Activity-dependent wiring of the developing hippocampal neuronal circuit

In the developing hippocampus, functional excitatory synaptic connections seem to be recruited from a preformed, initially silent synaptic network. This functional synapse induction requires presynaptic action potentials paired with postsynaptic depolarization, thus obeying Hebb's rule of association. During early postnatal development the hippocampus exhibits an endogenous form of patterned neuronal activity that is driven by GABAergic depolarization. We propose that this recurrent activity promotes the input-specific induction of functional synapses in the CA1 region. Thus, activity-dependent synaptic reorganization in the developing hippocampus appears to be dominated by an active recruitment of new synapses rather than an active elimination of redundant connections.     

Molecular diversity of Dscam: recognition of molecular identity in neuronal wiring

Our understanding of how the enormously complex task of interconnecting millions of nerve cells is accomplished remains rudimentary. What molecular mechanisms control its exquisite specificity? Can we pinpoint single molecular interactions that might help to explain some of the specificity requirements that underlie neuronal wiring? A series of recent studies on the molecular diversity of the Drosophila melanogaster cell-surface receptor Down syndrome cell-adhesion molecule (Dscam) provide one exceptional example of a novel mechanistic model of neuronal-wiring specificity, progressing from structural studies of single protein-protein interactions to biochemical analysis in vitro and to an understanding of complex neuronal differentiation at the single-cell or tissue levels.     

Systematic identification of genes that regulate neuronal wiring in the Drosophila visual system

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring.     

The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila

Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity.     

Variability v.s. synchronicity of neuronal activity in local cortical network models with different wiring topologies

Dynamical behavior of a biological neuronal network depends significantly on the spatial pattern of synaptic connections among neurons. While neuronal network dynamics has extensively been studied with simple wiring patterns, such as all-to-all or random synaptic connections, not much is known about the activity of networks with more complicated wiring topologies. Here, we examined how different wiring topologies may influence the response properties of neuronal networks, paying attention to irregular spike firing, which is known as a characteristic of in vivo cortical neurons, and spike synchronicity. We constructed a recurrent network model of realistic neurons and systematically rewired the recurrent synapses to change the network topology, from a localized regular and a “small-world” network topology to a distributed random network topology. Regular and small-world wiring patterns greatly increased the irregularity or the coefficient of variation (Cv) of output spike trains, whereas such an increase was small in random connectivity patterns. For given strength of recurrent synapses, the firing irregularity exhibited monotonous decreases from the regular to the random network topology. By contrast, the spike coherence between an arbitrary neuron pair exhibited a non-monotonous dependence on the topological wiring pattern. More precisely, the wiring pattern to maximize the spike coherence varied with the strength of recurrent synapses. In a certain range of the synaptic strength, the spike coherence was maximal in the small-world network topology, and the long-range connections introduced in this wiring changed the dependence of spike synchrony on the synaptic strength moderately. However, the effects of this network topology were not really special in other properties of network activity. Action Editor: Xiao-Jing Wang     

Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species.     

Class-specific antibody in human dermatophytosis reactive withTrichophyton rubrum derived antigen

Dermatophyte infections induce a humoral immune response and an enzyme linked immunosorbent assay was used to detect specific antibody classes against antigen derived fromTrichophyton rubrum. Sera from 19 acute patients, 18 chronic patients, and 27 normal controls were evaluated. Mean IgG titers against dermatophyte antigen were significantly higher in all patients than in controls. Mean IgM levels were significantly higher in acute patients than in controls. No significant difference was detected in IgE titers between the patients and controls. These results do not reveal whether the humoral immune response has a role in the progression of the infection.Abbreviations CMI cell-mediated immunity - PBS phosphate buffered saline - Tr antigen Trichophyton rubrum antigen     

Class-specific effects of selenium on PWM-driven human antibody synthesis in vitro

