We are particularly interested in understanding how abused drugs, stress and social interactions modulate reward systems to ultimately modify voluntary behavior.
Experimentally, we are approaching these questions with a diverse set of techniques that include whole-genome transcriptional profiling, high-resolution neuroanatomy, and analysis of high-content behavioral outputs.
Ulrike Heberlein Group Leader
Pavan Agrawal Research Staff
Mark Eddison Research Staff
Clement Kent Senior Scientist
Yong-Kyu Kim Research Staff
Mathias Saver Graduate Student
Lisha Shao Postdoctoral Associate
Jasper Simon Postdoctoral Associate
The brain's reward systems reinforce behaviors required for species survival, including sex, food consumption, and social interaction. Drugs of abuse co-opt these neural pathways, which can lead to addiction. Here, we used Drosophila melanogaster to investigate the relationship between natural and drug rewards. In males, mating increased, whereas sexual deprivation reduced, neuropeptide F (NPF) levels. Activation or inhibition of the NPF system in turn reduced or enhanced ethanol preference. These results thus link sexual experience, NPF system activity, and ethanol consumption. Artificial activation of NPF neurons was in itself rewarding and precluded the ability of ethanol to act as a reward. We propose that activity of the NPF-NPF receptor axis represents the state of the fly reward system and modifies behavior accordingly.
Prior Publications (21 of 89)
Animal studies have been instrumental in providing knowledge about the molecular and neural mechanisms underlying drug addiction. Recently, the fruit fly Drosophila melanogaster has become a valuable system to model not only the acute stimulating and sedating effects of drugs but also their more complex rewarding properties. In this review, we describe the advantages of using the fly to study drug-related behavior, provide a brief overview of the behavioral assays used, and review the molecular mechanisms and neural circuits underlying drug-induced behavior in flies. Many of these mechanisms have been validated in mammals, suggesting that the fly is a useful model to understand the mechanisms underlying addiction.
Understanding sensory systems that perceive environmental inputs and neural circuits that select appropriate motor outputs is essential for studying how organisms modulate behavior and make decisions necessary for survival. Drosophila melanogaster oviposition is one such important behavior, in which females evaluate their environment and choose to lay eggs on substrates they may find aversive in other contexts. We employed neurogenetic techniques to characterize neurons that influence the choice between repulsive positional and attractive egg-laying responses toward the bitter-tasting compound lobeline. Surprisingly, we found that neurons expressing Gr66a, a gustatory receptor normally involved in avoidance behaviors, receive input for both attractive and aversive preferences. We hypothesized that these opposing responses may result from activation of distinct Gr66a-expressing neurons. Using tissue-specific rescue experiments, we found that Gr66a-expressing neurons on the legs mediate positional aversion. In contrast, pharyngeal taste cells mediate the egg-laying attraction to lobeline, as determined by analysis of mosaic flies in which subsets of Gr66a neurons were silenced. Finally, inactivating mushroom body neurons disrupted both aversive and attractive responses, suggesting that this brain structure is a candidate integration center for decision-making during Drosophila oviposition. We thus define sensory and central neurons critical to the process by which flies decide where to lay an egg. Furthermore, our findings provide insights into the complex nature of gustatory perception in Drosophila. We show that tissue-specific activation of bitter-sensing Gr66a neurons provides one mechanism by which the gustatory system differentially encodes aversive and attractive responses, allowing the female fly to modulate her behavior in a context-dependent manner.
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.
Prenatal exposure to ethanol in humans results in a wide range of developmental abnormalities, including growth deficiency, developmental delay, reduced brain size, permanent neurobehavioral abnormalities and fetal death. Here we describe the use of Drosophila melanogaster as a model for exploring the effects of ethanol exposure on development and behavior. We show that developmental ethanol exposure causes reduced viability, developmental delay and reduced adult body size. We find that flies reared on ethanol-containing food have smaller brains and imaginal discs, which is due to reduced cell division rather than increased apoptosis. Additionally, we show that, as in mammals, flies reared on ethanol have altered responses to ethanol vapor exposure as adults, including increased locomotor activation, resistance to the sedating effects of the drug and reduced tolerance development upon repeated ethanol exposure. We have found that the developmental and behavioral defects are largely due to the effects of ethanol on insulin signaling; specifically, a reduction in Drosophila insulin-like peptide (Dilp) and insulin receptor expression. Transgenic expression of Dilp proteins in the larval brain suppressed both the developmental and behavioral abnormalities displayed by ethanol-reared adult flies. Our results thus establish Drosophila as a useful model system to uncover the complex etiology of fetal alcohol syndrome.
A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.
Drosophila tao controls mushroom body development and ethanol-stimulated behavior through par-1.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 2011
I. King, L. T-Y. Tsai, R. Pflanz, A. Voigt, S. Lee, H. Jäckle, B. Lu, and U. Heberlein The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 31:1139-48 (2011)
In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.
Alk is a transcriptional target of LMO4 and ERα that promotes cocaine sensitization and reward.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 2011
A. W. Lasek, J. Gesch, F. Giorgetti, V. Kharazia, and U. Heberlein The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 31:14134-41 (2011)
Previously, we showed that the mouse LIM-domain only 4 (Lmo4) gene, which encodes a protein containing two zinc-finger LIM domains that interact with various DNA-binding transcription factors, attenuates behavioral sensitivity to repeated cocaine administration. Here we show that transcription of anaplastic lymphoma kinase (Alk) is repressed by LMO4 in the striatum and that Alk promotes the development of cocaine sensitization and conditioned place preference, a measure of cocaine reward. Since LMO4 is known to interact with estrogen receptor α (ERα) at the promoters of target genes, we investigated whether Alk expression might be controlled by a similar mechanism. We found that LMO4 and ERα are associated with the Alk promoter by chromatin immunoprecipitation and that Alk is an estrogen-responsive gene in the striatum. Moreover, we show that ERα knock-out mice exhibit enhanced cocaine sensitization and conditioned place preference and an increase in Alk expression in the nucleus accumbens. These data define a novel regulatory network involved in behavioral responses to cocaine. Interestingly, sex differences in several behavioral responses to cocaine in humans and rodents have been described, and estrogen is thought to mediate some of these differences. Our data suggest that estrogen regulation of Alk may be one mechanism responsible for sexually dimorphic responses to cocaine.
Alcohol addiction is a common affliction with a strong genetic component . Although mammalian studies have provided significant insight into the molecular mechanisms underlying ethanol consumption , other organisms such as Drosophila melanogaster are better suited for unbiased, forward genetic approaches to identify novel genes. Behavioral responses to ethanol, such as hyperactivity, sedation, and tolerance, are conserved between flies and mammals [3, 4], as are the underlying molecular pathways [5-9]. However, few studies have investigated ethanol self-administration in flies . Here we characterize ethanol consumption and preference in Drosophila. Flies prefer to consume ethanol-containing food over regular food, and this preference increases over time. Flies are attracted to the smell of ethanol, which partially mediates ethanol preference, but are averse to its taste. Preference for consuming ethanol is not entirely explained by attraction to either its sensory or caloric properties. We demonstrate that flies can exhibit features of alcohol addiction. First, flies self-administer ethanol to pharmacologically relevant concentrations. Second, flies will overcome an aversive stimulus in order to consume ethanol. Third, flies rapidly return to high levels of ethanol consumption after a period of imposed abstinence. Thus, ethanol preference in Drosophila provides a new model for studying aspects of addiction.
Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.Proceedings of the National Academy of Sciences of the United States of America 2009
R. M. Joseph, A. V. Devineni, I. F G. King, and U. Heberlein Proceedings of the National Academy of Sciences of the United States of America, 106:11352-7 (2009)
Selection of appropriate oviposition sites is essential for progeny survival and fitness in generalist insect species, such as Drosophila melanogaster, yet little is known about the mechanisms regulating how environmental conditions and innate adult preferences are evaluated and balanced to yield the final substrate choice for egg-deposition. Female D. melanogaster are attracted to food containing acetic acid (AA) as an oviposition substrate. However, our observations reveal that this egg-laying preference is a complex process, as it directly opposes an otherwise strong, default behavior of positional avoidance for the same food. We show that 2 distinct sensory modalities detect AA. Attraction to AA-containing food for the purpose of egg-laying relies on the gustatory system, while positional repulsion depends primarily on the olfactory system. Similarly, distinct central brain regions are involved in AA attraction and repulsion. Given this unique situation, in which a single environmental stimulus yields 2 opposing behavioral outputs, we propose that the interaction of egg-laying attraction and positional aversion for AA provides a powerful model for studying how organisms balance competing behavioral drives and integrate signals involved in choice-like processes.
The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.
Glycogen synthase kinase-3/Shaggy mediates ethanol-induced excitotoxic cell death of Drosophila olfactory neurons.Proceedings of the National Academy of Sciences of the United States of America 2009
R. L. French, and U. Heberlein Proceedings of the National Academy of Sciences of the United States of America, 106:20924-9 (2009)
It has long been known that heavy alcohol consumption leads to neuropathology and neuronal death. While the response of neurons to an ethanol insult is strongly influenced by genetic background, the underlying mechanisms are poorly understood. Here, we show that even a single intoxicating exposure to ethanol causes non-cell-autonomous apoptotic death specifically of Drosophila olfactory neurons, which is accompanied by a loss of a behavioral response to the smell of ethanol and a blackening of the third antennal segment. The Drosophila homolog of glycogen synthase kinase-3 (GSK-3)beta, Shaggy, is required for ethanol-induced apoptosis. Consistent with this requirement, the GSK-3beta inhibitor lithium protects against the neurotoxic effects of ethanol, indicating the possibility for pharmacological intervention in cases of alcohol-induced neurodegeneration. Ethanol-induced death of olfactory neurons requires both their neural activity and functional NMDA receptors. This system will allow the investigation of the genetic and molecular basis of ethanol-induced apoptosis in general and provide an understanding of the molecular role of GSK-3beta in programmed cell death.
In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.
We identified moody in a genetic screen for Drosophila mutants with altered cocaine sensitivity. Hypomorphic mutations in moody cause an increased sensitivity to cocaine and nicotine exposure. In contrast, sensitivity to the acute intoxicating effects of ethanol is reduced. The moody locus encodes two novel GPCRs, Moody-alpha and Moody-beta. While identical in their membrane-spanning domains, the two Moody proteins differ in their long carboxy-terminal domains, which are generated by use of alternative reading frames. Both Moody forms are required for normal cocaine sensitivity, suggesting that they carry out distinct but complementary functions. Moody-alpha and Moody-beta are coexpressed in surface glia that surround the nervous system, where they are actively required to maintain the integrity of the blood-brain barrier in the adult fly. We propose that a Moody-mediated signaling pathway functions in glia to regulate nervous system insulation and drug-related behaviors.
The insulin signaling pathway regulates multiple physiological processes, including energy metabolism, organismal growth, aging and reproduction. Here we show that genetic manipulations in Drosophila melanogaster that impair the function of insulin-producing cells or of the insulin-receptor signaling pathway in the nervous system lead to increased sensitivity to the intoxicating effects of ethanol. These findings suggest a previously unknown role for this highly conserved pathway in regulating the behavioral responses to an addictive drug.
Repeated alcohol consumption leads to the development of tolerance, simply defined as an acquired resistance to the physiological and behavioural effects of the drug. This tolerance allows increased alcohol consumption, which over time leads to physical dependence and possibly addiction. Previous studies have shown that Drosophila develop ethanol tolerance, with kinetics of acquisition and dissipation that mimic those seen in mammals. This tolerance requires the catecholamine octopamine, the functional analogue of mammalian noradrenaline. Here we describe a new gene, hangover, which is required for normal development of ethanol tolerance. hangover flies are also defective in responses to environmental stressors, such as heat and the free-radical-generating agent paraquat. Using genetic epistasis tests, we show that ethanol tolerance in Drosophila relies on two distinct molecular pathways: a cellular stress pathway defined by hangover, and a parallel pathway requiring octopamine. hangover encodes a large nuclear zinc-finger protein, suggesting a role in nucleic acid binding. There is growing recognition that stress, at both the cellular and systemic levels, contributes to drug- and addiction-related behaviours in mammals. Our studies suggest that this role may be conserved across evolution.
Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LN(v)s), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LN(v)function,Lmomutants also show defects in circadian rhythms of behavior. However, the role for LN(v)s in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LN(v)s reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LN(v)s to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.
High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 2002
F. W. Wolf, A. R. Rodan, L. T-Y. Tsai, and U. Heberlein The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 22:11035-44 (2002)
Understanding how ethanol influences behavior is key to deciphering the mechanisms of ethanol action and alcoholism. In mammals, low doses of ethanol stimulate locomotion, whereas high doses depress it. The acute stimulant effect of ethanol has been proposed to be a manifestation of its rewarding effects. In Drosophila, ethanol exposure transiently potentiates locomotor activity in a biphasic dose- and time-dependent manner. An initial short-lived peak of activity corresponds to an olfactory response to ethanol. A second, longer-lasting period of increased activity coincides with rising internal ethanol concentrations; these closely parallel concentrations that stimulate locomotion in mammals. High-resolution analysis of the walking pattern of individual flies revealed that locomotion consists of bouts of activity; bout structure can be quantified by bout frequency, bout length, and the time spent walking at high speeds. Ethanol exposure induces both dramatic and dynamic changes in bout structure. Mutants with increased ethanol sensitivity show distinct changes in ethanol-induced locomotor behavior, as well as genotype-specific changes in activity bout structure. Thus, the overall effect of ethanol on locomotor behavior in Drosophila is caused by changes in discrete quantifiable parameters of walking pattern. The effects of ethanol on locomotion are comparable in flies and mammals, suggesting that Drosophila is a suitable model system to study the underlying mechanisms.
Functional dissection of neuroanatomical loci regulating ethanol sensitivity in Drosophila.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 2002
A. R. Rodan, J. A. Kiger, and U. Heberlein The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 22:9490-501 (2002)
Ethanol has complex but similar effects on behavior in mammals and the fruit fly Drosophila melanogaster. In addition, genetic and pharmacological approaches have implicated the cAMP pathway in the regulation of ethanol-induced behaviors in both flies and rodents. Here we examine the neuroanatomical loci that modulate ethanol sensitivity in Drosophila by targeting the expression of an inhibitor of cAMP-dependent protein kinase (PKA) to specific regions in the fly's brain. Expression of the inhibitor in most brain regions or in muscle has no effect on behavior. In contrast, inhibition of PKA in a relatively small number of cells, possibly neurosecretory cells, in the fly's brain is sufficient to decrease sensitivity to the incoordinating effects of ethanol. Additional brain areas are, however, also involved. The mushroom bodies, brain structures where cAMP signaling is required for olfactory classical conditioning, are dispensable for the regulation of ethanol sensitivity. Finally, different behavioral effects of ethanol, motor incoordination and sedation, appear to be regulated by PKA function in distinct brain regions. We conclude that the regulation of ethanol-induced behaviors by PKA involves complex interactions among groups of cells that mediate either increased or reduced sensitivity to the acute intoxicating effects of ethanol.
In humans, repeated alcohol consumption leads to the development of tolerance, manifested as a reduced physiological and behavioral response to a particular dose of alcohol. Here we show that adult Drosophila develop tolerance to the sedating and motor-impairing effects of ethanol with kinetics of acquisition and dissipation that mimic those seen in mammals. Importantly, this tolerance is not caused by changes in ethanol absorption or metabolism. Rather, the development of tolerance requires the functional and structural integrity of specific central brain regions. Mutants unable to synthesize the catecholamine octopamine are also impaired in their ability to develop tolerance. Taken together, these data show that Drosophila is a suitable model system in which to study the molecular and neuroanatomical bases of ethanol tolerance.
Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown.
Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.