Octopamine integrates the status of internal energy supply into the formation of food-related memories.

The brain regulates food intake in response to internal energy demands and food availability. However, can internal energy storage influence the type of memory that is formed? We show that the duration of starvation determines whether Drosophila melanogaster forms appetitive short-term or longer-lasting intermediate memories. The internal glycogen storage in the muscles and adipose tissue influences how intensely sucrose-associated information is stored. Insulin-like signaling in octopaminergic reward neurons integrates internal energy storage into memory formation. Octopamine, in turn, suppresses the formation of long-term memory. Octopamine is not required for short-term memory because octopamine-deficient mutants can form appetitive short-term memory for sucrose and to other nutrients depending on the internal energy status. The reduced positive reinforcing effect of sucrose at high internal glycogen levels, combined with the increased stability of food-related memories due to prolonged periods of starvation, could lead to increased food intake.

Deciding what and how much to eat is a complex biological process which involves balancing many types of information such as the levels of internal energy storage, the amount of food previously available in the environment, the perceived value of certain food items, and how these are remembered. At the molecular level, food contains carbohydrates that are broken down to produce glucose, which is then delivered to cells under the control of a hormone called insulin. There, glucose molecules are either immediately used or stored as glycogen until needed. Insulin signalling is also known to interact with the brain's decision-making systems that control eating behaviors; however, how our brains balance food intake with energy storage is poorly understood. Berger et al. set out to investigate this question using fruit flies as an experimental model. These insects also produce insulin-like molecules which help to relay information about glycogen levels to the brain's decision-making system. In particular, these signals reach a population of neurons that produce a messenger known as octopamine similar to the human noradrenaline, which helps regulate how much the flies find consuming certain types of foods rewarding. Berger et al. were able to investigate the role of octopamine in helping to integrate information about internal and external resource levels, memory formation and the evaluation of different food types. When the insects were fed normally, increased glycogen levels led to foods rich in carbohydrates being rated as less rewarding by the decision-making cells, and therefore being consumed less. Memories related to food intake were also short-lived ­ in other words, long-term 'food memory' was suppressed, re-setting the whole system after every meal. In contrast, long periods of starvation in insects with high carbohydrates resources produced a stable, long-term memory of food and hunger which persisted even after the flies had fed again. This experience also changed their food rating system, with highly nutritious foods no longer being perceived as sufficiently rewarding. As a result, the flies overate. This study sheds new light on the mechanisms our bodies may use to maintain energy reserves when food is limited. The persistence of 'food memory' after long periods of starvation may also explain why losing weight is difficult, especially during restrictive diets. In the future, Berger et al. hope that this knowledge will contribute to better strategies for weight management.

The function of ethanol in olfactory associative behaviors in Drosophila melanogaster larvae.

Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae.

From Natural Behavior to Drug Screening: Invertebrates as Models to Study Mechanisms Associated with Alcohol Use Disorders.

Humans consume ethanol-containing beverages, which may cause an uncontrollable or difficult-to-control intake of ethanol-containing liquids and may result in alcohol use disorders. How the transition at the molecular level from "normal" ethanol-associated behaviors to addictive behaviors occurs is still unknown. One problem is that the components contributing to normal ethanol intake and their underlying molecular adaptations, especially in neurons that regulate behavior, are not clear. The fruit fly Drosophila melanogaster and the earthworm Caenorhabditis elegans show behavioral similarities to humans such as signs of intoxication, tolerance, and withdrawal. Underlying the phenotypic similarities, invertebrates and vertebrates share mechanistic similarities. For example in Drosophila melanogaster, the dopaminergic neurotransmitter system regulates the positive reinforcing properties of ethanol and in Caenorhabditis elegans, serotonergic neurons regulate feeding behavior. Since these mechanisms are fundamental molecular mechanisms and are highly conserved, invertebrates are good models for uncovering the basic principles of neuronal adaptation underlying the behavioral response to ethanol. This review will focus on the following aspects that might shed light on the mechanisms underlying normal ethanol-associated behaviors. First, the current status of what is required at the behavioral and cellular level to respond to naturally occurring levels of ethanol is summarized. Low levels of ethanol delay the development and activate compensatory mechanisms that in turn might be beneficial for some aspects of the animal's physiology. Repeated exposure to ethanol however might change brain structures involved in mediating learning and memory processes. The smell of ethanol is already a key component in the environment that is able to elicit behavioral changes and molecular programs. Minimal networks have been identified that regulate normal ethanol consumption. Other environmental factors that influence ethanol-induced behaviors include the diet, dietary supplements, and the microbiome. Second, the molecular mechanisms underlying neuronal adaptation to the cellular stressor ethanol are discussed. Components of the heat shock and oxidative stress pathways regulate adaptive responses to low levels of ethanol and in turn change behavior. The adaptive potential of the brain cells is challenged when the organism encounters additional cellular stressors caused by aging, endosymbionts or environmental toxins or excessive ethanol intake. Finally, to underline the conserved nature of these mechanisms between invertebrates and higher organisms, recent approaches to identify drug targets for ethanol-induced behaviors are provided. Already approved drugs regulate ethanol-induced behaviors and they do so in part by interfering with cellular stress pathways. In addition, invertebrates have been used to identify new compounds targeting molecules involved in the regulation in ethanol withdrawal-like symptoms. This review primarily highlights the advances of the last 5 years concerning Drosophila melanogaster, but also provides intriguing examples of Caenorhabditis elegans and Apis mellifera in support.

