Because of its genetic, molecular, and behavioral tractability, Drosophila has emerged as a powerful model system for studying molecular and cellular mechanisms underlying the development and function of nervous systems. The Drosophila nervous system has fewer neurons and exhibits a lower glia:neuron ratio than is seen in vertebrate nervous systems. Despite the simplicity of the Drosophila nervous system, glial organization in flies is as sophisticated as it is in vertebrates. Furthermore, fly glial cells play vital roles in neural development and behavior. In addition, powerful genetic tools are continuously being created to explore cell function in vivo. In taking advantage of these features, the fly nervous system serves as an excellent model system to study general aspects of glial cell development and function in vivo. In this article, we review and discuss advanced genetic tools that are potentially useful for understanding glial cell biology in Drosophila.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans.

NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.

Parallel processing is an organizing principle of many neural circuits. In the retina, parallel neuronal pathways process signals from rod and cone photoreceptors and support vision over a wide range of light levels. Toward this end, rods and cones form triad synapses with dendrites of distinct bipolar cell types, and the axons or dendrites, respectively, of horizontal cells (HCs). The molecular cues that promote the formation of specific neuronal pathways remain largely unknown. Here, we discover that developing and mature HCs express the leucine-rich repeat (LRR)-containing protein netrin-G ligand 2 (NGL-2). NGL-2 localizes selectively to the tips of HC axons, which form reciprocal connections with rods. In mice with null mutations in Ngl-2 (Ngl-2⁻/⁻), many branches of HC axons fail to stratify in the outer plexiform layer (OPL) and invade the outer nuclear layer. In addition, HC axons expand lateral territories and increase coverage of the OPL, but establish fewer synapses with rods. NGL-2 can form transsynaptic adhesion complexes with netrin-G2, which we show to be expressed by photoreceptors. In Ngl-2⁻/⁻ mice, we find specific defects in the assembly of presynaptic ribbons in rods, indicating that reverse signaling of complexes involving NGL-2 regulates presynaptic maturation. The development of HC dendrites and triad synapses of cone photoreceptors proceeds normally in the absence of NGL-2 and in vivo electrophysiology reveals selective defects in rod-mediated signal transmission in Ngl-2⁻/⁻ mice. Thus, our results identify NGL-2 as a central component of pathway-specific development in the outer retina.

SUMMARY: Sequence database searches are an essential part of molecular biology, providing information about the function and evolutionary history of proteins, RNA molecules and DNA sequence elements. We present a tool for DNA/DNA sequence comparison that is built on the HMMER framework, which applies probabilistic inference methods based on hidden Markov models to the problem of homology search. This tool, called nhmmer, enables improved detection of remote DNA homologs, and has been used in combination with Dfam and RepeatMasker to improve annotation of transposable elements in the human genome. AVAILABILITY: nhmmer is a part of the new HMMER3.1 release. Source code and documentation can be downloaded from http://hmmer.org. HMMER3.1 is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X. CONTACT: wheelert@janelia.hhmi.org.

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of α4β2-type nicotinic acetylcholine receptors (α4β2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required α4β2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotine’s addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger “ministacks”, similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of α4β2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2 neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2 neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

Metal ion homeostasis is critical to the survival of all cells. Regulation of nickel concentrations in Escherichia coli is mediated by the NikR repressor via nickel-induced transcriptional repression of the nickel ABC-type transporter, NikABCDE. Here, we report two crystal structures of nickel-activated E. coli NikR, the isolated repressor at 2.1 A resolution and in a complex with its operator DNA sequence from the nik promoter at 3.1 A resolution. Along with the previously published structure of apo-NikR, these structures allow us to evaluate functional proposals for how metal ions activate NikR, delineate the drastic conformational changes required for operator recognition, and describe the formation of a second metal-binding site in the presence of DNA. They also provide a rare set of structural views of a ligand-responsive transcription factor in the unbound, ligand-induced, and DNA-bound states, establishing a model system for the study of ligand-mediated effects on transcription factor function.

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso & Rubin 2016). Here we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in . NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

Nitric oxide (NO) is produced in mammals by different isoforms of NO synthase (NOS), including the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochondria. We show that mitochondrially derived NO mediates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the impaired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L-NAME. These observations suggest that mitochondria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochondrial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.