Bidirectional regulation of hippocampal mossy fiber filopodial motility by kainate receptors: a two-step model of synaptogenesis

The rapid motility of axonal filopodia and dendritic spines is prevalent throughout the developing CNS, although the function of this motility remains controversial. Using two-photon microscopy, we imaged hippocampal mossy fiber axons in slice cultures and discovered that filopodial extensions are highly motile. Axonal filopodial motility is actin based and is downregulated with development, although it remains in mature cultures. This motility is correlated with free extracellular space yet is inversely correlated with contact with postsynaptic targets, indicating a potential role in synaptogenesis. Filopodial motility is differentially regulated by kainate receptors: synaptic stimulation of kainate receptors enhances motility in younger slices, but it inhibits it in mature slices. We propose that neuronal activity controls filopodial motility in a developmentally regulated manner, in order to establish synaptic contacts in a two-step process. A two-step model of synaptogenesis can also explain the opposite effects of neuronal activity on the motility of dendritic protrusions.     

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.     

Regulation of Dendritic Filopodial Interactions by ZO-1 and Implications for Dendrite Morphogenesis

Neuronal dendrites dynamically protrude many fine filopodia in the early stages of neuronal development and gradually establish complex structures. The importance of the dendritic filopodia in the formation of axo-dendritic connections is established, but their role in dendrite morphogenesis remains unknown. Using time-lapse imaging of cultured rat hippocampal neurons, we revealed here that many filopodia dynamically protruded from dendrites and transiently interacted with each other to form dendritic filopodia-filopodia contacts in the early stages of neuronal development. The MAGUK family member, Zonula Occludens-1 (ZO-1), which is known to be associated with the nectin and cadherin cell adhesion systems, was concentrated at these dendritic filopodia-filopodia contact sites and also at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology. Conversely, knockdown of ZO-1 decreased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology which was different from that induced by the overexpression of ZO-1. The components of the nectin and cadherin systems were co-localized with ZO-1 at the dendritic filopodia-filopodia contact sites, but not at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the accumulation of these cell adhesive components at the dendritic filopodia-filopodia contact sites and stabilized their interactions, whereas knockdown of ZO-1 reduced their accumulation at the dendritic filopodia-filopodia contact sites. These results indicate that ZO-1 regulates dendritic filopodial dynamics, which is implicated in dendrite morphogenesis cooperatively with the nectin and cadherin systems in cultured neurons.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

Dynamics of dendritic spines and their afferent terminals: spines are more motile than presynaptic boutons

Previous work has established that dendritic spines, sites of excitatory input in CNS neurons, can be highly dynamic, in later development as well as in mature brain. Although spine motility has been proposed to facilitate the formation of new synaptic contacts, we have reported that spines continue to be dynamic even if they bear synaptic contacts. An outstanding question related to this finding is whether the presynaptic terminals that contact dendritic spines are as dynamic as their postsynaptic targets. Using multiphoton time-lapse microscopy of GFP-labeled Purkinje cells and DiI-labeled granule cell parallel fiber afferents in cerebellar slices, we monitored the dynamic behavior of both presynaptic terminals and postsynaptic dendritic spines in the same preparation. We report that while spines are dynamic, the presynaptic terminals they contact are quite stable. We confirmed the relatively low levels of presynaptic terminal motility by imaging parallel fibers in vivo. Finally, spine motility can occur when a functional presynaptic terminal is apposed to it. These analyses further call into question the function of spine motility, and to what extent the synapse breaks or maintains its contact during the movement of the spine.     

Calcium helps neurons identify synaptic targets during development

Correct wiring of brain circuitry during development involves the selective formation and retention of synaptic connections between neurons. In this issue of Neuron, Lohmann and Bonhoeffer show that dendritic filopodia can distinguish among prospective presynaptic axonal targets during development. Contact with the appropriate target triggers local calcium signals that stabilize the filopodia and tell them to form mature synapses.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

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.     

Paralemmin-1, a modulator of filopodia induction is required for spine maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.     

Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.     

Proteins that promote filopodia stability, but not number, lead to more axonal-dendritic contacts

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.     

A real-time analysis of growth cone-target cell interactions during the formation of stable contacts between hippocampal neurons in culture.

