The Role of Toll and Nonnuclear NF-κB Signaling in the Response to Alcohol.

An understanding of neuroimmune signaling has become central to a description of how alcohol causes addiction and how it damages people with an AUD. It is well known that the neuroimmune system influences neural activity via changes in gene expression. This review discusses the roles played by CNS Toll-like receptor (TLR) signaling in the response to alcohol. Also discussed are observations in Drosophila that show how TLR signaling pathways can be co-opted by the nervous system and potentially shape behavior to a far greater extent and in ways different than generally recognized. For example, in Drosophila, TLRs substitute for neurotrophin receptors and an NF-κB at the end of a TLR pathway influences alcohol responsivity by acting non-genomically.

A Non-Nuclear NF-κB Modulates Alcohol Sensitivity But Not Immunity.

NF-κB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-κBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. Here, we demonstrate that, in Drosophila melanogaster, NF-κB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NF-κB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NF-κB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-κB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse.SIGNIFICANCE STATEMENT NF-κBs are transcription factors used by innate immune signaling pathways to protect against infection. Alcohol abuse also activates these pathways, which contributes to the addictive process and the health consequences associated with alcohol abuse. In the mammalian nervous system, NF-κBs localize to synapses, but it is axiomatic that they effect change by acting in the nucleus. However, for the Drosophila Dif gene, immune and neural function segregate into different protein isoforms. Whereas the nuclear isoform (DifA) activates immune genes in response to infection, the CNS isoform acts nongenomically to modulate alcohol sensitivity. Immunohistochemical and biochemical assays localize DifB to synapse-rich regions. Direct synaptic action would provide a novel and rapid way for NF-κB signaling to modulate behavior.

Alcohol-induced Aggression.

Intraspecies aggression is commonly focused on securing reproductive resources such as food, territory, and mates, and it is often males who do the fighting. In humans, individual acts of overt physical aggression seem maladaptive and probably represent dysregulation of the pathways underlying aggression. Such acts are often associated with ethanol consumption. The Drosophila melanogaster model system, which has long been used to study how ethanol affects the nervous system and behavior, has also been used to study the molecular origins of aggression. In addition, ethanol-induced aggression has been demonstrated in flies. Recent publications show that ethanol stimulates Drosophila aggression in 2 ways: the odor of ethanol and the consumption of ethanol both make males more aggressive. These ethanol effects occur at concentrations that flies likely experience in the wild. A picture emerges of males arriving on their preferred reproductive site-fermenting plant matter-and being stimulated by ethanol to fight harder to secure the site for their own use. Fly fighting assays appear to be a suitable bioassay for studying how low doses of ethanol reshape neural signaling.

Alcohol-induced aggression in Drosophila.

Alcohol-induced aggression is a destructive and widespread phenomenon associated with violence and sexual assault. However, little is understood concerning its mechanistic origin. We have developed a Drosophila melanogaster model to genetically dissect and understand the phenomenon of sexually dimorphic alcohol-induced aggression. Males with blood alcohol levels of 0.04-mg/ml BAC were less aggressive than alcohol-naive males, but when the BAC had dropped to ~0.015 mg/ml, the alcohol-treated males showed an increase in aggression toward other males. This aggression-promoting treatment is referred to as the post-ethanol aggression (PEA) treatment. Females do not show increased aggression after the same treatment. PEA-treated males also spend less time courting and attempt to copulate earlier than alcohol-naive flies. PEA treatment induces expression of the FruM transcription factor (encoded by a male-specific transcript from the fruitless gene), whereas sedating doses of alcohol reduce FruM expression and reduce male aggression. Transgenic suppression of FruM induction also prevents alcohol-induced aggression. In male flies, alcohol-induced aggression is dependent on the male isoform of the fruitless transcription factor (FruM). Low-dose alcohol induces FruM expression and promotes aggression, whereas higher doses of alcohol suppress FruM and suppress aggression.

Alcohol potentiates a pheromone signal in flies.

