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.

Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, Drosophila show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. Here we describe the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of foraging (for) encoding a cGMP-dependent kinase. We show this allele of for reduces expression of a for-T1 isoform expressed in the head and functions normally to inhibit OSH. We localize for-T1 function to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of for-T1 in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; for-T1 may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to our understanding of the genes and neurons underlying olfactory habituation in Drosophila.

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.

ro(Dom) is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. We found that the ro(Dom) stop-furrow phenotype was sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. We demonstrate that ro(Dom) interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and that normal eye size can be restored by providing excess Ato protein. We used ro(Dom) as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, we recovered multiple alleles of groucho (gro) and Hairless (H). Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern.

In the fruit fly Drosophila melanogaster, as in mammals, acute exposure to a high dose of ethanol leads to stereotypical behavioral changes beginning with increased activity, followed by incoordination, loss of postural control, and eventually, sedation. The mechanism(s) by which ethanol impacts the CNS leading to ethanol-induced sedation and the genes required for normal sedation sensitivity remain largely unknown. Here we identify the gene apontic (apt), an Myb/SANT-containing transcription factor that is required in the nervous system for normal sensitivity to ethanol sedation. Using genetic and behavioral analyses, we show that apt mediates sensitivity to ethanol sedation by acting in a small set of neurons that express Corazonin (Crz), a neuropeptide likely involved in the physiological response to stress. The activity of Crz neurons regulates the behavioral response to ethanol, as silencing and activating these neurons affects sedation sensitivity in opposite ways. Furthermore, this effect is mediated by Crz, as flies with reduced crz expression show reduced sensitivity to ethanol sedation. Finally, we find that both apt and crz are rapidly upregulated by acute ethanol exposure. Thus, we have identified two genes and a small set of peptidergic neurons that regulate sensitivity to ethanol-induced sedation. We propose that Apt regulates the activity of Crz neurons and/or release of the neuropeptide during ethanol exposure.

Disrupted-in-Schizophrenia-1 (DISC1) is a genetic susceptibility locus for major mental illness, including schizophrenia and depression. The Disc1 protein was recently shown to interact with the Wnt signaling protein, DIX domain containing 1 (Dixdc1). Both proteins participate in neural progenitor proliferation dependent on Wnt signaling, and in neural migration independently of Wnt signaling. Interestingly, their effect on neural progenitor proliferation is additive. By analogy to Disc1, mutations in Dixdc1 may lead to abnormal behavior in mice, and to schizophrenia or depression in humans. To explore this hypothesis further, we generated mice mutant at the Dixdc1 locus and analyzed their behavior. Dixdc1(-/-) mice had normal prepulse inhibition, but displayed decreased spontaneous locomotor activity, abnormal behavior in the elevated plus maze and deficits in startle reactivity. Our results suggest that Dixdc1(-/-) mice will be a useful tool to elucidate molecular pathophysiology involving Disc1 in major mental illnesses.

In mammalian and insect models of ethanol intoxication, low doses of ethanol stimulate locomotor activity whereas high doses induce sedation. Sex differences in acute ethanol responses, which occur in humans, have not been characterized in Drosophila. In this study, we find that male flies show increased ethanol hyperactivity and greater resistance to ethanol sedation compared with females. We show that the sex determination gene transformer (tra) acts in the developing nervous system, likely through regulation of fruitless (fru), to at least partially mediate the sexual dimorphism in ethanol sedation. Although pharmacokinetic differences may contribute to the increased sedation sensitivity of females, neuronal tra expression regulates ethanol sedation independently of ethanol pharmacokinetics. We also show that acute activation of fru-expressing neurons affects ethanol sedation, further supporting a role for fru in regulating this behavior. Thus, we have characterized previously undescribed sex differences in behavioral responses to ethanol, and implicated fru in mediating a subset of these differences.

Alcohol abuse is a pervasive problem known to be influenced by genetic factors, yet our understanding of the mechanisms underlying alcohol addiction is far from complete. Drosophila melanogaster has been established as a model for studying the molecular mechanisms that mediate the acute and chronic effects of alcohol. However, the Drosophila model has not yet been extended to include more complex alcohol-related behaviors such as self-administration. We recently established a paradigm to characterize ethanol consumption and preference in flies. We demonstrated that flies prefer to consume ethanol-containing food over regular food, and this preference exhibits several features of alcohol addiction: flies increase ethanol consumption over time, they consume ethanol to pharmacologically relevant concentrations, they will overcome an aversive stimulus in order to consume ethanol, and they exhibit relapse after a period of ethanol deprivation. Thus, ethanol preference in flies provides a new model for studying important aspects of addiction and their underlying mechanisms. One mutant that displayed decreased ethanol preference, krasavietz, may represent a first step toward uncovering those mechanisms.

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.