Dscam diversity is essential for neuronal wiring and self-recognition

Neurons are thought to use diverse families of cell-surface molecules for cell recognition during circuit assembly. In Drosophila, alternative splicing of the Down syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 closely related transmembrane proteins of the immunoglobulin superfamily, each comprising one of 19,008 alternative ectodomains linked to one of two alternative transmembrane segments. These ectodomains show isoform-specific homophilic binding, leading to speculation that Dscam proteins mediate cell recognition. Genetic studies have established that Dscam is required for neural circuit assembly, but the extent to which isoform diversity contributes to this process is not known. Here we provide conclusive evidence that Dscam diversity is essential for circuit assembly. Using homologous recombination, we reduced the entire repertoire of Dscam ectodomains to just a single isoform. Neural circuits in these mutants are severely disorganized. Furthermore, we show that it is crucial for neighbouring neurons to express distinct isoforms, but that the specific identity of the isoforms expressed in an individual neuron is unimportant. We conclude that Dscam diversity provides each neuron with a unique identity by which it can distinguish its own processes from those of other neurons, and that this self-recognition is essential for wiring the Drosophila brain.     

Dscam2 mediates axonal tiling in the Drosophila visual system

Sensory processing centres in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. Here we show that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. Our data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. We propose that Dscam2 is a tiling receptor for L1 neurons.     

Neurite arborization and mosaic spacing in the mouse retina require DSCAM

To establish functional circuitry, retinal neurons occupy spatial domains by arborizing their processes, which requires the self-avoidance of neurites from an individual cell, and by spacing their cell bodies, which requires positioning the soma and establishing a zone within which other cells of the same type are excluded. The mosaic patterns of distinct cell types form independently and overlap. The cues that direct these processes in the vertebrate retina are not known. Here we show that some types of retinal amacrine cells from mice with a spontaneous mutation in Down syndrome cell adhesion molecule (Dscam), a gene encoding an immunoglobulin-superfamily member adhesion molecule, have defects in the arborization of processes and in the spacing of cell bodies. In the mutant retina, cells that would normally express Dscam have hyperfasciculated processes, preventing them from creating an orderly arbor. Also, their cell bodies are randomly distributed or pulled into clumps rather than being regularly spaced mosaics. Our results indicate that mouse DSCAM mediates isoneuronal self-avoidance for arborization and heteroneuronal self-avoidance within specific cell types to prevent fasciculation and to preserve mosaic spacing. These functions are analogous to those of Drosophila DSCAM (ref. 6) and DSCAM2 (ref. 7). DSCAM may function similarly in other regions of the mammalian nervous system, and this role may extend to other members of the mammalian Dscam gene family.     

Structural basis of Dscam isoform specificity

The Dscam gene gives rise to thousands of diverse cell surface receptors thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.     

Teneurins instruct synaptic partner matching in an olfactory map

Neurons are interconnected with extraordinary precision to assemble a functional nervous system. Compared to axon guidance, far less is understood about how individual pre- and postsynaptic partners are matched. To ensure the proper relay of olfactory information in the fruitfly Drosophila, axons of ～50 classes of olfactory receptor neurons (ORNs) form one-to-one connections with dendrites of ～50 classes of projection neurons (PNs). Here, using genetic screens, we identified two evolutionarily conserved, epidermal growth factor (EGF)-repeat containing transmembrane Teneurin proteins, Ten-m and Ten-a, as synaptic-partner-matching molecules between PN dendrites and ORN axons. Ten-m and Ten-a are highly expressed in select PN-ORN matching pairs. Teneurin loss- and gain-of-function cause specific mismatching of select ORNs and PNs. Finally, Teneurins promote homophilic interactions in vitro, and Ten-m co-expression in non-partner PNs and ORNs promotes their ectopic connections in vivo. We propose that Teneurins instruct matching specificity between synaptic partners through homophilic attraction.     

Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons

Neurons in culture can have fundamentally distinct morphologies which permit their cytological identification and the recognition of their neurites as axons or dendrites. Microtubules may have a role in determining morphology by the selective stabilization of spatially distinct microtubule subsets. The plasticity of a neurite correlates inversely with the stability of its component microtubules: microtubules in growth cones are very dynamic, and in initial neurites there is continuous incorporation of labelled subunits, whereas in mature neurites, microtubules are highly stabilized. The binding of microtubule-associated proteins to the microtubules very probably contributes to this stability. Cerebellar neurons in dissociated culture initially extend exploratory neurites and, after a relatively constant interval, become polarized. Polarity becomes evident when a single neurite exceeds the others in length. These stable neurites cease to undergo the retractions and extensions characteristic of initial neurites and assume many features of axons and dendrites. We have now studied the role of the neuronal microtubule-associate protein tau in neurite polarization by selectively inhibiting tau expression by the addition of antisense oligonucleotides to the culture media. Although the extension of initial exploratory neurites occurred normally, neurite asymmetry was inhibited by the failure to elaborate an axon.     

The role of neuronal identity in synaptic competition

In developing mammalian muscle, axon branches of several motor neurons co-innervate the same muscle fibre. Competition among them results in the strengthening of one and the withdrawal of the rest. It is not known why one particular axon branch survives or why some competitions resolve sooner than others. Here we show that the fate of axonal branches is strictly related to the identity of the axons with which they compete. When two neurons co-innervate multiple target cells, the losing axon branches in each contest belong to the same neuron and are at nearly the same stage of withdrawal. The axonal arbor of one neuron engages in multiple sets of competitions simultaneously. Each set proceeds at a different rate and heads towards a common outcome based on the identity of the competitor. Competitive vigour at each of these sets of local competitions depends on a globally distributed resource: neurons with larger arborizations are at a competitive disadvantage when confronting neurons with smaller arborizations. An accompanying paper tests the idea that the amount of neurotransmitter released is this global resource.     

UNC-6/netrin and its receptor UNC-5 locally exclude presynaptic components from dendrites

Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored. Here we show that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the Caenorhabditis elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this anti-synaptogenic activity is interchangeable with that of LIN-44/Wnt despite being transduced through different receptors, suggesting that extracellular cues such as netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion.     

Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones

Outgrowth of distinct axonal and dendritic processes is essential for the development of the functional polarity of nerve cells. In cultures of neurons from the hippocampus, where the differential outgrowth of axons and dendrites is readily discernible, we have sought molecules that might underlie the distinct modes of elongation of these two types of processes. One particularly interesting protein is GAP-43 (also termed B-50, F1 or P-57), a neuron-specific, membrane-associated phosphoprotein whose expression is dramatically elevated during neuronal development and regeneration. GAP-43 is among the most abundant proteins in neuronal growth cones, the motile structures that form the tips of advancing neurites, but its function in neuronal growth remains unknown. Using immunofluorescence staining, we show that GAP-43 is present in axons and concentrated in axonal growth cones of hippocampal neurons in culture. Surprisingly, we could not detect GAP-43 in growing dendrites and dendritic growth cones. These results show that GAP-43 is compartmentalized in developing nerve cells and provide the first direct evidence of important molecular differences between axonal and dendritic growth cones. The sorting and selective transport of GAP-43 may give axons and axonal growth cones certain of their distinctive properties, such as the ability to grow rapidly over long distances or the manner in which they recognize and respond to cues in their environment.     

Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites

Information in neurons flows from synapses, through the dendrites and cell body (soma), and, finally, along the axon as spikes of electrical activity that will ultimately release neurotransmitters from the nerve terminals. However, the dendrites of many neurons also have a secretory role, transmitting information back to afferent nerve terminals. In some central nervous system neurons, spikes that originate at the soma can travel along dendrites as well as axons, and may thus elicit secretion from both compartments. Here, we show that in hypothalamic oxytocin neurons, agents that mobilize intracellular Ca(2+) induce oxytocin release from dendrites without increasing the electrical activity of the cell body, and without inducing secretion from the nerve terminals. Conversely, electrical activity in the cell bodies can cause the secretion of oxytocin from nerve terminals with little or no release from the dendrites. Finally, mobilization of intracellular Ca(2+) can also prime the releasable pool of oxytocin in the dendrites. This priming action makes dendritic oxytocin available for release in response to subsequent spike activity. Priming persists for a prolonged period, changing the nature of interactions between oxytocin neurons and their neighbours.     