Mechanisms of cell-cell recognition and structural changes of growth cones (g.c.) and target membranes during contact formation are poorly understood. To examine these issues, we obtained a high magnification, real-time record of stable contact formation in cultured cells from the hippocampal CA1 area in the newborn rat. We used differential interference contrast (DIC) optics coupled to a video microscope for periods of over 24 h of continuous time-lapse recording. Our goal was to observe the sequential changes exhibited by afferent and target cells as they form a stable contact. Understanding the process of how stable contacts are made is important because such contacts are the first step in synapse formation. Four principal observations emerged from our study: (1) The target cell was receptive to a contact on a specific patch on its surface defined by the presence of lamellae and filopodia. This specific patch (named target site) was invariably present on the target cell surface before the time the growth cone arrived. (2) Stable adhesion between filopodia on the two cells initiated events leading to cell-cell contact formation. Specifically, the remaining filopodia on the growth cone and target cell were redirected toward the adhering filopodia, and the growth cone size decreased dramatically. (3) The axonal process then grew at a significantly accelerated rate (up to 50 times its baseline growth rate). (4) In addition, a number of observations were obtained on axonal turns towards the target cell, induction of target sites, and architectural remodelling of cells after the formation of a new contact. Our findings indicate that in this neuronal system, filopodia are the means used by cells to interact at stages prior to and during contact formation. We speculate that the molecules involved in cell recognition and the machinery that initiates contact formation are embedded in the fine structure of filopodia. Finally, our results provide possible clues as to some of the stages that may be involved in synapse formation in the mammalian central nervous system.     

Extracellular matrix molecules, their receptors, and secreted proteases in synaptic plasticity

Neural cells secrete diverse molecules, which accumulate in the extracellular space and form the extracellular matrix (ECM). Interactions between cells and the ECM are well recognized to play the crucial role in cell migration and guidance of growing axons, whereas formation of mature neural ECM in the form of perineuronal nets is believed to restrict certain forms of developmental plasticity. On the other hand, major components of perineuronal nets and other ECM molecules support induction of functional plasticity, the most studied form of which is long-term potentiation. Here, we review the underlying mechanisms by which ECM molecules, their receptors and remodeling proteases regulate the induction and maintenance of synaptic modifications. In particular, we highlight that activity-dependent secretion and activation of proteases leads to a local cleavage of the ECM and release of signaling proteolytic fragments. These molecules regulate transmitter receptor trafficking, actin cytoskeleton, growth of dendritic spines, and formation of dendritic filopodia.     

Interneurite affinity is regulated by heterophilic nectin interactions in concert with the cadherin machinery

Neurites recognize their specific partners during the formation of interneuronal connections. In hippocampal pyramidal neurons, axons attach to dendrites for their synaptogenesis, but the dendrites do not form stable contacts with each other, suggesting the presence of a mechanism to allow their selective associations. Nectin-1 (N1), an immunoglobulin domain adhesive protein, is preferentially localized in axons, and its heterophilic partner, N3, is present in both axons and dendrites; we tested their potential roles in interneurite recognition. The overexpression of N1, causing its mislocalization to dendrites, induced atypical dendrodendritic as well as excessive axodendritic associations. On the contrary, the genetic deletion of N1 loosened the contacts between axons and dendritic spines. Those actions of nectins required cadherin-catenin activities, but the overexpression of cadherin itself could not accelerate neurite attachment. These results suggest that the axon-biased localization of N1 and its trans-interaction with N3 in cooperation with the cadherin machinery is critical for the ordered association of axons and dendrites.     

A real-time analysis of growth cone–target cell interactions during the formation of stable contacts between hippocampal neurons in culture

Mechanisms of cell-cell recognition and structural changes of growth cones (g.c.) and target membranes during contact formation are poorly understood. To examine these issues, we obtained a high magnification, realtime record of stale contact formation in cultured cells from the hippocampal CA1 area in the newborn rat. We used differential interference contrast (DIC) optics coupled to a video microscope for periods of over 24 h of continuous time-lapse recording. Our goal was to observe the sequential changes exhibited by afferent and target cells as they form a stable contact. Understanding the process of how stable contacts are made is important because such contacts are the first step in synapse formation. Four principal observations emerged from our study: (1) The target cell was receptive to a contact on a specific patch on its surface defined by the presence of lamellae and filopodia. This specific patch (named target site) was invariably present on the target cell surface before the time the growth cone arrived. (2) Stable adhesion between filopodia on the two cells initiated events leading to cell–cell contact formation. Specifically, the remaining filopodia on the growth cone and target cell were redirected toward the adhering filopodia, and the growth cone size decreased dramatically. (3) The axonal process then grew at a significantly accelerated rate (up to 50 times its baseline growth rate). (4) In addition, a number of observations were obtained on axonal turns towards the target cell, induction of target sites, and architectural remodelling of cells after the formation of a new contact. Our findings indicate that in this neuronal system, filopodia are the means used by cells to interact at stages prior to and during contact formation. We speculate that the molecules involved in cell recognition and the machinery that initiates contact formation are embedded in the fine structure of filopodia. Finally, our results provide possible clues as to some of the stages that may be involved in synapse formation in the mammalian central nervous system. © 1992 John Wiley & Sons, Inc.     

