Drosophila Stat92E Signaling Following Pre-exposure to Ethanol.

Repeated exposure to alcohol alters neuromolecular signaling that influences acute and long-lasting behaviors underlying Alcohol Use Disorder (AUD). Recent animal model research has implicated changes in the conserved JAK/STAT pathway, a signaling pathway classically associated with development and the innate immune system. How ethanol exposure impacts STAT signaling within neural cells is currently unclear. Here, we investigated the role of Drosophila Stat92E in ethanol-induced locomotion, signaling activity, and downstream transcriptional responses. Findings suggest that expressing Stat92E-RNAi causes enhanced ethanol-induced hyperactivity in flies previously exposed to ethanol. Furthermore, alternative splicing of Stat92E itself was detected after repeated ethanol exposure, although no changes were found in downstream transcriptional activity. This work adds to our growing understanding of altered neuromolecular signaling following ethanol exposure and suggests that STAT signaling may be a relevant target to consider for AUD treatment.

Using the Drosophila Transcriptional Reporter of Intracellular Calcium (TRIC) to examine lasting ethanol-induced changes in neuroexcitability.

The Drosophila transcriptional reporter of intracellular calcium (TRIC) is a genetic tool used to measure lasting changes in neuroexcitability. Both pan-neuronal and dopaminergic cells were examined with TRIC to test the hypothesis that ethanol exposure causes lasting changes in adult brain neuroexcitability. We found little to no impact on TRIC signal following acute and repeated ethanol vapor exposures. This work shows that TRIC may be useful in future investigations such as developmental or chronic drug exposure paradigms.

Receptors and Channels Associated with Alcohol Use: Contributions from Drosophila.

Alcohol Use Disorder (AUD) is a debilitating disorder that manifests as problematic patterns of alcohol use. At the core of AUD's behavioral manifestations are the profound structural, physiological, cellular, and molecular effects of alcohol on the brain. While the field has made considerable progress in understanding the neuromolecular targets of alcohol we still lack a comprehensive understanding of alcohol's actions and effective treatment strategies. Drosophila melanogaster is a powerful model for investigating the neuromolecular targets of alcohol because flies model many of the core behavioral elements of AUD and offer a rich genetic toolkit to precisely reveal the in vivo molecular actions of alcohol. In this review, we focus on receptors and channels that are often targeted by alcohol within the brain. We discuss the general roles of these proteins, their role in alcohol-associated behaviors across species, and propose ways in which Drosophila models can help advance the field.

Alcohol Causes Lasting Differential Transcription in Drosophila Mushroom Body Neurons.

Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. New methods to analyze gene expression within precise neuronal cell types can provide further insight toward AUD prevention and treatment. Here, we used genetic tools in Drosophila melanogaster to investigate the lasting consequences of ethanol on transcription in memory-encoding neurons. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Differential expression analyses revealed that distinct transcripts, but not genes, in the MB were associated with experiencing ethanol alone compared to forming a memory of an odor cue associated with ethanol. Adult MB-specific knockdown of spliceosome-associated proteins demonstrated the necessity of RNA-processing in ethanol memory formation. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting effect of ethanol on transcript usage during memory formation.

Significance of DopEcR, a G-protein coupled dopamine/ecdysteroid receptor, in physiological and behavioral response to stressors.

Organisms respond to various environmental stressors by modulating physiology and behavior to maintain homeostasis. Steroids and catecholamines are involved in the highly conserved signaling pathways crucial for mounting molecular and cellular events that ensure immediate or long-term survival under stress conditions. The insect dopamine/ecdysteroid receptor (DopEcR) is a dual G-protein coupled receptor for the catecholamine dopamine and the steroid hormone ecdysone. DopEcR acts in a ligand-dependent manner, mediating dopaminergic signaling and unconventional "nongenomic" ecdysteroid actions through various intracellular signaling pathways. This unique feature of DopEcR raises the interesting possibility that DopEcR may serve as an integrative hub for complex molecular cascades activated under stress conditions. Here, we review previously published studies of Drosophila DopEcR in the context of stress response and also present newly discovered DopEcR loss-of-function phenotypes under different stress conditions. These findings provide corroborating evidence that DopEcR plays vital roles in responses to various stressors, including heat, starvation, alcohol, courtship rejection, and repeated neuronal stimulation in Drosophila. We further discuss what is known about DopEcR in other insects and DopEcR orthologs in mammals, implicating their roles in stress responses. Overall, this review highlights the importance of dual GPCRs for catecholamines and steroids in modulating physiology and behavior under stress conditions. Further multidisciplinary studies of Drosophila DopEcR will contribute to our basic understanding of the functional roles and underlying mechanisms of this class of GPCRs.

Insights from intoxicated Drosophila.

Our understanding of alcohol use disorder (AUD), particularly alcohol's effects on the nervous system, has unquestionably benefited from the use of model systems such as Drosophila melanogaster. Here, we briefly introduce the use of flies in alcohol research, and highlight the genetic accessibility and neurobiological contribution that flies have made to our understanding of AUD. Future fly research offers unique opportunities for addressing unresolved questions in the alcohol field, such as the neuromolecular and circuit basis for cravings and alcohol-induced neuroimmune dysfunction. This review strongly advocates for interdisciplinary approaches and translational collaborations with the united goal of confronting the major health problems associated with alcohol abuse and addiction.

Alcohol Activates Scabrous-Notch to Influence Associated Memories.

Drugs of abuse, like alcohol, modulate gene expression in reward circuits and consequently alter behavior. However, the in vivo cellular mechanisms through which alcohol induces lasting transcriptional changes are unclear. We show that Drosophila Notch/Su(H) signaling and the secreted fibrinogen-related protein Scabrous in mushroom body (MB) memory circuitry are important for the enduring preference of cues associated with alcohol's rewarding properties. Alcohol exposure affects Notch responsivity in the adult MB and alters Su(H) targeting at the dopamine-2-like receptor (Dop2R). Alcohol cue training also caused lasting changes to the MB nuclear transcriptome, including changes in the alternative splicing of Dop2R and newly implicated transcripts like Stat92E. Together, our data suggest that alcohol-induced activation of the highly conserved Notch pathway and accompanying transcriptional responses in memory circuitry contribute to addiction. Ultimately, this provides mechanistic insight into the etiology and pathophysiology of alcohol use disorder.

The Unique Dopamine/Ecdysteroid Receptor Modulates Ethanol-Induced Sedation in Drosophila.

Steroids profoundly influence behavioral responses to alcohol by activating canonical nuclear hormone receptors and exerting allosteric effects on ion channels. Accumulating evidence has demonstrated that steroids can also trigger biological effects by directly binding G-protein-coupled receptors (GPCRs), yet physiological roles of such unconventional steroid signaling in controlling alcohol-induced behaviors remain unclear. The dopamine/ecdysteroid receptor (DopEcR) is a GPCR that mediates nongenomic actions of ecdysteroids, the major steroid hormones in insects. Here, we report that Drosophila DopEcR plays a critical role in ethanol-induced sedation.DopEcR mutants took longer than control flies to become sedated during exposure to ethanol, despite having normal ethanol absorption or metabolism. RNAi-mediated knockdown of DopEcR expression revealed that this receptor is necessary after eclosion, and is required in particular neuronal subsets, including cholinergic and peptidergic neurons, to mediate this behavior. Additionally, flies ubiquitously overexpressing DopEcR cDNA had a tendency to become sedated quickly upon ethanol exposure. These results indicate that neuronal subset-specific expression of DopEcR in adults is required for normal sedation upon exposure to ethanol. We also obtained evidence indicating that DopEcR may promote ethanol sedation by suppressing epidermal growth factor receptor/extracellular signal-regulated kinase signaling. Last, genetic and pharmacological analyses suggested that in adult flies ecdysone may serve as an inverse agonist of DopEcR and suppress the sedation-promoting activity of DopEcR in the context of ethanol exposure. Our findings provide the first evidence for the involvement of nongenomic G-protein-coupled steroid receptors in the response to alcohol, and shed new light on the potential roles of steroids in alcohol-use disorders. SIGNIFICANCE STATEMENT: Alcohol abuse is an alarming personal and societal burden. The improvement of prevention and treatment strategies for alcohol-use disorders requires a better understanding of their biological basis. Steroid hormones profoundly affect alcohol-induced behaviors, but the contribution of their unconventional, nongenomic actions during these responses has not yet been elucidated. We found that Drosophila DopEcR, a unique G-protein-coupled receptor (GPCR) with dual specificity for dopamine and steroids, mediates noncanonical steroid actions to promote ethanol-induced sedation. Because steroid signaling and the behavioral response to alcohol are evolutionarily well conserved, our findings suggest that analogous mammalian receptors likely play important roles in alcohol-use disorders. Our work provides a foundation for further characterizing the function and mechanisms of action of nonclassical steroid GPCR signaling.

Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy.

Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun et al. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABAA receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.