Actin in the nervous system

Since synaptic plasticity is an important property of the brain, it is timely to try to understand the possible mechanisms underlying this phenomenon. The role of the cytoplasm for neuronal functions has until now been largely overlooked, the main emphases being on the plasma membrane for fast electrical events and on cytoplasmic organelles for the slower metabolic processes. However, recent studies on the cytoplasm of non-muscle cells have stressed the importance of contractile proteins, like actin, on maintaining the cell shape and a number of vital cellular functions, which may be related to the phase transitions in the cytoplasm. The necessary versatility is conferred on the actin networks by actin-associated proteins and by the free cytosolic calcium. In the nervous system, in addition to actin and myosin, a number of actin regulatory proteins was recently isolated, and they were shown to have properties similar to those of other non-muscle cells. Consequently, actin networks in neurons like those in non-muscle cells may be capable of contraction and phase transitions. The phase transitions have a rapid onset, and they may be quickly terminated or they may last over extended periods of time. In this way actin networks may gain control over the state of the cytoplasm and hence over the function of the neuron. Actin may be, therefore, uniquely suited to regulate various plastic reactions. The cytoplasm of growth cones and dendritic spines contains solely actin networks and is devoid of microtubules and neurofilaments. Since both these structures contain myosin and since growth cones are endowed with a considerable motility, dendritic spines also may have a likewise property. The necessary regulation of the levels of free cytosolic calcium may be provided by the spine apparatus in addition to calcium pumps in the plasma membrane and calcium regulatory proteins in the spine cytoplasm. Various types of stimulation which change the level of free cytosolic calcium may induce contraction of the spine actin network which may be responsible for the morphometric changes observed following different experimental interventions and pathological conditions. Although most of the conclusions in this review are rather speculative, they may provide directions for future research in the spine and synaptic plasticity.     

In situ localization of myosin and actin in dendritic spines with the immunogold technique

The in situ detection of macromolecules by means of immunoelectron microscopy provides information about their ultrastructural localization in cellular compartments. With this technique, we have demonstrated that the contractile proteins actin and myosin are both localized in dendritic spines at densities exceeding those of other neuronal compartments. Myosin was associated with actin filaments, with spine plasma membrane, and with membranes of the spine apparatus. Given the dynamic properties of actin and myosin, these data suggest that these proteins may be involved in the mechanism of synaptic plasticity in general and in morphometric change resulting from intense synaptic activation in particular.     

Myosin IIb controls actin dynamics underlying the dendritic spine maturation

Precise control of the formation and development of dendritic spines is critical for synaptic plasticity. Consequently, abnormal spine development is linked to various neurological disorders. The actin cytoskeleton is a structural element generating specific changes in dendritic spine morphology. Although mechanisms underlying dendritic filopodia elongation and spine head growth are relatively well understood, it is still not known how spine heads are enlarged and stabilized during dendritic spine maturation. By using rat hippocampal neurons, we demonstrate that the size of the stable actin pool increases during the neuronal maturation process. Simultaneously, the treadmilling rate of the dynamic actin pool increases. We further show that myosin IIb controls dendritic spine actin cytoskeleton by regulating these two different pools of F-actin via distinct mechanisms. The findings indicate that myosin IIb stabilizes the stable F-actin pool through actin cross-linking. Simultaneously, activation of myosin IIb contractility increases the treadmilling rate of the dynamic pool of actin. Collectively, these data show that myosin IIb has a major role in the regulation of actin filament stability in dendritic spines, and elucidate the complex mechanism through which myosin IIb functions in this process. These new insights into the mechanisms underlying dendritic spine maturation further the model of dendritic spine morphogenesis.     

β-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle γ- and β-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that γ-actin is expressed at a constant level throughout development, while the level of β-actin expression rapidly decreases with age. At the light microscopic level, γ-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, β-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, γ-actin is found in the cell body, neurites and growth cones, while β-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565–9572; Bassell et al. (1998), J. Neuroscience, 18, 251–265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of γ-actin, and also reveals an increased concentration of γ-actin in dendritic spines in the adult. During development, β-actin accumulation is observed in actively growing structures, e.g. growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, β-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.     