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.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

A QUANTITATIVE STUDY OF SYNAPSES ON MOTOR NEURON DENDRITIC GROWTH CONES IN DEVELOPING MOUSE SPINAL CORD

The proportion of synaptic contacts occurring on dendrites as well as on dendritic growth cones and filopodia was determined from electron micrographs of developing mouse (C57BL/6J) spinal cord. Comparable areas of the marginal zone adjacent to the lateral motor nucleus were sampled from specimens on the 13th–16th days of embryonic development (E13–E16). At the beginning of this period, synapses upon growth cones and filopodia comprise about 80% of the observed synaptic junctions, but this proportion decreases with developmental time so that in E16 specimens growth cone synapses account for slightly less than 30% of the synaptic population. Conversely, at E13, synapses upon dendrites comprise less than 20% of the total number of synapses, but increase with developmental time so that they account for about 65% of the synaptic population of E16 specimens. From these data, we suggest the following temporal sequence for the formation of synaptic junctions on motor neuron dendrites growing into the marginal zone. New synapses are initially made upon the filopodia of dendritic growth cones. A synaptically contacted filopodium expands to become a growth cone while the original growth cone begins to differentiate into a dendrite. This process is repeated as the dendrite grows farther into the marginal zone so that synapses originally made with filopodia come to be located upon dendrites. This speculation is briefly discussed in relation to the work and ideas of others concerning synaptogenesis and dendritic development.     

Competitive interactions between retinal ganglion cells during prenatal development

In the mammalian visual system, retinal ganglion cell axons terminate within the LGN in a series of alternating eye-specific layers. These layers are not present initially during development. In the cat they emerge secondarily following a prenatal period in which originally intermixed inputs from the two eyes gradually segregate from each other to give rise to the characteristic set of layers by birth. Many lines of evidence suggest that activity-dependent competitive interactions between ganglion cell axons from the two eyes for LGN neurons play an important role in the final patterning of retinogeniculate connections. Studies of the branching patterns of individual ganglion cell axons suggest that during the period when inputs from the two eyes are intermixed, axons from one eye send side branches into territory later occupied exclusively by axons from the other eye. Ultrastructural studies indicate that these branches in fact are sites of synaptic contacts, which are later eliminated since the side branches disappear as axons form their mature terminal arbors in appropriate territory. In vitro microelectrode recordings from LGN neurons indicate that they can receive convergent synaptic excitation from electrical stimulation of the optic nerves before but not after the eye-specific layers form, suggesting that at least some of the synaptic contacts seen at the ultrastructural level are functonal. Finally, experiments in which tetrodotoxin was infused intracranially during the two week period during which the eye-specific layers normally form demonstrate that it is possible to prevent, or at least delay, the formation of the layers. Accordingly, individual axons fail to develop their restricted terminal arbor branching pattern and instead branch widely throughout the LGN. These results indicate that all of the machinery necessary for synaptic function and competition is present during fetal life. Moreover, it is highly likely that neuronal activity is required for the formation of the eye-specific layers. If so, then activity would have to be present in the form of spontaneously generated action potentials, since vision is not possible at these early ages. Thus, the functioning of the retinogeniculate system many weeks before it is put to the use for which it is ultimately designed may contribute to the final patterning of connections present in the adult.     

Development of the dendritic branching pattern of the Medial Giant Interneuron in the grasshopper embryo

The embryonic development of the grasshopper's Medial Giant Interneuron (MGI) was examined by injecting the cell with the fluorescent dye Lucifer Yellow at a series of stages in its growth. Particular attention was given to the way in which this neuron constructs its stereotyped dendritic branching pattern. The MGI's dendrites originate as secondary processes which sprout at characteristic points along the neurite after the primary growth cone has passed. These processes then arborize to form a miniature version of their adult branching pattern before the end of embryonic life. While growing, the dendritic branches are covered with a radiant profusion of filopodia; however, these filopodia are ephemeral structures and disappear once the cell matures. By contrast there is no significant reduction in either the number or the spatial extent of the actual dendrites at any embryonic stage. This implies that the stereotyped branching pattern of the mature MGI is primarily determined by a precise pattern of initial growth, and that secondary pruning of branches does not play an important role in shaping the final form of this cell. The coordinate ingrowth of the first cercal sensory axons was examined by cobalt filling the embryonic nerve, and the means by which these sensory axons make their initial contacts with the MGI's dendrites is herein discussed. The following paper considers the degree to which this sensory innervation regulates dendritic growth and branching.