DREADDs in Drosophila: A Pharmacogenetic Approach for Controlling Behavior, Neuronal Signaling, and Physiology in the Fly

SummaryWe have translated a powerful genetic tool, designer receptors exclusively activated by designer drugs (DREADDs), from mammalian systems to Drosophila melanogaster to selectively, rapidly, reversibly, and dose-dependently control behaviors and physiological processes in the fly. DREADDs are muscarinic acetylcholine G protein-coupled receptors evolved for loss of affinity to acetylcholine and for the ability to be fully activated by an otherwise biologically inert chemical, clozapine-N-oxide. We demonstrate its ability to control a variety of behaviors and processes in larvae and adults, including heart rate, sensory processing, diurnal behavior, learning and memory, and courtship. The advantages of this particular technology include the dose-responsive control of behaviors, the lack of a need for specialized equipment, and the capacity to remotely control signaling in essentially all neuronal and nonneuronal fly tissues

The Serotonin 5-HT7Dro Receptor Is Expressed in the Brain of Drosophila, and Is Essential for Normal Courtship and Mating

The 5-HT7 receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly, Drosophila melanogaster, is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT7 receptor, as well as homologs for the mammalian 5-HT1A, and 5-HT2, receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and have been demonstrated to mediate similar behavioral responses. Here, we report on the expression and function of the 5-HT7Dro receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, we have found that 5-HT7Dro activity is essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly

Xyloglucan endotransglucosylase/hydrolases of Arabidopsis: Expression and function of a gene family

The plant cell wall is a complex structure composed in part of cellulose microfibrils interconnected by a network of hemicellulose. The most abundant hemicellulose of dicotyledonous plants is xyloglucan. Xyloglucan endotransglucosylase/hydrolases (XTHs) cleave xyloglucan polymers and religate the newly generated reducing ends to other xyloglucan polymers (Rose et al., 2002). Analysis of the Arabidopsis database reveals an extensive gene family that encodes thirty-three XTH proteins. These proteins are highly similar and share a conserved motif that is predicted to be necessary for their activity, as well as N-linked glycosylation sites and putative signal sequences for translation at ER membranes. Although the biochemical activity of this enzyme is well defined, the physiological consequences of XTH activity in vivo remain undetermined. XTH biochemical activity, however, predicts a role in modification of the cell wall. An attempt was started to elucidate the physiological functions of the 33 XTHs of Arabidopsis. From this work, 47 mutations were identified in 28 XTH genes. Mutations in XTH15 were chosen for detailed study. Theses mutants exhibit a shorter phenotype than wild type when grown at higher temperatures. This difference may be due to the misshapen cell morphology observed in the xth15 mutants through microscopy. XTH gene expression was examine through the use of reporter-gene fusions with the beta-glucuronidase (GUS) gene as well as by compiling evidence of expression from various on-line databases, such as the Genevestigator website. Overall, divergent XTH expression patterns are observed from the earliest stages of seed germination through flowering. XTH::GUS patterns and data from other expression sources show some overlap which may result in novel combinations of XTH activities in distinct cells or organs

Serotonin 5-hydroxytryptamine(2A) receptor activation suppresses tumor necrosis factor-alpha-induced inflammation with extraordinary potency

ABSTRACT The G protein-coupled serotonin 5-hydroxytryptamine (5-HT) 2A receptor is primarily recognized for its role in brain neurotransmission, where it mediates a wide variety of functions, including certain aspects of cognition. However, there is significant expression of this receptor in peripheral tissues, where its importance is largely unknown. We have now discovered that activation of 5-HT 2A receptors in primary aortic smooth muscle cells provides a previously unknown and extremely potent inhibition of tumor necrosis factor (TNF)-␣-mediated inflammation. 5-HT 2A receptor stimulation with the agonist (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(R)-DOI] rapidly inhibits a variety of TNF-␣-mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin (IL)-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor B, with IC 50 values of only 10 to 20 pM. It is significant that proinflammatory markers can also be inhibited by (R)-DOI hours after treatment with TNF-␣. With the exception of a few natural toxins, no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. TNF-␣-mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, depression, schizophrenia, and Alzheimer's disease. Our results indicate that activation of 5-HT 2A receptors represents a novel, and extraordinarily potent, potential therapeutic avenue for the treatment of disorders involving TNF-␣-mediated inflammation. Note that because (R)-DOI can significantly inhibit the effects of TNF-␣ many hours after the administration of TNF-␣, potential therapies could be aimed not only at preventing inflammation but also treating inflammatory injury that has already occurred or is ongoing. Serotonin, 5-hydroxytryptamine (5-HT), is a small monoamine molecule primarily known for its role as a neurotransmitter. Within the brain, it modulates a variety of behaviors including cognition, mood, aggression, mating, feeding, and sleep . These behaviors are mediated through interactions at seven different receptor families (5-HT 1-7 ) comprised of 14 distinct subtypes . Each of these are G protein-coupled receptors, with the exception of the 5-HT 3 receptor, which is a ligandgated ion channel. Of all the serotonin receptors, the 5-HT 2A receptor, which is known to primarily couple to the G␣q effector pathwa

Cell Reports Resource DREADDs in Drosophila: A Pharmacogenetic Approach for Controlling Behavior, Neuronal Signaling, and Physiology in the Fly

SUMMARY We have translated a powerful genetic tool, designer receptors exclusively activated by designer drugs (DREADDs), from mammalian systems to Drosophila melanogaster to selectively, rapidly, reversibly, and dose-dependently control behaviors and physiological processes in the fly. DREADDs are muscarinic acetylcholine G protein-coupled receptors evolved for loss of affinity to acetylcholine and for the ability to be fully activated by an otherwise biologically inert chemical, clozapine-N-oxide. We demonstrate its ability to control a variety of behaviors and processes in larvae and adults, including heart rate, sensory processing, diurnal behavior, learning and memory, and courtship. The advantages of this particular technology include the dose-responsive control of behaviors, the lack of a need for specialized equipment, and the capacity to remotely control signaling in essentially all neuronal and nonneuronal fly tissues

TITLE PAGE Serotonin 5-HT 2A receptor activation suppresses TNF-α-induced inflammation with extraordinary potency RUNNING TITLE PAGE Running Title: 5-HT 2A receptor stimulation inhibits TNF-α inflammation Corresponding Author

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