γ-Aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila : Adult RNA interference and pharmacological evidence

Abstract

In addition to their physiological function, metabotropic receptors for neurotransmitter ␥-aminobutyric acid (GABA), the GABAB receptors, may play a role in the behavioral actions of addictive compounds. Recently, GABAB receptors were cloned in fruit flies (Drosophila melanogaster), indicating that the advantages of this experimental model could be applied to GABAB receptor research. RNA interference (RNAi) is an endogenous process triggered by double-stranded RNA and is being used as a tool for functional gene silencing and functional genomics. Here we show how cell-nonautonomous RNAi can be induced in adult fruit flies to silence a subtype of GABAB receptors, GABABR1, and how RNAi combined with pharmacobehavioral techniques (including intraabdominal injections of active compounds and a computer-assisted quantification of behavior) can be used to functionally characterize these receptors. We observed that injection of double-stranded RNA complementary to GABABR1 into adult Drosophila selectively destroys GABABR1 mRNA and attenuates the behavioral actions of the GABAB agonist, 3-aminopropyl-(methyl)phosphinic acid. Moreover, both GABABR1 RNAi and the GABAB antagonist CGP 54626 reduced the behavior-impairing effects of ethanol, suggesting a putative role for the Drosophila GABAB receptors in alcohol's mechanism of action. The Drosophila model we have developed can be used for further in vivo functional characterization of GABAB receptor subunits and their involvement in the molecular and systemic actions of addictive substances.fruit fly ͉ GABAB receptors ͉ ethanol ͉ CGP 54626 ͉ 3-APMPA F ruit flies are used for research primarily because of the homology of Drosophila genes to those of mammals and because Drosophila is amenable to genetic manipulation including gene silencing through a process known as RNA interference (RNAi) (1-7). Typically, RNAi is triggered by double-stranded RNA (dsRNA), which is first processed by an RNase, Dicer (8), into 21-to 23-nt fragments. These fragments form a silencing complex that binds specifically to the dsRNA-complementary endogenous mRNA and leads to the destruction of the mRNA (1, 9). Injecting adult Drosophila intraabdominally with dsRNA results in the cell-nonautonomous silencing of the complementary endogenous mRNA throughout the body, including the CNS (10). Because this method does not interfere in the normal development of the animal but can replicate typical phenotypes produced by gene mutations (11), here we have used adult RNAi to silence ␥-aminobutyric acid (GABA) B receptors.The slow inhibitory GABA synaptic neurotransmission is mediated by the metabotropic G protein-coupled and cAMPlinked GABA B receptors (12, 13) that have been cloned in humans (14) and also recently in Drosophila (15). In addition to their physiological function, the GABA B receptors may play a role in the behavioral actions of addictive compounds such as ethanol (16)(17)(18)(19). Although Drosophila has been used to study the mechanisms of action of alcohol (20)(21)(22)(23), as yet, this model ...

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training

“…These reports include mammalian systems: hypothalamic neurons transfected with dsRNA in vivo (Bhargava et al 2004), neuroblastoma cells transfected with dsRNA in vitro (Gan et al 2002), myotubes transfected with plasmids that express a 1-kb dsRNA in vitro (Yi et al 2003), and adult muscle fibers injected with dsRNA in vivo (Kong et al 2004). Similar results have also been reported for dsRNA introduced into neurons in invertebrates (Dzitoyeva et al 2001(Dzitoyeva et al , 2003Korneev et al 2002).…”

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

“…It is currently believed that insects lack a pathway for the systemic spreading of RNAi. Nevertheless, injected dsRNA elicits cell nonautonomous RNAi in adult Drosophila, juvenile grasshopper, Tribolium castaneum (flour beetle) and Anopheles gambiae 9,[31][32][33] . In plants, it seems that systemic spread relies on the plasmodesmal channel system, which connects all the cells in the plant 34,35 .…”

Acute ethanol ingestion produces dose-dependent effects on motor behavior in the honey bee (Apis mellifera)

“…Indeed, previous studies have shown that one of ethanol's primary effects on locomotion occurs via its interaction on the pathways involving these same neuromodulators (for reviews see Phillips and Shen 1996;Wolf and Heberlein, 2002). For example, studies using GABA A receptor knock out mice (see Boehm, et al, 2004 for a review) or Drosophila with reduced levels of GABA B receptors (Dzitoyeva, et al, 2003) suggest that ethanol affects locomotion and posture via modulation of GABAergic signaling. In addition, tyramine which influences Drosophila locomotion via CPGs (Fox, et al, 2006) also plays a role in ethanol induced hyperactivity (Scholz, 2005).…”