Drosophila Short Neuropeptide F Regulates Food Intake and Body Size

Abstract

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster. Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster. Neuropeptides regulate a wide range of animal behavior. In vertebrates, neuropeptide Y (NPY) 1The abbreviations used are: NPY, neuropeptide Y; NPF, neuropeptide F; sNPF, short NPF; dNPF, Drosophila NPF; CNS, central nervous system; PNS, peripheral nervous system; Dig, digoxigenin.1The abbreviations used are: NPY, neuropeptide Y; NPF, neuropeptide F; sNPF, short NPF; dNPF, Drosophila NPF; CNS, central nervous system; PNS, peripheral nervous system; Dig, digoxigenin. regulates food consumption, circadian rhythms, anxiety, and other physiological processes (1Zimanyi I.A. Fathi Z. Poindexter G.S. Curr. Pharm. Des. 1998; 4: 349-366PubMed Google Scholar). NPY, a 36-amino acid neuromodulator, is expressed abundantly in the mammalian brain and controls feeding (2Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 309-352Crossref PubMed Google Scholar). The NPY injection into the hypothalamus of rat brain resulted in hyperphagia and obesity, whereas the NPY-deficient mouse in the leptin mutant background (NPY–/–; ob/ob) showed the less obese phenotype (3Erickson J.C. Hollopeter G. Palmiter R.D. Science. 1996; 274: 1704-1707Crossref PubMed Scopus (758) Google Scholar). In invertebrates, neuropeptide F (NPF) peptides share structural similarity with the vertebrate NPY (4Maule A.G. Halton D.W. Shaw C. Hydrobiologia. 1995; 305: 297-303Crossref Scopus (23) Google Scholar). NPF peptides isolated from various invertebrate animals have the conserved (A/L)R(P/L)RF amide sequence at their C-terminal ends (5Spittaels K. Verhaert P. Shaw C. Johnston R.N. Devreese B. Van Beeumen J. De Loof A. Insect. Biochem. Mol. Biol. 1996; 26: 375-382Crossref PubMed Scopus (87) Google Scholar, 6Huang Y. Brown M.R. Lee T.D. Crim J.W. Insect. Biochem. Mol. Biol. 1998; 28: 345-356Crossref PubMed Scopus (44) Google Scholar). From a completely sequenced Drosophila melanogaster genome, short NPF (sNPF; CG13968) and Drosophila NPF (dNPF; CG10342) were found. The dNPF peptide was isolated by the radioimmunoassay. The preprotein is processed to the 36-amino acid peptide containing RVRF amide in the C terminus. dNPF is considered the homolog of the vertebrate NPY. dNPF mRNA and peptide are expressed in the brain and midgut of Drosophila larvae and adults (7Brown M.R. Crim J.W. Arata R.C. Cai H.N. Chun C. Shen P. Peptides. 1999; 20: 1035-1042Crossref PubMed Scopus (202) Google Scholar). The dNPF neural network in the larval central nervous system (CNS) is changed by the gustatory stimulation of sugar, indicating that dNPF is an integral part of the chemosensory system that regulates eating behavior (8Shen P. Cai H.N. J. Neurobiol. 2001; 47: 16-25Crossref PubMed Scopus (110) Google Scholar). Drosophila larvae eat continuously and grow in a relatively short period. About 5 days after egg laying, they stop feeding, leave the food, and enter the wandering stage to prepare for pupation (9Ashburner M. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 140-298Google Scholar). Recently, Wu et al. (10Wu Q. Wen T. Lee G. Park J.H. Cai H.N. Shen P. Neuron. 2003; 39: 147-161Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) showed that dNPF is expressed in the larval brain during the feeding stage but not in the older larval brain during the wandering stage. Loss-of-function dNPF mutation in the larval feeding stage leads to the premature behavioral phenotypes similar to wandering larvae, whereas overexpression of dNPF in the wandering larval stage shows the continuous feeding phenotype. These indicate that the neuropeptide F system, which consists of dNPF and its receptor, regulates larval feeding behavior (10Wu Q. Wen T. Lee G. Park J.H. Cai H.N. Shen P. Neuron. 2003; 39: 147-161Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). The DmNPFR, the receptor for dNPF, was cloned and showed the homology with the vertebrate NPY receptor family, which are the seven transmembrane G protein-coupled receptors (11Blomqvist A.G. Herzog H. Trends. Neurosci. 1997; 20: 294-298Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). DmNPFR1 mRNA is expressed in larval CNS and midgut. In the binding assay with Chinese hamster ovary cells, the dNPF binds to the DmNPFR1 receptor (12Garczynski S.F. Brown M.R. Shen P. Murray T.F. Crim J.W. Peptides. 