presynaptic axon or the postsynaptic dendritic spine. Synaptic plasticity is accompanied by a change in the molecular composition of the synapse that results in either more or less efficient synaptic transmission, sometimes being regulated by changes in the morphology of dendritic spines[1].

Dendritic spines are micron-sized actin-rich protrusions on dendrites that serve as the primary source of synaptic contacts for excitatory neurons in the nervous system. Dendritic protrusions have generally been classified morphologically into several types, including filopodia, stubby spines, thin spines, mushroom-shaped spines and complex spines [2-7]. However, these shapes are not fixed. In the rodent brain most spines maintain their relative shape throughout life, although spines are capable of converting into different shapes or even appearing/disappearing [8-10]. Regardless of the ability of spines to morph into different shapes or remain stable, it is clear that the turnover of proteins, lipids and organelles in spines is an ongoing and dynamic process[11,12].

Dynamics of both actin filaments and microtubules are important for providing the structure for cellular shape changes. Moreover, changes in structure influence function. Since dendritic spines are rich in actin filaments that undergo dynamic assembly and disassembly, it follows that both the structure and function of spines is dictated by actin dynamics. But what about the function of microtubule dynamics in spine function? In this review we will present new evidence for a complex actin organization and dynamics in spines and also evaluate several recent studies that demonstrate microtubules are capable of entering dendritic spines in a dynamic, activity-dependent fashion.

Actin organization and dynamics in dendritic spines

Filamentous actin (f-actin) is the primary cytoskeletal element in dendritic spines and there are a plethora of actin-associated proteins whose functional interactions with the actin cytoskeleton are an active area of investigation [13-18]. Here we will touch upon several new studies that have focused directly on actin architecture and dynamics. The enrichment of actin in dendritic spines has been known since the early 1980’s [19-21]. One of these studies showed, with myosin-S1 decoration, that f-actin was primarily arrayed with the barbed (fast growing) end toward the plasma membrane and the actin filaments were generally short and arranged in a crosshatched and branched formation [21].

A recent study has confirmed and extended these studies using platinum replica electron microscopy to show that dendritic filopodia, which can mature into spines, have a very different actin network structure from most other filopodia, including those in non-neuronal cells and axonal growth cones [22*]. Instead of having parallel actin filaments that are oriented with barbed ends toward the tip of the filopodium, dendritic filopodia appear to form from a mixed network of both linear and branched actin filaments that extend to form a filopodium. Furthermore, in contrast to other filopodia, they appear to contain concentrations of Arp2/3, capping protein and myosin II, while lacking fascin [22*]. Fascin bundles linear actin filaments that are oriented in the same direction, while myosin II can only organize actin filaments of opposite polarity. Arp2/3 and capping protein are necessary to form branched actin filaments and maintain their short length, respectively. These data led the authors to propose a model in which dendritic filopodia and spines contain a mixed array of linear and branched actin filaments throughout the spine head, neck and base. Interestingly, they also discovered that actin filaments appear to be interdigitated among the microtubules present in the dendrite shaft, which may have implications for microtubule entry into dendritic spines.

Although electron micrographs provide extremely high resolution images of dendritic spines, they can only hint at how dynamic processes such as actin polymerization and depolymerization might be regulated in spines. Previous studies have shown that actin is in a very dynamic state in spines, with a turnover time of less than a minute [23]. Actin dynamics are also important for spine plasticity. Several previous studies have documented that actin polymerization is necessary for long-term potentiation (LTP) and is reduced following induction of long-term depression (LTD) [24,25]. Thus, determining where actin is polymerized and depolymerized within spines is critical for understanding how it regulates spine structure and function. However, these studies did not have the ability to determine how actin dynamics were regulated in sub-regions of spines.

