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 ...

RNA interference (RNAi) is a gene silencing mechanism that can be triggered by introducing double-stranded RNA (dsRNA) into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. RNAi can be used for determining gene function and creating functional 'knockout' organisms. Here we show for the first time that RNAi can be induced in adult fruit flies by injecting dsRNA into the abdomen of anesthetized Drosophila, and that this method can also target genes expressed in the central nervous system (CNS). Two genes were targeted to investigate the effects of dsRNA injection on their mRNA content; lacZ transgene (expressed either in the gut or in the CNS), and GM06434, the Drosophila homologue of the C. elegans gene nrf (nose resistant to fluoxetine). Both the transgene and the endogenous gene were successfully silenced in adult Drosophila by intra-abdominal injection of their respective dsRNA. We propose that our method of RNAi in adult flies can be used to characterize gene functioning in the CNS without the typical interference in development found in most gene mutation studies. Molecular Psychiatry (2001) 6, 665-670.

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A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX; 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, 6 daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 μg per 100 mg body-weight per day, required three weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings.Keywords 5-lipoxygenase (5-LOX, 5LO); antidepressant; depression; atherosclerosis; GluR1 cardiovascular Recent studies pointed out an association between depressive symptoms and the progression of atherosclerosis [7,26]. It was proposed that a common biological mechanism contributes to triggering and/or maintenance of these two pathological conditions [19].A prominent role in the pathobiology of atherosclerosis is played by 5-lipoxygenase (5-LOX; 5LO), an enzyme involved in the metabolism of arachidonic acid in 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and leukotrienes [16,25]. Whereas an Correspondence: Radmila Manev, MD, Department of Psychiatry, Heart and Mind Clinic, University of Illinois at Chicago, 1601 West Taylor Street, MC912, Chicago, IL 60612, USA, e-mail: Rmanev@psych.uic.edu; phone: 312-413-3970; fax: 312-413-4569. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the...

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