Ulrike Heberlein

Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila

Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli are sparsely represented by ∼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection. DOI: http://dx.doi.org/10.7554/eLife.04580.001

Characterization of drosophila transcription factors that activate the tandem promoters of the alcohol dehydrogenase gene

Fractionation of a nuclear extract derived from Drosophila tissue culture cells reveals the presence of multiple components involved in accurate transcription of both distal and proximal promoters of the alcohol dehydrogenase (Adh) gene. Transcription of deletion mutants indicates that a region between -24 and -85 upstream of the distal start site contains sequences required for RNA synthesis in vitro. Moreover, sequences that overlap this same upstream control region are specifically bound and protected from DNAase digestion by a promoter-specific transcription factor, Adf-1. Analysis of proximal promoter mutants identified multiple upstream elements that influence transcription, and DNAase footprint analysis detected three specific binding regions. Adf-1 binds at least one of these proximal promoter regions but interaction at this site is not specifically required for transcription. Our results suggest that multiple sequence-specific DNA binding proteins interact differentially with the proximal and distal promoters of Adh to activate transcription.

A Drosophila model for alcohol reward

The rewarding properties of drugs contribute to the development of abuse and addiction. We developed a new assay for investigating the motivational properties of ethanol in the genetically tractable model Drosophila melanogaster. Flies learned to associate cues with ethanol intoxication and, although transiently aversive, the experience led to a long-lasting attraction for the ethanol-paired cue, implying that intoxication is rewarding. Temporally blocking transmission in dopaminergic neurons revealed that flies require activation of these neurons to express, but not develop, conditioned preference for ethanol-associated cues. Moreover, flies acquired, consolidated and retrieved these rewarding memories using distinct sets of neurons in the mushroom body. Finally, mutations in scabrous, encoding a fibrinogen-related peptide that regulates Notch signaling, disrupted the formation of memories for ethanol reward. Our results thus establish that Drosophila can be useful for understanding the molecular, genetic and neural mechanisms underling the rewarding properties of ethanol.

Competing dopamine neurons drive oviposition choice for ethanol in Drosophila

Significance Flies use fermenting fruit as a food source and a site for oviposition. One of the main metabolites of fermentation is ethanol. When provided with different choices in the laboratory, female flies prefer to lay their eggs on food supplemented with ecologically relevant concentrations of ethanol. We show that different subsets of dopaminergic neurons have opposing effects on oviposition preference. We propose that in the wild, this may be a mechanism by which flies choose oviposition sites that are optimal for offspring fitness and survival and that this choice is highly dependent on context. The neural circuits that mediate behavioral choice evaluate and integrate information from the environment with internal demands and then initiate a behavioral response. Even circuits that support simple decisions remain poorly understood. In Drosophila melanogaster, oviposition on a substrate containing ethanol enhances fitness; however, little is known about the neural mechanisms mediating this important choice behavior. Here, we characterize the neural modulation of this simple choice and show that distinct subsets of dopaminergic neurons compete to either enhance or inhibit egg-laying preference for ethanol-containing food. Moreover, activity in α′β′ neurons of the mushroom body and a subset of ellipsoid body ring neurons (R2) is required for this choice. We propose a model where competing dopaminergic systems modulate oviposition preference to adjust to changes in natural oviposition substrates.

The Evolution of Drosophila melanogaster as a Model for Alcohol Research

Animal models have been widely used to gain insight into the mechanisms underlying the acute and long-term effects of alcohol exposure. The fruit fly Drosophila melanogaster encounters ethanol in its natural habitat and possesses many adaptations that allow it to survive and thrive in ethanol-rich environments. Several assays to study ethanol-related behaviors in flies, ranging from acute intoxication to self-administration and reward, have been developed in the past 20 years. These assays have provided the basis for studying the physiological and behavioral effects of ethanol and for identifying genes mediating these effects. In this review we describe the ecological relationship between flies and ethanol, the effects of ethanol on fly development and behavior, the use of flies as a model for alcohol addiction, and the interaction between ethanol and social behavior. We discuss these advances in the context of their utility to help decipher the mechanisms underlying the diverse effects of ethanol, including those that mediate ethanol dependence and addiction in humans.

A Subset of Serotonergic Neurons Evokes Hunger in Adult Drosophila.

Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. One immediate response to hunger is increased food consumption. Hunger also modulates behaviors related to food seeking such as increased locomotion and enhanced sensory sensitivity in both insects and vertebrates. In addition, hunger can promote the expression of food-associated memory. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. Here, we use Drosophila melanogaster to identify neurons encoding hunger. We found a small group of neurons that, when activated, induced a fed fly to eat as though it were starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. We determined that the neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend our understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit.

The C-terminus of the homeodomain is required for functional specificity of the Drosophila rough gene

In contrast to most Drosophila homeobox genes, which are required during embryogenesis, the rough gene is involved in photoreceptor cell specification in the compound eye. Taking advantage of the viability of null rough alleles and the small size of the rough gene, we have combined in vivo and in vitro mutagenesis to define important functional domains in the rough protein. All missense mutations found to disrupt rough function mapped to highly conserved amino acids in the homeodomain (HD), suggesting that the nature of few, if any, single amino acids outside the HD is critical for rough activity. The analysis of chimeric proteins, in which the whole HD or parts of it were swapped between the rough and Antennapedia (Antp) proteins, revealed that the C-terminus of the rough HD is important for rough activity in vivo. This C-terminal region was also found to be required for the recognition of rough binding sites in vitro. Our data suggest that amino acids located in the C-terminus of the homeodomain may play important roles in selective binding site recognition.

Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay

Significance The perception and processing of rewarding events are essential for organismal survival. In Drosophila, several groups of neurons have been shown to mediate reward perception or processing. However, a complete description of the reward circuit is missing. Here, we describe a simple two-choice, high-throughput assay suitable for performing large neuronal activation screens for neural circuits involved in reward perception/processing. We characterized this assay using activation of neuropeptide F (NPF) neurons, a known rewarding experience for flies. Furthermore, using genetic intersectional strategies, we subdivided the NPF neurons into different classes and showed that the activation of a subset of small NPF neurons located in the dorsomedial brain is sufficient to trigger preference. In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies’ preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

Social isolation-induced epigenetic and transcriptional changes in Drosophila dopaminergic neurons

Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. Here we report first use of a modified INTACT method in behavioral epigenetics of Drosophila: a method we call mini-INTACT. Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, we identified epigenetic signatures in socially-isolated and socially-enriched Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations were predicted by expression changes of four transcription factors and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep. Our work enables the use of Drosophila as a model for cell-type-specific behavioral epigenetics.