The roles of dendritic spine shapes in Purkinje cells

Shapes of dendritic spines are changed by various physiological or pathological states. The high degree of spine shape heterogeneity suggests that they would be the morphological basis for synaptic plasticity. An increasing number of proteins and signal transduction pathways have recently been shown to be associated with structural modifications of spines. Here, we review the possible functional roles of spine shapes in cerebellar Purkinje neurons. Several studies have suggested that spine shapes in Purkinje cells are regulated by both intrinsic and environmental factors, and different spine shapes could have significantly different consequences for brain function. Clearly constricted necks observed in thin, mushroom-shaped, and branched spines serve for compartmentalization of calcium and other second messenger molecules, influencing different signaling mechanisms and synaptic plasticity. Mushroom-shaped spines frequently have perforated postsynaptic density and the area of the spine head is much larger than simple spines, implying that membrane dynamics and receptor turnover are occurring. Branched spines might form additional synapses with afferent inputs resulting in the modification of neuronal circuits. Taken together, all these studies suggest that each spine shape is likely to have a distinct role in Purkinje cell function.     

Actin filament organization within dendrites and dendritic spines during development

The myosin S-1 subfragment was used to label actin filaments in the developing rat brain. The results show actin filaments present throughout the dendritic region with highest concentrations within growth cones and regions of spine development. Between 6 and 25 days postnatal, spines became more complex and actin filaments within them increased in number and formed a complex network. The observed organization of actin supports the hypothesis that actin has a role in the protrusion of spines from the dendrite during development.     

Interactions between drebrin and Ras regulate dendritic spine plasticity

Dendritic spines are major sites of morphological plasticity in the CNS, but the molecular mechanisms that regulate their dynamics remain poorly understood. Here we show that the association of drebrin with actin filaments plays a major role in regulating dendritic spine stability and plasticity. Overexpressing drebrin or the internal actin-binding site of drebrin in rat hippocampal neurons destabilized mature dendritic spines so that they lost synaptic contacts and came to resemble immature dendritic filopodia. Drebrin-induced spine destabilization was dependent on Ras activation: expression of constitutively active Ras destabilized spine morphology whereas drebrin-induced spine destabilization was rescued by co-expressing dominant negative Ras. Conversely, RNAi-mediated drebrin knockdown prevented Ras-induced destabilization and promoted spine maturation in developing neurons. Together these data demonstrate a novel mechanism in which the balance between stability and plasticity in dendritic spines depends on binding of drebrin to actin filaments in a manner that is regulated by Ras.     

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single‐spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.     

Subcellular distribution of the Rho-GEF Lfc in primate prefrontal cortex: effect of neuronal activation

The strength of synaptic connections in the brain varies with activity, and this plasticity depends on remodeling of the actin cytoskeleton in dendritic spines. Critical to this are the Rho family GTPases, whose activity is controlled by various modulatory proteins, including the Rho-GEF Lfc. In cultured neurons and nonneuronal cells, Lfc has been shown both to bind to microtubules and to regulate the actin cytoskeleton. Significantly, Lfc was found to be concentrated in the dendritic shafts of cultured hippocampal neurons under control conditions but then translocated into spines when neural activity was stimulated. In this study, we used immunohistochemistry and electron microscopy to examine activity-dependent changes in the distribution of Lfc in the neuropil of monkey prefrontal cortex. We found that, although Lfc was concentrated in dendrites, it also had a complex distribution in the neuropil, including being present in spines, axons, terminals, and glial processes. Moreover, Lfc distribution varied in different layers of cortex. By using an in vitro slice preparation of monkey prefrontal cortex, we demonstrated an activity-dependent translocation of Lfc from dendritic shafts to spines. The results of this study support a role for Lfc in activity-dependent spine plasticity and demonstrate the feasibility of studying activity-dependent changes in protein localization in tissue slices.     

Abl2:Cortactin Interactions Regulate Dendritic Spine Stability via Control of a Stable Filamentous Actin Pool

Dendritic spines act as the receptive contacts at most excitatory synapses. Spines are enriched in a network of actin filaments comprised of two kinetically distinct pools. The majority of spine actin is highly dynamic and regulates spine size, structural plasticity, and postsynaptic density organization. The remainder of the spine actin network is more stable, but the function of this minor actin population is not well understood, as tools to study it have not been available. Previous work has shown that disruption of the Abl2/Arg nonreceptor tyrosine kinase in mice compromises spine stability and size. Here, using cultured hippocampal neurons pooled from both sexes of mice, we provide evidence that binding to cortactin tethers Abl2 in spines, where Abl2 and cortactin maintain the small pool of stable actin required for dendritic spine stability. Using fluorescence recovery after photobleaching of GFP-actin, we find that disruption of Abl2:cortactin interactions eliminates stable actin filaments in dendritic spines, significantly reducing spine density. A subset of spines remaining after Abl2 depletion retain their stable actin pool and undergo activity-dependent spine enlargement, associated with increased cortactin and GluN2B levels. Finally, tonic increases in synaptic activity rescue spine loss following Abl2 depletion by promoting cortactin enrichment in vulnerable spines. Together, our findings strongly suggest that Abl2:cortactin interactions promote spine stability by maintaining pools of stable actin filaments in spines.SIGNIFICANCE STATEMENT Dendritic spines contain two kinetically distinct pools of actin. The more abundant, highly dynamic pool regulates spine shape, size, and plasticity. The function of the smaller, stable actin network is not well understood, as tools to study it have not been available. We demonstrate here that Abl2 and its substrate and interaction partner, cortactin, are essential to maintain the stable pool in spines. Depletion of the stable actin pool via disruption of Abl2 or cortactin, or interactions between the proteins, significantly reduces spine stability. We also provide evidence that tonic increases in synaptic activity promote spine stability via enrichment of cortactin in spines, suggesting that synaptic activity acts on the stable actin pool to stabilize dendritic spines.     

Change in the shape and density of dendritic spines caused by overexpression of acidic calponin in cultured hippocampal neurons

Dendritic spines are morphing structures believed to provide a cellular substrate for synaptic plasticity. It has been suggested that the actin cytoskeleton is the target of molecular mechanisms regulating spine morphology. Here we hypothesized that acidic calponin, an actin-binding protein, is one of the key regulators of actin filaments during spine plasticity. Our data showed that the overexpression of acidic calponin-GFP (green fluorescent protein) in primary cultures of rat hippocampal neurons causes an elongation of spines and an increase of their density as compared with those of GFP-expressing neurons. These effects required the actin-binding domains of acidic calponin. The close apposition of the presynatic marker synaptophysin to these long spines and the presence of specific postsynaptic markers actin, PSD-95, NR1, and GluR1 suggested the existence of functional excitatory synaptic contacts. Indeed, electrophysiological data showed that the postsynaptic overexpression of acidic calponin enhanced the frequency of miniature excitatory postsynaptic currents as compared with that of GFP-expressing neurons, but did not affect their properties such as amplitude, rise time, and half width. Studies in heterologous cells revealed that acidic calponin reorganized the actin filaments and stabilized them. Taken together, these findings show that acidic calponin regulates dendritic spine morphology and density, likely via regulation of the actin cytoskeleton reorganization and dynamic. Furthermore, the acidic calponin-induced spines are able to establish functional glutamatergic synapses. Such data suggest that acidic calponin is a key factor in the regulation of spine plasticity and synaptic activity.     

Nogo‐A controls structural plasticity at dendritic spines by rapidly modulating actin dynamics

Nogo‐A and its receptors have been shown to control synaptic plasticity, including negatively regulating long‐term potentiation (LTP) in the cortex and hippocampus at a fast time scale and restraining experience‐dependent turnover of dendritic spines over days. However, the molecular mechanisms and the precise time course mediating these actions of Nogo‐A are largely unexplored. Here we show that Nogo‐A signaling in the adult nervous system rapidly modulates the spine actin cytoskeleton within minutes to control structural plasticity at dendritic spines of CA3 pyramidal neurons. Indeed, acute Nogo‐A loss‐of‐function transiently increases F‐actin stability and results in an increase in dendritic spine density and length. In addition, Nogo‐A acutely restricts AMPAR insertion and mEPSC amplitude at hippocampal synaptic sites. These data indicate a crucial function of Nogo‐A in modulating the very tight balance between plasticity and stability of the neuronal circuitry underlying learning processes and the ability to store long‐term information in the mature CNS. © 2016 Wiley Periodicals, Inc.     

Cellular and subcellular distribution of spinophilin, a PP1 regulatory protein that bundles F-actin in dendritic spines

Spinophilin is an actin binding protein that positions protein phosphatase 1 next to its substrates in dendritic spines. It contains a single PDZ domain and has the biochemical characteristics of a cytoskeletal scaffolding protein. Previous studies suggest that spinophilin is present in most spines, but the concentration of spinophilin varies from brain region to region in a manner that does not simply reflect differences in spine density. Here, we show that spinophilin is enriched in the great majority of dendritic spines in cerebral cortex, caudatoputamen, hippocampal formation, and cerebellum, irrespective of regional differences in spinophilin concentration. In addition, spinophilin is present postsynaptic to asymmetrical contacts on interneuronal dendritic shafts. We further show that, in hippocampus and ventral pallidum, spinophilin is occasionally present in dendritic shafts adjacent to gamma-aminobutyric acid-containing contacts. Thus, the functional role of spinophilin may not be exclusively restricted to excitatory synapses and may be significant at a small fraction of inhibitory contacts. These data also suggest that the concentration of spinophilin per spine is variable and is likely regulated by local physiological factors and/or regional influences.     

Fast confocal imaging of calcium released from stores in dendritic spines

The emerging significance of calcium stores in neuronal plasticity and the assumed involvement of dendritic spines in long-term plastic properties of neurons have led us to examine the presence and possible regulation of calcium stores in dendritic spines. Immunohistochemical staining for ryanodine receptors was found in dendritic spines of cultured hippocampal neurons. Confocal microscopic imaging of calcium transients in dendritic spines of these neurons in response to caffeine allowed us to demonstrate an independent and unique calcium store in spines. The response to caffeine was blocked by thapsigargin and ryanodine, and maintained in calcium-free medium. The calcium stores were depleted faster in the spines than the dendrites. Furthermore, when calcium was released from stores under calcium-free conditions, and diffused passively between the spine and the dendrite, the length of the spine neck determined the degree of spine independence. Finally, the caffeine-sensitive ryanodine receptor-linked calcium store was instrumental in regulating the response of neurons to glutamate. These results have important implications for understanding the roles of dendritic spines in neuronal integration and plasticity.     

Actin dynamics in dendritic spines: a form of regulated plasticity at excitatory synapses

Dendritic spines form the postsynaptic element at most excitatory synapses in the brain. The spine cytoskeleton consists of actin filaments which, in time-lapse recordings of living neurons expressing actin labeled with green fluorescent protein, can be seen to undergo rapid, dynamic changes. Because actin dynamics are associated with changes in cell shape, these cytoskeletal rearrangements may form a molecular basis for the morphological plasticity at brain synapses. The rapidity of these dynamic events in dendritic spines raises new questions. First, do the changes in actin cytoskeleton that are visible by light microscopy really correspond to changes in spine morphology, or do they represent changes in the relationship between actin and its many binding partners at postsynaptic sites? Second, how are these changes regulated by synaptic transmission? Third, to what extent do these changes occur in organized brain tissue? Answers to these questions are now beginning to emerge.     

Developmentally regulated changes in cellular compartmentation and synaptic distribution of actin in hippocampal neurons

Actin dynamics and actin-based motility are important for neurite outgrowth and synapse plasticity. Recent work implicates actin in synapse assembly, but the morphological relationship between actin and synapses during development is unclear. Here we used developing hippocampal neurons grown in culture to examine the relationship between F- and G-actin and clusters of synaptic proteins. Both F- and G-actin are most enriched in dendritic and axonal growth cones, but only G-actin is present within the distal tips of filopodia. Outside of growth cones, F-actin levels are greater in dendrites than in axons, whereas G-actin levels are slightly greater in axons than in dendrites. The distribution of both F- and G-actin is consistent with their presence at synapses, but only F-actin levels become detectably enhanced at synaptic sites. Quantitative analyses suggest that first-forming synapses are associated with enhanced levels of pre- and postsynaptic F-actin that do not necessarily remain elevated during synapse maturation. However, nearly all mature excitatory synapses become associated with high, mostly postsynaptic concentrations of F-actin contained principally within dendritic spines. Mature shaft and GABAergic synapses are also associated with enhanced levels of F-actin, but to a lesser degree. Thus, although F-actin is essential for function and maintenance of young synapses, it need not be highly concentrated at every site. The large increase in postsynaptic F-actin concentration observed in mature neurons is likely to reflect actin's role in dendritic spine morphology and in synapse plasticity.     

Alterations in Purkinje cell spines of calbindin D-28 k and parvalbumin knock-out mice

The second messenger Ca2+ is known to act in a broad spectrum of fundamental cell processes, including modifications of cell shape and motility, through the intermediary of intracellular calcium-binding proteins. The possible impact of the lack of the intracellular soluble Ca2+-binding proteins parvalbumin (PV) and calbindin D-28 k (CB) was tested on spine morphology and topology in Purkinje cell dendrites of genetically modified mice. Three different genotypes were studied, i.e. PV or CB single knock-out (PV-/-, CB-/-) and PV and CB double knock-out mice (PV-/-CB-/-). Purkinje cells were microinjected with Lucifer Yellow and terminal dendrites scanned at high resolution with a confocal laser microscope followed by three-dimensional (3-D) reconstruction. The absence of PV had no significant effect on spine morphology, whereas the absence of CB resulted in a slight increase of various spine parameters, most notably spine length. In double knock-out mice, the absence of both PV and CB entailed a doubling of spine length, an increase in spine volume and spine surface, a higher spine density along the dendrites, as well as a more clustered spine distribution. In all three genotypes, a reduction in the number of stubby spines was observed compared with wild-type animals. These results suggest a morphological compensation for the lack of the soluble calcium buffers in the cytoplasm of Purkinje cell dendritic spines. The increase in various spine parameters, particularly volume, may counteract the lack of the calcium buffers, such as to adjust Ca2+-transients at the transitional zone between spines and dendrites.     

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2–3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon–spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2–3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2–3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon–spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.     

Actin-associated protein synaptopodin in the rat hippocampal formation: localization in the spine neck and close association with the spine apparatus of principal neurons

Dendritic spines are sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. However, little is known about the cellular mechanisms that regulate spine morphology and motility. Synaptopodin is a recently described actin-associated protein found in renal podocytes and dendritic spines (Mundel et al. J Cell Biol. [1997] 139:193-204), which is believed to play a role in spine plasticity. The present study analyzed the distribution of synaptopodin in the hippocampal formation. In situ hybridization histochemistry revealed a high constitutive expression of synaptopodin mRNA in the principal cell layers. Light microscopic immunohistochemistry showed that the protein is distributed throughout the hippocampal formation in a region- and lamina-specific manner. Postembedding immunogold histochemistry demonstrated that synaptopodin is exclusively present in dendrites and spines, specifically in the spine neck in close association with the spine apparatus. Spines lacking a spine apparatus are not immunoreactive for synaptopodin. These data suggest that synaptopodin links the spine apparatus to actin and may thus be involved in the actin-based plasticity of spines.