Several recent studies have shed light on where actin is polymerized and depolymerized in different regions of the spine. An elegant study by the Kasai lab, using two photon uncaging of photoactivatable actin (PA-β-actin) in hippocampal slices, showed that there are at least three pools of f-actin in spines [26**]. The first is a dynamic pool (time constant ~40 sec.) that polymerizes near the tip of the spine and treadmills toward the center of the spine (Fig. 1a). This was the first direct demonstration that actin in spines undergoes treadmilling in a manner similar to actin in growth cones of cortical neurons [27] and newly differentiated hippocampal neurons [28]. However, the actin retrograde flow in spines (~1μm/min) is slower than that of actin in younger mammalian CNS neurons (6-8μm/min). This slower flow could be due to a different structure of actin filaments or fewer or less efficient coupling of myosin II motors [29,30] to the actin cytoskeleton. The second pool of actin was a relatively stable pool (time constant ~17 min.) that was located at the base of the spine and varied according to spine volume [26**]. The third pool of actin, termed the enlargement pool, was induced by uncaging glutamate on an individual spine and turned over relatively slowly (2-15 min.). The increase in this pool of actin was necessary but not sufficient for a long-term increase in spine size, as it was also dependent on the confinement of the pool by the spine neck.

Figure 1.

Figure 1

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Model for dynamism of f-actin and microtubules in dendritic spines.

(a) Model of a dendritic spine showing the possible arrangement of actin filaments in the spine head, neck and dendritic shaft. Actin takes the form of short filaments with several of the filaments having their barbed (fast growing) ends near the membrane. Note that actin filaments do not have uniform polarity either in the head or shaft. The actin polymerizes at the membrane forcing a flow of actin toward the center of the spine. Myosin may play a role in this actin flow as well. (b) Image of a dendrite from a hippocampal neurons transfected with DsRed and EGFP-tubulin (DIV18). (c) Time-lapse images of the spine demarcated by the box in (b). DsRed label shows the morphology of the spine, while EGFP-tubulin labels microtubules. One dynamic microtubule extends into and retracts from the spine over a period of 40 seconds. (d) Model showing how a microtubule polymerizes into and retracts from a dendritic spine. Microtubule entry is due to polymerization of the dynamic end of the microtubule into the spine. EB3, an important microtubule +TIP protein, binds to the ends of polymerizing microtubules in both the spine and the dendrite shaft. Note that microtubules can extend to the most distal regions of the spine and have even been shown to deform the spine head membrane, forming a transient spine head protrusion.

Using a barbed-end polymerization assay and FRAP analysis another study showed that actin was capable of polymerizing at the spine tip, confirming the study by Kasai and colleagues [26**], but also demonstrating that actin can be polymerized at the base of the spine [31*]. However, actin polymerization in the base of the protrusion occurred more frequently in filopodia from less mature cultures and much less frequently in dendritic spines. Recently, another study, using single molecule tracking of fluorescent actin monomers that polymerized within otherwise non-fluorescent actin filaments, showed that actin filament treadmilling within dendritic spines is a complex and varied process that takes place throughout the spine head and shaft [32*]. However, unlike the polarized actin filament array observed in the lamellipodium of fibroblasts [32*] or newly differentiated neurons [28] these filaments were much less polarized, with only a small portion of filaments exhibiting flow (0.5-2μm/min) in any one direction. This study is both consistent with the older study that used electron microscopy [21] and the speed of actin treadmilling measured with confocal microscopy [26**].

Blanpied and colleagues have recently expanded on these findings, using single particle tracking and photoactivated localization microscopy (PALM) [33,34] of a photoswitchable actin probe (actin-mEos) to track the movements of individual actin molecules in different subregions of dendritic spines [35**]. Using this approach, the authors found that actin filaments participate in retrograde flow from the periphery towards a central region of the spine, roughly where the spine’s head meets its neck. Importantly, they found that actin filaments are particularly dynamic near the post-synaptic density (PSD) but not near endocytic zones, suggesting that stability of the PSD is conferred by an active process of continuous turnover in the actin network. Whereas, clathrin-mediated endocytosis depends on either a relatively stable f-actin network or on transient, sparsely occurring actin polymerization events.