Mutation of the Drosophila melanogaster serotonin transporter dSERT impacts sleep, courtship, and feeding behaviors.

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.

Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain.

Alcohol intoxication at early ages is a risk factor for the development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope-labeled mice combined with quantitative mass spectrometry to screen more than 2,000 hippocampal proteins, of which 72 changed synaptic abundance up to twofold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naïve mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila flies. This study introduces mitochondrial trafficking as a process implicated in reward learning and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.

Ethanol-guided behavior in Drosophila larvae.

Chemosensory signals allow vertebrates and invertebrates not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. Ethanol is a substance found in the natural environment of Drosophila melanogaster. Accordingly, D. melanogaster has evolved specific sensory systems, physiological adaptations, and associated behaviors at its larval and adult stage to perceive and process ethanol. To systematically analyze how D. melanogaster larvae respond to naturally occurring ethanol, we examined ethanol-induced behavior in great detail by reevaluating existing approaches and comparing them with new experiments. Using behavioral assays, we confirm that larvae are attracted to different concentrations of ethanol in their environment. This behavior is controlled by olfactory and other environmental cues. It is independent of previous exposure to ethanol in their food. Moreover, moderate, naturally occurring ethanol concentration of 4% results in increased larval fitness. On the contrary, higher concentrations of 10% and 20% ethanol, which rarely or never appear in nature, increase larval mortality. Finally, ethanol also serves as a positive teaching signal in learning and memory and updates valence associated with simultaneously processed odor information. Since information on how larvae perceive and process ethanol at the genetic and neuronal level is limited, the establishment of standardized assays described here is an important step towards their discovery.

ERR and dPECR Suggest a Link Between Neuroprotection and the Regulation of Ethanol Consumption Preference.

Reconsumption of ethanol after withdrawal is a hallmark for relapse in recovering patients with alcohol use disorders. We show that the preference of Drosophila melanogaster to reconsume ethanol after abstinence shares mechanistic similarities to human behavior by feeding the antirelapse drug acamprosate to flies and reducing the ethanol consumption preference. The Drosophila cellular stress mutant hangover also reduced ethanol consumption preference. Together with the observation that an increasing number of candidate genes identified in a genome-wide association study on alcohol use disorders are involved in the regulation of cellular stress, the results suggest that cellular stress mechanisms might regulate the level of ethanol reconsumption after abstinence. To address this, we analyzed mutants of candidate genes involved in the regulation of cellular stress for their ethanol consumption level after abstinence and cellular stress response to free radicals. Since hangover encodes a nuclear RNA-binding protein that regulates transcript levels, we analyzed the interactions of candidate genes on transcript and protein level. The behavioral analysis of the mutants, the analysis of transcript levels, and protein interactions suggested that at least two mechanisms regulate ethanol consumption preference after abstinence-a nuclear estrogen-related receptor-hangover-dependent complex and peroxisomal trans-2-enoyl-CoA reductase (dPECR)-dependent component in peroxisomes. The loss of estrogen-like receptor and dPECR in neurons share a protective function against oxidative stress, suggesting that the neuroprotective function of genes might be a predictor for genes involved in the regulation of ethanol reconsumption after abstinence.

