The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α'/β' and α/β) and 7 cell subtypes (γd, γm, α'/β'ap, α'/β'm, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker and the α'/β' class marker Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the MB provides a valuable resource for the fly neuroscience community.

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 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 brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in 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 co-expression, 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.

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.

The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior.

Evolution has tuned the nervous system of most animals to produce stereotyped behavioural responses to ethologically relevant stimuli. For example, female Drosophila avoid laying eggs in the presence of geosmin, an odorant produced by toxic moulds. Using this system, we now identify third order olfactory neurons that are essential for an innate aversive behaviour. Connectomics data place these neurons in the context of a complete synaptic circuit from sensory input to descending output. We find multiple levels of valence-specific convergence, including a novel form of axo-axonic input onto second order neurons conveying another danger signal, the pheromone of parasitoid wasps. However we also observe a massive divergence as geosmin-responsive second order olfactory neurons connect with a diverse array of ∼75 cell types. Our data suggest a transition from a labelled line organisation in the periphery to one in which olfactory information is mapped onto many different higher order populations with distinct behavioural significance.

The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. We screened over 5000 Drosophila melanogaster GAL4 lines for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and used multicolor stochastic labelling to analyze the morphology, polarity and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. We identified nine NO and three AB cell types and describe them here. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. We demonstrate that the AB is a bilateral structure which exhibits asymmetry in size between the left and right bodies. We show that the AB neurons directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. We propose that the AB be considered a central complex neuropil in Drosophila. Finally, we present highly restricted GAL4 lines for most identified protocerebral bridge, NO and AB cell types. These lines, generated using the split-GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments. 

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individuals neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time.

The development of the adult optic lobe (OL) of is directed by a wave of ingrowth of the photoreceptors over a two day period at the outset of metamorphosis which is accompanied by the appearance of the pupal-specific transcription factor Broad-Z3 (Br-Z3) and expression of early drivers in OL neurons. During this time, there are pulses of ecdysteroids that time the metamorphic events. At the outset, the transient appearance of juvenile hormone (JH) prevents precocious development of the OL caused by the ecdysteroid peak that initiates pupariation, but the artificial maintenance of JH after this time misdirects subsequent development. Axon ingrowth, Br-Z3 appearance and the expression of early drivers were unaffected, but aspects of later development such as the dendritic expansion of the lamina monopolar neurons and the expression of late drivers were suppressed. This effect of the exogenous JH mimic (JHM) pyriproxifen is lost by 24 hr after pupariation. Part of this effect of JHM is due to its suppression of the appearance of ecdysone receptor EcR-B1 that occurs after pupation and during early adult development.

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Nervous systems combine lower-level sensory signals to detect higher-order stimulus features critical to survival, such as the visual looming motion created by an imminent collision or approaching predator. Looming-sensitive neurons have been identified in diverse animal species. Different large-scale visual features such as looming often share local cues, which means loom-detecting neurons face the challenge of rejecting confounding stimuli. Here we report the discovery of an ultra-selective looming detecting neuron, lobula plate/lobula columnar, type II (LPLC2) in Drosophila, and show how its selectivity is established by radial motion opponency. In the fly visual system, directionally selective small-field neurons called T4 and T5 form a spatial map in the lobula plate, where they each terminate in one of four retinotopic layers, such that each layer responds to motion in a different cardinal direction. Single-cell anatomical analysis reveals that each arm of the LPLC2 cross-shaped primary dendrites ramifies in one of these layers and extends along that layer's preferred motion direction. In vivo calcium imaging demonstrates that, as their shape predicts, individual LPLC2 neurons respond strongly to outward motion emanating from the centre of the neuron's receptive field. Each dendritic arm also receives local inhibitory inputs directionally selective for inward motion opposing the excitation. This radial motion opponency generates a balance of excitation and inhibition that makes LPLC2 non-responsive to related patterns of motion such as contraction, wide-field rotation or luminance change. As a population, LPLC2 neurons densely cover visual space and terminate onto the giant fibre descending neurons, which drive the jump muscle motor neuron to trigger an escape take off. Our findings provide a mechanistic description of the selective feature detection that flies use to discern and escape looming threats.