Together these studies paint a picture of a relatively dynamic and complex actin filament array that is essential for the functional readout of the dendritic spine. Generally, it has been thought that microtubules, which are rich in the dendrite shaft, do not make excursions into dendritic spines. Consequently, microtubules were not thought to play any role in dendritic spines directly. In many other systems however, actin filaments and microtubules participate intimately in many processes [36,37]. Modern advances in high-resolution live-cell microscopy have permitted a reinvestigation of cytoskeletal dynamics in dendritic spines. Indeed, several new studies have now shown that microtubules do in fact enter dendritic spines, opening up the possibility that actin/microtubule interactions are essential for spine function as well.

History of microtubule involvement in dendritic spines

The first studies to hint at a role for microtubules in dendritic spines were by Gray and Westrum in the early 1980’s [38-40]. These studies showed with electron microscopy that microtubules could penetrate dendritic spines of neurons within intact regions of cortex, sometimes extending directly into the electron-dense post-synaptic density (PSD) [38,40]. However, these results were called into question for a number of reasons. First, the regions of cortex were dissected directly into a hypotonic solution of 20% BSA in water. This unusual procedure may have caused spurious polymerization of microtubules into spines. Furthermore, several recent studies failed to show microtubule entry into spines under physiological conditions.

In one study Harris and colleagues were able to show, using serial reconstruction electron microscopy, that microtubules could enter spines, but only the uniquely branched spines within the CA3 region in the hippocampus [41]. Another study by the same group using similar techniques showed that dendritic microtubules depolymerize after sectioning at 4°C, but after warming of the slices they appear in approximately 10-15% of hippocampal spines within the first half-hour after cutting the slices [42]. However, this study failed to find microtubules in dendritic spines of similar slices at three hours after slice equilibration or in hippocampi of animals that were directly perfused with fixative. These results led them to conclude that although microtubule entry into dendritic spines could occur, spines were devoid of microtubules under physiological conditions. Nonetheless it is possible that a population of highly dynamic, unstable microtubules were depolymerized during the fixation process, making the subcellular localization of these microtubules impossible to determine.

The absence of microtubules in spines was corroborated by two studies from Matus and colleagues, who examined mature dissociated hippocampal neurons transfected with actin and the microtubule associated protein MAP2c fused to different fluorescent proteins [43,44]. In both fixed and live cells these authors observed that MAP2c-labeled microtubules did not enter dendritic spines. However, they also found little microtubule dynamics in dendrite shafts. This may have been due to a stabilizing effect of overexpression of a structural MAP. Alternatively, MAP2c may preferentially label stable regions of microtubule that do not undergo dynamic bouts of polymerization or depolymerization. More recently, the microtubule end binding protein EB3 (fused to EGFP) was imaged in 10DIV hippocampal neurons, revealing that microtubules are indeed dynamic in younger dendrites, though this study made no mention of microtubule tip entry into dendritic protrusions [45]. In light of this study, it is likely that the methods employed in earlier electron microscopy and fluorescence imaging studies precluded detection of highly dynamic microtubules, resulting in an incomplete understanding of the activities of microtubules in neuronal dendrites and spines.