Serotonin transporter dependent modulation of food-seeking behavior.

The olfactory pathway integrates the odor information required to generate correct behavioral responses. To address how changes of serotonin signaling in two contralaterally projecting, serotonin-immunoreactive deutocerebral neurons impacts key odorant attraction in Drosophila melanogaster, we selectively alter serotonin signaling using the serotonin transporter with mutated serotonin binding sites in these neurons and analyzed the consequence on odorant-guided food seeking. The expression of the mutated serotonin transporter selectively changed the odorant attraction in an odorant-specific manner. The shift in attraction was not influenced by more up-stream serotonergic mechanisms mediating behavioral inhibition. The expression of the mutated serotonin transporter in CSD neurons did not influence other behaviors associated with food seeking such as olfactory learning and memory or food consumption. We provide evidence that the change in the attraction by serotonin transporter function might be achieved by increased serotonin signaling and by different serotonin receptors. The 5-HT1B receptor positively regulated the attraction to low and negatively regulated the attraction to high concentrations of acetic acid. In contrast, 5-HT1A and 5-HT2A receptors negatively regulated the attraction in projection neurons to high acetic acid concentrations. These results provide insights into how serotonin signaling in two serotonergic neurons selectively regulates the behavioral response to key odorants during food seeking.

Unraveling the Mechanisms of Behaviors Associated With AUDs Using Flies and Worms.

Alcohol use disorders (AUDs) are very common worldwide and negatively affect both individuals and societies. To understand how normal behavior turns into uncontrollable use of alcohol, several approaches have been utilized in the last decades. However, we still do not completely understand how AUDs evolve or how they are maintained in the brains of affected individuals. In addition, efficient and effective treatment is still in need of development. This review focuses on alternative approaches developed over the last 20 years using Drosophila melanogaster (Drosophila) and Caenorhabditis elegans (C. elegans) as genetic model systems to determine the mechanisms underlying the action of ethanol (EtOH) and behaviors associated with AUDs. All the results and insights of studies over the last 20 years cannot be comprehensively summarized. Thus, a few prominent examples are provided highlighting the principles of the genes and mechanisms that have been uncovered and are involved in the action of EtOH at the cellular level. In addition, examples are provided of the genes and mechanisms that regulate behaviors relevant to acquiring and maintaining excessive alcohol intake, such as decision making, reward and withdrawal, and/or relapse regulation. How the insight gained from the results of Drosophila and C. elegans models can be translated to higher organisms, such as rodents and/or humans, is discussed, as well as whether these insights have any relevance or impact on our understanding of the mechanisms underlying AUDs in humans. Finally, future directions are presented that might facilitate the identification of drugs to treat AUDs.

Octopamine Shifts the Behavioral Response From Indecision to Approach or Aversion in Drosophila melanogaster.

Animals must make constant decisions whether to respond to external sensory stimuli or not to respond. The activation of positive and/or negative reinforcers might bias the behavioral response towards approach or aversion. To analyze whether the activation of the octopaminergic neurotransmitter system can shift the decision between two identical odor sources, we active in Drosophila melanogaster different sets of octopaminergic neurons using optogenetics and analyze the choice of the flies using a binary odor trap assay. We show that the release of octopamine from a set of neurons and not acetylcholine acts as positive reinforcer for one food odor source resulting in attraction. The activation of a subset of these neurons causes the opposite behavior and results in aversion. This aversion is due to octopamine release and not tyramine, since in Tyramine-ß-hydroxylase mutants (Tßh) lacking octopamine, the aversion is suppressed. We show that when given the choice between two different attractive food odor sources the activation of the octopaminergic neurotransmitter system switches the attraction for ethanol-containing food odor to a less attractive food odor. Consistent with the requirement for octopamine in biasing the behavioral outcome, Tßh mutants fail to switch their attraction. The execution of attraction does not require octopamine but rather initiation of the behavior or a switch of the behavioral response. The attraction to ethanol also depends on octopamine. Pharmacological increases in octopamine signaling in Tßh mutants increase ethanol attraction and blocking octopamine receptor function reduces ethanol attraction. Taken together, octopamine in the central brain orchestrates behavioral outcomes by biasing the decision of the animal towards food odors. This finding might uncover a basic principle of how octopamine gates behavioral outcomes in the brain.

