Nikola Dimitrijevic

Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system

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.

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

In addition to their physiological function, metabotropic receptors for neurotransmitter γ-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 alcohols 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.

Techniques: fruit flies as models for neuropharmacological research.

An unlikely animal model is gaining popularity in neuropharmacological research: the 2-mm fruit fly (Drosophila melanogaster). Drugs have been administered to adult flies in their food and, more recently, via gasses and injections. Pharmacological tools have introduced behavioral alterations in Drosophila reminiscent of human behavior, rescued flies from gene-alteration-triggered neuropathologies, and triggered gene silencing. Combined, these methods hold promise for significant neuropharmacological advancement.

Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi)

BackgroundRNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.ResultsBeltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.ConclusionsWe have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice

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, six 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 micro/(100 mg(body-weight)day), required 3 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.

Caffeic acid attenuates the decrease of cortical BDNF transcript IV mRNA induced by swim stress in wild-type but not in 5-lipoxygenase-deficient mice

Caffeic acid is a natural compound that inhibits 5-lipoxygenase (5-LOX). In mice, caffeic acid produces antidepressant-like effects and attenuates the decrease in cortical brain-derived neurotrophic factor (BDNF) mRNA induced by forced swimming. We used wild-type and 5-LOX-deficient mice and found that swimming reduced the cortical content of BDNF exon IV but not exon I mRNA. The BDNF transcript IV decrease was attenuated by caffeic acid in wild-type but not in 5-LOX-deficient mice, suggesting a role for 5-LOX in BDNF regulation.

5-Lipoxygenase inhibitor MK-886 increases GluR1 phosphorylation in neuronal cultures in vitro and in the mouse cortex in vivo

Modifications of AMPA glutamate receptor GluR1 phosphorylation are critical for neuroplastic mechanisms. Previous in vitro studies in brain slices employed MK-886, a functional inhibitor of the enzyme 5-lipoxygenase (5-LOX), and found increased GluR1 phosphorylation. Since slice preparations have accompanying postmortem phosphorylation changes, e.g., decreased GluR1 phosphorylation, it remains to be clarified whether MK-886 can affect GluR1 phosphorylation in intact neurons and in the brain in vivo. We used primary neuronal cultures prepared from embryonic mouse brain and in vivo drug administration to investigate the effects of MK-886 on GluR1 phosphorylation using quantitative Western immunoblotting assays. In vitro, MK-886 increased GluR1 phosphorylation at both serine 831 and serine 845. In vivo, repeated but not a single MK-886 injection increased GluR1 phosphorylation in the prefrontal cortex. These findings indicate that MK-886 has an intrinsic effect on neuronal phosphorylation both in vitro and in vivo and support the use of MK-886 as a pharmacological tool in studies of not only the 5-LOX pathway but also neuronal GluR1 functioning.

An automated assay of the behavioral effects of cocaine injections in adult Drosophila.

Introducing Drosophila models in neuropharmacological research helps to discover new mechanisms of drug action. In fruit flies, the pharmacobehavioral approach has been used to evaluate the effects of drugs of abuse including cocaine. Standard procedures of cocaine administration to flies employ drug vaporization whereas behavior is evaluated either by trained observers or by videotaping followed by analysis via a computer-operated tracking system. Because in mammalian studies cocaine is typically administered by injection, a procedure that ensures precise and timely dose delivery, we developed a method for injecting cocaine into adult Drosophila. To objectively measure the behavioral response of flies to cocaine injections, we adapted the standard Drosophila Activity Monitoring System (Trikinetics). We found that in wild-type Canton S flies, cocaine injections produce a dose-dependent increase in the number of hyperactivity bursts and that repeated injections of cocaine produce behavioral sensitization. Acute responses to cocaine were observed also in period null (per(0)) mutant flies, but in these flies, repeated injections of cocaine did not produce sensitization. In conclusion, we developed a method for accurately measuring the behavioral effects of cocaine in adult fruit flies that can be applied to studies of the mechanisms of behavioral sensitization.

Drosophila metabolize 1,4-butanediol into γ-hydroxybutyric acid in vivo

Abstract A solvent, 1,4-butanediol, is also abused as a recreational drug. In mammals, 1,4-butanediol is metabolized into γ-hydroxybutyric acid (GHB), which stimulates metabotropic γ-aminobutyric acid (GABA) GABA B and putative GHB receptors. Here we show that in vivo injection of 1,4-butanediol into adult Drosophila leads to GHB synthesis (GHB was detectable 5 min after 1,4-butanediol injection and increased dramatically 1–2 h later). This synthesis of GHB was accompanied by an impairment of locomotor activity that was mimicked by a direct injection of GHB into flies. We propose Drosophila as a model to study the molecular actions of 1,4-butanediol and GHB.