2002; 23: 773-780Crossref PubMed Scopus (139) Google Scholar). NPFR76F, the receptor for sNPF, was also cloned and showed the similarity with NPYR2. It is expressed in the CNS of embryos and larvae. In the electrophysiological assay using Xenopus oocytes and in the bioluminescence assay using Chinese hamster ovary cells, the receptor was maximally activated by the putative sNPF peptides (13Mertens I. Meeusen T. Huybrechts R. De Loof A. Schoofs L. Biochem. Biophys. Res. Commun. 2002; 297: 1140-1148Crossref PubMed Scopus (108) Google Scholar, 14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar). Although the functions of dNPF and DmNPFR1 genes in the dNPF system are well characterized in feeding behavior (10Wu Q. Wen T. Lee G. Park J.H. Cai H.N. Shen P. Neuron. 2003; 39: 147-161Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), the function of the sNPF gene is unknown. In this report, we present the cloning, RNA and peptide localizations, and functional characterization of the sNPF gene from Drosophila. We demonstrated that the sNPF gene encodes the preprotein containing putative RLRF amide peptides and sNPF mRNA and peptide were expressed in the nervous system of late stage embryos, larvae, and adult brain. From the sNPF antibody staining, we revealed the sNPF neural circuit from neurons in the brain to thoracic axons and to connective axons of the ventral ganglion in the larval CNS. Using the feeding assay, we showed that the sNPF overexpressing mutants in nervous systems promoted food intake, whereas the sNPF loss-of-function mutants suppressed food intake. We also showed that sNPF overexpressing flies seemed to increase appetite and produced bigger and heavier flies. Cloning of the sNPF Gene and Sequencing—The forward (5′-GAATTCATGTTTCATTTGAAGCGGGGA-3′) and reverse (5′-TCTAGATTAGTTCTGTGTCTTTGGTG-3′) primers were used to clone the coding region of the sNPF gene. The sequences of primers came from the predicted sNPF mRNA in the Drosophila genome data base. To perform PCR-based cloning, total RNA from wild-type (Oregon-R) adult heads was isolated with the RNeasy Midi kit (Qiagen), and mRNA was purified with Oligotex mRNA Midi kit (Qiagen). From 0.5 μg of purified mRNA, first strand cDNA was synthesized with the avian myeloblastosis virus reverse transcriptase kit (Roche Applied Science) according to the manufacturer's instruction. To isolate the coding region of the sNPF gene, the PCR containing 100 pm of the primers, 1 μl of first strand cDNA, and 25 μl of High Fidelity Master Mix (Roche Applied Science) was performed in 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 10 min. The 0.85-kb PCR product was subcloned into the pGEM-T easy vector (Promega) and was sequenced using an ABI automated sequencer (Applied Biosystem). Northern Blot Analysis—The antisense sNPF digoxigenin (Dig)-labeled probe was generated with linearized pGEM-sNPF, SP6 RNA polymerase, and NTP/Dig-UTP mixture (Roche Applied Science). The sense sNPF Dig-labeled probe for the negative control was generated with T7 RNA polymerase. The Northern blot analysis was performed as previously described (14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar) with the Dig-labeled antisense sNPF RNA probe. The rp49 were used as the control. After post-hybridization washes, the signal was detected with the anti-Dig antibody conjugated with alkaline phosphatase and visualized by chemiluminescent reagent (Roche Applied Science). In Situ Hybridization—In situ hybridization in whole mount embryos or larval CNS with the Dig-labeled antisense sNPF RNA probe was performed as previously described (15Yu K. Sturtevant M.A. Biehs B. Francois V. Padgett R.W. Blackman R.K. Bier E. Development. 1996; 122: 4033-4044PubMed Google Scholar). The embryos and larval CNS were mounted on the Gary's Magic mounting medium. Generation of the sNPF Antiserum and Blot Analysis—The sNPF was produced by the of a with the peptide to the acid of the blot analysis was performed as previously described B. H. T. H. Y. Lee K. Biochem. J. 2001; PubMed Google Scholar) with the in whole mount was performed according to Yu et al. K. Biehs B. Bier E. Development. PubMed Google Scholar). The embryos, the larval CNS, and adult brain were mounted in the Gary's Magic mounting medium. and coding sequence of the sNPF gene was subcloned into the of the vector and produced the The was generated and subcloned into the vector to the The was performed according to and Science. PubMed Scopus Google Scholar). and were and and wild-type (Oregon-R) flies or wild-type feeding larvae after egg were in the for into the containing the with and for 10 J. Biol. PubMed Google Scholar). The with in their were for the analysis and to the of with a at the flies were with the body size the from the of a to the of in the was were performed in assay, and were used in data are as the and analysis of was used for the and was as the and the have cloned the cDNA that the coding region of the sNPF gene from the PCR-based encodes the acid that the acid signal peptide in the and putative RLRF peptides by of In the Drosophila genome data are predicted for the sNPF is and the other is The acid sequences of the sNPF preprotein have with of However, the sNPF gene not have a vertebrate The PCR with the of primers produced the 0.85-kb from adult cDNA and the from adult The sequence the from the Drosophila genome data and the sNPF cDNA that the coding region of the sNPF and the was produced by of the sNPF preprotein the putative acid and acid we peptide sequences dNPF, and NPY peptides The acid sequences of and peptides are with the vertebrate NPY other C-terminal whereas dNPF not the of of but also homology with NPY. It that the dNPF not sNPF is considered as the homolog of the vertebrate dNPF, and vertebrate NPY acid sequences in a of sNPF during and in the of and of sNPF mRNA, we performed the developmental Northern blot analysis with the Dig-labeled sNPF antisense RNA probe. The similar of the sNPF were expressed all of the developmental stages To the of the sNPF mRNA, we performed the in situ hybridization in the whole mount embryos and the CNS of the feeding with the Dig-labeled sNPF antisense RNA probe. of the sNPF was in the nervous system from stage embryos not In stage embryos, was in the CNS of brain and ventral ganglion and the peripheral nervous system In the brain, the sNPF was expressed in neural in the region In the ventral the sNPF was expressed in the of neural the ventral and with a in of the ventral ganglion the of the sNPF in the brain and ventral is similar to that of the sNPF receptor, (14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar). In the PNS, the sNPF was expressed in the and which is not with the of (14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar). In the brain from the feeding the sNPF was expressed in neural in the region of the In the larval ventral the sNPF was expressed in neural in the region the ventral and in thoracic and sNPF in various neural that the sNPF gene in a wide range of neural the of the sNPF with the Drosophila neuropeptide F in the ganglion (8Shen P. Cai H.N. J. Neurobiol. 2001; 47: 16-25Crossref PubMed Scopus (110) Google Scholar) that of the sNPF functions is feeding behavior. sNPF during have generated the sNPF antibody peptide peptide to the sNPF peptide during In the developmental blot analysis with the sNPF the processed sNPF peptide were during all developmental stages The of the sNPF mRNA and the peptide during all developmental stages that sNPF various during developmental stages other sNPF in the of CNS, and the localization of the sNPF peptide was by the using the sNPF in embryos, larval CNS, and the adult brain. In stage embryos, the localization of the sNPF peptide was in the CNS and the In the stage embryonic CNS, the sNPF peptide was localized the with in of the ventral In the stage embryonic CNS, the sNPF peptide was localized in the axons and neural in the In the embryonic PNS, the sNPF peptide was localized in the of and In the larval CNS, the sNPF peptide was localized in the brain and the ventral ganglion In the brain the sNPF peptide was localized in in the region of the and the axons In the ventral the sNPF peptide was localized in the connective thoracic axons neural which are and from the in and the axons which are in the of connective axons The neural of the sNPF from thoracic axons to the brain indicate that the sNPF function related to Also, the localization of the sNPF in the neural that the sNPF peptide functions in the The localization of the sNPF in and was not not In the adult brain, the sNPF peptide was in the medulla and and the mushroom body and The mushroom body is the for after Neuron. 