Temporal dynamics of NMDA receptor-induced changes in spine morphology and AMPA receptor recruitment to spines

Recent studies have shown that the activation of NMDA receptors can induce rapid changes in dendritic morphology and synaptic recruitment of AMPA receptors in dendritic spines. Here, we analyze the time course of NMDA receptor-induced changes in dendrite morphology and recruitment of AMPA receptors to synapses in cultured neurons. Activation of NMDA receptors causes a rapid transient increase in the size of preexisting spines and then the gradual formation of new dendritic protrusions and spines. NMDA receptor activation also induced GFP-tagged AMPA receptors to cluster in dendrites and to be inserted into the surface of dendritic spines. These results indicate that NMDA receptor activation induces several phases of dendritic plasticity, initial expansion of dendritic spines, followed by the de novo formation of spines and AMPA receptor dendritic clustering and surface expression on spines. Each of these forms of plasticity may have significant effects on the efficacy of synaptic transmission.     

Structural and functional plasticity of dendritic spines – root or result of behavior?

Dendritic spines are multifunctional integrative units of the nervous system and are highly diverse and dynamic in nature. Both internal and external stimuli influence dendritic spine density and morphology on the order of minutes. It is clear that the structural plasticity of dendritic spines is related to changes in synaptic efficacy, learning and memory and other cognitive processes. However, it is currently unclear whether structural changes in dendritic spines are primary instigators of changes in specific behaviors, a consequence of behavioral changes, or both. In this review, we first examine the basic structure and function of dendritic spines in the brain, as well as laboratory methods to characterize and quantify morphological changes in dendritic spines. We then discuss the existing literature on the temporal and functional relationship between changes in dendritic spines in specific brain regions and changes in specific behaviors mediated by those regions. Although technological advancements have allowed us to better understand the functional relevance of structural changes in dendritic spines that are influenced by environmental stimuli, the role of spine dynamics as an underlying driver or consequence of behavior still remains elusive. We conclude that while it is likely that structural changes in dendritic spines are both instigators and results of behavioral changes, improved research tools and methods are needed to experimentally and directly manipulate spine dynamics in order to more empirically delineate the relationship between spine structure and behavior.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.     

Electrical advantages of dendritic spines

Many neurons receive excitatory glutamatergic input almost exclusively onto dendritic spines. In the absence of spines, the amplitudes and kinetics of excitatory postsynaptic potentials (EPSPs) at the site of synaptic input are highly variable and depend on dendritic location. We hypothesized that dendritic spines standardize the local geometry at the site of synaptic input, thereby reducing location-dependent variability of local EPSP properties. We tested this hypothesis using computational models of simplified and morphologically realistic spiny neurons that allow direct comparison of EPSPs generated on spine heads with EPSPs generated on dendritic shafts at the same dendritic locations. In all morphologies tested, spines greatly reduced location-dependent variability of local EPSP amplitude and kinetics, while having minimal impact on EPSPs measured at the soma. Spine-dependent standardization of local EPSP properties persisted across a range of physiologically relevant spine neck resistances, and in models with variable neck resistances. By reducing the variability of local EPSPs, spines standardized synaptic activation of NMDA receptors and voltage-gated calcium channels. Furthermore, spines enhanced activation of NMDA receptors and facilitated the generation of NMDA spikes and axonal action potentials in response to synaptic input. Finally, we show that dynamic regulation of spine neck geometry can preserve local EPSP properties following plasticity-driven changes in synaptic strength, but is inefficient in modifying the amplitude of EPSPs in other cellular compartments. These observations suggest that one function of dendritic spines is to standardize local EPSP properties throughout the dendritic tree, thereby allowing neurons to use similar voltage-sensitive postsynaptic mechanisms at all dendritic locations.     

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.     

GSK-3β and MMP-9 Cooperate in the Control of Dendritic Spine Morphology

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3β (GSK-3β) has been implicated. Despite much evidence of the involvement of GSK-3β in functional synaptic plasticity, it is unclear how GSK-3β controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3β in neurons to investigate how GSK-3β affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3β activity increased the number of thin spines, whereas lacking GSK-3β increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3β activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3β overexpressing mice. Administration of the nonspecific GSK-3β inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3β and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3β activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3β-induced synaptic alterations.     

Rho-GTPase-activating Protein Interacting with Cdc-42-interacting Protein 4 Homolog 2 (Rich2): A NEW Ras-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (Rac1) GTPase-ACTIVATING PROTEIN THAT CONTROLS DENDRITIC SPINE MORPHOGENESIS*

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.     

Dendritic spine geometry: functional implication and regulation

Dendritic spines are tiny protrusions on dendritic shafts where most excitatory synapses are located. Recent advances in imaging technologies have given us great insight into the function of spines as biochemical compartments. Here we review recent evidence suggesting that the geometry of dendritic spines controls postsynaptic calcium signaling and is bidirectionally regulated during synaptic plasticity.     

