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.     

Synapse formation proceeds independently of dendritic elongation in cultured hippocampal neurons

In central neurons, dendritic differentiation begins well after axonal elongation and is accompanied by the compartmentation of the microtubule-associated protein 2 (MAP2) in the somatodendritic domain. Whether MAP2 plays a role in the morphological and functional maturation of dendrites remains an open question and is the focus of this study. Cultured hippocampal neurons depleted of MAP2 by means of antisense oligonucleotides failed to elongate their dendrites. On the other hand, MAP2-depleted neurons were capable of receiving synapses within the same time course as their control counterparts. However, both the number of synapses per cell and the synaptic density were markedly reduced in neurons in which dendritic elongation has been impaired. Taken collectively, these results suggest that the expression of MAP2 is required for the morphological differentiation of dendrites. Dendritic elongation, however, is not a prerequisite for synapse formation in cultured hippocampal neurons.     

Long-term depression of kainate receptor-mediated synaptic transmission

Kainate receptors (KARs) have been shown to be involved in hippocampal mossy fiber long-term potentiation (LTP); however, it is not known if KARs are involved in the induction or expression of long-term depression (LTD), the other major form of long-term synaptic plasticity. Here we describe LTD of KAR-mediated synaptic transmission (EPSC(KA) LTD) in perirhinal cortex layer II/III neurons that is distinct from LTD of AMPAR-mediated transmission, which also coexists at the same synapses. Induction of EPSC(KA) LTD requires a rise in postsynaptic Ca(2+) but is independent of NMDARs or T-type voltage-gated Ca(2+) channels; however, it requires synaptic activation of inwardly rectifying KARs and release of Ca(2+) from stores. The synaptic KARs are regulated by tonically activated mGluR5, and expression of EPSC(KA) LTD occurs via a mechanism involving mGluR5, PKC, and PICK1 PDZ domain interactions. Thus, we describe the induction and expression mechanism of a form of synaptic plasticity, EPSC(KA) LTD.     

A cooperative switch determines the sign of synaptic plasticity in distal dendrites of neocortical pyramidal neurons

Pyramidal neurons in the cerebral cortex span multiple cortical layers. How the excitable properties of pyramidal neuron dendrites allow these neurons to both integrate activity and store associations between different layers is not well understood, but is thought to rely in part on dendritic backpropagation of action potentials. Here we demonstrate that the sign of synaptic plasticity in neocortical pyramidal neurons is regulated by the spread of the backpropagating action potential to the synapse. This creates a progressive gradient between LTP and LTD as the distance of the synaptic contacts from the soma increases. At distal synapses, cooperative synaptic input or dendritic depolarization can switch plasticity between LTD and LTP by boosting backpropagation of action potentials. This activity-dependent switch provides a mechanism for associative learning across different neocortical layers that process distinct types of information.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

Molecular constraints on synaptic tagging and maintenance of long-term potentiation: a predictive model

Protein synthesis-dependent, late long-term potentiation (LTP) and depression (LTD) at glutamatergic hippocampal synapses are well characterized examples of long-term synaptic plasticity. Persistent increased activity of protein kinase M ζ (PKMζ) is thought essential for maintaining LTP. Additional spatial and temporal features that govern LTP and LTD induction are embodied in the synaptic tagging and capture (STC) and cross capture hypotheses. Only synapses that have been "tagged" by a stimulus sufficient for LTP and learning can "capture" PKMζ. A model was developed to simulate the dynamics of key molecules required for LTP and LTD. The model concisely represents relationships between tagging, capture, LTD, and LTP maintenance. The model successfully simulated LTP maintained by persistent synaptic PKMζ, STC, LTD, and cross capture, and makes testable predictions concerning the dynamics of PKMζ. The maintenance of LTP, and consequently of at least some forms of long-term memory, is predicted to require continual positive feedback in which PKMζ enhances its own synthesis only at potentiated synapses. This feedback underlies bistability in the activity of PKMζ. Second, cross capture requires the induction of LTD to induce dendritic PKMζ synthesis, although this may require tagging of a nearby synapse for LTP. The model also simulates the effects of PKMζ inhibition, and makes additional predictions for the dynamics of CaM kinases. Experiments testing the above predictions would significantly advance the understanding of memory maintenance.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.     

