Dynamic microtubules promote synaptic NMDA receptor-dependent spine enlargement

Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines.     

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.     

Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.     

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.     

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.     

M1 muscarinic receptors boost synaptic potentials and calcium influx in dendritic spines by inhibiting postsynaptic SK channels

Acetylcholine release and activation of muscarinic cholinergic receptors (mAChRs) enhance synaptic plasticity in?vitro and cognition and memory in?vivo. Within the hippocampus, mAChRs promote NMDA-type glutamate receptor-dependent forms of long-term potentiation. Here, we use calcium (Ca) imaging combined with two-photon laser glutamate uncaging at apical spines of CA1 pyramidal neurons to examine postsynaptic mechanisms of muscarinic modulation of glutamatergic transmission. Uncaging-evoked excitatory postsynaptic potentials and Ca transients are increased by muscarinic stimulation; however, this is not due to direct modulation of glutamate receptors. Instead, mAChRs modulate a negative feedback loop in spines that normally suppresses synaptic signals. mAChR activation reduces the Ca sensitivity of small conductance Ca-activated potassium (SK) channels that are found in the spine, resulting in increased synaptic potentials and Ca transients. These effects are mediated by M1-type muscarinic receptors and occur in a casein kinase-2-dependent manner. Thus, muscarinic modulation regulates synaptic transmission by tuning the activity of nonglutamatergic postsynaptic ion channels.     

The life cycle of Ca(2+) ions in dendritic spines

Spine Ca(2+) is critical for the induction of synaptic plasticity, but the factors that control Ca(2+) handling in dendritic spines under physiological conditions are largely unknown. We studied [Ca(2+)] signaling in dendritic spines of CA1 pyramidal neurons and find that spines are specialized structures with low endogenous Ca(2+) buffer capacity that allows large and extremely rapid [Ca(2+)] changes. Under physiological conditions, Ca(2+) diffusion across the spine neck is negligible, and the spine head functions as a separate compartment on long time scales, allowing localized Ca(2+) buildup during trains of synaptic stimuli. Furthermore, the kinetics of Ca(2+) sources governs the time course of [Ca(2+)] signals and may explain the selective activation of long-term synaptic potentiation (LTP) and long-term depression (LTD) by NMDA-R-mediated synaptic Ca(2+).     

Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes

Dendritic spines are micron-sized membrane protrusions receiving most excitatory synaptic inputs in the mammalian brain. Spines form and grow during long-term potentiation (LTP) of synaptic strength. However, the source of membrane for spine formation and enlargement is unknown. Here we report that membrane trafficking from recycling endosomes is required for the growth and maintenance of spines. Using live-cell imaging and serial section electron microscopy, we demonstrate that LTP-inducing stimuli promote the mobilization of recycling endosomes and vesicles into spines. Preventing recycling endosomal transport abolishes LTP-induced spine formation. Using a pH-sensitive recycling cargo, we show that exocytosis from recycling endosomes occurs locally in spines, is triggered by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. Thus, recycling endosomes provide membrane for activity-dependent spine growth and remodeling, defining a novel membrane trafficking mechanism for spine morphological plasticity and providing a mechanistic link between structural and functional plasticity during LTP.     

Ca2+ signalling in postsynaptic dendrites and spines of mammalian neurons in brain slice.

Postsynaptic Ca2+ changes are involved in control of cellular excitability and induction of synaptic long-term changes. We monitored Ca2+ changes in dendrites and spines during synaptic and direct stimulation using high resolution microfluorometry of fura-2 injected into CA3 pyramidal neurons in guinea pig hippocampal slice. When driven by current injection from an intracellular electrode or with synaptic stimulation, postsynaptic Ca2+ accumulations were highest in the proximal dendrites with a pronounced fall-off towards the soma and some fall-off towards more distal dendrites. Muscarinic activation by low concentrations of carbachol strongly increased intradendritic Ca2+ accumulation during directly-evoked repetitive firing. This enhancement occurred in large part because muscarinic activation suppressed the normal Ca(2+)-dependent activation of K-channels that mediates adaptation of firing. Repetitive firing of cholinergic fibers in the slice reproduced the effects of carbachol. Inhibition of acetylcholine-esterase activity by eserine enhanced the effects of repetitive stimulation of chlolinergic fibers. All effects were reversible and were blocked by the muscarinic antagonist atropine. Ca2+ accumulations in postsynaptic spines might be the basis of specificity of synaptic plasticity. With selective stimulation of few associative/comissural fibers, Ca2+ accumulated in single postsynaptic spines but not in the parent dendrite. With strong stimulation, dendrite levels also increased but spine levels were considerably higher. The NMDA-receptor antagonist AP-5 blocked Ca(2+)-peaks in spines, but left Ca2+ changes in dendrite shafts largely unaffected. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, developed with repeated stimulation. Our results demonstrate a spine entity that can act independent from the dendrite with respect to Ca(2+)-dependent processes. Muscarinic augmentation of dendritic Ca2+ levels might reduce diffusional loss of Ca2+ from hot spines into the parent dendrite, thus supporting cooperativity and associativity of synaptic plasticity.     