Evidence for microtubule entry into dendritic spines

Recently, four independent studies published within a one year period showed that microtubules enter spines under physiological conditions in cultured hippocampal [46**-48**] and cortical neurons [47**], as well as after tetanic stimulation of hippocampal slices [49*]. Using different methodologies, each of these studies showed that microtubule entry into spines was likely to be dependent on synaptic activity. Mitsuyama and colleagues demonstrated via electron microscopy that strong tetanic stimulation in the CA1 region of hippocampus caused dendrites to assume an undulating shape and an enlargement of postsynaptic density (PSD) [49*]. Accompanying these morphological changes to CA1 dendrites was the presence of microtubules in dendritic spines, oftentimes protruding into the PSD itself. Many studies have documented changes in spines after strong stimulation protocols but have not shown microtubule entry. What made this study different? The authors suggested that using a microtubule-stabilizing fixative was essential for preserving the otherwise labile microtubules that entered dendritic protrusions, while the use of conventional fixatives did not preserve this population of microtubules [49*]. This was similar in reasoning, although different in methodology, to the earlier studies of Gray and Westrum [38-40]. Another unanswered question from these studies was whether microtubules polymerized into spines from the dendrite shaft or were locally nucleated within the spine and then polymerized into the dendritic shaft, as suggested by others [50].

To address this issue by direct observation within living neurons, another group used time-lapse total internal reflection fluorescence microscopy (TIRFM) to directly observe microtubule polymerization from the dendritic shaft into spines [47**]. By labeling microtubules with EGFP-tubulin and imaging them at high resolution and quick succession Dent and colleagues showed that microtubule entry into dendritic protrusions (both spines and filopodia) occurred in only a small percentage of protrusions at any one time (~1% of spines) and was transient, averaging about three minutes per invasion (ranging from 20 seconds to ~30 min.) (Fig. 1b-c). By imaging microtubule dynamics, with either EGFP-tubulin or EB3-EGFP, a microtubule end binding protein, they documented that 10% of dendritic protrusions were targeted by microtubules in an hour [47**] (Fig. 1d). Extrapolating these results suggests that most dendritic protrusions would be targeted by microtubules over the course of a day. However, it is not known if some spines are preferentially targeted or if others are never targeted. Surprisingly, microtubule invasion could induce transient extensions from spine heads in a small number of cases [47**]. Microtubule invasion of spines appears to be an ongoing process because it occurred in very mature hippocampal neurons (DIV63). This is surprising because maintaining microtubules in a dynamic state is an energy intensive process governed by GTP hydrolysis, suggesting that microtubule dynamics probably serves important functions in mature dendrites. It remains unknown whether microtubule invasion of spines occurs in an intact animal throughout its lifespan, although with techniques such as two-photon imaging of cortical dendrites in living mice this hypothesis could be tested.

Microtubule entry into dendritic spines is activity-dependent

Based on the results of the Mitsuyama study [49*] it is likely that microtubule entry into dendritic spines is dependent on synaptic activity and contributes to synapse plasticity and/or formation. Three separate studies, using different methods of stimulating activity, showed that this is indeed the case [46**-48**]. Zheng and colleagues documented the presence of microtubules in dendritic spines with confocal microscopy of fixed cultures, and used BDNF to stimulate activity in these cultures [46**]. In two week old hippocampal cultures they showed that the increase in density of dendritic spines induced by BDNF treatment was augmented by pharmacological stabilization of microtubules and mitigated by pharmacological inhibition of microtubule dynamics [46**]. These results were corroborated through shRNA knockdown of the +TIP protein EB3, which also decreased spine density [46**,51]. However, in a study by Hoogenraad and colleagues, EB3 knockdown with shRNA, EB3 overexpression and pharmacological inhibition of microtubule dynamics resulted in no change to spine density. Rather, EB3 shRNA caused an increase in filopodia at the expense of spines and EB3 overexpression promoted spine development at the expense of filopodia [48**]. At present, it is unclear why these two studies, using similar techniques to disrupt EB3, obtained conflicting results with regard to spine formation and maintenance.