For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. In this study, we find that when male flies inhabit ethanol-containing food substrates they become more aggressive. We identify a possible mechanism for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, we observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors.

F654A and K558Q Mutations in NMDA Receptor 1 Affect Ethanol-Induced Behaviors in Drosophila.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH. METHODS: Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects. RESULTS: We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant. CONCLUSIONS: Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila.

Monitoring food preference in Drosophila by oligonucleotide tagging.

Drosophila melanogaster is a powerful model organism for dissecting the neurogenetic basis of appetitive and aversive behaviors. However, some methods used to assay food preference require or cause starvation. This can be problematic for fly ethanol research because it can be difficult to dissociate caloric preference for ethanol from pharmacological preference for the drug. We designed BARCODE, a starvation-independent assay that uses trace levels of oligonucleotide tags to differentially mark food types. In BARCODE, flies feed ad libitum, and relative food preference is monitored by qPCR of the oligonucleotides. Persistence of the ingested oligomers within the fly records the feeding history of the fly over several days. Using BARCODE, we identified a sexually dimorphic preference for ethanol. Females are attracted to ethanol-laden foods, whereas males avoid consuming it. Furthermore, genetically feminizing male mushroom body lobes induces preference for ethanol. In addition, we demonstrate that BARCODE can be used for multiplex diet measurements when animals are presented with more than two food choices.

Alcohol-Induced Neuroadaptation Is Orchestrated by the Histone Acetyltransferase CBP.

Homeostatic neural adaptations to alcohol underlie the production of alcohol tolerance and the associated symptoms of withdrawal. These adaptations have been shown to persist for relatively long periods of time and are believed to be of central importance in promoting the addictive state. In Drosophila, a single exposure to alcohol results in long-lasting alcohol tolerance and symptoms of withdrawal following alcohol clearance. These persistent adaptations involve mechanisms such as long-lasting changes in gene expression and perhaps epigenetic restructuring of chromosomal regions. Histone modifications have emerged as important modulators of gene expression and are thought to orchestrate and maintain the expression of multi-gene networks. Previously genes that contribute to tolerance were identified as those that show alcohol-induced changes in histone H4 acetylation following a single alcohol exposure. However, the molecular mediator of the acetylation process that orchestrates their expression remains unknown. Here we show that the Drosophila ortholog of mammalian CBP, nejire, is the histone acetyltransferase involved in regulatory changes producing tolerance-alcohol induces nejire expression, nejire mutations suppress tolerance, and transgenic nejire induction mimics tolerance in alcohol-naive animals. Moreover, we observed that a loss-of-function mutation in the alcohol tolerance gene slo epistatically suppresses the effects of CBP induction on alcohol resistance, linking nejire to a well-established alcohol tolerance gene network. We propose that CBP is a central regulator of the network of genes underlying an alcohol adaptation.

Genetics and genomics of alcohol responses in Drosophila.

Drosophila melanogaster has become a significant model organism for alcohol research. In flies, a rich variety of behaviors can be leveraged for identifying genes affecting alcohol responses and adaptations. Furthermore, almost all genes can be easily genetically manipulated. Despite the great evolutionary distance between flies and mammals, many of the same genes have been implicated in strikingly similar alcohol-induced behaviors. A major problem in medical research today is that it is difficult to extrapolate from any single model system to humans. Strong evolutionary conservation of a mechanistic response between distantly related organisms, such as flies and mammals, is a powerful predictor that conservation will continue all the way to humans. This review describes the state of the Drosophila alcohol research field. It describes common alcohol behavioral assays, the independent origins of resistance and tolerance, the results of classical genetic screens and candidate gene analysis, and the outcomes of recent genomics studies employing GWAS, transcriptome, miRNA, and genome-wide histone acetylation surveys. This article is part of the Special Issue entitled "Alcoholism".

Immediate-early alcohol-responsive miRNA expression in Drosophila.

At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in the Drosophila model organism. In our analysis, we combine the power of next-generation sequencing with Drosophila genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 known Drosophila miRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.