Neuronal Synapse Formation Induced by Microglia and Interleukin 10

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.     

Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.     

Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus

The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.     

Rap1-induced p38 mitogen-activated protein kinase activation facilitates AMPA receptor trafficking via the GDI.Rab5 complex. Potential role in (S)-3,5-dihydroxyphenylglycene-induced long term depression

Recent evidence has emphasized the importance of p38 mitogen-activated protein kinase (MAPK) in the induction of metabotropic glutamate receptor (mGluR)-dependent long term depression (LTD) at hippocampal CA3-CA1 synapses. However, the cascade responsible of mGluR to activate p38 MAPK and the signaling pathway immediately downstream from it to induce synaptic depression is poorly understood. Here, we show that transient activation of group I mGluR with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) activates p38 MAPK through G protein betagamma-subunit, small GTPase Rap1, and MAPK kinase 3/6 (MKK3/6), thus resulting in mGluR5-dependent LTD. Furthermore, our data clearly show that an accelerating AMPA receptor endocytosis by stimulating the formation of guanyl nucleotide dissociation inhibitor-Rab5 complex is a potential downstream processing of p38 MAPK activation to mediate DHPG-LTD. These results suggest an important role for Rap1-MKK3/6-p38 MAPK pathway in the induction of mGluR-dependent LTD by directly coupling to receptor trafficking machineries to facilitate the loss of synaptic AMPA receptors.     

Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: a mechanism for organizational sex differences

The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the rat as a model of sexual differentiation is that treatment of neonatal females with estradiol will permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a nongenomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases, thereby inducing dendritic spine formation. These results reveal a transneuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.     

Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.     

Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.     

Interaction between long-term potentiation and depression in CA1 synapses: temporal constrains, functional compartmentalization and protein synthesis

Information arriving at a neuron via anatomically defined pathways undergoes spatial and temporal encoding. A proposed mechanism by which temporally and spatially segregated information is encoded at the cellular level is based on the interactive properties of synapses located within and across functional dendritic compartments. We examined cooperative and interfering interactions between long-term synaptic potentiation (LTP) and depression (LTD), two forms of synaptic plasticity thought to be key in the encoding of information in the brain. Two approaches were used in CA1 pyramidal neurons of the mouse hippocampus: (1) induction of LTP and LTD in two separate synaptic pathways within the same apical dendritic compartment and across the basal and apical dendritic compartments; (2) induction of LTP and LTD separated by various time intervals (0-90 min). Expression of LTP/LTD interactions was spatially and temporally regulated. While they were largely restricted within the same dendritic compartment (compartmentalized), the nature of the interaction (cooperation or interference) depended on the time interval between inductions. New protein synthesis was found to regulate the expression of the LTP/LTD interference. We speculate that mechanisms for compartmentalization and protein synthesis confer the spatial and temporal modulation by which neurons encode multiplex information in plastic synapses.     

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.     

Active dendrites,potassium channels and synaptic plasticity

The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons.     

A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis

Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.     

Local dendritic activity sets release probability at hippocampal synapses

The arrival of an action potential at a synapse triggers neurotransmitter release with a limited probability, p(r). Although p(r) is a fundamental parameter in defining synaptic efficacy, it is not uniform across all synapses, and the mechanisms by which a given synapse sets its basal release probability are unknown. By measuring p(r) at single presynaptic terminals in connected pairs of hippocampal neurons, we show that neighboring synapses on the same dendritic branch have very similar release probabilities, and p(r) is negatively correlated with the number of synapses on the branch. Increasing dendritic depolarization elicits a homeostatic decrease in p(r), and equalizing activity in the dendrite significantly reduces its variability. Our results indicate that local dendritic activity is the major determinant of basal release probability, and we suggest that this feedback regulation might be required to maintain synapses in their operational range.