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

Dendritic spine geometry can localize GTPase signaling in neurons

Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.     

Spine motility. Phenomenology,mechanisms, and function

Throughout the history of neuroscience, dendritic spines have been considered stable structures, but in recent years, imaging techniques have revealed that spines are constantly changing shape. Spine motility is difficult to categorize, has different forms, and possibly even represents multiple phenomena. It is influenced by synaptic transmission, intracellular calcium, and a multitude of ions and other molecules. An actin-based cascade mediates this phenomenon, and while the precise signaling pathways are still unclear, the Rho family of GTPases could well be a "common denominator" controlling spine morphology. One role of spine motility might be to enable a searching function during synaptogenesis, allowing for more efficacious neuronal connectivity in the neuronal thicket. This idea revisits concepts originally formulated by Cajal, who proposed over a hundred years ago that spines might help to increase and modify synaptic connections.     

Do thin spines learn to be mushroom spines that remember?

Dendritic spines are the primary site of excitatory input on most principal neurons. Long-lasting changes in synaptic activity are accompanied by alterations in spine shape, size and number. The responsiveness of thin spines to increases and decreases in synaptic activity has led to the suggestion that they are 'learning spines', whereas the stability of mushroom spines suggests that they are 'memory spines'. Synaptic enhancement leads to an enlargement of thin spines into mushroom spines and the mobilization of subcellular resources to potentiated synapses. Thin spines also concentrate biochemical signals such as Ca(2+), providing the synaptic specificity required for learning. Determining the mechanisms that regulate spine morphology is essential for understanding the cellular changes that underlie learning and memory.     

Ca(2+) signaling in dendritic spines

Recent studies have revealed that Ca(2+) signals evoked by action potentials or by synaptic activity within individual dendritic spines are regulated at multiple levels. Ca(2+) influx through glutamate receptors and voltage-sensitive Ca(2+) channels located on spines depends on the channel subunit composition, the activity of kinases and phosphatases, the local membrane potential and past patterns of activity. Furthermore, sources of spine Ca(2+) interact nonlinearly such that activation of one Ca(2+) channel can enhance or depress the activity of others. These studies have revealed that each spine is a complex and partitioned Ca(2+) signaling domain capable of autonomously regulating the electrical and biochemical consequences of synaptic activity.     

Reversible, activity-dependent targeting of profilin to neuronal nuclei

The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.     

The ventral lateral geniculate nucleus,pars lateralis of the rat

Summary The synaptic organization of the pars lateralis portion of the ventral lateral geniculate nucleus is similar to that of other thalamic nuclei. There are four types of synaptic knobs (RL, RS, F1, F2). RL knobs are large and irregularly shaped, contain round synaptic vesicles and make multiple asymmetrical junctions. They are found primarily in synaptic islands making contact with gemmules, spines, small dendrites, and other synaptic profiles containing pleiomorphic synaptic vesicles (F2). Smaller RS knobs contain round vesicles and make asymmetrical junctions with the same type of elements as RL knobs, with the exception of the F2 profiles, but are seldom found in synaptic islands. F1 knobs contain flattened synaptic vesicles and form symmetrical junctions with F2 knobs, gemmules, spines, and small-medium dendrites in synaptic islands, throughout the neuropil, and on the proximal dendrites and soma of the largest type of neuron. F2 knobs are irregularly shaped, contain pleiomorphic synaptic vesicles and make symmetrical junctions primarily with gemmules and spines in synaptic islands. They are postsynaptic to RL and F1 knobs. Occipital decortication indicates that cortical terminals are of the RS type. Bilateral enucleation indicates that retinal terminals are of both the RL and RS type. The large amount of geographic overlap of retinal and cortical terminals on gemmules, spines, and small dendrites found in the neuropil outside of synaptic islands logically would maximize axonal sprouting between these two sources.We would like to thank Mr. Peter Rossetti for his excellent technical assistance on a major portion of this project, Ms. Judith Strauss for photographic assistance, and Ms. Nancy Wood for typing. Supported by grants NS 10579, NS 08724, 5 S01 RR 05402, and 2 T01 GM 00326     