How the Ganetzky lab drove me to alcohol.

This is a brief reminiscence of my time in the Ganetzky lab from 1986-1990 and its effect on my scientific trajectory.

A DNA element in the slo gene modulates ethanol tolerance.

In Drosophila, the slo gene encodes BK-type Ca(2+)-activated K(+) channels and is involved in producing rapid functional tolerance to sedation with ethanol. Drosophila are ideal for the study of functional ethanol tolerance because the adult does not acquire metabolic ethanol tolerance (Scholz, Ramond, Singh, & Heberlein, 2000). It has been shown that mutations in slo block the capacity to acquire tolerance, that sedation with ethanol vapor induces slo gene expression in the nervous system, and that transgenic induction of slo can phenocopy tolerance (Cowmeadow, Krishnan, & Atkinson, 2005; Cowmeadow et al., 2006). Here we use ethanol-induced histone acetylation to map a DNA regulatory element in the slo transcriptional control region and functionally test the element for a role in producing ethanol tolerance. Histone acetylation is commonly associated with activating transcription factors. We used the chromatin immunoprecipitation assay to map histone acetylation changes following ethanol sedation to identify an ethanol-responsive DNA element. Ethanol sedation induced an increase in histone acetylation over a 60 n DNA element called 6b, which is situated between the two ethanol-responsive neural promoters of the slo gene. Removal of the 6b element from the endogenous slo gene affected the production of functional ethanol tolerance as assayed in an ethanol-vapor recovery from sedation assay. Removal of element 6b extended the period of functional ethanol tolerance from ∼10 days to more than 21 days after a single ethanol-vapor sedation. This study demonstrates that mapping the position of ethanol-induced histone acetylation is an effective way to identify DNA regulatory elements that help to mediate the response of a gene to ethanol. Using this approach, we identified a DNA element, which is conserved among Drosophila species, and which is important for producing a behaviorally relevant ethanol response.

A histone modification identifies a DNA element controlling slo BK channel gene expression in muscle.

The slo gene encodes the BK-type Ca(2+)-activated K(+) channels. In Drosophila, expression of slo is induced by organic solvent sedation (benzyl alcohol and ethanol), and this increase in neural slo expression contributes to the production of functional behavioral tolerance (inducible resistance) to these drugs. Within the slo promoter region, we observed that benzyl alcohol sedation produces a localized spike of histone acetylation over a 65-nucleotide (65-n) conserved DNA element called 55b. Changes in histone acetylation are commonly the consequence of transcription factor activity, and previously, a localized histone acetylation spike was used to successfully map a DNA element involved in benzyl alcohol-induced slo expression. To determine whether the 55b element was also involved in benzyl alcohol-induced neural expression of slo, we deleted it from the endogenous slo gene by homologous recombination. Flies lacking the 55b element were normal with respect to basal and benzyl alcohol-induced neural slo expression, the capacity to acquire and maintain functional tolerance, their threshold for electrically-induced seizures, and most slo-related behaviors. Removal of the 55b element did however increase the level of basal expression from the muscle/tracheal cell-specific slo core promoter and produced a slight increase in overall locomotor activity. We conclude that the 55b element is involved in control of slo expression from the muscle and tracheal-cell promoter but is not involved in the production of functional benzyl alcohol tolerance.

Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals.

Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.

Susceptibility to ethanol withdrawal seizures is produced by BK channel gene expression.

Alcohol withdrawal seizures are part of the symptomatology of severe alcohol dependence and are believed to originate from long-term neural adaptations that counter the central nervous system depressant effects of alcohol. Upon alcohol withdrawal, however, the increased neural excitability that was adaptive in the presence of alcohol becomes counter-adaptive and produces an imbalanced hyperactive nervous system. For some individuals, the uncovering of this imbalance by alcohol abstention can be sufficient to generate a seizure. Using the Drosophila model organism, we demonstrate a central role for the BK-type Ca(2+) -activated K(+) channel gene slo in the production of alcohol withdrawal seizures.

