Simulating cortical development as a self constructing process: a novel multi-scale approach combining molecular and physical aspects.

Current models of embryological development focus on intracellular processes such as gene expression and protein networks, rather than on the complex relationship between subcellular processes and the collective cellular organization these processes support. We have explored this collective behavior in the context of neocortical development, by modeling the expansion of a small number of progenitor cells into a laminated cortex with layer and cell type specific projections. The developmental process is steered by a formal language analogous to genomic instructions, and takes place in a physically realistic three-dimensional environment. A common genome inserted into individual cells control their individual behaviors, and thereby gives rise to collective developmental sequences in a biologically plausible manner. The simulation begins with a single progenitor cell containing the artificial genome. This progenitor then gives rise through a lineage of offspring to distinct populations of neuronal precursors that migrate to form the cortical laminae. The precursors differentiate by extending dendrites and axons, which reproduce the experimentally determined branching patterns of a number of different neuronal cell types observed in the cat visual cortex. This result is the first comprehensive demonstration of the principles of self-construction whereby the cortical architecture develops. In addition, our model makes several testable predictions concerning cell migration and branching mechanisms.

Pathways of attention: synaptic relationships of frontal eye field to V4, lateral intraparietal cortex, and area 46 in macaque monkey.

The frontal eye field (FEF) of the primate neocortex occupies a pivotal position in the matrix of inter-areal projections. In addition to its role in directing saccadic eye movements, it is the source of an attentional signal that modulates the activity of neurons in extrastriate and parietal cortex. Here, we tested the prediction that FEF preferentially excites inhibitory neurons in target areas during attentional modulation. Using the anterograde tracer biotinylated dextran amine, we found that the projections from FEF terminate in all cortical layers of area 46, lateral intraparietal area (LIP), and visual area V4. Axons in layer 1 spread extensively, those in layer 2/3 were most numerous, individual axons in layer 4 formed sprays of collaterals, and those of the deep layers were the finest caliber and irregular. All labeled synapses were the typical asymmetric morphology of excitatory synapses of pyramidal neurons. Dendritic spines were the most frequent synaptic target in all areas (95% in area 46, 89% in V4, 84% in LIP, 78% intrinsic local FEF). The remaining targets were one soma and dendritic shafts, most of which showed characteristics of inhibitory neurons with smooth dendrites (5% of all targets in area 46, 2% in V4, 9% in LIP, and 13% in FEF).

An application of multivariate statistical analysis for Query-Driven Visualization.

Driven by the ability to generate ever-larger, increasingly complex data, there is an urgent need in the scientific community for scalable analysis methods that can rapidly identify salient trends in scientific data. Query-Driven Visualization (QDV) strategies are among the small subset of techniques that can address both large and highly complex data sets. This paper extends the utility of QDV strategies with a statistics-based framework that integrates nonparametric distribution estimation techniques with a new segmentation strategy to visually identify statistically significant trends and features within the solution space of a query. In this framework, query distribution estimates help users to interactively explore their query's solution and visually identify the regions where the combined behavior of constrained variables is most important, statistically, to their inquiry. Our new segmentation strategy extends the distribution estimation analysis by visually conveying the individual importance of each variable to these regions of high statistical significance. We demonstrate the analysis benefits these two strategies provide and show how they maybe used to facilitate the refinement of constraints over variables expressed in a user's query. We apply our method to data sets from two different scientific domains to demonstrate its broad applicability.

Smooth, volume-accurate material interface reconstruction.

A new material interface reconstruction method for volume fraction data is presented. Our method is comprised of two components: first, we generate initial interface topology; then, using a combination of smoothing and volumetric forces within an active interface model, we iteratively transform the initial material interfaces into high-quality surfaces that accurately approximate the problem's volume fractions. Unlike all previous work, our new method produces material interfaces that are smooth, continuous across cell boundaries, and segment cells into regions with proper volume. These properties are critical during visualization and analysis. Generating high-quality mesh representations of material interfaces is required for accurate calculations of interface statistics, and dramatically increases the utility of material boundary visualizations.

The synaptic connections between cortical areas V1 and V2 in macaque monkey.

The primary visual cortex (V1) and V2 together form approximately 24% of the total neocortex of the macaque monkey and have each other as their major partners. The major target of the V1 projection to V2 is layer 4, where it forms clusters of boutons, which form asymmetric (excitatory) synapses mainly with dendritic spines (75%). The remainder form synapses with dendritic shafts. The synapses found on spines were often more complex, perforated postsynaptic densities than those found on dendritic shafts. The reciprocal projection from V2 to V1 targeted layers 1, 2/3, and 5 and was formed of axons of different morphologies. One axon type, originating from superficial layer pyramidal cells, had a morphology resembling those of local pyramidal cell collaterals. These axons arborized in layers 1, 2/3, and 5 of V1. Another type of axon, arborizing in layer 1, was slender (0.3 microm), unbranched, unmyelinated, and uniformly covered with boutons terminaux and formed asymmetric synapses mainly with slender spines. Yet a third type of axon also confined to layer 1, was thick (>1 microm), branched, heavily myelinated, and formed separate small clusters of large ( approximately 1 microm) en passant multisynaptic boutons that formed asymmetric synapses mainly with large flat spines. These data show the existence of a reciprocal excitatory loop between V1 and V2 that is formed by different axonal types, each with preferred layers of termination.

The W cell pathway to cat primary visual cortex.

The thalamic input to area 17 in the cat can be divided into at least three parallel pathways, the W, X, and Y. Although the latter two are some of the best studied synaptic connections in the brain, the former remains poorly understood both in structure and in function. By combining light and electron microscopy, we have reconstructed in 3-D single W axons and described quantitatively the synapses that they form. We have also made a structural comparison of reconstructed synapses from the three visual pathways. Thalamic axons were labeled in vivo by injections of biotinylated dextran amine into the dLGN. W axons originating from C laminae injections arborized in layers 1, 2/3, and 5. Axons that traversed layer 1 supplied a few descending collaterals to layer 2/3, but the most extensive innervation in layer 2/3 was provided by axons ascending from the white matter. Most W boutons formed a single synapse, dendritic spines being the most common target, with dendritic shafts forming the remaining targets. In layer 1, the area of the postsynaptic density of spine synapses (0.16 microm(2)) was significantly larger than that of layers 2/3 (0.11 microm(2)) and 5 (0.09 microm(2)). Synapses from X and Y axons in layer 4 were similar in size to synapses formed by W boutons in layer 1. In layer 1, the main targets of the W axons are likely the apical dendrites of pyramidal cells, so that both proximal and distal regions of pyramidal cell dendritic trees can be excited by the W pathway.

Query-driven visualization of time-varying adaptive mesh refinement data.

The visualization and analysis of AMR-based simulations is integral to the process of obtaining new insight in scientific research. We present a new method for performing query-driven visualization and analysis on AMR data, with specific emphasis on time-varying AMR data. Our work introduces a new method that directly addresses the dynamic spatial and temporal properties of AMR grids that challenge many existing visualization techniques. Further, we present the first implementation of query-driven visualization on the GPU that uses a GPU-based indexing structure to both answer queries and efficiently utilize GPU memory. We apply our method to two different science domains to demonstrate its broad applicability.

Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons.

Activity-dependent morphological plasticity of neurons is central to understanding how the synaptic network of the CNS becomes reconfigured in response to experience. In recent years, several studies have shown that synaptic activation that leads to the induction of long-term potentiation also drives the growth of new dendritic spines, raising the possibility that new synapses are made. We examine this directly by correlating time-lapse two-photon microscopy of newly formed spines on CA1 pyramidal neurons in organotypic hippocampal slices with electron microscopy. Our results show that, whereas spines that are only a few hours old rarely form synapses, older spines, ranging from 15 to 19 h, consistently have ultrastructural hallmarks typical of synapses. This is in agreement with a recent in vivo study that showed that, after a few days, new spines consistently form functional synapses. In addition, our study provides a much more detailed understanding of the first few hours after activity-dependent spinogenesis. Within tens of minutes, physical contacts are formed with existing presynaptic boutons, which slowly, over the course of many hours, mature into new synapses.

Fly photoreceptors demonstrate energy-information trade-offs in neural coding.

Trade-offs between energy consumption and neuronal performance must shape the design and evolution of nervous systems, but we lack empirical data showing how neuronal energy costs vary according to performance. Using intracellular recordings from the intact retinas of four flies, Drosophila melanogaster, D. virilis, Calliphora vicina, and Sarcophaga carnaria, we measured the rates at which homologous R1-6 photoreceptors of these species transmit information from the same stimuli and estimated the energy they consumed. In all species, both information rate and energy consumption increase with light intensity. Energy consumption rises from a baseline, the energy required to maintain the dark resting potential. This substantial fixed cost, approximately 20% of a photoreceptor's maximum consumption, causes the unit cost of information (ATP molecules hydrolysed per bit) to fall as information rate increases. The highest information rates, achieved at bright daylight levels, differed according to species, from approximately 200 bits s(-1) in D. melanogaster to approximately 1,000 bits s(-1) in S. carnaria. Comparing species, the fixed cost, the total cost of signalling, and the unit cost (cost per bit) all increase with a photoreceptor's highest information rate to make information more expensive in higher performance cells. This law of diminishing returns promotes the evolution of economical structures by severely penalising overcapacity. Similar relationships could influence the function and design of many neurons because they are subject to similar biophysical constraints on information throughput.

Synaptic connection from cortical area V4 to V2 in macaque monkey.

The major target of the V4 projection in V2 is layer 1, where it forms a tangential spread of asymmetric (excitatory) synapses. This is characteristic of a "feedback" projection. Some axons formed discrete clusters of bouton terminaux between lengths of myelinated axon, while others were unbranched and formed a continuous distribution of en passant boutons with no intercalated myelin. Minor projections were found in layers 2/3 and 6. Dendritic spines were the most frequently encountered targets of the V4 projection (80% in layer 1 and layer 2/3, 94% in layer 6). The remaining targets were dendritic shafts. In layer 1, 69% of target dendrites (12% of all targets) had characteristics identifying them as smooth (GABAergic) cells. In layer 2/3 and layer 6 virtually all the shaft synapses were on smooth dendrites (86% and 100%, respectively). Multisynaptic boutons were rare (mean 1.1 synapses per bouton). Synapses formed in layer 6 were smaller than those of layer 1 (mean area 0.073 microm(2) vs. 0.117 microm(2)). Synapses formed with spines had a more complex postsynaptic density than those formed with dendritic shafts. With respect to targets and synaptic type and size and morphology of synapses, the feedback projection from V4 to V2 resembles those of feedforward projections. The principal difference between the feedforward and feedback projection is in the lamina location of their terminal boutons. The concentration of the V4 projection on layer 1, where it forms asymmetric synapses mainly with spines, suggests that it excites the distal apical dendrites of pyramidal cells.

Connection from cortical area V2 to V3 A in macaque monkey.

The V2 projection to V3 A was labeled by pressure microinjecting biotinylated dextran amine (BDA) and Phaseolus vulgaris lectin (PHA-L) into V2 just posterior to the lunate sulcus. Dense terminal labeling in clusters was found in layer 4, with a weaker terminal projection in layer 3. About 3.5--4.1% of the synapses in the densest bouton clusters in layer 4 were made by labeled boutons. All were asymmetric (Gray's type 1) synapses, made by spiny, excitatory neurons. The most frequently encountered synaptic targets were spines (76% in layer 4, 98% in layer 2/3). The remainder of the synaptic targets were dendritic shafts, of which just less than half (44%) had the characteristic ultrastructure of smooth (inhibitory) cells. Multisynaptic boutons were rare (mean synapses per bouton for layer 4 1.2, for layer 2/3 1.1). The mean size of the postsynaptic densities found on spines (0.11 microm(2)) was not significantly different from that for dendrites (0.09 microm(2)). In terms of their type, laminar location, number, and targets, the synapses that formed the V2 projection to V3 A are typical of a major, excitatory, feedforward projection of macaque visual cortex.

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins.

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.

Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA.

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

Connection from cortical area V2 to MT in macaque monkey.

The extrastriate visual area of the macaque monkey called MT or V5, receives its input from multiple sources. We have previously examined the synaptic connections made by V1 cells that project to MT (Anderson et al., 1998). Here, we provide a similar analysis of the projection from V2 to MT. The major target of the V2 projection in MT is layer 4, where it forms clusters of asymmetric (excitatory) synapses. Unlike the V1 projection, it also forms synapses in layers 1 and 2 and does not form synapses in layer 6. The most frequently encountered targets of boutons labeled from V2 were spines (67% in layer 4; 82% in layer 2/3). Unusually, only 5/12 boutons examined in layer 1 actually formed synapses. Unlike the V1 projection, multisynaptic boutons were rare (mean, 1.1 synapses per bouton vs. 1.7 for the V1 projection). Like the V1 projection, the input to MT from any point in V2 is sparse (contributing approximately 4-6% of the asymmetric synapses in the densest clusters in layer 4). The synapses of the V2 projection were similar in size to those of the V1 projection (0.1 microm(2) vs. 0.09 microm(2)) and both formed more complex postsynaptic densities on spines than on dendritic shafts. The clear differences between the V1 and V2 projection to MT indicate that their functions are complementary rather than completely overlapping.

Chance or design? Some specific considerations concerning synaptic boutons in cat visual cortex.

To understand the rules by which axons lay down their synaptic boutons we analyzed the linear bouton distributions in 39 neurons (23 spiny, 13 smooth) and 3 thalamic axons, which were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The variation of the total number of boutons and the total axonal length was large (789-7,912 boutons, 12-126 mm). The overall linear bouton density for smooth cells was higher than that of spiny cells and thalamic afferents (mean +/- sd, 110 +/- 21 and 78 +/- 27 boutons per mm of axonal length). The distribution of boutons varied according to their location on the tree. Distal axon collaterals (first and second order segments in Horton-Strahler ordering) of smooth neurons had a 3.5 times higher, spiny cells and thalamic afferents a 2 times higher bouton density compared to the higher order (more proximal) segments. The distribution of interbouton intervals was positively skewed and similar for cells of the same type. In most cases a gamma-distribution fitted well, but the distributions had a tendency to have a heavier tail. To a first approximation these bouton distributions are consistent with both diffuse and specific models of interneuronal connections. Quite simple rules can explain these distributions and the connections between the different classes of neurons.

The measurement of glycosylated haemoglobins by the cation exchange chromatographic method in subjects with abnormal haemoglobins

Glycosylated haemoglobins were estimated in subjects with normal and abnormal haemoglobins by a cation exchange chromatographic method. Lower values were obtained in the subjects with abnormal haemoglobins. Diabetic subjects tend to have higher values. In interpreting the values in subjects with abnormal haemoglobins either different reference ranges must be used or a correction factor can be employed (AU)