Key Odorants Regulate Food Attraction in Drosophila melanogaster.

In insects, the search for food is highly dependent on olfactory sensory input. Here, we investigated whether a single key odorant within an odor blend or the complexity of the odor blend influences the attraction of Drosophila melanogaster to a food source. A key odorant is defined as an odorant that elicits a difference in the behavioral response when two similar complex odor blends are offered. To validate that the observed behavioral responses were elicited by olfactory stimuli, we used olfactory co-receptor Orco mutants. We show that within a food odor blend, ethanol functions as a key odorant. In addition to ethanol other odorants might serve as key odorants at specific concentrations. However, not all odorants are key odorants. The intensity of the odor background influences the attractiveness of the key odorants. Increased complexity is only more attractive in a concentration-dependent range for single compounds in a blend. Orco is necessary to discriminate between two similarly attractive odorants when offered as single odorants and in food odor blends, supporting the importance of single odorant recognition in odor blends. These data strongly indicate that flies use more than one strategy to navigate to a food odor source, depending on the availability of key odorants in the odor blend and the alternative odor offered.

Genetic Contribution to Alcohol Dependence: Investigation of a Heterogeneous German Sample of Individuals with Alcohol Dependence, Chronic Alcoholic Pancreatitis, and Alcohol-Related Cirrhosis.

The present study investigated the genetic contribution to alcohol dependence (AD) using genome-wide association data from three German samples. These comprised patients with: (i) AD; (ii) chronic alcoholic pancreatitis (ACP); and (iii) alcohol-related liver cirrhosis (ALC). Single marker, gene-based, and pathway analyses were conducted. A significant association was detected for the ADH1B locus in a gene-based approach (puncorrected = 1.2 × 10-6; pcorrected = 0.020). This was driven by the AD subsample. No association with ADH1B was found in the combined ACP + ALC sample. On first inspection, this seems surprising, since ADH1B is a robustly replicated risk gene for AD and may therefore be expected to be associated also with subgroups of AD patients. The negative finding in the ACP + ALC sample, however, may reflect genetic stratification as well as random fluctuation of allele frequencies in the cases and controls, demonstrating the importance of large samples in which the phenotype is well assessed.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

Correction: A Single Pair of Serotonergic Neurons Counteracts Serotonergic Inhibition of Ethanol Attraction in Drosophila.

[This corrects the article DOI: 10.1371/journal.pone.0167518.].

Hangover Links Nuclear RNA Signaling to cAMP Regulation via the Phosphodiesterase 4d Ortholog dunce.

The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, we analyzed Hangover function on a cellular and neuronal level. We provide evidence that Hangover acts as a nuclear RNA binding protein and we identified the phosphodiesterase 4d ortholog dunce as a target RNA. We generated a transcript-specific dunce mutant that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes.

A Single Pair of Serotonergic Neurons Counteracts Serotonergic Inhibition of Ethanol Attraction in Drosophila.

Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling-the serotonin transporter-in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior.

XRCC5 as a risk gene for alcohol dependence: evidence from a genome-wide gene-set-based analysis and follow-up studies in Drosophila and humans.

Genetic factors have as large role as environmental factors in the etiology of alcohol dependence (AD). Although genome-wide association studies (GWAS) enable systematic searches for loci not hitherto implicated in the etiology of AD, many true findings may be missed owing to correction for multiple testing. The aim of the present study was to circumvent this limitation by searching for biological system-level differences, and then following up these findings in humans and animals. Gene-set-based analysis of GWAS data from 1333 cases and 2168 controls identified 19 significantly associated gene-sets, of which 5 could be replicated in an independent sample. Clustered in these gene-sets were novel and previously identified susceptibility genes. The most frequently present gene, ie in 6 out of 19 gene-sets, was X-ray repair complementing defective repair in Chinese hamster cells 5 (XRCC5). Previous human and animal studies have implicated XRCC5 in alcohol sensitivity. This phenotype is inversely correlated with the development of AD, presumably as more alcohol is required to achieve the desired effects. In the present study, the functional role of XRCC5 in AD was further validated in animals and humans. Drosophila mutants with reduced function of Ku80-the homolog of mammalian XRCC5-due to RNAi silencing showed reduced sensitivity to ethanol. In humans with free access to intravenous ethanol self-administration in the laboratory, the maximum achieved blood alcohol concentration was influenced in an allele-dose-dependent manner by genetic variation in XRCC5. In conclusion, our convergent approach identified new candidates and generated independent evidence for the involvement of XRCC5 in alcohol dependence.