Alcohol-induced histone acetylation reveals a gene network involved in alcohol tolerance.

Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

A DNA element regulates drug tolerance and withdrawal in Drosophila.

Drug tolerance and withdrawal are insidious responses to drugs of abuse; the first increases drug consumption while the second punishes abstention. Drosophila generate functional tolerance to benzyl alcohol sedation by increasing neural expression of the slo BK-type Ca(2+) activated K(+) channel gene. After drug clearance this change produces a withdrawal phenotype-increased seizure susceptibility. The drug-induced histone modification profile identified the 6b element (60 nt) as a drug responsive element. Genomic deletion of 6b produces the allele, slo (Δ6b), that reacts more strongly to the drug with increased induction, a massive increase in the duration of tolerance, and an increase in the withdrawal phenotype yet does not alter other slo-dependent behaviors. The 6b element is a homeostatic regulator of BK channel gene expression and is the first cis-acting DNA element shown to specifically affect the duration of a drug action.

Circadian genes differentially affect tolerance to ethanol in Drosophila.

BACKGROUND: There is a strong relationship between circadian rhythms and ethanol (EtOH) responses. EtOH consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Alcohol Clin Exp Res 2005b, 29, 1550). The Drosophila central circadian pacemaker is composed of proteins encoded by the per, tim, cyc, and Clk genes. Using Drosophila mutant analysis, we asked whether these central components of the circadian clock make the equivalent contribution toward EtOH tolerance and whether rhythmicity itself is necessary for tolerance. METHODS: We tested flies carrying mutations in core clock genes for the capacity to acquire EtOH tolerance. Tolerance was assayed by comparing the sedation curves of populations during their first and second sedation. Animals that had acquired tolerance sedated more slowly. Movement was also monitored as the flies breathe the EtOH vapor to determine if other facets of the EtOH response were affected by the mutations. Gas chromatography was used to measure internal EtOH concentration. Constant light was used to nongenetically destabilize the PER and TIM proteins. RESULTS: A group of circadian mutations, all of which eliminate circadian rhythms, do not disrupt tolerance identically. Mutations in per, tim, and cyc completely block tolerance. However, a mutation in Clk does not interfere with tolerance. Constant light also disrupts the capacity to acquire tolerance. These lines did not differ in EtOH absorption. CONCLUSIONS: Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid EtOH tolerance. However, the role of circadian genes in EtOH tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire EtOH tolerance by some circadian mutations is not merely a downstream effect of a nonfunctional circadian clock; instead, these circadian genes play an independent role in EtOH tolerance.

Drosophila larvae as a model to study physiological alcohol dependence.

Alcohol addiction is a disease that includes a diverse set of phenotypes. Functional alcohol tolerance is an adaptation to the effects of alcohol that restores neuronal homeostatic balance while the drug is present. When the drug is suddenly withheld, these adaptations unbalance the nervous system and are thought to be the origin of some withdrawal symptoms. Withdrawal symptoms, which can be a motivating factor for alcoholics to relapse, are taken as evidence of physiological ethanol dependence. Both tolerance and withdrawal symptoms are diagnostic criteria for alcoholism. Recent studies have demonstrated that the larvae of Drosophila show conserved alcohol tolerance and withdrawal phenotypes indicating that Drosophila genetics can now be used in studying this endophenotype of alcohol addiction.

Is alcoholism learned? Insights from the fruit fly.

Alcohol addiction is a complex, unique human disease. Breaking addiction down into contributing endophenotypes enables its study in a variety of model systems. The Drosophila model system has been most often used to study alcohol sensitivity, tolerance, and physiological dependence. However, none of these endophenotypes can account for the near-permanent quality of the addicted state. It has been recently discussed that addictive drugs may hijack the learning-and-memory machinery to produce persistent behavioral changes. Learning and memory is amenable to experimental study, and provides us with a window into how alcohol affects higher-order mental functions that are likely to contribute compulsive drug use. Here, we review the Drosophila literature that links alcohol-related behaviors to learning and memory.