I. A. Alcohol and Alcoholism
Alcohol is one of the most widely abused drugs in the world, and alcohol use disorders have grave consequences for both the affected individual and society at large. Our understanding of what changes in the brain that cause a person to go from casual consumption to uncontrolled use is still lacking. One of the reasons for this is the fact that unlike most other drugs of abuse, ethanol does not have a primary high affinity target in the brain, but rather, alcohol binds to many proteins at relatively low affinities and thus many neurochemical systems may be affected by ethanol and contribute to alcoholism.
Many studies have indicated that alcoholism has a large genetic component (reviewed by Hill, this issue). This makes genetic model organisms like the mouse (Buck, this issue) or Drosophila very relevant as tools to isolate candidate genes altering behavioral alcohol responses and drinking patters, and to understand how these genes work and how they might act to alter brain function in addicted individuals. This review focuses on the advances in our understanding of the genes and biochemical pathways that mediate the behavioral responses to alcohol in flies.
I. B. Drosophila as a Model Organism
Drosophila melanogaster vinegar flies have been used for over a hundred years as a model organism to study the laws and mechanisms of heredity. In this review, the terms Drosophila, or flies, will refer to this species, unless specifically noted otherwise. The main reasons why Thomas Hunt Morgan’s research group decided to focus on Drosophila a hundred years ago were threefold: first, flies are easily and inexpensively cultured in glass bottles with banana pulp. Second, their life cycle is fast, requiring only about 12 days at room temperature to go from freshly laid egg to reproducing adult. And third, a single female can have over a hundred offspring. In addition to that, Drosophila larvae have giant salivary gland chromosomes, allowing the visualization of subsegments of the four chromosomes. This was the basis for the accumulation and characterization of many fly strains with cytologically defined chromosomal aberrations, like deficiencies and duplications. These were, and still are, invaluable tools for genetic mapping and stock maintenance techniques. All these reasons have made Drosophila an excellent model organism for the study of the basics of genetic inheritance.
Many fundamental discoveries were made in flies, including genetic recombination, or the fact that X-rays are mutagenic (Sturtevant, 1967). The genomic sequence of Drosophila melanogaster was completed in 2000 (Adams et al., 2000), and currently, 192 wild-derived recombinant inbred lines are being fully sequenced, some of which have already been used to associate genetic variation with behavioral alcohol responses (Morozova et al., 2009) (see section III.B.) Current estimates are that ~75% of genes associated with a human disease have an obvious ortholog in flies (Chien et al., 2002). This high degree of conservation became obvious in the 1980s when many developmental genes found in Drosophila were shown to have similar function in humans (Gehring et al., 2009). However, even in such complex behaviors as circadian rhythm there is a very high degree of conservation between flies and mammals, both in gene structure and molecular function (Collins and Blau, 2007).
While forward genetics, i.e. going from phenotype to gene, has traditionally been the approach taken in Drosophila, genetic transformation was established in 1982 (Rubin and Spradling, 1982). This allows both the reintroduction of a gene to confirm that its mutation causes a phenotype, and also allows for introduction of transgenes mis-, or overexpressing specific proteins or their altered derivatives. One of the most widely used tools in that regard is the binary Gal4/UAS system (Brand and Perrimon, 1993). One transgene carries the yeast transcriptional activator Gal4 under the control of a specific promoter, allowing for spatially controlled expression. The second transgene carries a cDNA of interest under the control of the Gal4-responsive upstream activating sequence (UAS). This allows testing of hypotheses such as: is CNS-specific expression of a cDNA sufficient to rescue the phenotype caused by a given mutation? In addition, certain proteins can be expressed in neurons that induce temperature-sensitive neuronal silencing (Kitamoto, 2001), or action potentials (Pulver et al., 2009), allowing questions like: is a given set of neurons necessary, or even sufficient for a certain behavioral response? Given that a substantial effort is being made to produce 5000 unique Gal4 drivers expressing in a small subset of CNS neurons (Pfeiffer et al., 2008), we might well learn much more about the neurons and circuits mediating these behaviors in the near future.
In addition to the binary Gal4/UAS system, other reverse genetic tools have been developed in flies that allow going from gene to phenotype. One of them is the systematic generation of UAS-lines expressing interfering RNA constructs for knock-down of every Drosophila gene (Dietzl et al., 2007). And in the last few years, it has also become easier, and more common to use homologous recombination to generate targeted gene knock-outs, or replacement knock-ins of any given fly gene (Maggert et al., 2008). Lastly, large-scale efforts have been undertaken to generate a library of fly strains that contain mutations in every single gene (Matthews et al., 2005).