Mechanisms of dendritic maturation

The highly complex geometry of dendritic trees is crucial for neural signal integration and the proper wiring of neuronal circuits. The morphogenesis of dendritic trees is regulated by innate genetic factors, neuronal activity, and external molecular cues. How each of these factors contributes to dendritic maturation has been addressed in the developing nervous systems of animals ranging from insects to mammals. The results of such investigations have shown that the contribution of intrinsic and extrinsic factors and activity, however, appear to be weighted differentially in different types of neurons, in different brain areas, and especially in different species. Moreover, it appears that dozens of molecules have been found to regulate dendritic maturation, but it is almost certain that each molecule plays only a specific role in this formidable cooperative venture. This article reviews our current knowledge and understanding of the role of various factors in the establishment of the architecture of mature dendritic trees.     

Electron-microscopic study of the maturing rat red nucleus. I. The large-neuron population

The maturing large neurons of the rat red nucleus in animals ranging in age from 1 to 21 days of postnatal life were studied ultrastructurally. Days 1--6 were characterized by rapid morphologic maturation occurring concomitantly with the onset of synaptogenesis. Morphogenesis was confined to the soma, while the first synaptic contacts were also formed in relationship to the soma. Days 6--9 demonstrated continued somal morphogenesis exemplified by cytoplasmic expansion and by the conspicuous presence of perisomatic and growth cone processes. Proximal dendritic morphogenesis was initiated, and synaptogenesis became complex with synaptic sites occurring in relation to the neuronal soma, the perisomatic processes and proximal dendrites. Days 9--15 were characterized by the completion of somal and proximal dendritic morphogenesis and by a massive degree of synaptogenic activity. During this interval, the soma lost perisomatic and growth cone processes, while somatic spines appeared. By the end of this period the neuronal soma and the proximal dendrites appeared mature in terms of both morphology and synaptic input. Complete neuronal maturation was ultimately attained by day 21 of postnatal life.     

The principle of the minimal support of a functional system. The neuronal mechanisms]

Developing nervous cells are characterized by an important trait: a capability for functioning long before the full maturity. During this period, the excitable membrane of a neuron has specific characteristics, the dendritic apparatus is not fully developed, and the level of afferent input is low. All this complicates the initiation of synaptic and impulse potentials. It is suggested that the functioning of neurons at the early developmental stages is underlain by a system of adaptation including: 1) the phenomenon of redundancy (increased number of neurons, spines, and synapses, increased branching); 2) factors increasing the probability to receive the expected afferentation (orientation and growth of dendrites towards the afferent input, structure and localization of branching foci); 3) factors facilitating the initiation of neuronal discharge (juvenile channels generating ion currents, electric interaction between cells, additional triggering zones). The common goal of this whole group of factors is to facilitate the response of a cell to a single or weak signal. This is the principal condition ensuring the realization of neuronal interaction at early stages of brain development. Thus, the essence of the principle of the minimal provision of the early functional systems in Anokhin's theory of systemogenesis that the early functional system involves receptor cells and neurons of hierarchical brain structures that have been the first to mature heterochronously and are at the earliest maturation stage. Achieving the result analogous to that achieved by an adult organism is subserved by additional mechanisms characteristic exclusively for the early maturation stages of neurons and functional systems, which enable the nervous cells to function efficiently even at the early stages of maturity.     

Pharmacological Characterization of Cultivated Neuronal Networks: Relevance to Synaptogenesis and Synaptic Connectivity

Mental disorders, such as schizophrenia or Alzheimer’s disease, are associated with impaired synaptogenesis and/or synaptic communication. During development, neurons assemble into neuronal networks, the primary supracellular mediators of information processing. In addition to the orchestrated activation of genetic programs, spontaneous electrical activity and associated calcium signaling have been shown to be critically involved in the maturation of such neuronal networks. We established an in vitro model that recapitulates the maturation of neuronal networks, including spontaneous electrical activity. Upon plating, mouse primary hippocampal neurons grow neurites and interconnect via synapses to form a dish-wide neuronal network. Via live cell calcium imaging, we identified a limited period of time in which the spontaneous activity synchronizes across neurons, indicative of the formation of a functional network. After establishment of network activity, the neurons grow dendritic spines, the density of which was used as a morphological readout for neuronal maturity and connectivity. Hence, quantification of neurite outgrowth, synapse density, spontaneous neuronal activity, and dendritic spine density allowed to study neuronal network maturation from the day of plating until the presence of mature neuronal networks. Via acute pharmacological intervention, we show that synchronized network activity is mediated by the NMDA-R. The balance between kynurenic and quinolinic acid, both neuro-active intermediates in the tryptophan/kynurenine pathway, was shown to be decisive for the maintenance of network activity. Chronic modulation of the neurotrophic support influenced the network formation and revealed the extreme sensitivity of calcium imaging to detect subtle alterations in neuronal physiology. Given the reproducible cultivation in a 96-well setup in combination with fully automated analysis of the calcium recordings, this approach can be used to build a high-content screening assay usable for neurotoxicity screening, target identification/validation, or phenotypic drug screening.     

Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.     

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.     

Loss of sensory input causes rapid structural changes of inhibitory neurons in adult mouse visual cortex

A fundamental property of neuronal circuits is the ability to adapt to altered sensory inputs. It is well established that the functional synaptic changes underlying this adaptation are reflected by structural modifications in excitatory neurons. In contrast, the degree to which structural plasticity in inhibitory neurons accompanies functional changes is less clear. Here, we use two-photon imaging to monitor the fine structure of inhibitory neurons in mouse visual cortex after deprivation induced by retinal lesions. We find that a subset of inhibitory neurons carry dendritic spines, which form glutamatergic synapses. Removal of visual input correlates with a rapid and lasting reduction in the number of inhibitory cell spines. Similar to the effects seen for dendritic spines, the number of inhibitory neuron boutons dropped sharply after retinal lesions. Together, these data suggest that structural changes in inhibitory neurons may precede structural changes in excitatory circuitry, which ultimately result in functional adaptation following sensory deprivation.     

Synaptic integration in dendritic trees

Most neurons have elaborate dendritic trees that receive tens of thousands of synaptic inputs. Because postsynaptic responses to individual synaptic events are usually small and transient, the integration of many synaptic responses is needed to depolarize most neurons to action potential threshold. Over the past decade, advances in electrical and optical recording techniques have led to new insights into how synaptic responses propagate and interact within dendritic trees. In addition to their passive electrical and morphological properties, dendrites express active conductances that shape individual synaptic responses and influence synaptic integration locally within dendrites. Dendritic voltage-gated Na(+) and Ca(2+) channels support action potential backpropagation into the dendritic tree and local initiation of dendritic spikes, whereas K(+) conductances act to dampen dendritic excitability. While all dendrites investigated to date express active conductances, different neuronal types show specific patterns of dendritic channel expression leading to cell-specific differences in the way synaptic responses are integrated within dendritic trees. This review explores the way active and passive dendritic properties shape synaptic responses in the dendrites of central neurons, and emphasizes their role in synaptic integration.     

Thin dendrites of cerebellar interneurons confer sublinear synaptic integration and a gradient of short-term plasticity

Interneurons are critical for neuronal circuit function, but how their dendritic morphologies and membrane properties influence information flow within neuronal circuits is largely unknown. We studied the spatiotemporal profile of synaptic integration and short-term plasticity in dendrites of mature cerebellar stellate cells by combining two-photon guided electrical stimulation, glutamate uncaging, electron microscopy, and modeling. Synaptic activation within thin (0.4?μm) dendrites produced somatic responses that became smaller and slower with increasing distance from the soma, sublinear subthreshold input-output relationships, and a somatodendritic gradient of short-term plasticity. Unlike most studies showing that neurons employ active dendritic mechanisms, we found that passive cable properties of thin dendrites determine the sublinear integration and plasticity gradient, which both result from large?dendritic depolarizations that reduce synaptic driving force. These integrative properties allow stellate cells to act as spatiotemporal filters of synaptic input patterns, thereby biasing their output in favor of sparse presynaptic activity.     

alpha2-Chimaerin is an essential EphA4 effector in the assembly of neuronal locomotor circuits

The assembly of neuronal networks during development requires tightly controlled cell-cell interactions. Multiple cell surface receptors that control axon guidance and synapse maturation have been identified. However, the signaling mechanisms downstream of these receptors have remained unclear. Receptor signals might be transmitted through dedicated signaling lines defined by specific effector proteins. Alternatively, a single cell surface receptor might couple to multiple effectors with overlapping functions. We identified the neuronal RacGAP alpha2-chimaerin as an effector for the receptor tyrosine kinase EphA4. alpha2-Chimaerin interacts with activated EphA4 and is required for ephrin-induced growth cone collapse in cortical neurons. alpha2-Chimaerin mutant mice exhibit a rabbit-like hopping gait with synchronous hindlimb movements that phenocopies mice lacking EphA4 kinase activity. Anatomical and functional analyses of corticospinal and spinal interneuron projections reveal that loss of alpha2-chimaerin results in impairment of EphA4 signaling in vivo. These findings identify alpha2-chimaerin as an indispensable effector for EphA4 in cortical and spinal motor circuits.     