Facilitation of a dendritic calcium conductance by 5-hydroxytryptamine in the substantia nigra

Within the substantia nigra, the dendrites of dopaminergic neurons that project to the striatum appear to play an active and nonclassical role in the physiology of the neuron in that they release transmitter and protein, but little is known of the factors controlling release of substances from these dendrites. In this study, we show that 5-hydroxytryptamine, which is contained in afferent fibres to the substantia nigra, is present in terminals making direct synaptic contact with dopaminergic neurons and also that it has a site-dependent, receptor-mediated, facilitatory effect on a specific dendritic calcium-dependent potential in nigrostriatal neurons in vitro. The ionic and spatial features of this response, which is insensitive to blockade by three different K+-channel antagonists, could correspond to those underlying the dendritic release of dopamine.     

Rapid BDNF-induced retrograde synaptic modification in a developing retinotectal system

In cultures of hippocampal neurons, induction of long-term synaptic potentiation or depression by repetitive synaptic activity is accompanied by a retrograde spread of potentiation or depression, respectively, from the site of induction at the axonal outputs to the input synapses on the dendrites of the presynaptic neuron. We report here that rapid retrograde synaptic modification also exists in an intact developing retinotectal system. Local application of brain-derived neurotrophic factor (BDNF) to the Xenopus laevis optic tectum, which induced persistent potentiation of retinotectal synapses, led to a rapid modification of synaptic inputs at the dendrites of retinal ganglion cells (RGCs), as shown by a persistent enhancement of light-evoked excitatory synaptic currents and spiking activity of RGCs. This retrograde effect required TrkB receptor activation, phospholipase Cgamma activity and Ca2+ elevation in RGCs, and was accounted for by a selective increase in the number of postsynaptic AMPA-subtype glutamate receptors at RGC dendrites. Such retrograde information flow in the neuron allows rapid regulation of synaptic inputs at the dendrite in accordance to signals received at axon terminals, a process reminiscent of back-propagation algorithm for learning in neural networks.     

两种染色方法对小脑浦肯野细胞显示效果的比较

用常规Nissl染色和成年动物Golgi染色方法分别标记小脑浦肯野细胞(Purkinje cell,PC),比较其染色效果,结果显示,常规Nissl染色只能观察到PC胞体,胞体内的胞核及核仁也清晰可见,但树突和轴突不着色;成年动物Golgi染色能清晰地显示PC树突、轴突及树突棘的形态结构,但胞体结构不清楚。     

Selective dendritic transport of RNA in hippocampal neurons in culture

Typical neurons of the central nervous system (CNS) elaborate tens of thousands of membrane specializations at sites of synaptic contacts on their dendrites. To construct, maintain, and modify these specializations, neurons must produce and deliver the appropriate molecular constituents to particular synaptic sites. Previous studies have revealed that polyribosomes are selectively positioned beneath postsynaptic sites, suggesting that in neurons, as in other cell types, protein synthetic machinery is located at or near the sites where particular proteins are needed. The mechanisms that deliver ribosomes and messenger RNA to their specific destinations in cells are therefore of considerable interest. Here we describe a system for RNA transport in dendrites that could provide a mechanism for the delivery of ribosomes and mRNA to synaptic sites in dendrites. Hippocampal neurons grown in culture incorporate 3H-uridine in the nucleus, then selectively transport the newly synthesized RNA into dendrites at a rate of about 0.5 mm day-1. The transport is inhibited by metabolic poisons, suggesting that it is an active, energy-dependent process. The RNA may be transported in association with the cytoskeleton.     

Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers

The peptide GsMTx4, isolated from the venom of the tarantula Grammostola spatulata, is a selective inhibitor of stretch-activated cation channels (SACs). The mechanism of inhibition remains unknown; but both GsMTx4 and its enantiomer, enGsMTx4, modify the gating of SACs, thus violating a trademark of the traditional lock-and-key model of ligand-protein interactions. Suspecting a bilayer-dependent mechanism, we examined the effect of GsMTx4 and enGsMTx4 on gramicidin A (gA) channel gating. Both peptides are active, and the effect increases with the degree of hydrophobic mismatch between bilayer thickness and channel length, meaning that GsMTx4 decreases the energy required to deform the boundary lipids adjacent to the channel. GsMTx4 decreases inward SAC single-channel currents but has no effect on outward currents, suggesting it is located within a Debye length of the outer vestibule of the SACs, but significantly farther from the inner vestibule. Likewise, GsMTx4 decreases gA single-channel currents. Our results suggest that modulation of membrane proteins by amphipathic peptides--mechanopharmacology--involves not only the protein itself but also the surrounding lipids. The surprising efficacy of the d form of GsMTx4 peptide has important therapeutic implications, because d peptides are not hydrolysed by endogenous proteases and may be administered orally.     