New spines, new memories

For more than a century dendritic spines have been a source of fascination and speculation. The long-held belief that these anatomical structures are involved in learning and memory are addressed. Specifically, two lines of evidence that support this claim are reviewed. In the first, we review evidence that experimental manipulations that affect dendritic spine number in the hippocampus also affect learning processes of various sorts. In the second, we review evidence that learning itself affects the presence of dendritic spines in the hippocampus. Based on these observations, we propose that the presence of spines enhances synaptic efficacy and thereby the excitability of the network involved in the learning process. With this scheme, learning is not dependent on changes in spine density but rather changes in the presence of dendritic spines provide anatomical support for the processing of novel information used in memory formation.     

Quantitative studies on the neuron structure of the rat fascia dentata]

1. In 6 month old male rats the structure of dendrites and the distribution of spines on the apical dendrites of granular cells of the dentate gyrus were investigated by light microscopy and statistical methods. 2. The number of dendrites of the first, second and third order of granular cells increases in this sequence in a ratio of 1:2:3; the total length of the dendrites increases correspondently in a ratio of 1:4:5. 3. The mean number of origin points of dendritic branches is 10, the mean number of free dendritic endings is 12. 4. The number of spines per a 25 mum dendritic segment near to the pericaryon (dendritic segment A), in the middle of the dendrite (dendritic segment B) and in the peripheral dendritic part (dendritic segment C) as well as the distribution of spines in the whole apical dendrite was evaluated. The total mean of spines of granular cell apical dendrites of the dentate gyrus (superior respectively inferior) is 12 respectively 10 for the dendritic segment A, 18 respectively 17 for the dendritic segment B and 17 respectively 15 for the dendritic segment C calculated for a dendritic length of 25 mum. 5. The spine density in each case depends upon the distance pericaryondendritic segment and is in close relation to the adjacent layers with their specific afferents. 6. The averaged total number of spines per 1 mum of dendritic length is 0,62 spines/mum for the dentate gyrus (superior) and 0,57 spines/mum for the dentate gyrus (inferior). 7. The granular cells of the dentate gyrus (superior) have a mean dendritic length of a total of 357 mum with a total of 226 visible spines; the granular cells of the dentate gyrus (inferior) have a mean dendritic length of a total of 450 mum with a total of 258 visible spines.     

Membrane specializations of dentritic spines and glia in the weaver mouse cerebellum: a freeze-fracture study

Electron microscopy of thin-sectioned and freeze-fractured preparations of the cerebellum of the weaver mouse indicates that the dendritic spines are morphologically identical to those of their normal littermates. The weaver dendritic spines have been characterized as "unattached" since the synaptic input from the parallel fibers is absent (8-10). The entire region around the dendritic spines is taken up by astrocytic processes in the weaver. The outer fracture face of a normal dendritic spine contains aggregations of 10-nm wide particles in the immediate postsynaptic region. Similar particle aggregations occur in the unattached spines of the weaver. Freeze-fracture preparations reveal rectilinear arrays of particles, having a 7-nm center-to-center distance in the glial membranes. Rectilinear arrays are apparently distributed throughout the astrocyte membrane.     

Sampling issues in quantitative analysis of dendritic spines morphology

ABSTRACT: BACKGROUND: Quantitative analysis of changes in dendritic spine morphology has become an interesting issue in contemporary neuroscience. However, the diversity in dendritic spines population might seriously influence the results of measurements in which their morphology is studied, the detection of differences in spine morphology between control and test group is often compromised by the number of dendritic spines taken for analysis. In order to estimate how severe is such an impact we have performed Monte Carlo simulations examining various experimental setups and statistical approaches. The confocal images of dendritic spines from hippocampal dissociated cultures have been used to create a set of variables exploited as the simulation resources. RESULTS: The tabulated results of simulations are given, providing the number of dendritic spines required for the detection of hidden morphological differences between control and test group, in spine head-width, length and area. It turns out that this is the head-width among these three variables, where the changes are most easily detected. Simulation of changes occurring in a subpopulation of spines reveal the strong dependence of detectability on the statistical approach applied. The analysis based on comparison of percentage of spines in subclasses is less sensitive than the direct comparison of relevant variables describing spines morphology. CONCLUSIONS: We evaluated the sampling aspect and effect of systematic morphological variation on detecting the differences in spine morphology. Provided results may serve as a guideline in selecting the number of samples to be studied in a planned experiment. Our simulations might be a step towards the development of a standardized method of quantitative comparison of dendritic spines morphology, in which different sources of errors are considered.     

Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.     

Dendritic spines and long-term plasticity

A recent flurry of time-lapse imaging studies of live neurons have tried to address the century-old question: what morphological changes in dendritic spines can be related to long-term memory? Changes that have been proposed to relate to memory include the formation of new spines, the enlargement of spine heads and the pruning of spines. These observations also relate to a more general question of how stable dendritic spines are. The objective of this review is to critically assess the new data and to propose much needed criteria that relate spines to memory, thereby allowing progress in understanding the morphological basis of memory.     

The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.     

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.     

Dendritic spines: from structure to in vivo function

Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines.