Sex-specific regulation of Lgr3 in Drosophila neurons.

The development of sexually dimorphic morphology and the potential for sexually dimorphic behavior in Drosophila are regulated by the Fruitless (Fru) and Doublesex (Dsx) transcription factors. Several direct targets of Dsx have been identified, but direct Fru targets have not been definitively identified. We show that Drosophila leucine-rich repeat G protein-coupled receptor 3 (Lgr3) is regulated by Fru and Dsx in separate populations of neurons. Lgr3 is a member of the relaxin-receptor family and a receptor for Dilp8, necessary for control of organ growth. Lgr3 expression in the anterior central brain of males is inhibited by the B isoform of Fru, whose DNA binding domain interacts with a short region of an Lgr3 intron. Fru A and C isoform mutants had no observed effect on Lgr3 expression. The female form of Dsx (Dsx(F)) separately up- and down-regulates Lgr3 expression in distinct neurons in the abdominal ganglion through female- and male-specific Lgr3 enhancers. Excitation of neural activity in the Dsx(F)-up-regulated abdominal ganglion neurons inhibits female receptivity, indicating the importance of these neurons for sexual behavior. Coordinated regulation of Lgr3 by Fru and Dsx marks a point of convergence of the two branches of the sex-determination hierarchy.

Joint control of Drosophila male courtship behavior by motion cues and activation of male-specific P1 neurons.

Sexual behaviors in animals are governed by inputs from multiple external sensory modalities. However, how these inputs are integrated to jointly control animal behavior is still poorly understood. Whereas visual information alone is not sufficient to induce courtship behavior in Drosophila melanogaster males, when a subset of male-specific fruitless (fru)- and doublesex (dsx)-expressing neurons that respond to chemosensory cues (P1 neurons) were artificially activated via a temperature-sensitive cation channel (dTRPA1), males followed and extended their wing toward moving objects (even a moving piece of rubber band) intensively. When stationary, these objects were not courted. Our results indicate that motion input and activation of P1 neurons are individually necessary, and under our assay conditions, jointly sufficient to elicit early courtship behaviors, and provide insights into how courtship decisions are made via sensory integration.

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors.

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. Here we report studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. We show that the sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (Fru(M)) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (Dsx(F)). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (Dsx(M)). Finally, we (1) demonstrate that the roundabout (robo) paralogs also regulate midline crossing by these neurons, and (2) provide evidence that Fru(M) exerts its effect on midline crossing by directly or indirectly regulating Robo signaling.

Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

A searchable image resource of Drosophila GAL4 driver expression patterns with single neuron resolution.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

Mapping Neurotransmitter Identity in the Whole-Mount Drosophila Brain Using Multiplex High-Throughput Fluorescence in Situ Hybridization.

Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter coexpression, reveal neurotransmitter phenotypes of specific cell types, and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large-scale FISH detection of any mRNA in whole-mount fly brains.

Blueprints for behavior: genetic specification of neural circuitry for innate behaviors.

Innate behaviors offer a unique opportunity to use genetic analysis to dissect and characterize the neural substrates of complex behavioral programs. Courtship in Drosophila involves a complex series of stereotyped behaviors that include numerous exchanges of multimodal sensory information over time. As we will discuss in this review, recent work has demonstrated that male-specific expression of Fruitless transcription factors (Fru(M) proteins) is necessary and sufficient to confer the potential for male courtship behaviors. Fru(M) factors program neurons of the male central and peripheral nervous systems whose function is dedicated to sexual behaviors. This circuitry seems to integrate sensory information to define behavioral states and regulate conserved neural elements for sex-specific behavioral output. The principles that govern the circuitry specified by Fru(M) expression might also operate in subcortical networks that govern innate behaviors in mammals.

Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

Central brain neurons expressing doublesex regulate female receptivity in Drosophila.

Drosophila melanogaster females respond to male courtship by either rejecting the male or allowing copulation. The neural mechanisms underlying these female behaviors likely involve the integration of sensory information in the brain. Because doublesex (dsx) controls other aspects of female differentiation, we asked whether dsx-expressing neurons mediate virgin female receptivity to courting males. Using intersectional techniques to manipulate the activities of defined subsets of dsx-expressing neurons, we found that activation of neurons in either the pCd or pC1 clusters promotes receptivity, while silencing these neurons makes females unreceptive. Furthermore, pCd and pC1 neurons physiologically respond to the male-specific pheromone cis-vaccenyl acetate (cVA), while pC1 neurons also respond to male courtship song. The pCd and pC1 neurons expressing dsx in females do not express transcripts from the fruitless (fru) P1 promoter. Thus, virgin female receptivity is controlled at least in part by neurons that are distinct from those governing male courtship.

A small subset of fruitless subesophageal neurons modulate early courtship in Drosophila.

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless Male (fru M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru M expression or inhibition of synaptic transmission in these fru M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.

Functional dissection of the neural substrates for sexual behaviors in Drosophila melanogaster.

The male-specific Fruitless proteins (FruM) 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 FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM 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 results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM 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 FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.

Reversal of motor learning in the vestibulo-ocular reflex in the absence of visual input.

Motor learning in the vestibulo-ocular reflex (VOR) and eyeblink conditioning use similar neural circuitry, and they may use similar cellular plasticity mechanisms. Classically conditioned eyeblink responses undergo extinction after prolonged exposure to the conditioned stimulus in the absence of the unconditioned stimulus. We investigated the possibility that a process similar to extinction may reverse learned changes in the VOR. We induced a learned alteration of the VOR response in rhesus monkeys using magnifying or miniaturizing goggles, which caused head movements to be accompanied by visual image motion. After learning, head movements in the absence of visual stimulation caused a loss of the learned eye movement response. When the learned gain was low, this reversal of learning occurred only when head movements were delivered, and not when the head was held stationary in the absence of visual input, suggesting that this reversal is mediated by an active, extinction-like process.