The influence of inorganic and organic forms of selenium (Se) on human antibody production was studied in a Pokeweed Mitogen (PWM)-driven in vitro system. Mitogen-stimulated peripheral blood mononuclear cells (PBMC) of eight healthy donors were cultured with different Se compounds at concentrations between 10(-3) and 10(-9) M. At high Se levels (10(-3)-10(-4) M), IgM and IgG production of all donors were strongly inhibited owing to reduced cell viability. However, in five of eight donors, low levels of Se enhanced IgG secretion. This was most effective in the presence of inorganic Se, whereas selenomethionine and selenocystine were less effective. In contrast to IgG, IgM synthesis was significantly reduced by low Se levels in five donors. No significant correlation between donor serum Se levels and antibody production in vitro was found. The addition of low levels of Se to PBMC, stimulated with PHA or PWM, showed no effect on proliferation, whereas a high concentration (5 x 10(-3) M) of sodium selenite and selenocystine suppressed proliferation owing to reduced cell viability. Thus, the present results show that Se supplementation can enhance human antibody production and, moreover, suggest some selectivity of Se action on human immune responses that may result in increased switching from IgM to IgG production.     

Specific features of neuronal size and shape are regulated by tropomyosin isoforms

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.     

Molecular features of an alcohol binding site in a neuronal potassium channel

Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K(+) channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 micros) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K(+) channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only approximately 2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K(+) channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the alpha-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.     

Geometry-induced features of current transfer in neuronal dendrites with tonically activated conductances

The impact of dendritic geometry on somatopetal transfer of the current generated by steady uniform activation of excitatory synaptic conductance distributed over passive, or active (Hodgkin-Huxley type), dendrites was studied in simulated neurons. Such tonic activation was delivered to the uniform dendrite and to the dendrites with symmetric or asymmetric branching with various ratios of branch diameters. Transfer effectiveness of the dendrites with distributed sources was estimated by the core current increment directly related to the total membrane current per unit path length. The effectiveness decreased with increasing path distance from the soma along uniform branches. The primary reason for this was the asymmetry of somatopetal vs somatofugal input core conductance met by synaptic current due to a greater leak conductance at the proximal end of the dendrite. Under these conditions, an increasing somatopetal core current and a corresponding drop of the depolarization membrane potential occurred. The voltage-dependent extrasynaptic conductances, if present, followed this depolarization. Consequently, the driving potential and membrane current densities decreased with increasing path distance from the soma. All path profiles were perturbed at bifurcations, being identical in symmetrical branches and diverging in asymmetrical ones. These perturbations were caused by voltage gradient breaks (abrupt change in the profile slope) occurring at the branching node due to coincident inhomogeneity of the dendritic core cross-section area and its conductance. The gradient was greater on the side of the smaller effective cross-section. Correspondingly, the path profiles of the somatopetal current transfer effectiveness were broken and/or diverged. The dendrites, their paths, and sites which were more effective in the current transfer from distributed sources were also more effective in the transfer from single-site inputs. The effectiveness of the active dendrite depended on the activation-inactivation kinetics of its voltage-gated conductances. In particular, dendrites with the same geometry were less effective with the Hodgkin-Huxley membrane than with the passive membrane, because of the effect of the noninactivating K⁺-conductance associated with the hyperpolarization equilibrium potential. Such electrogeometrical coupling may form a basis for path-dependent input-output conversion in the dendritic neurons, as the output discharge rate is defined by the net current delivered to the soma. Received: 18 December 1997 / Accepted in revised form: 12 June 1998     

Neural development features: spatio-temporal development of the Caenorhabditis elegans neuronal network

The nematode Caenorhabditis elegans, with information on neural connectivity, three-dimensional position and cell linage, provides a unique system for understanding the development of neural networks. Although C. elegans has been widely studied in the past, we present the first statistical study from a developmental perspective, with findings that raise interesting suggestions on the establishment of long-distance connections and network hubs. Here, we analyze the neuro-development for temporal and spatial features, using birth times of neurons and their three-dimensional positions. Comparisons of growth in C. elegans with random spatial network growth highlight two findings relevant to neural network development. First, most neurons which are linked by long-distance connections are born around the same time and early on, suggesting the possibility of early contact or interaction between connected neurons during development. Second, early-born neurons are more highly connected (tendency to form hubs) than later-born neurons. This indicates that the longer time frame available to them might underlie high connectivity. Both outcomes are not observed for random connection formation. The study finds that around one-third of electrically coupled long-range connections are late forming, raising the question of what mechanisms are involved in ensuring their accuracy, particularly in light of the extremely invariant connectivity observed in C. elegans. In conclusion, the sequence of neural network development highlights the possibility of early contact or interaction in securing long-distance and high-degree connectivity.     

