The anatomy of many neural circuits is being characterized with increasing resolution, but their molecular properties remain mostly unknown. Here, we characterize gene expression patterns in distinct neural cell types of the visual system using genetic lines to access individual cell types, the TAPIN-seq method to measure their transcriptomes, and a probabilistic method to interpret these measurements. We used these tools to build a resource of high-resolution transcriptomes for 100 driver lines covering 67 cell types, available at http://www.opticlobe.com. Combining these transcriptomes with recently reported connectomes helps characterize how information is transmitted and processed across a range of scales, from individual synapses to circuit pathways. We describe examples that include identifying neurotransmitters, including cases of apparent co-release, generating functional hypotheses based on receptor expression, as well as identifying strong commonalities between different cell types.

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA deficient flies were previously generated using tissue selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by L-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, since existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, revealing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.

Assigning valence-appeal or aversion-to gustatory stimuli and relaying it to higher-order brain regions to guide flexible behaviors is crucial to survival. Yet the neural circuits that transform taste into motivationally relevant signals remain poorly defined in any model system. In Drosophila melanogaster, substantial progress has been made in mapping the sensorimotor pathways encoding intrinsic valence for feeding and the architecture of the dopaminergic reinforcement system. However, where and how "effective" (i.e., real-time) valence is first imposed on a taste has long been a mystery. Here, we identified a pair of subesophageal zone interneurons in Drosophila, termed Fox, that impart reinforcing positive valence to sweet taste and convey this signal to the mushroom body, the fly's associative learning center. We show that Fox neuron activity is necessary and sufficient to drive appetitive behaviors and can override a tastant's intrinsic neutral or aversive valence without impairing taste quality discrimination. Furthermore, Fox neurons relay the positive valence to specific dopaminergic neurons that mediate appetitive memory formation. Our findings reveal a circuit mechanism through which effective valence is bestowed upon sweet sensation and transformed into a reinforcing signal that supports learned sugar responses. The Fox neurons form a convergent-divergent "hourglass" circuit motif, acting as a bottleneck for valence assignment and distributing motivational signals to higher-order centers. This architecture confers both robustness and flexibility in reward processing-an organizational principle that may generalize across species.

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

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.

Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.

Over 6,000 fragments from the genome of Drosophila melanogaster were analyzed for their ability to drive expression of GAL4 reporter genes in the third-instar larval imaginal discs. About 1,200 reporter genes drove expression in the eye, antenna, leg, wing, haltere, or genital imaginal discs. The patterns ranged from large regions to individual cells. About 75% of the active fragments drove expression in multiple discs; 20% were expressed in ventral, but not dorsal, discs (legs, genital, and antenna), whereas \~{}23% were expressed in dorsal but not ventral discs (wing, haltere, and eye). Several patterns, for example, within the leg chordotonal organ, appeared a surprisingly large number of times. Unbiased searches for DNA sequence motifs suggest candidate transcription factors that may regulate enhancers with shared activities. Together, these expression patterns provide a valuable resource to the community and offer a broad overview of how transcriptional regulatory information is distributed in the Drosophila genome.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium’s nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations.