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      Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay


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          The perception and processing of rewarding events are essential for organismal survival. In Drosophila, several groups of neurons have been shown to mediate reward perception or processing. However, a complete description of the reward circuit is missing. Here, we describe a simple two-choice, high-throughput assay suitable for performing large neuronal activation screens for neural circuits involved in reward perception/processing. We characterized this assay using activation of neuropeptide F (NPF) neurons, a known rewarding experience for flies. Furthermore, using genetic intersectional strategies, we subdivided the NPF neurons into different classes and showed that the activation of a subset of small NPF neurons located in the dorsomedial brain is sufficient to trigger preference.


          In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies’ preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

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          Most cited references 35

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          Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain.

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            Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

             A H Brand,  N Perrimon (1993)
            We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
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              Neuropeptide transmission in brain circuits.

              Neuropeptides are found in many mammalian CNS neurons where they play key roles in modulating neuronal activity. In contrast to amino acid transmitter release at the synapse, neuropeptide release is not restricted to the synaptic specialization, and after release, a neuropeptide may diffuse some distance to exert its action through a G protein-coupled receptor. Some neuropeptides such as hypocretin/orexin are synthesized only in single regions of the brain, and the neurons releasing these peptides probably have similar functional roles. Other peptides such as neuropeptide Y (NPY) are synthesized throughout the brain, and neurons that synthesize the peptide in one region have no anatomical or functional connection with NPY neurons in other brain regions. Here, I review converging data revealing a complex interaction between slow-acting neuromodulator peptides and fast-acting amino acid transmitters in the control of energy homeostasis, drug addiction, mood and motivation, sleep-wake states, and neuroendocrine regulation. Copyright © 2012 Elsevier Inc. All rights reserved.

                Author and article information

                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                19 September 2017
                5 September 2017
                5 September 2017
                : 114
                : 38
                : E8091-E8099
                aJanelia Research Campus, Howard Hughes Medical Institute , Ashburn, VA 20147;
                bDepartment of Biology, York University, Toronto, ON, Canada M3J 1P3
                Author notes
                2To whom correspondence may be addressed. Email: shaol10@ 123456janelia.hhmi.org or heberleinu@ 123456janelia.hhmi.org .

                Contributed by Ulrike Heberlein, August 8, 2017 (sent for review June 12, 2017; reviewed by Ping Shen and Paul Taghert)

                Author contributions: L.S., M.S., P.C., and U.H. designed research; L.S., M.S., and P.C. performed research; Q.R. and T.L. contributed new reagents/analytic tools; L.S., M.S., and C.F.K. analyzed data; and L.S., M.S., and U.H. wrote the paper.

                Reviewers: P.S., University of Georgia; and P.T., Washington University School of Medicine.

                1L.S. and M.S. contributed equally to this work.

                PMC5617300 PMC5617300 5617300 201710552

                Freely available online through the PNAS open access option.

                Page count
                Pages: 9
                Funded by: Howard Hughes Medical Institute (HHMI) 100000011
                Award ID: NA
                PNAS Plus
                Biological Sciences
                PNAS Plus


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