Eph/ephrin对树突棘的调控与中枢神经系统疾病

树突棘是神经元树突上的功能性突起结构,通常作为突触后成份与投射来的轴突共同构成完整的突触连接。树突棘的形态与结构具有明显的可塑性,其变化通常会引起突触功能的改变。Eph受体酪氨酸激酶家族分子与其配体ephrin都是重要的神经导向因子,同时对树突棘结构也有直接的调控作用。Eph受体的活化可以促进树突棘的发生并影响树突棘的形态及内部结构;而Eph受体的异常也往往会损害正常的突触功能,甚至导致许多与树突棘结构异常相关的神经系统病变的发生。     

Activation of Ca2+/calmodulin-dependent protein kinase II within post-synaptic dendritic spines of cultured hippocampal neurons

To understand the cell signaling of protein kinases, it is essential to monitor their activity in each of the subcellular compartments. Here we developed a method to visualize the activities of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the cytoplasm, plasma membrane, and nucleus, separately, by utilizing targeted phosphorylation motifs and phosphorylation-specific antibodies. This approach was used to monitor the activities of post-synaptic CaMKII in cultured hippocampal neurons. Strong stimulation of the neurons by N-methyl-d-aspartate led to global activations of CaMKII in the cell bodies and dendrites. On the other hand, weak stimulation by removal of Mg(2+) block of N-methyl-d-aspartate receptors induced CaMKII signaling localized within single dendritic spines. Post-synaptic CaMKII is thought to modify synaptic efficiency. The present data for the first time demonstrate the activation of CaMKII localized within single dendritic spines and are consistent with the notion that synaptic efficiency is modified by CaMKII in single or multiple spine level depending on the strength of receptor activation.     

Structural Plasticity Can Produce Metaplasticity

Background

Synaptic plasticity underlies many aspect of learning memory and development. The properties of synaptic plasticity can change as a function of previous plasticity and previous activation of synapses, a phenomenon called metaplasticity. Synaptic plasticity not only changes the functional connectivity between neurons but in some cases produces a structural change in synaptic spines; a change thought to form a basis for this observed plasticity. Here we examine to what extent structural plasticity of spines can be a cause for metaplasticity. This study is motivated by the observation that structural changes in spines are likely to affect the calcium dynamics in spines. Since calcium dynamics determine the sign and magnitude of synaptic plasticity, it is likely that structural plasticity will alter the properties of synaptic plasticity.

Methodology/Principal Findings

In this study we address the question how spine geometry and alterations of N-methyl-D-aspartic acid (NMDA) receptors conductance may affect plasticity. Based on a simplified model of the spine in combination with a calcium-dependent plasticity rule, we demonstrated that after the induction phase of plasticity a shift of the long term potentiation (LTP) or long term depression (LTD) threshold takes place. This induces a refractory period for further LTP induction and promotes depotentiation as observed experimentally. That resembles the BCM metaplasticity rule but specific for the individual synapse. In the second phase, alteration of the NMDA response may bring the synapse to a state such that further synaptic weight alterations are feasible. We show that if the enhancement of the NMDA response is proportional to the area of the post synaptic density (PSD) the plasticity curves most likely return to the initial state.

Conclusions/Significance

Using simulations of calcium dynamics in synaptic spines, coupled with a biophysically motivated calcium-dependent plasticity rule, we find under what conditions structural plasticity can form the basis of synapse specific metaplasticity.