We developed a multicolor neuron labeling technique in Drosophila melanogaster that combines the power to specifically target different neural populations with the label diversity provided by stochastic color choice. This adaptation of vertebrate Brainbow uses recombination to select one of three epitope-tagged proteins detectable by immunofluorescence. Two copies of this construct yield six bright, separable colors. We used Drosophila Brainbow to study the innervation patterns of multiple antennal lobe projection neuron lineages in the same preparation and to observe the relative trajectories of individual aminergic neurons. Nerve bundles, and even individual neurites hundreds of micrometers long, can be followed with definitive color labeling. We traced motor neurons in the subesophageal ganglion and correlated them to neuromuscular junctions to identify their specific proboscis muscle targets. The ability to independently visualize multiple lineage or neuron projections in the same preparation greatly advances the goal of mapping how neurons connect into circuits.

Parallel circuits throughout the CNS exhibit distinct sensitivities and responses to sensory stimuli. Ambiguities in the source and properties of signals elicited by physiological stimuli, however, frequently obscure the mechanisms underlying these distinctions. We found that differences in the degree to which activity in two classes of Off retinal ganglion cell (RGC) encode information about light stimuli near detection threshold were not due to obvious differences in the cells’ intrinsic properties or the chemical synaptic input the cells received; indeed, differences in the cells’ light responses were largely insensitive to block of fast ionotropic glutamate receptors. Instead, the distinct responses of the two types of RGCs likely reflect differences in light-evoked electrical synaptic input. These results highlight a surprising strategy by which the retina differentially processes and routes visual information and provide new insight into the circuits that underlie responses to stimuli near detection threshold.

We took advantage of the unusual genomic organization of the ciliate Oxytricha trifallax to screen for eukaryotic non-coding RNA (ncRNA) genes. Ciliates have two types of nuclei: a germ line micronucleus that is usually transcriptionally inactive, and a somatic macronucleus that contains a reduced, fragmented and rearranged genome that expresses all genes required for growth and asexual reproduction. In some ciliates including Oxytricha, the macronuclear genome is particularly extreme, consisting of thousands of tiny ’nanochromosomes’, each of which usually contains only a single gene. Because the organism itself identifies and isolates most of its genes on single-gene nanochromosomes, nanochromosome structure could facilitate the discovery of unusual genes or gene classes, such as ncRNA genes. Using a draft Oxytricha genome assembly and a custom-written protein-coding genefinding program, we identified a subset of nanochromosomes that lack any detectable protein-coding gene, thereby strongly enriching for nanochromosomes that carry ncRNA genes. We found only a small proportion of non-coding nanochromosomes, suggesting that Oxytricha has few independent ncRNA genes besides homologs of already known RNAs. Other than new members of known ncRNA classes including C/D and H/ACA snoRNAs, our screen identified one new family of small RNA genes, named the Arisong RNAs, which share some of the features of small nuclear RNAs.

Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, “leuco” intermediates.

MOTIVATION: Homology search for RNAs can use secondary structure information to increase power by modeling base pairs, as in covariance models, but the resulting computational costs are high. Typical acceleration strategies rely on at least one filtering stage using sequence-only search. RESULTS: Here we present the multi-segment CYK (MSCYK) filter, which implements a heuristic of ungapped structural alignment for RNA homology search. Compared to gapped alignment, this approximation has lower computation time requirements (O(N⁴) reduced to O(N³), and space requirements (O(N³) reduced to O(N²). A vector-parallel implementation of this method gives up to 100-fold speed-up; vector-parallel implementations of standard gapped alignment at two levels of precision give 3- and 6-fold speed-ups. These approaches are combined to create a filtering pipeline that scores RNA secondary structure at all stages, with results that are synergistic with existing methods.

Synaptic plasticity in response to changes in physiologic state is coordinated by hormonal signals across multiple neuronal cell types, but the significance and underlying mechanisms are unclear. Here, we combine cell type-specific electrophysiological, pharmacological, and optogenetic techniques to dissect neural circuits and molecular pathways controlling synaptic plasticity onto AGRP neurons, a population that regulates feeding. We find that food deprivation elevates excitatory synaptic input, which is mediated by a presynaptic positive feedback loop involving AMP-activated protein kinase. Potentiation of glutamate release was triggered by the orexigenic hormone ghrelin and exhibited hysteresis, persisting for hours after ghrelin removal. Persistent activity was reversed by the anorexigenic hormone leptin, and optogenetic photostimulation demonstrated involvement of opioid release from POMC neurons. Based on these experiments, we propose a memory storage device for physiological state constructed from bistable synapses that are flipped between two sustained activity states by transient exposure to hormones signaling energy levels. Supported by: Howard Hughes Medical Institute.

Optogenetics is routinely used to activate and inactivate genetically defined neuronal populations in vivo. A second optogenetic revolution will occur when spatially distributed and sparse neural assemblies can be precisely manipulated in behaving animals.

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI(N)) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI(N) values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.

The male-specific Fruitless proteins (Fru(M)) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened  1000 GAL4 lines, using assays for general courtship, male-male interactions, and male fertility to determine the phenotypes resulting from the GAL4 driven inhibition of Fru(M) expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups based on additional neurobiological and behavioral criteria. For example, in some lines restoration of Fru(M) expression in cholinergic neurons restores fertility or reduces male-male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately whereas in other lines this phenotype result from apparent habituation deficits. Inhibition of ectopic Fru(M) expression in females, in populations of neurons where Fru(M) is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous Fru(M) expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with Fru(M) expression in many regions of the nervous system suggesting likely redundant Fru(M)-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.

Research in the fruit fly Drosophila melanogaster has led to insights in neural development, axon guidance, ion channel function, synaptic transmission, learning and memory, diurnal rhythmicity, and neural disease that have had broad implications for neuroscience. Drosophila is currently the eukaryotic model organism that permits the most sophisticated in vivo manipulations to address the function of neurons and neuronally expressed genes. Here, we summarize many of the techniques that help assess the role of specific neurons by labeling, removing, or altering their activity. We also survey genetic manipulations to identify and characterize neural genes by mutation, overexpression, and protein labeling. Here, we attempt to acquaint the reader with available options and contexts to apply these methods.