Note: Characterization of electrode materials for dielectric spectroscopy.

When measuring the dielectric properties of aqueous samples, the impedance of the electrode/sample interface can limit low frequency measurements. The electrode polarization problem can be reduced by increasing the effective surface area of the electrodes. In this work, impedance spectroscopy was used to characterize and compare three different electrode surfaces that can be used to mitigate this effect: platinum black, iridium oxide, and [polypyrrole/poly(styrenesulphonate)] (PPy/PSS) conducting polymer. All three materials were directly compared with a bright platinum electrode. Equivalent circuit models were used to extract the increase in the effective surface area of the electrodes: platinum black, iridium oxide and PPy/PSS increase the effective capacitance of the electrode by factors of approximately 240, 75, and 790, respectively. The practical aspects of all electrode materials are discussed. These results suggest that iridium oxide and PPy/PSS are good alternatives to the commonly used platinum black, which is prone to mechanical damage (scratches) and is potentially toxic to cells.

Inbred mouse strains differ in multiple hippocampal activity traits.

A major challenge in neuroscience is to identify genes that influence specific behaviors and to understand the intermediary neuronal mechanisms. One approach is to identify so-called endophenotypes at different levels of neuronal organization from synapse to brain activity. An endophenotype is a quantitative trait that is closer to the gene action than behavior, and potentially a marker of neuronal mechanisms underlying behavior. Hippocampal activity and, in particular, hippocampal oscillations have been suggested to underlie various cognitive and motor functions. To identify quantitative traits that are potentially useful for identifying genes influencing hippocampal activity, we measured gamma oscillations and spontaneous activity in acute hippocampal slices from eight inbred mouse strains under three experimental conditions. We estimated the heritability of more than 200 quantitative traits derived from this activity. We observed significant differences between the different mouse strains, particularly in the amplitude of the activity and the correlation between activities in different hippocampal subregions. Interestingly, these traits had a low genetic correlation between the three experimental conditions, which suggests that different genetic components influence the activity in different conditions. Our findings show that several traits of hippocampal gamma oscillations and spontaneous activity are heritable and could thus be potentially useful in gene-finding strategies based on endophenotypes.

The need for integrating neuronal morphology databases and computational environments in exploring neuronal structure and function.

Neurons connect to each other through a myriad of dendritic and axonal arborisations. Dendritic structures provide the substrate for integration of postsynaptic potentials and control of action potential generation. Axonal structures provide the substrate for action potential dissemination and signalling to target neurons. The morphological complexity of dendritic arborisations is assumed to play a critical role in the transformation of spatio-temporal patterns of postsynaptic potentials into time-structured series of action potentials. Although these transformations lie at the basis of information processing in the brain, it is still far from understood how their details are influenced by dendritic shape. To facilitate research in this area, it is necessary that data on both the morphology and electrical properties of neurons, as well as computational tools for analysis, become available in an integrated way. This requires a combined effort from the fields of informatics and neurosciences (together called neuroinformatics) in order to create data acquisition, databasing and computational tools. Focusing on neuronal morphology, this chapter will give a brief review of the current neuroinformatics developments in both reconstruction techniques, morphological quantification, modeling of morphological complexity, modeling of function and the need for databasing neuronal morphologies. Additionally, one of the dendritic modeling approaches is described in more detail in the Appendix.

The role of calcium signaling in early axonal and dendritic morphogenesis of rat cerebral cortex neurons under non-stimulated growth conditions.

The effects of depolarizing stimuli on neurite outgrowth have been shown to depend on an influx of extracellular calcium. However, the role of calcium under non-stimulated growth conditions is less well established. Here we investigated the contribution of calcium signaling to early neuronal morphogenesis of rat cerebral cortex neurons at three levels by blocking L-type voltage sensitive calcium channels, by depleting intracellular calcium or by blocking myosin light chain kinase. Detailed quantitative morphological analysis of neurons treated for 1 day revealed that depletion of intracellular calcium strongly decreased the density of filopodia, arrested axonal outgrowth and strongly decreased dendritic branching. Preventing calcium influx through L-type voltage sensitive calcium channels and blocking of myosin light chain kinase activity selectively decreased dendritic branching. Our observations support an essential role for basal intracellular calcium levels in axonal elongation. Furthermore, under non-stimulated conditions calcium entry through L-type voltage sensitive calcium channels and myosin light chain kinase play an important role in dendritic branching.

Competition in the development of nerve connections: a review of models.

The establishment and refinement of neural circuits involve both the formation of new connections and the elimination of already existing connections. Elimination of connections occurs, for example, in the development of mononeural innervation of muscle fibres and in the formation of ocular dominance columns in the visual cortex. The process that leads to the elimination of connections is often referred to as axonal or synaptic competition. Although the notion of competition is commonly used, the process is not well understood-with respect to, for example, the type of competition, what axons and synapses are competing for, and the role of electrical activity. This article reviews the types of competition that have been distinguished and the models of competition that have been proposed. Models of both the neuromuscular system and the visual system are described. For each of these models, the assumptions on which it is based, its mathematical structure, and the extent to which it is supported by the experimental data are evaluated. Special attention is given to the different modelling approaches and the role of electrical activity in competition.

Development of nerve connections under the control of neurotrophic factors: parallels with consumer-resource systems in population biology.

The development of connections between neurons and their target cells involves competition between axons for target-derived neurotrophic factors. Although the notion of competition is commonly used in neurobiology, the process is not well understood, and only a few formal models exist. In population biology, in contrast, the concept of competition is well developed and has been studied by means of many formal models of consumer-resource systems. Here we show that a recently formulated model of axonal competition can be rewritten as a general consumer-resource system. This allows neurobiological phenomena to be interpreted in population biological terms and, conversely, results from population biology to be applied to neurobiology. Using findings from population biology, we have studied two extensions of our axonal competition model. In the first extension, the spatial dimension of the target is explicitly taken into account. We show that distance between axons on their target mitigates competition and permits the coexistence of axons. The model can account for the fact that in many types of neurons a positive correlation exists between the size of the dendritic tree and the number of innervating axons surviving into adulthood. In the second extension, axons are allowed to respond to more than one neurotrophic factor. We show that this permits competitive exclusion among axons of one type, while at the same time there is coexistence with axons of another type innervating the same target. The model offers an explanation for the innervation pattern found on cerebellar Purkinje cells, where climbing fibres compete with each other until only a single one remains, which coexists with parallel fibre input to the same Purkinje cell.

Lateral cell movement driven by dendritic interactions is sufficient to form retinal mosaics.

The formation of retinal mosaics is thought to involve lateral movement of retinal cells from their clonal column of origin. The forces underlying this lateral cell movement are currently unknown. We have used a model of neurite outgrowth combined with cell movement to investigate the hypothesis that lateral cell movement is guided by dendritic interactions. We have assumed that cells repel each other in proportion to the degree of dendritic overlap between neighbouring cells. Our results first show that small cell movements are sufficient to transform random cell distributions into regular mosaics, and that all cells within the population move. When dendritic fields are allowed to grow, the model produces regular mosaics across all cell densities tested. We also find that the model can produce constant coverage of visual space over varying cell densities. However, if dendritic field sizes are fixed, mosaic regularity is proportional to the cell density and dendritic field size. Our model suggests that dendritic mechanisms may therefore provide sufficient information for rearrangement of cells into regular mosaics. We conclude by mentioning possible future experiments that might suggest whether dendritic interactions are adaptive or fixed during mosaic formation.

Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme.

We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.

Isolation and functional characterisation of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous.

The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crt YB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to beta-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.

Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae.

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.

A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

Cloning and characterization of the astaxanthin biosynthetic gene encoding phytoene desaturase of Xanthophyllomyces dendrorhous.

The first carotenoid biosynthetic gene from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated by heterologous complementation in Escherichia coli. The isolated gene, denominated as crtI, was found to encode for phytoene desaturase. The coding region is interrupted by 11 introns. The deduced amino acid sequence showed significant homology with its bacterial and eukaryotic counterparts, especially those of fungal origin. A plasmid containing the geranylgeranyl diphosphate synthase and phytoene synthase encoding genes from Erwinia uredovora was introduced in E. coli together with the phytoene desaturase encoding cDNA from X. dendrorhous. As a result, lycopene accumulation was observed in these transformants. We conclude that in X. dendrorhous the four desaturase steps, by which phytoene is converted into lycopene, are carried out by a single gene product.

Competition for neurotrophic factor in the development of nerve connections.

The development of nerve connections is thought to involve competition among axons for survival promoting factors, or neurotrophins, which are released by the cells that are innervated by the axons. Although the notion of competition is widely used within neurobiology, there is little understanding of the nature of the competitive process and the underlying mechanisms. We present a new theoretical model to analyse competition in the development of nerve connections. According to the model, the precise manner in which neurotrophins regulate the growth of axons, in particular the growth of the amount of neurotrophin receptor, determines what patterns of target innervation can develop. The regulation of neurotrophin receptors is also involved in the degeneration and regeneration of connections. Competition in our model can be influenced by factors dependent on and independent of neuronal electrical activity. Our results point to the need to measure directly the specific form of the regulation by neurotrophins of their receptors.

Poly- and mononeuronal innervation in a model for the development of neuromuscular connections.

In the normal development of connections between motor neurons and muscle fibres, an initial stage of polyneuronal innervation is followed by withdrawal of connections until each muscle fibre is innervated by a single axon. However, polyneuronal innervation has been found to persist after prolonged nerve conduction block, in spite of the resumption of normal neuromuscular activity. Here we analyse in detail a model proposed for the withdrawal of nerve connections in developing muscle, based on competition between nerve terminals. The model combines competition for a pre-synaptic resource with competition for a post-synaptic resource. Using bifurcation and phase space analysis, we show that polyneuronal innervation, as well as mononeuronal innervation, can be stable. The model accounts for the development of mononeuronal innervation and for persistent polyneuronal innervation after prolonged nerve conduction block, which appears as a consequence of the general competitive interactions operating during normal development.

Models of axon guidance and bundling during development.

Diffusible chemoattractants and chemorepellants, together with contact attraction and repulsion, have been implicated in the establishment of connections between neurons and their targets. Here we study how such diffusible and contact signals can be involved in the whole sequence of events from bundling of axons, guidance of axon bundles towards their targets, to debundling and the final innervation of individual targets. By means of computer simulations, we investigate the strengths and weaknesses of a number of particular mechanisms that have been proposed for these processes.

Molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Phaffia rhodozyma.

The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.

Effects of inhibition on neural network development through activity-dependent neurite outgrowth.

Empirical studies have demonstrated that electrical activity of the neuron can directly affect the outgrowth of its neurites. In this paper, the implications of activity-dependent neurite outgrowth are studied in a simple two-cell model, containing one excitatory and one inhibitory cell. We show that activity-dependent outgrowth in combination with the presence of inhibition can account for biostability. The attractors, which can be both point and limit cycle attractors, may be associated with "normal" and "pathological" end states of network development. A slight modification of the model makes it applicable also to a range of other activity-dependent processes in neurons, such as changes in the number of efficacy of receptors. The main results of the previous model are also found in the modified model.

High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma.

This report describes a transformation system leading to stable high copy number integration into the ribosomal DNA (rDNA) of the astaxanthin-producing yeast Phaffia rhodozyma. A plasmid was constructed that contains the transposon Tn5 encoded kanamycin resistance gene (KmR) fused in frame to the 5'-terminal portion of the Phaffia actin gene. This marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that comprises the 18S rDNA and promotes high copy integration leading to stable Phaffia transformants that maintained the plasmid at high copy number after 15 generations of non-selective growth. Phaffia, strain CBS 6938, was found to contain the rDNA units in clusters distributed over three chromosomes with a total copy number of 61. Phaffia transformants were shown to have over 50 copies of pGB-Ph9 integrated in tandem in chromosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophoresis strongly suggest that Phaffia is haploid.

Structural and phylogenetic analysis of the actin gene from the yeast Phaffia rhodozyma.

The gene coding for actin from Phaffia rhodozyma was cloned and sequenced. The Phaffia actin gene contains four intervening sequences and the predicted protein consists of 375 amino acids. The structural features of the Phaffia actin introns were studied and compared with actin introns from seven fungi and yeasts with ascomycetous and basidiomycetous affinity. It was shown that the architecture of the Phaffia introns most resembles that of the basidiomycete Filobasidiella neoformans (perfect stage of Cryptococcus neoformans), whereas least resemblance occurs with the ascomycetous yeasts. Based on the intron structure, the ascomycetous yeasts can be accommodated in one group in that their splice site sequences are very similar and show less homology with the other fungi investigated, including Phaffia. It was demonstrated that the Phaffia actin introns cannot be spliced in Saccharomyces cerevisiae, which shows that the differences found in intron structure are significant. Alignment of the Phaffia actin gene with the actin sequences from the yeasts and fungi investigated showed a high level of homology both on the DNA level and on the protein level. Based on these alignments Phaffia showed highest homology with F. neoformans and both organisms were accommodated in the same cluster. In addition, the actin gene comparisons also supported the distant relationship of Phaffia with the ascomycetous yeasts. These results supported the usefulness of actin sequences for phylogenetic studies.

Complex periodic behaviour in a neural network model with activity-dependent neurite outgrowth.

Empirical studies have demonstrated that electrical activity of the neuron can directly affect neurite outgrowth. High levels of activity cause neurites to retract, whereas low levels allow further outgrowth. Previously we studied networks in which all the cells reacted in the same way on electrical activity. Since experiments have shown that neurons may in fact react differentially, we study in this paper networks in which the range of activity where outgrowth takes place varies among cells. We show that this can lead to complex periodic behaviour in electrical activity and connectivity of individual cells. The precise behaviour depends on the spatial distribution of the cells and the distribution of the outgrowth properties over the cells. Any other cellular property that adapts slowly to electrical activity such that neuronal activity is attempted to be maintained at a given level, can lead to similar results.