Morphological features of five neuronal classes in the gerbil lateral superior olive

Five morphologically distinct classes of neurons can be identified within the neuropil of the gerbil lateral superior olivary nucleus (LSO) by using a variety of histological techniques and electron microscopy. The physical features of these five classes resemble those found in the cat LSO and are identified, by using criteria and nomenclature established for the cat, as principal neurons, multiplanar neurons, marginal neurons, small neurons, and class 5 neurons. Principal cells compose approximately 75% of the total LSO neuronal population. They possess a discoid dendritic organization and are oriented rostrocaudally, perpendicular to the transverse curvatures of the LSO. Roughly 8% of the LSO population is composed of multiplanar neurons, whose dendritic fields are not restricted to any single plane of section. Both principal and multiplanar neurons share similar cytoplasmic features, and greater than 65% of their perikaryal surface is in contact with synaptic terminals. Small neurons compose approximately 11% of the LSO neurons, have the lowest percentage of their somal surface contacted by synaptic terminals (approximately 8%), and are found mostly in the middle/medial portions of the LSO. Marginal neurons, which compose approximately 6% of the LSO population, appear similar to principal neurons at the light microscopic level except that they are found along the contours of the LSO, oriented orthogonal to principal neurons. Approximately 28% of the somal surface of marginal neurons is in contact with synaptic terminals. The class 5 neuronal somata receive a similar number of axosomatic synaptic contacts as marginal neurons (approximately 31%) but are found well within the matrix of the LSO, aligned parallel to principal neurons. Class 5 neurons share the same light microscopic features as principal neurons and can be identified electron microscopically based only on the reduced percentage of somal surface occupied by synaptic terminals.     

Use of wiring configuration and wiring codes for estimating externally generated electric and magnetic fields

The relative locations and characteristics of the distribution lines feeding 434 residences in the Denver metropolitan area were recorded and classified according to the Wertheimer-Leeper code (WL code) as a part of an epidemiological study of the incidence of childhood cancer. The WL code was found to place the mean values of the fields in rank order. However, the standard deviations were approximately the same size as the means. Theoretical calculations indicate that a significant fraction of the low-power magnetic fields can be generated by the distribution lines, especially in the cases where the distribution lines are within 50 feet of the residence. Thus, the wiring code was shown to be a useful method for making a first-order approximation to predict long-term, low-level magnetic fields in residences.     

Brain wiring with composite instructions

The quest for molecular mechanisms that guide axons or specify synaptic contacts has largely focused on molecules that intuitively relate to the idea of an “instruction.” By contrast, “permissive” factors are traditionally considered background machinery without contribution to the information content of a molecularly executed instruction. In this essay, I recast this dichotomy as a continuum from permissive to instructive actions of single factors that provide relative contributions to a necessarily collaborative effort. Individual molecules or other factors do not constitute absolute instructions by themselves; they provide necessary context for each other, thereby creating a composite that defines the overall instruction. The idea of composite instructions leads to two main conclusions: first, a composite of many seemingly permissive factors can define a specific instruction even in the absence of a single dominant contributor; second, individual factors are not necessarily related intuitively to the overall instruction or phenotypic outcome.     

A wiring of the human nucleolus

Recent proteomic efforts have created an extensive inventory of the human nucleolar proteome. However, approximately 30% of the identified proteins lack functional annotation. We present an approach of assigning function to uncharacterized nucleolar proteins by data integration coupled to a machine-learning method. By assembling protein complexes, we present a first draft of the human ribosome biogenesis pathway encompassing 74 proteins and hereby assign function to 49 previously uncharacterized proteins. Moreover, the functional diversity of the nucleolus is underlined by the identification of a number of protein complexes with functions beyond ribosome biogenesis. Finally, we were able to obtain experimental evidence of nucleolar localization of 11 proteins, which were predicted by our platform to be associates of nucleolar complexes. We believe other biological organelles or systems could be "wired" in a similar fashion, integrating different types of data with high-throughput proteomics, followed by a detailed biological analysis and experimental validation.