MARCM (mosaic analysis with a repressible cell marker) involves specific labeling of GAL80-minus and GAL4-positive homozygous cells in otherwise heterozygous tissues. Here we demonstrate how the concurrent use of two independent binary transcriptional systems may facilitate complex MARCM studies in the Drosophila nervous system. By fusing LexA with the VP16 acidic activation domain (VP16) or the GAL4 activation domain (GAD), we obtained both GAL80-insensitive and GAL80-suppressible transcriptional factors. LexA::VP16 can mediate MARCM-independent binary transgene induction in mosaic organisms. The incorporation of LexA::GAD into MARCM, which we call dual-expression-control MARCM, permits the induction of distinct transgenes in different patterns among GAL80-minus cells in mosaic tissues. Lineage analysis with dual-expression-control MARCM suggested the presence of neuroglioblasts in the developing optic lobes but did not indicate the production of glia by postembryonic mushroom body neuronal precursors. In addition, dual-expression-control MARCM with a ubiquitous LexA::GAD driver revealed many unidentified cells in the GAL4-GH146-positive projection neuron lineages.

Identifying the genes underlying genetically complex traits is of fundamental importance for medicine, agriculture, and evolutionary biology. However, the level of resolution offered by traditional quantitative trait locus (QTL) mapping is usually coarse. We analyze here a trait closely related to fitness, ovariole number. Our initial interspecific mapping between Drosophila sechellia (8 ovarioles/ovary) and D. simulans (15 ovarioles/ovary) identified a major QTL on chromosome 3 and a minor QTL on chromosome 2. To refine the position of the major QTL, we selected 1038 additional recombinants in the region of interest using flanking morphological markers (selective phenotyping). This effort generated approximately one recombination event per gene and increased the mapping resolution by approximately seven times. Our study thus shows that using visible markers to select for recombinants can efficiently increase the resolution of QTL mapping. We resolved the major QTL into two epistatic QTL, QTL3a and QTL3b. QTL3a shows sign epistasis: it has opposite effects in two different genetic backgrounds, the presence vs. the absence of the QTL3b D. sechellia allele. This property of QTL3a allows us to reconstruct the probable order of fixation of the QTL alleles during evolution.

Janelia Farm, the new research campus of the Howard Hughes Medical Institute, is an ongoing experiment in the social engineering of research communities.

Although radial oblique dendrites are a major synaptic input site in CA1 pyramidal neurons, little is known about their integrative properties. We have used multisite two-photon glutamate uncaging to deliver different spatiotemporal input patterns to single branches while simultaneously recording the uncaging-evoked excitatory postsynaptic potentials and local Ca2+ signals. Asynchronous input patterns sum linearly in spite of the spatial clustering and produce Ca2+ signals that are mediated by NMDA receptors (NMDARs). Appropriately timed and sized input patterns ( approximately 20 inputs within approximately 6 ms) produce a supralinear summation due to the initiation of a dendritic spike. The Ca2+ signals associated with synchronous input were larger and mediated by influx through both NMDARs and voltage-gated Ca2+ channels (VGCCs). The oblique spike is a fast Na+ spike whose duration is shaped by the coincident activation of NMDAR, VGCCs, and transient K+ currents. Our results suggest that individual branches can function as single integrative compartments.

Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of our method and give example applications in ratiometric calcium imaging of bulk-stained olfactory bulb slices. Converting the fluorescence signals into [Ca2+] gives resting values consistent with earlier single-cell staining results, and odorant-induced [Ca2+] transients can be quantitatively compared in different cells. Using these examples we show that inaccurate background subtraction introduces large errors (easily in the range of 100%) in the assessment of both resting [Ca2+] and [Ca2+] dynamics. The proposed method allows us to avoid such errors.

Mean-Field theory is extended to recurrent networks of spiking neurons endowed with short-term depression (STD) of synaptic transmission. The extension involves the use of the distribution of interspike intervals of an integrate-and-fire neuron receiving a Gaussian current, with a given mean and variance, in input. This, in turn, is used to obtain an accurate estimate of the resulting postsynaptic current in presence of STD. The stationary states of the network are obtained requiring self-consistency for the currents-those driving the emission processes and those generated by the emitted spikes. The model network stores in the distribution of two-state efficacies of excitatory-to-excitatory synapses, a randomly composed set of external stimuli. The resulting synaptic structure allows the network to exhibit selective persistent activity for each stimulus in the set. Theory predicts the onset of selective persistent, or working memory (WM) activity upon varying the constitutive parameters (e.g. potentiated/depressed long-term efficacy ratio, parameters associated with STD), and provides the average emission rates in the various steady states. Theoretical estimates are in remarkably good agreement with data "recorded" in computer simulations of the microscopic model.

A sum-frequency generation (SFG) microscope that is sensitive toward molecular chirality was demonstrated for the first time. Optically active images of chiral 1,1’-bi-2-naphthol solutions were obtained with submicron spatial resolution. Three-dimensional sectioning capability of our microscope was also demonstrated. This optically active SFG microscopy can potentially become a powerful imaging technique for biological samples.

We pursue the hypothesis that neuronal placement in animals minimizes wiring costs for given functional constraints, as specified by synaptic connectivity. Using a newly compiled version of the Caenorhabditis elegans wiring diagram, we solve for the optimal layout of 279 nonpharyngeal neurons. In the optimal layout, most neurons are located close to their actual positions, suggesting that wiring minimization is an important factor. Yet some neurons exhibit strong deviations from "optimal" position. We propose that biological factors relating to axonal guidance and command neuron functions contribute to these deviations. We capture these factors by proposing a modified wiring cost function.

BACKGROUND: The best studied insect-symbiont system is that of aphids and their primary bacterial endosymbiont Buchnera aphidicola. Buchnera inhabits specialized host cells called bacteriocytes, provides nutrients to the aphid and has co-speciated with its aphid hosts for the past 150 million years. We have used a single microarray to examine gene expression in the pea aphid, Acyrthosiphon pisum, and its resident Buchnera. Very little is known of gene expression in aphids, few studies have examined gene expression in Buchnera, and no study has examined simultaneously the expression profiles of a host and its symbiont. Expression profiling of aphids, in studies such as this, will be critical for assigning newly discovered A. pisum genes to functional roles. In particular, because aphids possess many genes that are absent from Drosophila and other holometabolous insect taxa, aphid genome annotation efforts cannot rely entirely on homology to the best-studied insect systems. Development of this dual-genome array represents a first attempt to characterize gene expression in this emerging model system.

RESULTS: We chose to examine heat shock response because it has been well characterized both in Buchnera and in other insect species. Our results from the Buchnera of A. pisum show responses for the same gene set as an earlier study of heat shock response in Buchnera for the host aphid Schizaphis graminum. Additionally, analyses of aphid transcripts showed the expected response for homologs of known heat shock genes as well as responses for several genes with unknown functional roles.

CONCLUSION: We examined gene expression under heat shock of an insect and its bacterial symbiont in a single assay using a dual-genome microarray. Further, our results indicate that microarrays are a useful tool for inferring functional roles of genes in A. pisum and other insects and suggest that the pea aphid genome may contain many gene paralogs that are differentially regulated.

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.