Monitoring maturation of neural stem cell grafts within a host microenvironment

Neural stem cells (NSC) act as a versatile tool for neuronal cell replacement strategies to treat neurodegenerative disorders in which functional neurorestorative mechanisms are limited. While the beneficial effects of such cell-based therapy have already been documented in terms of neurodegeneration of various origins, a neurophysiological basis for improvement in the recovery of neurological function is still not completely understood. This overview briefly describes the cumulative evidence from electrophysiological studies of NSC-derived neurons, aimed at establishing the maturation of differentiated neurons within a host microenvironment, and their integration into the host circuits, with a particular focus on the neurogenesis of NSC grafts within the post-ischemic milieu. Overwhelming evidence demonstrates that the host microenvironment largely regulates the lineage of NSC grafts. This regulatory role, as yet underestimated, raises possibilities for the favoured maturation of a subset of neural phenotypes in order to gain timely remodelling of the impaired brain tissue and amplify the therapeutic effects of NSC-based therapy for recovery of neurological function.     

C. elegans bicd-1, homolog of the Drosophila dynein accessory factor Bicaudal D, regulates the branching of PVD sensory neuron dendrites

The establishment of cell type-specific dendritic arborization patterns is a key phase in the assembly of neuronal circuitry that facilitates the integration and processing of synaptic and sensory input. Although studies in Drosophila and vertebrate systems have identified a variety of factors that regulate dendrite branch formation, the molecular mechanisms that control this process remain poorly defined. Here, we introduce the use of the Caenorhabditis elegans PVD neurons, a pair of putative nociceptors that elaborate complex dendritic arbors, as a tractable model for conducting high-throughput RNAi screens aimed at identifying key regulators of dendritic branch formation. By carrying out two separate RNAi screens, a small-scale candidate-based screen and a large-scale screen of the ~3000 genes on chromosome IV, we retrieved 11 genes that either promote or suppress the formation of PVD-associated dendrites. We present a detailed functional characterization of one of the genes, bicd-1, which encodes a microtubule-associated protein previously shown to modulate the transport of mRNAs and organelles in a variety of organisms. Specifically, we describe a novel role for bicd-1 in regulating dendrite branch formation and show that bicd-1 is likely to be expressed, and primarily required, in PVD neurons to control dendritic branching. We also present evidence that bicd-1 operates in a conserved pathway with dhc-1 and unc-116, components of the dynein minus-end-directed and kinesin-1 plus-end-directed microtubule-based motor complexes, respectively, and interacts genetically with the repulsive guidance receptor unc-5.     

Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, we used the mosaic analysis with a repressible cell marker (MARCM) technique to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. We found that individual neurons in the peripheral nervous system (PNS) developed clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster developed a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis revealed that Flamingo did not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limited the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype was partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Our molecular analysis also indicated that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons.     

Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface

Neurons are highly polarized cells with distinct subcellular compartments, including dendritic arbors and an axon. The proper function of the nervous system relies not only on correct targeting of axons, but also on development of neuronal-class-specific geometry of dendritic arbors [1-4]. To study the intercellular control of the shaping of dendritic trees in vivo, we searched for cell-surface proteins expressed by Drosophila dendritic arborization (da) neurons [5-7]. One of them was Neuroglian (Nrg), a member of the Ig superfamily ; Nrg and vertebrate L1-family molecules have been implicated in various aspects of neuronal wiring, such as axon guidance, axonal myelination, and synapse formation [9-12]. A subset of the da neurons in nrg mutant embryos exhibited deformed dendritic arbors and abnormal axonal sprouting. Our functional analysis in a cell-type-selective manner strongly suggested that those da neurons employed Nrg to interact with the peripheral glia for suppressing axonal sprouting and for forming second-order dendritic branches. At least for the former role, Nrg functioned in concert with the intracellular adaptor protein Ankyrin (Ank) [13]. Thus, the neuron-glia interaction that is mediated by Nrg, together with Ank under some situations, contributes to axonal and dendritic morphogenesis.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.     

Impaired mitochondrial respiration promotes dendritic branching via the AMPK signaling pathway

Functional neuronal circuits require a constant remodeling of their network composed of highly interconnected neurons. The plasticity of synapses and the shaping of elaborated dendritic branches are energy demanding and therefore depend on an efficient mitochondrial oxidative phosphorylation (OXPHOS). The spatial and functional regulations of dendritic patterning occur also after cell fate specification; however, the molecular mechanisms underlying this complex process remain elusive. Here, we exploit the changes in dendritic architecture in highly branched neurons as a result of aberrant mitochondrial activity. In sensory neurons of Caenorhabditis elegans, genetic manipulations of mitochondrial complex I subunits cause an unexpected outgrowth of dendritic arbors and ectopic structures. The increased number of dendritic branches is coordinated through a specific signaling cascade rather than as a simple consequence of oxidative stress. On the basis of genetic and pharmacological evidence, we show that OXPHOS deficiency promotes branching through the activation of the AMP-activated protein kinase AMPK and the downstream target phosphoinositide 3-kinase PI3K. Taken together, our findings describe a well-defined signaling pathway that regulates dendritic outgrowth in conditions of compromised OXPHOS and the resulting AMPK activation.     

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. We have identified an ephrinB-EphB receptor trans-synaptic signaling pathway which regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin, or dominant-negative Rac1, or inhibition of PAK eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity.