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.     

Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis

It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of <10 NMDA receptors. The frequency of these miniature synaptic Ca2+ transients is not randomly distributed across synapses, as some synapses can display a >10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.     

Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.     

Prostaglandin E₂ regulates AMPA receptor phosphorylation and promotes membrane insertion in preoptic area neurons and glia during sexual differentiation

Sexual differentiation of the rodent brain is dependent upon the organizing actions of the steroid hormone, estradiol. In the preoptic area, a brain region critical for the expression of adult reproductive behavior, there are twice as many dendritic spine synapses per unit length on newborn male neurons compared to female neurons and this sex difference correlates with the expression of adult male copulatory behavior. The sex difference in the POA is achieved via estradiol''s upregulation of the membrane-derived lipid signaling molecule prostaglandin E₂ (PGE₂); PGE₂ is necessary and sufficient to masculinize both dendritic spine density and adult sexual behavior in rats. We have previously shown that PGE₂ activates EP₂ and EP₄ receptors which increases protein kinase A (PKA) activity and that masculinized dendritic spine density and sex behavior are both dependent upon PKA as well as activation of AMPA type glutamate receptors. In the current experiments, we build upon this signaling cascade by determining that PGE₂ induces phosphorylation of the AMPA receptor subunit, GluR1, which leads to increased AMPA receptor insertion at the membrane. Treating female pups on the day of birth with PGE₂ induced the phosphorylation of GluR1 at the PKA-sensitive site within 2 hours of treatment, an effect that was blocked by co-administration of the PKA/AKAP inhibitor, HT31 with PGE₂. Brief treatment of mixed neuronal/glial POA cultures with PGE₂ or the cAMP/PKA stimulator, forskolin, increased membrane associated GluR1 in both neurons and glia. We speculate that PGE₂ induced increases in AMPA receptor associated with the membrane underlies our previously observed increase in dendritic spine density and is a critical component in the masculinization of rodent sex behavior.     

Estradiol induces formation of dendritic spines in hippocampal neurons: functional correlates

Dissociated cultured rat hippocampal pyramidal neurons respond to estradiol with a time-dependent, twofold increase in density of their dendritic spines. This effect is mediated by an estrogen receptor, probably of the alpha nuclear receptor type. In searching for the molecular mechanisms leading from the initial activation of the estrogen receptor to the final formation of new dendritic spines, we found that estradiol acts on GABAergic interneurons expressing the estrogen receptor by decreasing their inhibitory tone. In culture, this is assumed to cause a shift in the balance between excitation and inhibition toward enhanced excitation, overactivation of the pyramidal neurons, and subsequent formation of novel dendritic spines. The action of estradiol on spine formation is mediated by phosphorylation of cyclic AMP response element binding protein in the pyramidal neurons and is blocked when inhibition is enhanced by diazepam and when excitation is blocked by tetrodotoxin. Progesterone blocks the effect of estradiol on dendritic spines through its conversion to tetrahydroprogesterone, which enhances GABAergic inhibition. Subsequent to formation of novel dendritic spines, there is an increase in the density of glutamatergic receptors in the affected cells, an increase in the cellular calcium response to glutamate, and an increase in network synaptic activity among the cultured neurons.     

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.     

Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses.

The protein brain-derived neurotrophic factor (BDNF) has been postulated to be a retrograde or paracrine synaptic messenger in long-term potentiation and other forms of activity-dependent synaptic plasticity. Although crucial for this concept, direct evidence for the activity-dependent synaptic release of BDNF is lacking. Here we investigate secretion of BDNF labelled with green fluorescent protein (BDNF-GFP) by monitoring the changes in fluorescence intensity of dendritic BDNF-GFP vesicles at glutamatergic synaptic junctions of living hippocampal neurons. We show that high-frequency activation of glutamatergic synapses triggers the release of BDNF-GFP from synaptically localized secretory granules. This release depends on activation of postsynaptic ionotropic glutamate receptors and on postsynaptic Ca(2+) influx. Release of BDNF-GFP is also observed from extrasynaptic dendritic vesicle clusters, suggesting that a possible spatial restriction of BDNF release to specific synaptic sites can only occur if the postsynaptic depolarization remains local. These results support the concept of BDNF being a synaptic messenger of activity-dependent synaptic plasticity, which is released from postsynaptic neurons.     

Backpropagating action potentials enable detection of extrasynaptic glutamate by NMDA receptors

Synaptic NMDA receptors (NMDARs) are crucial for neural coding and plasticity. However, little is known about the adaptive function of extrasynaptic NMDARs occurring mainly on dendritic shafts. Here, we find that in CA1 pyramidal neurons, back-propagating action potentials (bAPs) recruit shaft NMDARs exposed to ambient glutamate. In contrast, spine NMDARs are "protected," under baseline conditions, from such glutamate influences by peri-synaptic transporters: we detect bAP-evoked Ca(2+) entry through these receptors upon local synaptic or photolytic glutamate release. During theta-burst firing, NMDAR-dependent Ca(2+) entry either downregulates or upregulates an h-channel conductance (G(h)) of the cell depending on whether synaptic glutamate release is intact or blocked. Thus, the balance between activation of synaptic and extrasynaptic NMDARs can determine the sign of G(h) plasticity. G(h) plasticity in turn regulates dendritic input probed by local glutamate uncaging. These results uncover a metaplasticity mechanism potentially important for neural coding and memory formation.     

Differential regulation of dendrite complexity by AMPA receptor subunits GluR1 and GluR2 in motor neurons

Activity-dependent developmental mechanisms in many regions of the central nervous system are thought to be responsible for shaping dendritic architecture and connectivity, although the molecular mechanisms underlying these events remain obscure. Since AMPA glutamate receptors are developmentally regulated in spinal motor neurons, we have investigated the role of activation of AMPA receptors in dendritic outgrowth of spinal motor neurons by overexpression of two subunits, GluR1 and GluR2, and find that dendrite outgrowth is differentially controlled by expression of these subunits. Overexpression of GluR1 was associated with greater numbers of filopodia, and an increase in the length and complexity of dendritic arbor. In contrast, GluR2 expression did not alter dendritic complexity, but was associated with a moderate increase in length of arbor, and decreased numbers of filopodia. Neither GluR1 nor GluR2 had any effect on the motility of filopodia. In addition, GluR1 but not GluR2 expression increased the density of dendritic puncta incorporating a GFP-labeled PSD95, suggesting that GluR1 may mediate its effect in part by augmenting the number of excitatory synapses within motor neuron dendrites. Together these results suggest that in spinal motor neurons, AMPA receptors composed of GluR1 subunits may facilitate neurotrophic mechanisms in these neurons, permitting sustained dendrite outgrowth and synaptogenesis, whereas expression of AMPA receptors containing GluR2 acts to preserve existing dendritic arbor. Thus, the observed downregulation of GluR1 in motor neurons during postnatal development may limit the formation of new dendrite segments and synapses, promoting stabilized synaptic connectivity.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex

A key feature of the mammalian brain is its capacity to adapt in response to experience, in part by remodeling of synaptic connections between neurons. Excitatory synapse rearrangements have been monitored in vivo by observation of dendritic spine dynamics, but lack of a vital marker for inhibitory synapses has precluded their observation. Here, we simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbor of pyramidal neurons in the adult mammalian cortex using large-volume, high-resolution dual-color two-photon microscopy. We find that inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience. Further, we find inhibitory synapse and dendritic spine remodeling to be spatially clustered and that clustering is influenced by sensory input. Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.     

Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus

Recent evidence indicates that, in addition to their well known effects on neurons, gonadal steroids may exert part of their neural effects through astroglia. In adult female rats astroglia participate in the phasic remodelling of synapses that takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the influence of estradiol. Astroglia also appear to be involved in the genesis of sex differences in synaptic connectivity. Gonadal steroids influence hypothalamic astroglia differentiation in vitro and in vivo. In monolayer mixed neuronal-glial cultures from fetal rat hypothalami, estradiol induces a progressive differentiation of astrocytes from a flattened epithelioid morphology to bipolar, radial and stellate shapes. This effect of estradiol on astroglia is dependent on the expression of specific molecules on the neuronal surface, such as the polysialic acid-rich form of the neural cell adhesion molecule. In the rat arcuate nucleus in situ, perinatal androgen influences astroglia gene expression and differentiation, resulting in a sex difference in astroglia organization by postnatal day 20. By this day, the amount of neuronal surface covered by astroglial processes is higher in males than in females. This difference in the coverage of neuronal surface by astroglia may be directly related to the reduced number of synaptic contacts that is established on the soma of male neurons compared to females.     

Cellular mechanisms of estradiol-mediated masculinization of the brain

The sexual differentiation of reproductive physiology and behavior in the rodent brain is largely determined by estradiol aromatized from testicular androgens. The cellular mechanisms by which estradiol masculinizes the brain are beginning to emerge and revealing novel features of brain development that are highly region-specific. In the preoptic area, the major site controlling male sexual behavior, estradiol increases the level of the COX-2 enzyme and its product, prostaglandin E2 which promotes dendritic spine synaptogenesis. In the ventromedial nucleus of the hypothalamus, the major site controlling female reproductive behavior, estradiol promotes glutamate release from synaptic terminals, activating NMDA receptors and the MAP kinase pathway. In the arcuate nucleus, a major regulator of anterior pituitary function, estradiol increases GABA synthesis, altering the morphology of neighboring astrocytes and reducing formation of dendritic spines synapses. Glutamate, GABA and the importance of neuronal-astrocytic cross-talk are emerging as common aspects of masculinization. Advances are also being made in the mechanistic basis of female brain development, although the challenges are far greater.     

CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors

Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. Here, we show that cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. Our results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses.     

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.     

Dynamics of postsynaptic glutamate receptor targeting

Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.     

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。     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Casting a net on dendritic spines: the extracellular matrix and its receptors

Dendritic spines are dynamic structures that accommodate the majority of excitatory synapses in the brain and are influenced by extracellular signals from presynaptic neurons, glial cells, and the extracellular matrix (ECM). The ECM surrounds dendritic spines and extends into the synaptic cleft, maintaining synapse integrity as well as mediating trans-synaptic communications between neurons. Several scaffolding proteins and glycans that compose the ECM form a lattice-like network, which serves as an attractive ground for various secreted glycoproteins, lectins, growth factors, and enzymes. ECM components can control dendritic spines through the interactions with their specific receptors or by influencing the functions of other synaptic proteins. In this review, we focus on ECM components and their receptors that regulate dendritic spine development and plasticity in the normal and diseased brain.