Octopamine indirectly affects proboscis extension response habituation in Drosophila melanogaster by controlling sucrose responsiveness.

Octopamine is an important neurotransmitter in insects with multiple functions. Here, we investigated the role of this amine in a simple form of learning (habituation) in the fruit fly Drosophila melanogaster. Specifically, we asked if octopamine is necessary for normal habituation of a proboscis extension response (PER) to different sucrose concentrations. In addition, we analyzed the relationship between responsiveness to sucrose solutions applied to the tarsus and habituation of the proboscis extension response in the same individual. The Tyramine-ß-hydroxylase (Tßh) mutant lacks the enzyme catalyzing the final step of octopamine synthesis. This mutant was significantly less responsive to sucrose than controls. The reduced responsiveness directly led to faster habituation. Systemic application of octopamine or induction of octopamine synthesis by Tßh expression in a cluster of octopaminergic neurons within the suboesophageal ganglion restored sucrose responsiveness and habituation of octopamine mutants to control level. Further analyses imply that the reduced sucrose responsiveness of Tßh mutants is related to a lower sucrose preference, probably due to a changed carbohydrate metabolism, since Tßh mutants survived significantly longer under starved conditions. These findings suggest a pivotal role for octopamine in regulating sucrose responsiveness in fruit flies. Further, octopamine indirectly influences non-associative learning and possibly associative appetitive learning by regulating the evaluation of the sweet component of a sucrose reward.

A systems medicine research approach for studying alcohol addiction.

According to the World Health Organization, about 2 billion people drink alcohol. Excessive alcohol consumption can result in alcohol addiction, which is one of the most prevalent neuropsychiatric diseases afflicting our society today. Prevention and intervention of alcohol binging in adolescents and treatment of alcoholism are major unmet challenges affecting our health-care system and society alike. Our newly formed German SysMedAlcoholism consortium is using a new systems medicine approach and intends (1) to define individual neurobehavioral risk profiles in adolescents that are predictive of alcohol use disorders later in life and (2) to identify new pharmacological targets and molecules for the treatment of alcoholism. To achieve these goals, we will use omics-information from epigenomics, genetics transcriptomics, neurodynamics, global neurochemical connectomes and neuroimaging (IMAGEN; Schumann et al. ) to feed mathematical prediction modules provided by two Bernstein Centers for Computational Neurosciences (Berlin and Heidelberg/Mannheim), the results of which will subsequently be functionally validated in independent clinical samples and appropriate animal models. This approach will lead to new early intervention strategies and identify innovative molecules for relapse prevention that will be tested in experimental human studies. This research program will ultimately help in consolidating addiction research clusters in Germany that can effectively conduct large clinical trials, implement early intervention strategies and impact political and healthcare decision makers.

Inter-leg coordination in the control of walking speed in Drosophila.

Legged locomotion is the most common behavior of terrestrial animals and it is assumed to have become highly optimized during evolution. Quadrupeds, for instance, use distinct gaits that are optimal with regard to metabolic cost and have characteristic kinematic features and patterns of inter-leg coordination. In insects, the situation is not as clear. In general, insects are able to alter inter-leg coordination systematically with locomotion speed, producing a continuum of movement patterns. This notion, however, is based on the study of several insect species, which differ greatly in size and mass. Each of these species tends to walk at a rather narrow range of speeds. We have addressed these issues by examining four strains of Drosophila, which are similar in size and mass, but tend to walk at different speed ranges. Our data suggest that Drosophila controls its walking speed almost exclusively via step frequency. At high walking speeds, we invariably found tripod coordination patterns, the quality of which increased with speed as indicated by a simple measure of tripod coordination strength (TCS). At low speeds, we also observed tetrapod coordination and wave gait-like walking patterns. These findings not only suggest a systematic speed dependence of inter-leg movement patterns but also imply that inter-leg coordination is flexible. This was further supported by amputation experiments in which we examined walking behavior in animals after the removal of a hindleg. These animals show immediate adaptations in body posture, leg kinematics and inter-leg coordination, thereby maintaining their ability to walk.