Another study showed that microtubule entry of spines was increased three-fold after transient KCl applications to stimulate cultured hippocampal neurons [47**]. This increase in microtubule invasions was abolished by inclusion of TTX, indicating it was action potential driven. It is likely that more physiological changes in synaptic activity, such as LTP, homeostatic plasticity and BDNF application, also cause changes in the kinetics of microtubule invasion of spines (Merriam, Hu and Dent, unpublished observations). Indeed, pharmacological inhibition of microtubule dynamics in hippocampal slice cultures could inhibit the maintenance of LTP [48**]. Nevertheless, bath application of pharmacological compounds to inhibit microtubule dynamics or knockdown of microtubule-associated proteins, such as EB3, will affect microtubule dynamics throughout the entire neuron and surrounding glia and potentially disrupt microtubule-based transport as well [46**,48**]. Thus, it is not known what specific role microtubule entry into dendritic spines plays in either BDNF induced plasticity or LTP. Further experiments that disrupt microtubule dynamics locally within individual dendritic spines, or small numbers of spines, will be necessary to determine if microtubule invasion of spines is essential for different forms of synaptic plasticity.

Conclusions and future directions

Actin is clearly essential for dendritic spine structure and function. Recently, several elegant studies have confirmed this by providing images of actin structure and dynamics at unprecedented detail with platinum replica electron microscopy [22*], two-photon confocal imaging of photactivated actin [26**], single molecule imaging of actin dynamics [32*] and single particle tracking PALM [35**]. Taken together, these studies indicate that actin in dendritic spines forms a complex, dynamic meshwork that is likely to be regulated differentially in sub-compartments of the spine. Further work will be necessary to functionally integrate all of the actin-associated proteins that are present in the spine at this level of detail, but will undoubtedly yield new surprises in dendritic spine function.

Interestingly, new studies show that microtubules enter dendritic spines in both hippocampal and cortical neurons and may be important for their morphological and functional plasticity [46**-49*]. As with other interesting studies, the findings raise more questions than they answer. Two important questions that remain are 1) how do microtubules enter spines, and 2) what function do they serve upon entry? Although we now know that MTs polymerize from the dendrite shaft into spines [47**,48**], how do they polymerize from the dendritic shaft, through a thin spine neck, into the head of the spine? Clues from the aforementioned studies indicate that actin filaments and associated proteins are likely to play an important role [13]. In fact, one study has shown that the relatively little studied protein p140Cap and the actin associated protein cortactin are likely to form a complex that regulates interactions with microtubules through EB3 [48**]. It will be interesting to determine if actin filaments in dendritic spines act as a barrier for, a scaffold for, or do not affect microtubule invasion of spines. Likely, based on what is known about the complexity of actin dynamics in spines, there will prove to be very finely tuned and complex interactions between these two cytoskeletal elements.

The function microtubules serve by transiently targeting dendritic spines is likely to involve transport of essential proteins into and out of the spine, since microtubules are the major long-distance transport machinery inside all cells. It is possible that microtubule entry into the entire extent of dendritic protrusions, either spines or filopodia, may play an important role in “tagging” synapses, or synaptic precursors, for modification. This tagging could result in the specific kinesin-mediated delivery and/or dynein-mediated removal of receptors, structural proteins, mRNA, GTPase effectors or organelles that may be required for synaptic development or plasticity. Since kinesin and dynein driven transport is an order of magnitude faster than the rate of microtubule polymerization/depolymerization it would be possible to transport substantial cargo into or out of spines on microtubules during their relatively short excursions (minutes) into spines. This hypothesis does not imply that microtubule-driven delivery directly into spines is the only route for material destined for spines. For example, it is established that myosin motor-driven processes are essential for transport of material into spines [52,53]. However, the fact that microtubules remain dynamic and capable of entering spines throughout the lifetime of neurons suggests that they serve fundamental roles in synaptic architecture and functional plasticity.

Acknowledgments

We apologize to those authors whose work we were unable to cite due to space restrictions. We thank Witchuda Saengsawang and Thomas Fothergill for critical reading of the manuscript. Work in the authors’ laboratory is supported by grants from the National Institutes of Health (R01NS064014 to E.W.D and F31NS071816 to E.B.M), the Dana Foundation (E.W.D) and the Whitehall Foundation (E.W.D).

Footnotes

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