Transmitter-evoked local calcium release stabilizes developing dendrites

In the central nervous system, dendritic arborizations of neurons undergo dynamic structural remodelling during development. Processes are elaborated, maintained or eliminated to attain the adult pattern of synaptic connections. Although neuronal activity influences this remodelling, it is not known how activity exerts its effects. Here we show that neurotransmission-evoked calcium (Ca(2+)) release from intracellular stores stabilizes dendrites during the period of synapse formation. Using a ballistic labelling method to load cells with Ca(2+) indicator dyes, we simultaneously monitored dendritic activity and structure in the intact retina. Two distinct patterns of spontaneous Ca(2+) increases occurred in developing retinal ganglion cells--global increases throughout the arborization, and local 'flashes' of activity restricted to small dendritic segments. Blockade of local, but not global, activity caused rapid retraction of dendrites. This retraction was prevented locally by focal uncaging of caged Ca(2+) that triggered Ca(2+) release from internal stores. Thus, local Ca(2+) release is a mechanism by which afferent activity can selectively and differentially regulate dendritic structure across the developing arborization.     

基于关键参数反馈的神经元放电模式控制

针对神经元数学模型中相关参数的异常变化问题, 先通过无迹Kalman滤波器（unscented Kalman filter）对这些关键参数进行实时估计, 再使用这些参数作为控制器的反馈信号完成神经元放电的有效闭环控制. 使用该方法对Pinsky Rinzel（PR）神经元由于关键参数（胞体和树突间的电导gc及树突刺激电流Ic）的病变导致的异常放电进行MATLAB仿真控制, 分别实现了单参数病变及两个参数同时病变引起的异常放电控制. 实验结果证明了该方法的有效性.     

Motor neuron columnar fate imposed by sequential phases of Hox-c activity

The organization of neurons into columns is a prominent feature of central nervous system structure and function. In many regions of the central nervous system the grouping of neurons into columns links cell-body position to axonal trajectory, thus contributing to the establishment of topographic neural maps. This link is prominent in the developing spinal cord, where columnar sets of motor neurons innervate distinct targets in the periphery. We show here that sequential phases of Hox-c protein expression and activity control the columnar differentiation of spinal motor neurons. Hox expression in neural progenitors is established by graded fibroblast growth factor signalling and translated into a distinct motor neuron Hox pattern. Motor neuron columnar fate then emerges through cell autonomous repressor and activator functions of Hox proteins. Hox proteins also direct the expression of genes that establish motor topographic projections, thus implicating Hox proteins as critical determinants of spinal motor neuron identity and organization.     

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Experimentally induced alteration in the polarity of developing neurons

Despite the great diversity of shapes exhibited by different classes of nerve cells, nearly all neurons share one feature in that they have a single axon and several dendrites. The two types of processes differ in their morphology, in their rate of growth, in the macromolecular composition of their cytoskeletons and surface membranes, and in their synaptic polarity. When hippocampal neurons are dissociated from the embryonic brain and cultured, they reproducibly establish this basic form with a single axon and several dendrites, despite the absence of any spatially organized environmental cues, and without the need for cell to cell contact. We have cut the axons of young hippocampal neurons within a day of their development: in some cases the initial axon regenerated, but more frequently one of the other processes, which if undisturbed would have become a dendrite, instead became the axon. Frequently the stump of the original axon persisted following the transection and subsequently became a dendrite. Evidently the neuronal processes that first develop in culture have the capacity to form either axons or dendrites. The acquisition of axonal characteristics by one neuronal process apparently inhibits the others from becoming axons, so they subsequently become dendrites.