1993; Full Text PDF PubMed Scopus Google Scholar, K. M. T. Science. 1996; 274: PubMed Scopus Google Scholar, A. T. B. E. Curr. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). These in larval CNS and adult brain that the function of sNPF is related to the of in thoracic neurons and in brain. In the adult the localization of the sNPF peptide was not not The sNPF Gene the function of the sNPF gene in feeding we the gain-of-function and loss-of-function R.W. 18: PubMed Scopus Google Scholar) to the system Development. 1993; PubMed Google Scholar) and generated mutants Science. PubMed Scopus Google Scholar). feeding which is expressed in the CNS and the during larval to adult stages M.A. 1999; Google Scholar), and were used K. Biehs B. Bier E. Development. PubMed Google Scholar). we the of the sNPF peptide in the and gain-of-function mutants and in the loss-of-function and mutants produced sNPF peptide wild-type whereas mutants produced less sNPF peptide In the feeding assay, we food for 10 to the adults and feeding larvae A and In the overexpression of sNPF the of whereas of the loss-of-function mutants the of flies with controls In feeding larvae, the feeding showed the which are overexpression of sNPF the of larvae, whereas of the loss-of-function mutants the of larvae with controls However, in wandering larvae, overexpression of the gain-of-function or the loss-of-function sNPF mutants not the of larvae with the control not is with the function of the dNPF peptide the dNPF overexpression in wandering larvae feeding (10Wu Q. Wen T. Lee G. Park J.H. Cai H.N. Shen P. Neuron. 2003; 39: 147-161Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), that sNPF and dNPF are in the of feeding behavior during the larval stage. The of feeding by the food that with a showed the increase of the by the sNPF from to In Drosophila the appetite signal the of food in larvae and the in adults J. Biol. PubMed Google Scholar, I. M. J. 2002; PubMed Scopus Google Scholar). We the food intake to the of flies by overexpression of the sNPF gene in the first 10 the appetite or The of flies after in the wild-type whereas overexpression of sNPF flies after that the sNPF gene regulate appetite and control food intake. The sNPF Gene the of the sNPF gene in the adult we the size and of the sNPF gain-of-function and loss-of-function mutant flies. of showed heavier and bigger flies flies or wild-type flies and that the overexpressing sNPF gene hyperphagia and the phenotype However, flies not the size and with wild-type flies and that is in Drosophila. The Drosophila sNPF gene, which encodes the preprotein containing putative sNPF was expressed in the nervous system of late stage embryos and larvae. The localization of the sNPF peptide in embryonic and larval nervous systems the neural circuit from the brain to thoracic axons and to connective axons in the larval CNS. In the adult brain, the sNPF peptide was localized in the medulla and mushroom body. From the feeding with the sNPF the gain-of-function sNPF mutants promoted food intake, whereas of loss-of-function mutants suppressed food intake. Also, the sNPF overexpression produced bigger and heavier flies wild-type flies. These findings indicate that the sNPF is expressed in the nervous systems to regulate food intake and controls body size in melanogaster. The sNPF preprotein is predicted to putative RLRF peptides and putative peptides by are and J. Peptides. 2001; PubMed Scopus Google Scholar). In the electrophysiological assay using Xenopus oocytes the receptor, the of or short sNPF peptide showed whereas the of or showed (14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar). the to the electrophysiological all sNPF peptides in the bioluminescence assay using Chinese hamster ovary the receptor (13Mertens I. Meeusen T. Huybrechts R. De Loof A. Schoofs L. Biochem. Biophys. Res. Commun. 2002; 297: 1140-1148Crossref PubMed Scopus (108) Google Scholar). However, the of all putative sNPF peptides in was not Recently, the analysis with the Drosophila larval CNS using the followed by the the peptide and the The was considered as the product during peptide was the product after the of the in the of G. A. De Loof A. Schoofs L. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the analysis with adult CNS using detected and the peptide was the product after the of the in the of R. C. J. PubMed Scopus (139) Google Scholar). These indicate that and peptides were not detected in larval and adult CNS, and sNPF peptides were the in as and The neural circuit of the sNPF from the brain to thoracic axons in the larval CNS that of the sNPF functions is related to thoracic axons a signal for that is detected by chemosensory neurons in the Res. PubMed Scopus Google Scholar). The neural from the brain to the in the ventral ganglion that the sNPF gene other The neural from the thoracic axons to the neural A and which are a system in that the sNPF functions as a The neural were detected by the of the antibody the Drosophila amide neuropeptide D.W. I. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of functions in the related peptide is feeding I. 1998; PubMed Scopus Google Scholar). In the adult brain, the sNPF peptide was in the medulla and the mushroom body The adult brain of Drosophila is of neurons that are into The which consists of the and is in the of in the brain I.A. The of Drosophila melanogaster. Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar, Res. Scopus Google Scholar, of an Scholar). The are for the of J. Neurosci. Full Text PDF PubMed Scopus Google Scholar). The mushroom are in and and other behaviors Neuron. 1993; Full Text PDF PubMed Scopus Google Scholar, M. Scholar, M. A. J. PubMed Scopus Google Scholar, Rev. 1996; PubMed Scopus Google Scholar). The mushroom body is also the for and the mushroom body K. M. T. Science. 1996; 274: PubMed Scopus Google Scholar, A. T. B. E. Curr. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The neural circuit from thoracic axons to brain connective axons in the larval CNS and the mushroom body in the adult brain that the function of the sNPF peptide is related to in thoracic neurons and the to the brain to The localization of the sNPF peptide in ganglion in the larval CNS that the function of sNPF related to To this the loss-of-function sNPF flies were also generated using the The similar were demonstrated to have RNA in Drosophila and R.W. 18: PubMed Scopus Google Scholar). the of food intake, the phenotype of showed bigger and heavier flies with the wild-type However, the size increase was not detected in flies also showed the phenotype that of the sNPF gene is to body size overexpression of sNPF in the showed the size by the of not to the body size of flies is the of the increase in the body. that sNPF and dNPF peptides have The sNPF peptide was in nervous whereas the dNPF was as the Drosophila peptide (7Brown M.R. Crim J.W. Arata R.C. Cai H.N. Chun C. Shen P. Peptides. 1999; 20: 1035-1042Crossref PubMed Scopus (202) Google Scholar). The of sNPF and dNPF in the larval brain. the sNPF was in the neurons of the brain whereas the dNPF was detected in the neurons of larval brain (7Brown M.R. Crim J.W. Arata R.C. Cai H.N. Chun C. Shen P. Peptides. 1999; 20: 1035-1042Crossref PubMed Scopus (202) Google Scholar). In the feeding behavior overexpression of sNPF in wandering larvae not the feeding not to the of the feeding in the wandering larval stage by overexpression of dNPF (10Wu Q. Wen T. Lee G. Park J.H. Cai H.N. Shen P. Neuron. 2003; 39: 147-161Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). the receptor peptide in for the receptor is for sNPF peptides (13Mertens I. Meeusen T. Huybrechts R. De Loof A. Schoofs L. Biochem. Biophys. Res. Commun. 2002; 297: 1140-1148Crossref PubMed Scopus (108) Google Scholar, 14Feng G. Reale V. Chatwin H. Kennedy K. Venard R. Ericsson C. Yu K. Evans P.D. Hall L.M. Eur. J. Neurosci. 2003; 18: 227-238Crossref PubMed Scopus (80) Google Scholar), and the DmNPFR1 receptor is for the dNPF (12Garczynski S.F. Brown M.R. Shen P. Murray T.F. Crim J.W. Peptides. 2002; 23: 773-780Crossref PubMed Scopus (139) Google Scholar). These indicate that the dNPF and sNPF peptides function in neurons and regulate of feeding behaviors in Drosophila. other the sNPF peptide in various physiological processes other food intake the sNPF peptide and were expressed during all developmental stages and and the sNPF is localized in the mushroom body and medulla of the adult brain The mushroom body is in and Neuron. 1993; Full Text PDF PubMed Scopus Google Scholar). These of the sNPF peptide in various processes are the of We Park for with the